AU669857B2 - Mutated growth factor receptor as medicament and its use for treating cancer - Google Patents

Description

DPI DATE 05/04/93

AO

1 JP DATE 10/06/93 APPLN. ID 25185/92 I I 111 PCT NUMBER PCT/EP92/02058 BIl~iIIUIIII11111li 111 AU9225 185 (51) Initernationale Patentklassifikation C12N 9/12, C07K 15/00 A61IK 37/02, 9/127, 48/00 Internationale Verdffentliiungsnummer: Al 43)Internationales Vcrtiffentlichungsdatum: 18 WO 93/05148 M.Irz 1993 (18,03.93) (21) Internationales Aktenzeic (22) Internationakes Anmeldet Prioritartsdaten: P 4129 533.1 PCT/EP92/02058 7. September 1992 (07.09.92) 5. September 1991 (05.09.91) DE (74) Anwilte: BEZOLD, Gunter usw. Maximilianstr, 58, D- 8000 Mttnchen 22 (DE).

(81) Bestimmungsstaaten: AT, AU, BB, 13G, BR, CA, CH, CS, DE, DK, ES, Fl, G13, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, NO, PL, RO, RU, SD, SE, europttisches Patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, lE, IT, LU, MC, NL, SE), OAPI Patent (BF, Ri, CF, CG, Cl, CM, GA, GN, ML, MR, SN, TD, TG).

Ver6ffentlicht Alit intcrnationalen Recherclzcnberici (71) Anmelder: MAX- PLANC K-G ESELLSC HAFT ZUR FORDERUNG DER WISSENSCRAFTEN E.N. [DEl D 13*aseO-G tt-66fger-(E-).

(72) Erfinder: REDEMANN, Norbert ULLRICH, Axel Max-Planck-lnstitut fMr Biochemie, Am Klopferspitz 18A, D-8033 Martins~ied (DE).

669857 (54) Title: MUTATED GROWTH FACTOR RECEPTOR AS MEDICAMENT AND ITS USE FOR TREATING CANCER (54)Bezeichnung: MUTIERTER WACH-STUMSFAKTURREZEPTOR ALS ARZNEIMITTEL UND SEINE VERWEN- DUNG ZUR BEHANDLUNG VON KREBS (57) Abstract Mutated receptor tyrosine kinases are useful as medicaments. These mutated growth factor receptors are particularly advantageous for treating cancerous diseases, in particular those types of cancer in which the overactivity of growth factor receptors play a role in the formation of the cancer, as well as for treating other diseases caused by receptor overactivity. Mutants of the EGF receptors in which the tyrosine kinase activity of the wild type receptor has been eliminated by punctual mutation or deletion in the tyrosine kinase domain are particularly effective medicanments for treating cancer, (57) Zusammenfassung Die vorliegende Erfindung betrifft mutierte Rezeptortyrosinkinasen, die sich als Arzneimittel eignen. lBesonders vorteilnaft sind die mutierten Wachistumsfaktorrezeptoren zur Behandlung von Krebserkrankungen, insbesondere von solchen Krebsarten, bei denen die Clberfunktion von "Wachstumsfaktorrezeptoren eine Rolle bei der Krebsentstehung spielt sowie von anderen Krankheiten, die auf der (iberfunktion der Rezeptoren beruhen. Als besonders wirksames Arzneimittel zur Behandlung von Krebs werden Mutanten des EGF-Rezeptors offenba-t, bei denen die Tyrosinkinaseaktivitfit des Wildtyp-Rezeptors dureb eine Punktmutation oder Deletion in der Tyrosinkinase-Domlne elimniert worden ist.

MUTATED GROWTH FACTOR RECEPTOR AS A DRUG AND ITS USE FOR THE °O"v TREATMENT OF CANCER The present invention relates to mutated receptor tyrosine kinases that are defective in their sign°lling activity and have therapeutic properties, drugs containing at least one mutated receptor and the use of the mutated receptor(s) for the treatment of diseases associated with an uncontrolled hyperfunction of receptor tyrosine kinases, in particular cancer.

Cell growth is a carefully controlled process depending on the special needs of an organism. In a young organism, the cell division rate outweighs the dying rate of cells, which leads to an increase in size of the organism. In a fully grown organism, the new growth of cells and cell death are so balanced that a steady state is formed. However, in infrequent cases, the control of cell division collapes, and the cells begin to grow and to divide themselves, although there is no special need for a higher number of cells of this type in the organism. This uncontrolled cell growth is the cause of cancer: Factors which can cause the uncontrolled cell growth connected with metastasis are often of a chemical nature, but can also be of a physical nature such as radioactive radiation.

At present, two alternatives are substantially available for the treatment of cancer. Either one succeeds in completely removing the cancer cells from the diseased organism by surgical intervention or it is attempted to render the transformed cells in the organism innocuous, e.g. by the administration of drugs or by physical treatment methods such as radiotherapy.

Drugs are often used in chemotherapy, which interfere with the DNA metabolism in some way or other and damage rapidly growing cells, which must furnish a higher DNS metabolic efficiency, more strongly than cells which do not divide or only divide slowly. A serious disadvantage of many chemotherapies, however, is the 2 low specifity of the used active substances, the result of this being that healthy cells are also damaged in chemotherapy. This low specifity of the active substances requires furthermore that their dosage must in each case be made so that as few healthy cells as possible are damaged with the simultaneous killing of the cancer cells. This is often not possible, and the patient suffering from cancer dies due the increasingly spreading cancer cells which cause the failure of vital functions in the terminal stage.

