NZ244239A - Genetically engineered tyrosine kinase receptors - Google Patents

Description

24 4 23 Priority Date(s): X.V.Ij Complete Specification riled: Class: ten,- J Publication Date: ..J.(JL ^ I P.O. Journal. Nor ... AM>|. ' I N Z PAT F*J7 OFFICE -4 JUN 1993 RECEIVED NEW ZEALAND PATENTS ACT, 1953 No.: Date: COMPLETE SPECIFICATION MUTANT, SIGNALLING-INCOMPETENT, RECEPTOR TYROSINE KINASES AS A MEDICATION AND THEIR USE FOR THE TREATMENT OF CANCER We, MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV, incorporated in Germany, of Bunsenstr. 10, 3400 Gottingen, Germany, hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- (followed by page la) V. / o la- 24 4 23 9 fp^ I. Field of the Invention Thepresent invcntionconccrns mutant, signalling-incompetent, receptor tyrosine kinases, such as tyrosine kinase defective mutant growth factor receptors, with therapeutic properties, medications containing at least one type of such rrutant receptor, and the use of these mutant receptor(s) for the treatment of cancer.

II. Background of the Invention A. Cancer 1^ Cell growth is a carefully regulated process which depends on the specific needs of an organism. In a young organism, the cell division rate 15 exceeds the rate of destruction of cells, which leads to an increase in the size of the body. In a fully grown adult the formation of new cells and cell death ^ are balanced so that there is a "fluid equilibrium." In rare cases, however, the control of cell reproduction collapses and the cells begin to grow and divide even though there is no special need for a larger number of cells of this 20 particular cell type in the body. This uncontrolled cell growth is the cause of cancer. Factors that can induce uncontrolled cell growth are often chemical in nature but may also be physical in nature, such as radioactive radiation.

For the treatment of cancer, there are essentially two alternatives at the present. Either the cancer cells are completely removed from the diseased 25 body by means of a surgical procedure or an attempt is made to render the N.z. patent office 1 -4 JUN 1993 ' ..ved 244239 m malignant cells in the body harmless, e.g., by administering medication or by physical means of treatment such as radiation therapy.

In chemotherapy, drugs are often used that intervene in DNA metabolism in some way and damage rapidly growing cells (which must have a higher DNA turnover) to a greater extent than cells that do not divide at all or divide only slowly. One serious disadvantage of many chemotherapies, however, is the low specificity of the active agents which results from the fact that healthy cells are also damaged by the chemotherapy. The low specificity - of the active agents also requires dosing in such a way that the fewest possible 10 healthy cells are damaged while at the same time killing the cancer cells. This is often impossible and the cancer patient usually dies due to the repeated spread of cancer cells, which in the terminal stage results in the failure of vital functions.

B. Growth Factor Receptors Growth factor receptors play an important role in the development and reproduction of human cancer cells. With healthy cells, the growth factor receptors are involved in the regulation of cell growth. The actual signal for cell division is the growth factor which is formed in accordance with the body's needs. The receptor assumes the function of signal transmission, i.e., it is involved in the conversion of the extracellular growth signal into cell divisional activity in the interior of the cell. The ability of many growth factor receptors to transfer phosphate residues on tyrosine residues to proteins after the binding of the growth factor to the extracellular domains of their receptors plays a significant role. These receptors are also known as receptor tyrosine 25 kinases. A survey of receptor tyrosine kinases can be found in Yarden et at., Rev. