EP0667899A1

EP0667899A1 - Mutated growth factor receptor as medicament and its use for treating cancer

Info

Publication number: EP0667899A1
Application number: EP92918949A
Authority: EP
Inventors: Dr. Norbert Redemann; Axel Max-Planck-Institut für Biochemie ULLRICH
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date: 1991-09-05
Filing date: 1992-09-07
Publication date: 1995-08-23
Also published as: DE4129533A1; AU2518592A; CN1071586A; MX9205084A; NO940778D0; FI941053A; FI941053A0; NZ244239A; JPH07502884A; NO940778L; WO1993005148A1; AU669857B2; CA2117073A1; PT100844A

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 medicaments for treating cancer.

Description

Mutant growth factor receptor as a drug and its use in the treatment of cancer

The present invention relates to mutant receptor tyrosine kinases with defective signal transmission activity which have therapeutic properties, medicaments containing at least one mutant receptor and the use of the mutant receptor or receptors for the treatment of diseases which are associated with an uncontrolled overfunction of receptor tyrosine kinases , especially cancer.

Cell growth is a carefully regulated process depending on the special needs of an organism. In a young organism, the cell division rate outweighs the death rate of cells, which leads to an increase in the size of the organism. In an adult organism, the formation of new cells and cell death are balanced so that a "steady state" arises. In rare cases, however, cell proliferation control breaks down and cells begin to grow and divide, although there is no particular need in the organism for a higher number of cells of this type. This uncontrolled cell growth is the cause of cancer. Factors which can cause the uncontrolled cell growth associated with the formation of metastases are often chemical in nature, but can also be physical in nature, such as, for example, radioactive radiation.

There are currently essentially two alternatives for the treatment of cancer. Either the cancer cells can be completely removed from the diseased organism by a surgical intervention, or an attempt is made to destroy the degenerated cells in the organism to make, such as by administering medication or by physical therapy methods such as radiation.

In chemotherapy, drugs are often used which intervene in some form in the DNA metabolism and damage rapidly growing cells which have to produce a higher DNA metabolism than cells which do not or only slowly divide. A serious disadvantage of many chemotherapies is, however, the low specificity of the active ingredients used, which has the consequence that healthy cells are also damaged during chemotherapy. This low specificity of the active ingredients also requires that their dosage must be such that as few healthy cells as possible are damaged while the cancer cells are killed at the same time. This is often not possible, and the patient suffering from cancer dies due to the ever-expanding cancer cells, which in the final stage cause the loss of vital functions.

The object of the present invention is to provide a further active substance with valuable therapeutic properties, and a medicament containing the active substance, the active substance or the medicament being particularly advantageous in the treatment of cancer.

This object is achieved according to the invention by a mutated receptor tyrosine kinase with defective signal transmission activity, and by a medicament containing at least one mutant receptor tyrosine kinase.

To better understand the present invention, the terms used herein are explained in more detail below. "Receptor tyrosine kinase" means any receptor that has tyrosine kinase activity. The expression includes growth factor receptors which have tyrosine kinase activity, as well as HER2 or the met receptors.

"Defective signal transmission activity" means that a mutated receptor is no longer able to convert an extra-cellular growth signal or another signal into an intracellular signal, so that this signal is partially inhibited or completely blocked.

"Growth factor" means any mitogenic chemical, usually a polypeptide, which is secreted by normal and / or transformed mammalian cells and which plays an essential role in the regulation of cell growth, in particular in stimulating the proliferation of cells and maintaining it their viability. The term "growth factor" includes e.g. the epidermal growth factor (EGF), the platelet-derived growth factor (PDGF) and the nerve growth factor (NGF).

"Growth factor receptor" is understood to mean a polypeptide spanning the cell membrane, which binds a growth or differentiation factor and itself contains or is associated with a tyroinkinase activity in its intracellular portion.

“Mutant receptor tyrosine kinase” is understood to mean a tyrosine kinase receptor which contains a structural change in comparison with the wild-type receptor, so that the receptor no longer has the tyrosine kinase activity of the wild-type receptor.

Under "mutated growth factor receptor" a wax -. -

Understand factor receptor that contains a structural change compared to the wild-type receptor, so that the receptor no longer has the tyrosine kinase activity of the wild-type receptor.

"Wild type growth factor receptor" or other "wild type" receptor is understood to mean a naturally occurring growth factor receptor or other receptor which has tyrosine kinase activity and is therefore capable of signal transmission.

The “extracellular domain” of the growth factor receptor or other receptor is understood to mean the part of the receptor that normally protrudes from the cell into the extracellular environment. The extracellular domain includes, for example, the part of the receptor to which a growth factor or another molecule binds.

The "transmembrane region" of the growth factor receptor or other receptor is understood to mean the hydrophobic portion of the receptor which is normally located in the cell membrane of the cell which expresses the receptor.

“Tyrosine kinase domain” or “cytoplasmic domain” of the growth factor receptor or other receptor is understood to mean the part of the receptor which is normally located inside the cell and which brings about the transphosphorylation of tyrosine residues.

“An effective amount” means an amount of the composition according to the invention which can achieve the desired therapeutic effect.

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

"Epidermal growth factor (EGF)" is understood to mean a mitogenic polypeptide which usually generates a mitogenic response in fibroblasts and which stimulates the autophosphorylating protein tyrosine kinase activity of the EGF wild-type receptor.

“Disease based on hyperplasia” is understood to mean a disease of a tissue or organ, comprising, for example, the skin epidermis, the intestinal epithelium, hepatocytes, fibroplastics, bone marrow cells, other bone cells, cartilage and smooth muscles, the disease being characterized by an increase in the number of cells of the tissue or organ, such as Psoriasis and endometric hyperplasia.

"HER2" is understood to mean a receptor tyrosine kinase which shows sequence homology to the epidermal growth factor receptor.

"Liposomes" are understood to mean particles in an aqueous medium which are formed by lipid bilayers which enclose an aqueous environment.

"Hyperfunction" is understood to mean excess, uncontrolled activation of a signal transmission path mediated by growth factor receptors, which leads to excess cell division activity and other consequences, such as e.g. to those that occur in some cancer cells compared to normal cells of a similar cell type.

“Recombinant vectors” mean vectors which have been genetically modified using recombinant DNA technology in order to incorporate nucleic acid fragments which code for normal or mutated receptor tyrosine kinases. The recombinant vectors can infect target cells and cause the target cells to express the normal or mutated receptors.

“Recombinant retroviral vectors” are understood to mean recombinant vectors that are retroviruses.

According to the present invention, it has been possible to show that mutated receptor tyrosine kinases have valuable therapeutic properties which can be used for the treatment of diseases which are associated with an overfunction of receptor tyrosine kinases, the mutant receptors being particularly suitable for the treatment of cancer.

Growth factor receptors play a crucial role in the formation and proliferation of human cancer cells. In healthy cells they are

Growth factor receptors involved in the control of cell growth. The actual signal for cell division is the growth factor which is formed depending on the needs of the organism. The receptor assumes the function of signal transmission, ie it is involved in the conversion of the extracellular growth signal into cell division activity inside the cell. In many growth factor receptors, their ability to transfer phosphate residues to tyrosine residues in proteins after the growth factor has bound to the extracellular domain plays a decisive role. These receptors are also referred to as receptor tyrosine kinases. An overview of receptor tyrosine kinases can be found in Yarden, Y. and Ullrich A., Rev. Biochem. 1988, 57, 443-78. The dimerization of these growth factor receptors after Binding the growth factor is another important process in the signal transmission process. The conversion of an extracellular signal into an intracellular signal by means of growth factor receptors with tyrosine kinase activity can be broken down into the following five stages:

1. The binding of the growth factor (also referred to as ligand) to the extracellular domain of the receptor induces a change in conformation; this causes

2. Dimerization of receptors with changed conformation; With

3. simultaneous induction of an allosteric change in the cytoplasmic domain, which in turn induces the kinase activity;

4. transphosphorylation of tyrosine residues in the receptor dimer, which in turn generates and stabilizes an activated receptor conformation; and