It is the object of the present invention to make available a further active substance with valuable therapeutic properties and a drug containing the active substance, the active substance or the drug being especially advantageous in the treatment of cancer diseases.

This object is attained according to the invention by a mutated receptor tyrosine kinase that is defective in its signalling activity and by a drug containing at least one mutated receptor tyrosine kinase.

The terms which 're used in the present text shall now be explained in more detail for a better understanding of the present invention: 3 "Receptor tyrosine kinase" means any kind of receptor exhibiting tyrosine kinase activity. The term includes growth factor receptors exhibiting tyrosine kinase activity, as well as HER2 or met receptors.

"Defective in its signalling activity" means that a mutated receptor is no longer capable of converting an extracellular growth signal or another signal to an intracellular signal, so that said signal is partly inhibited or fully blocked.

"Growth factor" means any mitogenic chemical, normally a polypeptide which is secreted from normal and/or transformed mammalian cells and plays an important role in the regulation of cell growth, especially in the stimulation of the proliferation of cells and the maintenance of their viability. The term "growth factor" includes, the epidermal growth factor (EGF), the platelet-derived growth factor (PDGF) and the nerve growth factor (NGF).

"Growth factor receptor" means a polypeptide which spans the cell membrane and binds a growth or differentiation factor and has itself a tyrosine kinase activity in its intracellular part or is associated with such an activity.

4 "Mutated receptor tyrosine kinase" means a tyrosine kinase receptor which contains a structural change in comparison with the wild-type receptor, so that the receptor no longer possesses the tyrosine kinase activity of the wild-type receptor.

"Mutated growth factor receptor" means a growth factor receptor which contains a structural change in comparison with the wild-type receptor, so that the receptor no longer possesses the tyrosine kinase activity of the wild-type receptor.

"Wild-type growth factor receptor" or other "wild-type" receptor means a naturally occurring growth factor receptor or other receptor which exhibits tyrosine kinase activity and is thus capable of transmitting signals.

"Extracellular domain" of the growth factor receptor or other receptor means the part of the receptor that normally projects from the cell into the extracellular environment.

The extracellular domain comprises, for instance, the receptor part to which a growth factor or another molecule binds.

"Transmembrane region" of the growth factor receptor or other receptor means the hydrophobic part of the receptor that is normally localized in the cell membrane of the cell which expresses the receptor.

"Tyrosine kinase domain" or "cytoplasmatic domain" of the growth factor receptor or other receptor means the part of the receptor that is normally positioned within the cell and effects the transphosphorylation of tyrosine residues "An effective amount" means an amount of the composition of the invention which can achieve the desired therapeutic effect.

"Platelet-derived factor (PDGF)" means a mitogenic polypeptide which is contained in blood platelets and stimulates mesenchyme-derived cells and stimulates the autophosphorylating protein tyrosine kinase activity when it binds to the PDGF wild-type receptor.

"Epidermal growth factor (EGF)" means a mitogenic polypeptide which normally produces a mitogenic response in fibroblasts and which stimulates the autophosphorylating protein tyrosine kinase activity of the EGF wild-type receptor.

"Hyperplasia-based disease" means a disease of a tissue or organ, including, for instance, skin epidermis, the intestinal epithelium, hepatic cells, fibroblasts, marrow cells, other bone cells, cartilage, and unstriated muscles, the disease being characterized by an increase in the number of cells of the tissue or oroan, such as psoriasis and endometric hyperplasia.

6 "HER2" means a receptor tyrosine kinase which exhibits sequence homology to the epidermal growth factor receptor.

"Liposomes" mean particles in an aqueous medium which are formed by lipid bilayers that enclose an aqueous enviroment.

"Overactivity" means an excessive uncontrolled activation of a signal transmission path imparted by growth factor receptors, which results in an excessive cell division activity and other consequences such as those occurring in some cancer cells in comparison with the normal cells of a similar cell type.

"Recombinant vectors" mean vectors which were genetically changed using recombinant DNA technology to incorporate nucleic acid fragments that code for normal or mutated receptor tyrosine kinases. The recombinant vectors can infect target cells and induce the target cells to express normal or mutated receptors.

"Recombinant retroviral vectors" mean recombinant vectors which are retroviruses.

In accordance with the present invention, it was possible to show that mutated receptor tyrosine kinases have 7 valuable therapeutic properties which can be exploited for treating diseases associated with a hyperfunction of receptor tyrosine kinases, the mutated receptors being in particular suited for treating cancer diseases.

Growth factor receptors play a decisive role in the development and multiplication of human cancer cells.