5. Phosphorylation of polypeptide substrates and interaction with cellular factors.

An uncontrolled overfunction of this signal transmission chain due to overexpression or mutation can lead, among other things, to an excessive division activity of the corresponding cell and, in extreme cases, to a degenerate cancer cell. An overview of growth factor receptors and their function in signal transmission from the extracellular to the intracellular milieu, as well as the possible influence of abnormally expressed receptors on the development of cancer is in Ullrich, A. and Schlessinger, J. (1990) Cell 61, 203-212. The epidermal growth factor receptor (EGF-R) is a 170 kD glycoprotein with tyrosine kinase activity (Ullrich et al. (1984), Nature 309, 418-425). The molecular processes involved in binding its ligands and stimulating its kinase activity are described in detail in Ullrich et al. , Cell (1990) 61, 203-212. Although EGF normally produces a mitogenic response in fibroblasts, the overfunction of the signal transmission process by the EGF receptor, due to overexpressed receptors, causes 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. (1987), Cell 51, 1063-1070)). Intensive clinical studies have supported the function of this receptor in the development of certain carcinomas, such as breast cancer, ovarian cancer and lung cell cancer (Slamon et al. (1987), Science 235, 177-182 and (1989) Science 244, 707-712 and Kern et al. (1990) Cancer Res. 50, 5184-5191).

It has now surprisingly been found that the five-step signal transmission chain explained above can be blocked or inhibited by a mutated receptor tyrosine kinase if the mutated receptors are expressed together with the wild-type receptors by one cell. Mutated growth factor receptors with defective signal transmission activity are thus particularly suitable as medicaments with which diseases can be treated which are associated with an increased transmission of growth signals into the cell interior by corresponding receptors.

The tyrosine kinase activity of the wild-type receptor no longer has a preferred receptor mutant. This ligand is no longer able to phosphorylate tyrosine residues in the receptor dimer or in polypeptide substrates after ligand binding. The implementation of the extracellu- blocked growth signal into an intracellular signal or partially inhibited.

Even a point mutation in the wild-type receptor can suffice that the wild-type receptor is no longer functional if it has lost tyrosine kinase activity as a result of the point mutation. Such a point mutant (e.g. HERK721A) is particularly preferred.

Also preferred is a mutated receptor that carries a deletion in the tyrosine kinase domain that leads to a loss of tyrosine kinase activity.

It is preferred, however, that the receptor mutated by a deletion in the cytoplasmic domain still contains the transmembrane region (e.g. HERCD-533). Mutated receptors with an existing transmembrane region lead to a more effective inhibition of the growth signal transmission and thus show a better therapeutic effect than receptors without a transmembrane region, such as mutants which consist only of the extracellular domains (e.g. Fig. 1, HERCD-566).

Mutants of receptor tyrosine kinases, such as EGF, PDGF, IGF-I, MET receptors, EGF receptor-related receptors such as HER2, neu, C-erbB2 receptors or NGF receptors are particularly suitable as medicaments. The MET protein is described in detail, for example, in Giordano et al. (1988) Molec. Cell. Biol. 8, 3510-3517 and in Giordano et al. (1989) Nature, 339.

A mutant epidermal growth factor (EGF) receptor is particularly suitable.

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

The receptor mutants can be produced from the wild-type receptors by the customary genetic engineering methods, as described, for example, in Sa brook, J. et al (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press.

The medicament according to the invention contains at least one of the mutated receptors described above and the usual auxiliaries and carriers.

A drug is particularly preferred which contains the mutant receptor (s) packaged 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 selectively binding to them. The receptor mutants thus selectively reach the target tissue and can develop their desired effect there. The administration of active substances packaged in liposomes is a common form of administration today.

Also preferred is a drug that contains the receptor (s) in the form of one or more recombinant retroviral vectors. The recombinant vectors contain nucleic acid fragments that are suitable for the Encode receptors. After the drug has been administered to the patient, the retroviruses infect the target cell and result in the expression of the mutant receptors.

A particularly preferred medicament contains the retroviral vectors PNTK-HER-K721A and / or pNTK-HERCD-533 coding for EGF receptor mutants, which are deposited with the German Collection of Microorganisms, Mascheroden Weg 1B, D-3300 Braunschweig, under DSM 6678 bZW . DSM 6679.

The mutated receptors can be introduced into drugs in a conventional manner, as described, for example, in Remington's Pharmaceutical Sciences (Osol, A. Editor), Mack Publishing Company, Easton, PA (1980) and subsequent volumes.