In healthy cells, growth factor receptors are involved in the control of cell growth. The actual signal for cell division is the growth factor which is formed as a function of the needs of the organism. The receptor assumes the function of signal transmission, i.e. it is involved in the conversion of the extracellular growth signal to cell division activity in the interior of the cell. In many growth factor receptors, their ability to transfer phosphate residues to tyrosine residues in proteins after b;nding the growth factor to the extracellular domain represents a decisive roll. These receptors are also designated as receptor tyrosine kinases. An overview of receptor tyrosine kinases is found in Yarden, Y. and Ullrich Rev. Biochem. 1988, 57, 443-78. The dimerization of these growth factor receptors after binding of the growth factors is another important phase of the process of signal transmission. The conversion of an extracellular signal to an intracellular signal by means of growth factor receptors with tyrosine kinase activity can be divided into the following five steps: 8 1) The binding of the growth factor (also designated as ligand) to the extracellular domain of the receptor induces a change in conformation; the same causes 2) dimerization of receptors which changed conformation; with 3) simultaneous induction of an allosteric change in the cytoplasmatic domain, by means of which, on the other hand, the kinase activity is induced; 4) transphosphorylation of tyrosine residues in the receptor dimer, which, in turn, produces and stabilizes an activated receptor conformation; and phosphorylation of polypeptide substrates and interaction with cellular factors.

Uncontrolled overactivity of this signal transmission chain due to receptor overexpression or mutation can lead to an excessive division activity of the corresponding cell, and in the extreme case, to a transformed cancer cell.

An overview of growth factor receptors and their function in signal transmission from the extracellular to the intracellular environment and the possible influence of abnormally expressed receptors on the development of cancer are indicated in Ullrich, A. and Schlessinger, J.

(1990) Cell 61, 203 212.

9 The epidermal growth factor receptor (EGF-R) is a 170 kD glycoproteir. with tyrosine kinase activity (Ullrich et al.

(1984), Nature 309, 418 425). The molecular processes in the binding of its ligand and the stimulation of its kinase activity are described in detail in Ullrich et al., Cell (1990) 61, 203 212. Although EGF normally causes a mitogenic response in fibroblasts, an overactivity of the signal transmission process by the EGF receptor on account of overexpressed receptors leads to a ligand-dependent transformation of NIH 3T3 mouse cells (Riedel et al.

(1988), Proc. Natl. Acad. Sci., USA 85: 1477 1481 and Di Fiore et al. (12987), Cell 51, 1063 1070). Intensive clinical studies support the function of this receptor in the development of specific carcinomas, such as mastocarcinoma, ovarian carcinoma and pulmonary carcinoma (Slamon et al. (1987), Science 235, 177 182 and (1969) Science 244, 707 712 and Kern et al. (1990) Cancer Res.

5184 5191).

It was surprisingly found that the five-step signal transmission chain explained above can be blocked or inhibited by a mutated growth factor receptor, if the mutated receptors are simultaneously expressed with the wild-type receptors from one cell. Thus, mutated signalling defective growth factor receptors are suited as drugs with which diseases can be treated which are connected with an increased transmission of growth signals to the interior of the cell by corresponding receptors.

A preferred receptor mutant does no longer have the tyrosine kinase activity of the wild-type receptor.

This mutant is no longer capable of phosphorylating tyrosine residues in the receptor dimer or in the polypeptide substrates after ligand binding. Thus, the conversion of the extracellular growth signal to an intracellular signal is blocked or partly inhibited.

A point mutation in the wild-type receptor can already be sufficient so that the wild-type receptor does no longer function properly if it lost the tyrosine kinase activity due to point mutation. Such a point mutant (e.g.

HERK721A) is especially preferred.

A mdtated receptor is furthermore preferred, which carries a deletion in the tyrosine kinase domain, which leads to a loss of tyrosine kinase activity.

It is preferred that the receptor mutated by a deletion in the cytoplasmatic domain Hs, however, still the transmembrane regiozfi'f utated receptors with existing transmembrane region lead to a more effective inhibiton of growth signal transmission and thus exhibit a better therapeutic effect than receptors without transmembrane region such as mutants which only consist of the extracellular domains FIG. 1, HERCD-566).

11 Mutants of receptor tyrosine kinases such as of EGF, PDGF, IGF-I, MET receptors, EGF receptor-related receptors such as HER2, neu, C-erbB2 receptors or NGF receptors are especially suited as a drug. The MET protein, for instance, is described in detail in Giordano et al. (1988), Molec.

Cell. Biol. 8, 3510 3517 and in Giordano et al. (1989) Nature, 339.

A mutated receptor of the epidermal growth factor (EGF) is very particularly suited.

In a particularly, preferred mutant of the EGF receptor there is a point mutation at the amino acid position 721 of the wild-type receptor sequence. In a preferred mutant the lysine residue at position 721 is replaced by an alanins residue in the mutant. This mutant is deposited under DSM 6678 with the German Collection of Microorganisms and Cell Cultures GmbH under the Budapest Treaty. In a further preferred EGF receptor mutant the 533 C-terminal amino acids of the wild-type receptor are deleted. This mutant is deposited under DSM 6679.

The receptor mutants can be produced according to customary genetic engineering processes such as described in Sambrook, J. et al (1989) Molecular Cloning, Cold Spring Harbor Laboratory 7 .ess starting from the wild-type receptors.

12 The drug according to the invention contains at least one of the mutated growth factors described above and the customary adjuvants and carrier substances.

A drug is especially preferred which contains the receptor mutant(s) packed in liposomes. In order to bring the liposomes selectively to the target tissue it is advantageous if the liposomes contain antibodies in their membrane, the antibodies recognizing specific epitopes of the target cells and being bonded selectively to them.