The mutated receptors described above or the medicaments containing them are particularly suitable for treating cancer. Such types of cancer are particularly easy to treat as a result of an overfunction of growth factor receptors. These types of cancer include breast, ovarian and lung cancer in particular. 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 in Kern, J.A. et al (1990) Cancer Res., 50, 5184-5191.

The pharmaceutical composition may contain salts, buffers, additives and other substances which are desirable for improving the effectiveness of the mutant receptors.

Compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous suspensions for injection may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol 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.

Carriers or occlusive tapes can be used to increase skin permeability and dermal adsorption of the drug.

Liquid dosage forms can typically comprise a liposome solution which contains the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups and elixirs with inert diluents commonly used in the art, such as purified water.

In addition to the inert diluents, these compositions can also contain additives, wetting agents, emulsifying and suspending agents or fragrances. Examples of other materials suitable for use in the present pharmaceutical composition are given in Remington 'Pharmaceutical Sciences (Osol, A., editor), Mack Publishing Co., Easton, PA (1980) and subsequent volumes.

Treatment of an individual with a tumor involves administering an effective amount of the mutant receptors or recombinant vectors that produce the mutant receptors in a single dose, multiple doses, or by infusion to a patient or animal. According to the present invention, an "effective amount" of a pharmaceutical composition is an amount sufficient to achieve the desired biological effect. In general, the dosage required to provide an effective amount of the composition and which can be adjusted by a person skilled in the art depends on factors such as the specific receptor to be used, the presence and the type of other therapeutic agents Means, the age of the patient or animal, as well as their condition, gender and clinical status, including the extent of the disease and other variables.

The preferred dose of the pharmaceutical composition according to the invention in humans 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 route of administration of the pharmaceutical composition according to the invention is parenteral. The most preferred route is intravenous, intraperitoneal, topical or directly into the brain, the spinal fluid or the tumor itself.

Without being bound by any particular theory, it is believed that the described receptor mutants develop their effect in the target cells by incorporating the mutants next to the wild-type receptor into the membrane of the target cells, and the receptor mutant then impairs the function of the wild-type receptor by forming incompetent dimers for signal transmission, which consist of a wild-type or oncogenic receptor and a mutated receptor with defective signal transmission properties. The present invention is explained in more detail using mutants of the EGF receptor as a model.

It has surprisingly been found that the expression of EGF receptor mutants which no longer have tyrosine kinase activity in transformed cancer cells which express the EGF wild-type receptor can reverse the transformed phenotype.

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, CL et al, (1987) EMBO J., 6, 383-388; von Rüden, T. and Wagner, EF (1988), EMBO J., 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 generating a Clal site on both sides of the 2 kb fragment Xbal / Xhol of pLSXNA 8, described in Livneh, E. et al, J. Biol. Chem, 260, 12490-12497 , using conventional cloning methods, as described in Sambrook, J. et al (1989), Molecular Cloning, Cold Spring Harbor Laboratory Press, and then 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 is deposited under DSM 6680. 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 created using a modified infection Protocol as described in Miller, AD and Buttimore, C. (1986) Mol. Cell. Biol., 6, 2895-2902. Low titer amphotrophic virus was prepared by transient transfection of retroviral expression plasmids into the helper virus-free packaging cell line PA317, described in Miller, AD et al (1985) Mol. Cell. Biol., 5, 431-437, and was used to infect secondary packaging cells GP + E-86, followed by a selection of clones of the GP + E-86 producer line in G418 (1 mg / ml). The virus titer was determined by infecting NIH 3T3 cells with dilution series of retrovirus which contained cell-free GP + E-86 supernatants and determining the number of G418-resistant colonies. A retrovirus (S.2TGF_v) which contains the gene for the tower growth factor a (TGFα) is described in Blasband, AJ (1990), Oncogene, 5, 1213-1221.