Thus, the receptor mutants are targeted to the target tissue and can evolve their desired effect there. The administration of active substances packed in liposomes is a common form of administration nowadays.

A drug is furthermore preferred which contains the 15 receptor(s) in the form of one or several recombinant retroviral vectors. The recombinant vectors contain nucleic acid fragments coding for the receptor(s). After administration of the drug to the patient, the retroviruses infect the target cell and lead to the expression of the S 20 receptor mutants therein.

;An especially preferred drug contains the retroviral vectors pNTK-HER-K721A and/or pNTK-HERCD-533 coding for EGF receptor mutants, which were both deposited with DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, German Collection of Microorganisms), Mascheroden Weg 1B, D-3300 Braunschweig, under DSM 6678 and DSM 6679, respectively, on 22 August 1991.

S Helgo Schrapp Veronikastr. 81827 MOnchen TeI 0891439 67 57 Fax:0891439 68 58 13 The mutated receptors can be incorporated into a drug in the conventional manner, as is, for instance, described in Remington's Pharmaceutical Sciences (Osol, A. editor) Mack Publishing Company, Easton, PA (1980) and consecutive volumes.

The mutated receptors described above and/or the drugs containing them are especially suited for the treatment of cancer. Such types of cancer can be treated especially well, which are a result of an overactivity of growth factor receptors. These types of cancer include in particular mastocarcinoma, ovarian carcinoma and pulmonary Scarcinoma. The role of surface receptors in these cancer diseases is described in detail in Slamon, D.J.

et al (1987), Science, 235, 177 182 and (1989) Science, 244, 707 712 and Kern, J.A. et al (1990), Cancer Res., 50, 5184 5191.

The pharmaceutical composition can contain salts, buffers, additives and other substances that are desirable for improving the efficiency of the mutated receptors.

SHelga SchrapP Veronikastr. t e 81827 Munchn -Tel. 0891439 67 57 g SFax: O89/14398 5 8 14 Compositions for the parenteral administration comprise sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous suspensions for injection may contain substances increasing the viscosity of the suspension and comprise, for instance, sodium carboxymethylcellulose, sorbit and/or dextran. The suspension may optionally contain stabilizers. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters such as ethyl oleate.

Carrier substances or occlusive plasters may be used for increasing skin permeability as well as the dermal adsorption of the drug.

Liquid forms of dosage may typically include a liposome solution containing the liquid form of dosage. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrup and elixirs with inert dilutants which are normally used in this field, e.g., purified water.

Apart from the inert dilutants, these compositions may also include additives, wetting agents, emulsifying or suspending agents or aromatics. Examples of other materials suited for use in the present pharmaceutical composition are indicated in Remington' Pharmaceutical Sciences (Osol, editor), Mack Publishing Co., Easton, PA (1980) and consecutive volumes.

The treatment of an individual having a tumor includes the administration of an effective amount of the mutated receptors or recombinant vectors, which produce the mutated receptors, in a single dose, several doses or in the form of an infusion on a patient or an animal.

In accordance with the present invention an "effective amount" of a pharmaceutical composition is an amount which is sufficient to achieve the desired biological effect. As a rule, the dosage which is needed for providing an effective amount of the composition and which can be set by an expert will depend on factors, such as the receptor to be specifically used, the presence and kind of other therapeutical agents, the age of the patient or animal and the condition, sex and clinical state thereof, including the extent of the disease and other variables.

The preferred dose of the pharmaceutical composition of the invention in a human being is >10 9 plaque-forming units (pfu) per person and depends on the type of cancer and the extent of the receptor hyperfunction.

The preferred method of administering the pharmaceutical composition of the invention is a parenteral method. The most preferred way is an intravenous, intraperitoneal or topical one or directly into the brain, the spinal cord liquid or the tumor itself.

16 Without being committed to a specific theory, it is assumed that the described receptor mutants evolve their effect in the target cells in that the mutants are incorporated into the membrane of the target cells in addition to the wildtype receptor, and the receptor mutant then impairs the function of the wild-type receptor by forming signalling incompetent dimers consisting of one wild-type or oncogenic receptor and one signalling defective mutated receptor.

The present invention is described in more detail by means of mutants of the EGF receptor as a model.

It was surprisingly found that the expression of EGF receptor mutants which do no longer have any tyrosine kinase activity can reverse the transfomred phenotype in transformed cancer cells expressing the EGF wild-type receptor.

Material and Methods: Production of recombinant retroviruses The retroviral expression vectors pN2, pNTK2 and pNTK-HERc are described in detail in Keller, G. et al, (1985), Nature, 318, 149 154; Stewart, C.L. et al, (1987) EMBO 6, 383-388; von RUden, T. and Wagner, E.F. (1988), EMBO 7, 2749-2756. pNTK-HER-K 721A was produced by cloning a Bgl II fragment of CMVHER-K721A in pNTK-HERc. pNTK-HERCD-533 was produced by making a 17 Clal site at both sides of the 2 kb large fragment XbaI/XhoI of pLSXNA 8, described in Livneh, E. et al, J.