Gene transfer using retroviruses

Subconfluent NIH 3T3 cells (10 ⁵ cells / 6 cm plate) were used for 4 to with supernatants from GP + E-86 cells which released high titers of NTK-HERc virus ( ⁵ × 10 ⁵ G418 ^R colony-forming units per ml) Incubated for 12 hours in the presence of 4 μg / ml polybrene (Aldrich) and then in the supernatant of GP + E-86 cells, the high titer of either N2, NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566 Release viruses. The expression level of the receptors was increased by several rounds of infection, as described in Bordignon, C. et al (1989), Proc. Natl. Acad. Be. USA 86, 6748-6752. In the experiments described, the infection was carried out once with 1 ml of a diluted supernatant (1.25 x 10 - colony-forming units) or 1 to 4 times with the same volume of undiluted supernatant ( 5x10 5 colony forming egg) of GP + E-86 cells, the high titer of either N2, Release NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566 viruses.

Receptor phosphorylation in intact cells

The cells infected as above were cultured in 10 cm plates to 90% confluence, washed and cultured for 16 hours in methionine-free DMEM (Gibco) supplemented with 1% FCS containing 50 μCi / ml S-methionine (Amersham). The cells were stimulated for 10 min with 20 ng / ml EGF (Amgen Corp.) and in 0.5 ml lysis buffer (50 mM Hepes pH 7.2, 150 mM NaCl, 1.5 mM MgCl ₂ , 1 mM EGTA, 10 % Glycerine, 1% Triton X-100, ImM PMSF, 10 mg / ml aprotinin, 100 μM sodium orthovanadate) lysed at 4 ° C. The lysates were centrifuged in an Eppendorf centrifuge at around 12000 g for 10 min at 4 ° C. The supernatants were then treated with an excess of monoclonal antibody 108.1, described in Honegger, AM, Ullrich, A. and Schlessinger, J. (1989) Proc. Natl. Acad. Sei, USA, 86, pp. 925-929, and Protein A-Sepharose for 4 hours at 4 ° C. Immunoprecipitates were washed twice with HNTG (20 mM Hepes pH 7.3, 150 mM NaCl, 0.1% Triton X-100 and 10% glycerin). The pellet was resuspended in sample buffer, boiled for 5 min and analyzed using SDS-PAGE (7.5%). The proteins were transferred electrophoretically to nitrocellulose and then incubated with a monoclonal mouse antibody against phosphotyrosine (5 E2), described in Fendly, BM 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 Kodak X-Omat film, the nitrocellulose filters were washed with PBS containing 0.2% Tween20. The S-methionine was then labeled Proteins detected by autoradiography. The density of the bands was determined by densitometry.

[~ H] -Thymi.di.n-Ei.nbau

Subconfluent NIH 3T3 cells (10 ⁵ cells / 6 cm plate) were co-infected with NTK-HERc as described, followed by 4 rounds of infection with either N2, NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566. The cells were divided on 12-hole Costar plates. After confluence, the cell onolayers were starved for 24 hours in 0.5 ml DMEM, 0.5% FCS, and 18 hours after EGF addition, the cells were treated with 0.5 μCi methyl [ ³ H] -thymidine (Amersham) marked for 4 hours. The cells were washed twice with PBS and then precipitated on ice with 10% TCA for 1 hour. The precipitate was washed with 10% TCA and redissolved in 200 ul 0.2N NaOH / 0.2% SDS. The lysates were neutralized and the radioactivity incorporated was quantified by scintillation counting.

Transformation tests

To test the ability of NIH 3T3 cells to form colony in soft agar, subconfluent NIH 3T3 cells (10 ⁵ cells / 6 cm plate) were used with NTK-HERC, followed by 4 rounds of infection with either N2, NTK-HERK721A, NTK-HERCD -533, or NTK-HERCD-566 infected. In the cases where an autocrine stimulation was to produce, the cells were infected with ^"_, 2TGFα- virus (5x10 G418 ^R colony forming units per mL). NIH 3T3 cells (10) were dissolved in 6 cm plates plated in the Anwesen¬ Absence or absence of 10 ng / ml EGF in 3 ml medium for overlaying (top layer) of MEM, containing 10% FCS and 0.2% agar (Gibco). The bottom layer contained MEM, 10% FCS and 0.4% agar. Visible colonies were counted after 4 weeks.