Biol. Chem., 260, 12490-12497 by means of customary-cloning processes as described in Sambrook, J. et al (1989), Molecular Cloning, Cold Spring Harbour Laboratory Press, and subsequently the 2 kb Clal fragment was ligated with Clal-cleaved pNTK2. The NTK-HERCD-566 construct was produced by cloning a Clal fragment of CVNHERXCD into the Clal site of pNTK2. The construct was deposited with the German Collection of Microorganisms CDSM under DSM 6680 on 22 August 1991. Ecotrophic recombinant retroviruses were produced from the helper-virus-free producer line GP+E-86, described in Markowitz, D. (1988) J. Virol., 62, 1120-1124.

Stable GP+E-86 producer lines were produced by means of a S 15 modified infection instruction as described in Miller, A.D.

and Buttimore, C. (1986) Mol. Cell. Biol., 6, 2895-2902.

An amphotrophic virus with low titer was produced by the S" transient transfection of retroviral expression plasmids into the helper-virus-free packaging cell line PA317, S 20 described in Miller, A.D. et al (1985) Mol. Cell. Biol., :431-437, and was used to infect secondary packaging cells GP+E-86, followed by a selection of clones of the producer line GP+E-86 in G418 (1 mg/ml). The virus titer was o e determined by infecting NIH 3T3 cells with dilution series S 25 of retrovirus which contained cellfree GP+E-86 supernatants, and determination of the number of the G418 resistant colonies. A retrovirus (42TGFa) which contains the gene for the tumour growth factor a(TGFa) is described in Blasband, A.J. (1990), Oncogene, 5, 1213-1221.

7 Helga Schrapp S Violihaslr. 15 r 81827 Munchen A Tel. O4 398757 SFax 089/4396858 18 Gene transfer by means of retroviruses Subconfluent NIH 3T3 c-lls (105 cells/6 cm plate) were incubated with supernatants of GP+E-86 cells which release high titers of NTK-HERc virus (5x10 5

G

418

R

colony-forming units per ml) for 4 to 12 hours in the presence of 4 Ag/ml Polybrene (Aldrich) and subsequently in a supernatant of GP+E-86 cells which release high titers of either N2, NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566 viruses. The expression level of the receptors was increased by several infection cycles as described in Bordignon, C. et al (1989), Proc. Natl.

Acad. Sci, USA 86, 6748 6752. In the described experiments, the infection was once carried out with 1 ml of a diluted supernatant (1.25 x 105 colony-forming units) or 1 to 4 times with the same volume of undiluted supernatant (5 x 105 colony-forming units) of GP+E-86 cells which release high titers of either N2, NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566 viruses.

Receptor phosphorylation in intact cells The cells infected as indicated above were cultivated in 10cm plates up to a 90% confluence, washed and cultivated for 16 hours in methionine-free DMEM (Gibco) supplemented with 1% FCS containing 50Ci/ml 35 S-methionine (Amersham). The cells were stimulated with ng/ml EGF (Amgen Corp.) for 10 minutes and lysed in ml lyse buffer (50 mM Hepes pH 7.2, 150 mM NaCl, 1.5 mM MgC1 2 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 10 mg/ml Aprotinin 100 iM sodium orthovanadate) >t 4°C. The lysates were centrifuged in an Eppendorf 19 centrifuge at about 12,000 g at 4°C for 10 minutes. The supernatants were than incubated with an excess of monoclonal antibody 108.1, described in Honegger, A.M., Ullrich A. and Schlessinger, J. (1989) Proc. Natl.

Acad. Sci, USA, 86, pages 925 929, and protein A-sepharose at 4 0 C for 4 hours. Immuno- precipitates were washed twice with HNTG (20 mM Hepes pH 7.3, 150 mM NaCI, 0.1% Triton X-100 and 10% glycerol). The pellet was resuspended in a sample buffer, boiled for minutes and analysed by means of SDS-PAGE The proteins were electrophoretically transferred to nitrocellulose and subsequently incubated with a monoclonal mouse antibody against phosphotyrosine E2), described in Fendly, B.M. et al (1990) Cancer Research, 50, 1550-1558. For detection, the nitrocellulose filter was incubated with a peroxidase-coupled goat anti-mouse antibody, followed by an ECL substrate reaction (Amersham). After detection of the ECL substrate reaction with a Kodak X-Omat film, the nitrocellulose filters were washed with PBS containing 0.2% Tween 20. Subsequently the 35 S-methionine-marked proteins were detected by means of autoradiography. The density of the bands was ascertained by means of densitometry.

Helga Schrapp Veronikastr. 15 l 81827 MQnchen Tel 089/4396757 Fax; 089/4396858 3 H]-thymidine incorporation Subconfluent NIH cells 3T3 (105 cells/6cm- plate) were coinfected with NTK-HERc as described, followed by 4 infection cycles with either N2, NTK-HERK721A, NTK- HERCD-533 or NTK-HERCD-566. The cells were distributed to 12-well Costar plates. After the reaching of confluence, the cell monolayers were starved in 0.5 ml DMEM, 0.5% FCS for 24 hours, and, 18 hours after EGF addition, the cells were labeled with 0.5 uCi Mechyl-- 3 H]-thymidine (Amersham) for 4 hours. The cells were washed twice with PBS and subsequently precipitated on ice with 10% TCA for 1 hour. The precipitate was washed with 10% TCA and dissolved again in 200 pl 0.2 N NaOH/0.2% SDS. The lysates were neutralized, and the incorporated radioactivity was quantitatively determined by scintillation counting.

Transformation tests In order to examine the ability of NIH 3T3 cells for colony formation in soft agar, subconfluent NIH 3T3 cells (10 5 cells/6cm-plate) were infected with NTK-HERC, followed by 4 cycles of infection with either N2, NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566. In the cases in which an autocrine stimulation was to be caused, the cells were infected with Y2TGFa virus (5 x 104 G418

R

of colony-forming units per ml). NIH 3T3 Tel. 089/439 67 57 241 cells (105) were plated on 6cm- plates in the presence or absence of 10 ng/ml EGF in a top layer of 3 ml ME, containing 10%o FCS an 0.2% agar (Gibco).

The bottom layer contained MEM, 10% FCS and 0.4% agar.

Visible colonies were counted after 4 weeks.

For foci formation tests subconfluent NIH 3T3 cells 5 cells /6 cm-plate) were coinfected with NTK-HERc (1x10 4 G418R colony-forming units per ml), followed by 4 cycles of infection with either N2, NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566 viruses. In some experiments the cells were superinfected with 2TGFa virus (1 x 103 G418R colony-forming units per ml).

Infected cells were cultivated on 6 cm-plates with DMEM containing 4% FCS in the presence or absence of ng/ml EGF. The medium was replaced every 3 days. The plates were stained with crystal violet, and the foci were counted on day 18.

Legends for the Figs.

Fig. 1: Schematic representation of the human EGF wild-type receptor and mutated EGF receptors. The position of domains rich in cysteine (cys), of the tyrosine kinase (TK) and the transmembrane (TM) domains is indicated. The mutant HERK721A carries a point mutation at position 721 (an exchange of lysine to alanine), while HERCD-533 and HERCD-566 carry C-terminal deletions of 533 and 566 amino acids, respectively.

The mutants are described in detail in Livneh, et al ('1986) J. Biol. Chem., 260, 12490-12497 and Honegger A.M. et al (1987) Cell. 51, 199-209.

Fig. 2 Tyrosine phosphorylation of the wild-type and mutated EGF receptors. Cells which either S Helga Schrapp i Veronikastr. 15 T 81827 MOnchen S Tol. 089/4396757 Fax: 089/4396858 express the wild-type receptor alone or coexpress the wild-type receptor and mutated receptors were labeled with 35 S]-methionine overnight and subsequently incubated in the presence or absence of 2 ng/ml EGF for minutes. The cells were dissolved and precipated with anti-EGF receptor antibody (mAb 108), separated by SDS-PAGE and analysed immunologically with anti-phosphotyrosine antibodies (5E2), followed by an ECL substrate reaction.

Expression of the EGF receptor on NIH 3T3 cells. Cells which express either the wild-type receptor alone or coexpress the wild-type receptor and mutated receptors were labeled with [3 5 S]-methionine overnight and subsequently incubated in the presence or absence of 20 ng/ml EGF for 10 minutes. The cells were dissolved and precipitated with anti-EGF receptor antibody (mAb 108), separated by means of SDS-PAGE and immunologically detected with an anti-phosphotyrosine antibody (5E2), followed by an ECL substrate reaction. The ECL substrate was washed with PBS containing 0.2% Tween and the [35S]-methionine-marked proteins were detected by means of autoradiography.

Fig. 3: EGF-simulated [3 H-thymidine incorporation.

Cells which either expressed the wild-type receptor alone (dashed line) or coexpressed the wild-type receptor and mutated receptors as in A: wild-type EGF receptor K721A, B: wild-type EGF receptor +CD-533, C: wild-type EGF receptor CD-566 were cultivated in 12- well Costar plates up to confluence and starved for 2 days in DMEM containing 0.5% FCS. 10% FCS or different EGF concentrations were added, and, 18 hours after the EGF addition, 3 H]-thymidine (0.5 ACi/jell was added for 4 hours and its incorporation in DNA was determined. The -a 23 mitogenic response was recorded in order to show the relation between dose and response. The values were corrected by the basal thymidine incorporation and the maximally observed response to EGF was defined as 100%.

The filled triangles indicate the semi-maximal thymidine incorporation.

Results: Cells which express the wild-type receptor alone or together with the mutated receptors were labeled with 3 5 S]-methionine, incubated in the presence or absence of EGF for 10 minutes, lysed and immuno- precipated with a mouse antibody against human EGF receptor (mAb 108). The samples were separated with SDS-PAGE, transferrd to nitrocellulose filters, and the tyrosine phosphorylation was detected by means of the phosphotyrosine-specific mouse antibody 5E2 (Fig. 2A). The amount of the receptor present in the immunoprecipitate was detected by means of autoradiography of the same nitrocellulose filter (Fig. 2B).

As shown in Fig. 2A, lanes b and c, the EGF addition to intact NIH 3T3 cells, which are infected with the virus containing the wild-type EGF receptor, induces a strong tyrosine phosphorylation of the 170 kD EGF receptor band. Due to phosphorylation, the electrophoretic mobility of the EGF receptor decreases in the SDS-PAGE as compared with the unphosphorylated EGF receptor as can be seen from Fig. 2B, Lanes b and c. The level of the EGF-stimulated phosphorylation of the wild-type receptor was not reduced by the co-expression of the soluble extracellular domain of the EGF receptor as encoded by the NTK-HERCD-566 virus genome even if the extracellular domain was expressed in a 4-fold excess to the wild-type receptor (Fig. 2A, lanes d to f; Fig. 2B, LIAes d to f).