For focus formation tests, subconfluent NIH 3T3 cells (10 ⁵ cells / 6 cm plate) were co-infected with NTK-HERc (lxio ⁴ G418 colony-forming units per ml), followed by 4 rounds of infection with either N2, NTK-HERK721A, NTK-HERCD-533 , or NTK-HERCD-566 viruses. In some experiments the cells were superinfected with S_ ^* 2TGFc virus (lxlO ³ G418 ^R colony forming units per ml). Infected cells were cultured on 6 cm plates with DMEM containing 4% FCS in the presence or absence of 10 ng / ml EGF. The medium was changed every 3 days. The plates were stained with crystal violet and the foci were counted on day 18.

Legends for the figures

Fig. 1: Schematic representation of the human EGF wild-type receptor and mutant EGF receptors. The location of cysteine-rich domains (cys), tyrosine kinase (TK) and transmembrane (TM) domains are given. The mutant HERK721A carries a point mutation at position 721 (an exchange of lysine for alanine), while HERCD533 and HERCD-566 carry C-terminal deletions of 533 and 566 amino acids, respectively. The mutants are described in detail in Livneh, E., et al (1986) J. Biol. Chem., 260, 12490-12497 and Honegger, A.M. , et al (1987) Cell, 51, 199-209.

Figure 2 (A): Tyrosine phosphoryiation of the wild type and mutant EGF receptor. Cells that co-express either the wild-type receptor alone or the wild-type receptor and mutant receptors were co-expressed overnight [35S] Methionine marked and then incubated in the presence or absence of 2 ng / ml EGF for 10 min. The cells were brought into solution and precipitated with anti-EGF receptor antibody (mAb 108), separated by SDS-PAGE and immunologically analyzed with anti-phosphotyrosine antibodies (5E2), followed by an ECL substrate reaction.

(B): Expression of the EGF receptor on NIH 3T3 cells. Cells which either co-express the wild-type receptor alone or the wild-type receptor and mutant receptors were marked with [S] methionine overnight and then in the presence or absence of 20 ng / ml EGF for 10 min incubated. The cells were brought into solution and precipitated with anti-EGF receptor antibody (mAblOδ), separated using SDS-PAGE and immunologically detected with an anti-phosphotyrosine antibody (5E2), followed by an ECL substrate. Reaction. The ECL substrate was washed off with PBS containing 0.2% Tween 20, and the ³⁵ S methionine-labeled proteins were detected by car radio.

Fig. 3: EGF-simulated [ ³ H] thymidine incorporation. In cells that either co-expressed the wild-type receptor alone (dashed line) or wild-type receptor and mutant 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 to confluence in 12-hole Costar plates 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 μCi / well for 4 hours was added and its incorporation into DNA was determined. The mitogenic response was recorded to show the relationship between dose and response. The values were corrected for basal thymidine incorporation, and the maximum observed response to EGF was defined as 100%. The filled triangles indicate the half-maximum thymidine incorporation.

Results:

Cells expressing the wild-type receptor alone or together with the mutant receptors were also identified

Labeled, incubated in the presence or absence of EGF for 10 min, lysed and immunoprecipitated with a mouse antibody against human EGF receptor (mAbl08). The samples were separated on SDS-PAGE, transferred to nitrocellulose filters, and tyrosine phosphorylation was detected using the phosphotyrosine-specific mouse antibody 5E2 (FIG. 2A). The amount of the receptor present in the immunoprecipitate was detected by autoradiography of the same nitrocellulose filter (FIG. 2B).

As shown in Figure 2A, lanes b and c, EGF addition to intact NIH 3T3 cells infected with the virus containing the wild-type EGF receptor induces strong tyrosine phosphorylation of the 170 kD EGF receptor band. As a result of phosphorylation, the migration rate of the EGF receptor in the SDS-PAGE decreases compared to the unphosphorylated EGF receptor, as can be seen from FIG. 2B, lanes b and c. The level of EGF-stimulated phosphorylation of the wild-type receptor was not reduced by coexpression of the soluble extracellular domain of the EGF receptor, as encoded by the NTK-HERCD-566 virus genome, even if the extracellular domain was in 4-fold excess to the wild-type Receptor was expressed (Fig. 2A, lanes d to f; Fig. 2B, lanes d to f). On the contrary, in an analog experiment in which a virus expressing the EGF receptor deletion mutant HERCD-533 (FIG. 1) was used, there was a strong dose-dependent inhibition of EGF-induced phosphorylation of the wild-type EGF Receptor (Fig. 2A, lanes g to i), although the level of the 170 kD EGF receptor protein remained constant (Fig. 2B, lanes g to i). The intensity of the tyrosine-phosphorylated bands decreased from 100% to 71% and 30%, respectively. Under these conditions, the EGF receptor showed the same electrophoretic properties as a non-phosphorylated receptor (FIG. 2B, lane i), which was consistent with its state of tyrosine phosphorylation, as demonstrated by mAb 5E2 (FIG. 2A , Track i).