On the contrary, in an analogous experiment in which a virus which expresses the membrane-'anchored EGF receptor deletion mutant HERCD-533 (Fig. 1) was used, a strong dose-dependent inhibition of the EGF-induced phosphorylation of the wild-type EGF receptor was observed (Fig.2A, lanes g to although the level of the 170 kD EGF receptor protein remained constant (Fig.

2B, lanes g to The intensity of the tyrosinephosphorylated bands decreased from 100% to 71% or respectively. Under these conditions the EGF receptor had the same electrophoretic properties as a non-phosphorylated receptor (Fig. 2B, trace which is in conformity with its state of tyrosine phosphorylation, detected by mAb 5E2 (Fig. 2A, Lake i).

if the wild-type receptor was co-expressed with the kinase-negative mutant HERK721A, an increased tyrosine phosphorylation of the 120 kD band was detected (Fig.

2A, lanes k to The intensity of the signal of the tyrosine phosphorylation of the receptor increased from 251% to 337% or 450%, respectively, according to the densitometric analysis of the autoradiographs Since the wild-type receptor and the kinase-negative mutant are of the same size, the increased 170 kD signal in Fig. 2B, lanes k to m, represents the sum of the phosphorylation of both receptors since the mutated receptor can be transphorylated by the wild-type receptor.

Inhibition of the EGF-induced cell division rate.

SoJ0 EGF stimulates cell division in NIH 3T3 fibroblasts expressing the EGF receptor as described in Riedel, H.

et al (1988) Proc. Natl. .cad. Sci. USA, 85, 1477 1481 and Prywes, R. et al (1986) EMBO 5, 2179-2190.

The influence of mutated receptors on the cell division controlled by the EGF wild-type receptor was determined by means of the induction of the DNA synthesis.

The DNA synthesis was determined in cells infected with the NTK-HERc virus and the N2 virus control as [3H]-thymidine incorporation, and the synthesis was maximally stimulated at 2 ng/ml EGF, with a semimaximal stimulation (ED 50 at 0.66 ng/ml (Fig. In similar fashion as in earlier results as described in Honegger, A.M. et al (1988) EMBO 7, 3045 3052, and Riedel, et al (1988) Proc. Natl. Acad. Sci., USA, 85, 1477-1481 higher EGF concentrations led to lower levels of 3 H]-thymidine incorporation as shown in Fig. 3. The co-expression of the EGF receptor with HERCD-533 and HERK721A led to a marked shifting of the dose-dependent curve towards higher EGF concentrations after four cycles of infection with the corresponding viruses (Figs. 3A and This indicates that the cells have become less sensitive to the growth factor as compared with HERc/N2 cells. Both the deletion mutant HERCD-533 and the point mutant HERK721A (Fig. 1) showed similar effects on the cell division signal imparted by the EGF wild-type rceptor and caused a ten-fold increase of ED 50 to 6.6 ng/ml EGF. As opposed to this, the superinfectipn with NTK-HERCD-566 virus did not have any significant effect on the DNA synthesis stimulated by the wild-type receptor by EGF (Fig. 3C).

Anti-oncogenic activity of the EGF receptor mutants It is known that the overexpression of the EGF receptor causes an EGF-dependent cell transformation of NIH 3T3 cells as described in Di Fiore, P.P. et al (1987), Cell, 51, 1063-1070, Velu, T.J. et al (1987), Science, 237, 1408-1410 and Riedel, H. et al (1988) Proc. Natl.

Acad. Sci., USA 85, 1477-1481. In order to examine whether the transforming potential of overexpressed EGF receptor can be inhibited by EGF receptor mutants, the EGF receptor was co-expressed with receptor mutants, and subsequently their ability of producing colonies in soft agar or foci in a monolayer cell culture was examined. The stimulation of overexpressed EGF receptor was either achieved by the addition of EGF to the medium or by infection with a virus P2TGFa), which carries a TGF-a DNA in order to produce an autocrine activation system (table 1: average values from four experiments are shown).

After infection with the NTK-HERc virus and the N2 control, NIH 3T3 cells formed about 250 colonies in soft agar in the presence of 10 ng/ml EGF. By means of coinfection with Y2TGFG virus the formation of 148 colonies under otherwise identical conditions (table 1) was achieved. However, if the cells infected with the EGF receptor were superinfected either with NTK-HER- K721A or NTK-HERCD-533 viruses, the colony-forming capacity was suppressed almost completely. The co-expression of the EGF receptor with the extracellular domain HERCD-566 reduced the colony-forming ability by about 50% with stimulation with 10 ng/ml EGF in the agar layer and by about 33% with stimulation by the autocrine TGF after infection with the 4TGFa virus.

In similar fashion, the focus-forming potential of the NTK-HERc virus was determined in NIH 3T3 monolayer SHelga Schrapp S Veronikaslr. S 81827 MOnchen Tel. 089/4396757 .J F Fax 089/4396858 cultures, either in the presence of 10 ng/ml EGF, which led to 920 foci per 106 viruses or after coinfection with P2TGFa virus, which led to 480 foci per 106 NTK-HERC viruses. Superinfection with NTRK-HERK721A or NTK-HERCD-533 viruses suppressed the number of the foci by 100% or 90%, respectively, if the stimulation with EGF was effected and by 75% or 71%, respectively, in the case of stimulation with the VTGFa virus (table 2).