When 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 receptor tyrosine phosphorilization signal increased from 251% to 337% and 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 is by the wild-type receptor can be transphorylated.

Inhibition of EGF-induced cell division rate.

EGF stimulates cell division in NIH 3T3 fibroblasts that express the EGF receptor, as described in Riedel, H. et al (1988) Proc. Natl. Acad. Be. USA, 85, 1477-1481 and Prywes, R. et al (1986) EMBO J., 5, 2179-2190. The influence of mutant receptors on that of the EGF wild-type receptor controlled cell division was determined based on the induction of DNA synthesis.

DNA synthesis was determined as [H] -thymidine incorporation in cells infected with the NTK-HERc virus and the N2 virus control, and the synthesis was maximally stimulated at 2 ng / ml EGF, with a half-maximum stimulation (ED ₅₀ ) at 0.66 ng / ml (FIG. 3). Similar to previous results, as described in Honegger, AM et al (1988) EMBO J .. 7, 3045-3052, and Riedel, H., et al (1988) Proc. Natl. Acad. Sei., USA, 85, 1477-1481, higher EGF concentrations led to lower levels of [3H] -Thyι_ι.dι.n-Eι.nbau, as in Fi.g. 3 is displayed. After four rounds of infection with the corresponding viruses, the coexpression of the EGF receptor with HERCD-533 and HERK721A led to a significant shift in the dose-dependent curve towards higher EGF concentrations (FIGS. 3A and B). This indicates that the cells have become less sensitive to the growth factor compared to HERc / N2 cells. Both the deletion mutant HERCD-533 and the point mutant HERK721A (FIG. 1) showed similar effects on the cell division signal mediated by the EGF wild-type receptor and caused a ten-fold increase in the ED ₅₀ to 6.6 ng / ml EGF . In contrast, the superinfection with NTK-HERCD-566 virus had no significant effect on the DNA synthesis stimulated by the wild-type receptor by EGF (FIG. 3C).

Anionic activity of the EGF receptor mutants

Overexpression of the EGF receptor is known to cause EGF-dependent cell transformation of NIH 3T3 cells, as described in Di Fiore, PP et al (1987), Cell, 51, 1063-1070, Velu, T.. et al (1987), Science, 237, 1408-1410 and Riedel, H. et al (1988) Proc. Natl. - -

Acad. Sci., USA, 85, 1477-1481. In order to investigate whether the transforming potential of overexpressed EGF receptor can be inhibited by EGF receptor mutants, EGF receptor was coexpressed with receptor mutants, and subsequently their ability to colonies in soft agar or foci in a monolayer cell culture was increased generate, examined. Stimulation of overexpressed EGF receptor was achieved either by adding EGF to the medium or by infection with a virus (Sf.2TGFα) carrying TGF-α DNA 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 approximately 250 colonies in soft agar in the presence of 10 ng / ml EGF. Coinfection with _2TGFα virus resulted in the formation of 148 colonies under otherwise identical conditions (Table 1). However, when the cells infected with the EGF receptor were superinfected with either NTK-HER-K721A or NTK-HERCD-533 viruses, the colony-forming capacity was almost completely suppressed. Coexpression of the EGF receptor with the HERCD-566 extracellular domain reduced the colony-forming ability by approximately 50% when stimulated with 10 ng / ml EGF in the agar layer, and by approximately 33% when stimulated by the autocrine TGF after infection with ^* _ ^»" 2TGF_ virus.