Cells which co-expressed the EGF wild-type receptor and HERCD-566 showed the same number of foci as cells expressing the EGF receptor and infected with the control virus N2. This result was observed both in the stimulation with EGF and with the 42TGFa virus.

The aforementioned statements reveal that the EGF receptor mutants have both a marked antiproliferative and an anti-oncogenic potential and are thus excellently suited for the treatment of cancer.

Table 1: Colony-formation in soft agar 11 Number of colonies /106 CFU Infection-- ng/ml EGF 1V2 TGFoj N2 0 0 NTK-HERK721A 0 0 NTK-HERCD-533 0 0 NTK-HERCD-566 0 0 NTK--HERc/N2 246 148 NTK-HERc/NTK-HERK721A 8 2 NTK-HERc/NTK-HERCD-533 6 4 NTK-HERc/NTK-HERCD-566 128 100 The colonies were counted after 4 weeks. The values represent average values from four independent experiments. CFU means colony-forming units.

Table 2: NIH 3T3 focus formation Number of foci/10 6

CFU

Cell line-- rig/mi EGF 4f2 TGFu N2 0 0 NTK-HERK721A 0 0 NTK-HERCD-533 0 0 NTK-HERCD-566 0 0 NTK-HERc/N2 920 480 NTK-HERc/NTI(-HERK721A 40 18 NTK-HERc/NTK-HERCD-533 90 14 NTK-HERc/NTK-HERCD-566 910 500 The foci were counted after 14 to 16 days. The values represent average values from four independent experiments. CFU means colony-forming units.

Claims (22)

1. A mutated receptor tyrosine kinase that is defective in its signalling activity.

2. A mutated receptor according to claim 1, characterized in that it no longer has the tyrosine kinase activity of the corresponding wild-type receptor.

3. A mutated receptor according to claim 2, characterized in that it carries a deletion in its tyrosine kinase domain.

4. A mutated receptor according to claim 2, characterized in that it carries a point mutation in its tyrosine kinase domain. A mutated receptor according to claim 2, characterized in that the receptor contains its extracellular domain and its transmembrane region.

6. A mutated receptor according to claim 2, characterized in that the receptor includes an extracellular domain.

7. A mutated receptor according to claim 5, characterized in that the extracellular domain and the transmembrane region derive from the wild type. ,R-I 1-J rv- w L 31

8. A mutated receptor according to claim 6, characterized in that the extracellular domain derives front the wild type.

9. A mutated receptor according to any one of claims 1 to 8, characterized in that the mutated receptor is a growth factor receptor. A mutated receptor according to claim 9, characterized in that the receptor is a mutated receptor for the epidermal growth factor (EGF).

11. A mutated receptor according to claim 9, characterized in that the receptor is a mutated receptor for the platelet-derived growth factor (PDGF).

12. A mutated receptor according to any one of claims 1 to 8, characterized in that the receptor is a mutated HER2 receptor.

13. A mutated receptor according to any one of claims 1 t. 8, characterized in that the receptor is a met receptor.

14. A mutated receptor according to claim 4, characterized in that the point mutation is at the amino acid position 721 of the EGF wild-type receptor sequence. 32 A mutated receptou according to claim 14, characterized in that the point mutant carries an alanine residue at the amino acid position 721 and is deposited with the German Collection of Microorganisms under DSM 6678.

16. A mutated receptor according to claim 3, characterized in that the 533 C-terminal dinino acids of the EGF wild-type receptor are deleted.

17. A mutated receptor according to claim 3, 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.

18. A pharmaceutical composition comprising the receptor according to any one of claims 1 to 17.

19. A pharmaceutical composition according to claim 18, characterized in that the receptor is associated with liposomes. A pharmaceutical composition according to claim 18, characterized in that the composition contains the mutated receptors in the form of one or a plurality of recombinant vectors that carry the nucleic acid fragments coding for the receptor(s). 33

21. A pharmaceutical composition according to claim characterized in that the vector is a recombinant retroviral vector.

22. A pharmaceutical composition according to claim 21, characterized in that the retroviral vector is selected from pNTK-HER-K721A and pNTK-HERCD-533, deposited with the German Collection of Microorganisms under DSM 6678 and DSM 6679, respectively.

23. A method of treating man or animal affected by cancer, comprising the administration of an effective amount of the pharmaceutical composition according to any one of claims 18 to 22.

24. A method according to claim 23, characterized in that the cancer is the result of a hyperfunction of receptor tyrosine kinases. A method according to claim 24, characterized in that the cancer is selected from mastocarcinoma, ovarian carcinoma and pulmonary carcinoma.

26. A method of treating human or animal diseases based on hyperplasia, which are characterized by the hyperfunction of receptor tyrosine kinases, including Or 34 the administration of an effective amount of the pharmaceutical composition according to any one of claims 18 to 22.

27. Use according to claim 26, characterized in that the disease is psoriasis. Abstract The present invention relates to mutated growth factor receptors which are suitad 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 bveractivity 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.