Similarly, the focus-forming potential of the NTK-HERc virus was determined in NIH 3T3 monolayer cultures, either in the presence of 10 ng / ml EGF, which resulted in 920 foci per 10 viruses, or after co-infection with

Sf ^{r for} 2TGFc. virus, which resulted in 480 foci per 10 ⁶ NTK-HERC viruses. Superinfection with NTK-HERK721A or NTK-HERCD-533 viruses suppressed the number of foci by 100% or 90%, when stimulated with EGF and by 75% or 71% when stimulated with the \ f.2TGFc. virus (Table 2).

Cells that coexpressed the EGF wild-type receptor and HERCD566 showed the same number of foci as cells that expressed the EGF receptor and were infected with the control virus N2. This result was observed in both the stimulation with EGF as well with the f ^r 2TGFc-- irus.

It can be seen from the above explanations that the EGF receptor mutants have both a clear antiproliferative and anioncogenic potential and are therefore outstandingly suitable for the treatment of cancer.

Table 1: Colony formation in soft acrar

Number of colonies / 10 ⁶ CFU

infection

+10 ng / ml EGF + \ .2TGFc.

N2 0 0

NTK-HERK721A 0 0 NTK-HERCD-533 0 0 NTK-HERCD-566 0 0

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 Focusbildunσ

Number of foci / 10 ° CFU

Cell line

+10 ng / ml EGF + \ I.2TGFα

N2 0 0

NTK-HERK721A 0 0 NTK-HERCD-533 0 0 NTK-HERCD-566 0 0

NTK-HERC / NTK-HERK721A 40 18 NTK-HERc / NTK-HERCD-533 90 14 NTK-HERc / NTK-HERCD-566 910 500

The foci were counted after 14-16 days. The values represent average values from four independent experiments. CFU means colony-forming units.

Claims

1. Mutant receptor tyrosine kinase that no longer has signaling activity.

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

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

4. Mutant receptor according to claim 2, characterized in that it carries a point mutation in its tyrosine kinase domain.

5. Mutant receptor according to claim 2, characterized in that the receptor contains its extracellular domain and its transmembrane region.

6. Mutant receptor according to claim 2, characterized in that the receptor comprises an extracellular domain.

7. Mutant receptor according to claim 5, characterized in that the extracellular domain and the transmembrane region come from the wild type.

8. Mutant receptor according to claim 6, characterized in that the extracellular domain comes from the wild type.

9. Mutant receptor according to one of claims 1 to 8, characterized in that the mutant receptor Is growth factor receptor.

10. Mutant receptor according to claim 9, characterized in that the receptor is a mutant receptor for the epidermal growth factor (EGF).

11. Mutated receptor according to claim 9, characterized in that the receptor is a mutated receptor for the growth factor derived from platelets (PDGF).

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

13. Mutated receptor according to one of claims 1 to 8, characterized in that the receptor is a met receptor.

14. Mutated receptor according to claim 4, characterized in that the point mutation is in amino acid position 721 of the EGF wild-type receptor sequence.

15. Mutant receptor according to claim 14, characterized in that the point mutant carries an alanine residue in amino acid position 721 and is deposited under DSM 6678 at the German Collection of Microorganisms.

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

17. Mutant receptor according to claim 3, characterized in that the 566 C-terminal amino acids of the EGF wild-type receptor are deleted and is deposited under DSM 6680 at the German Collection of Microorganisms.

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

19. Pharmaceutical composition according to claim 18, characterized in that the receptor is associated with liposomes.

20. Pharmaceutical composition according to claim 18, characterized in that the composition contains the mutated receptors in the form of one or more recombinant vectors which carry nucleic acid fragments coding for the receptor or receptors.

21. Pharmaceutical composition according to claim 20, characterized in that the vector is a recombinant retroviral vector.

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

23. A method for treating a human or animal suffering from cancer, comprising administering an effective amount of the pharmaceutical composition according to any one of claims 18 to 22.

24. The method according to claim 23, characterized in that the cancer is the result of an overfunction of receptor tyrosine kinases.

25. The method according to claim 24, characterized in that the cancer is selected from breast cancer, ovarian cancer and lung cancer.

26. A method of treating hyperplasia-based human or animal diseases characterized by hyperfunction of receptor tyrosine kinases, comprising administering an effective amount of the pharmaceutical composition according to any one of claims 18 to 22.

27. The method according to claim 26, characterized in that the disease is psoriasis.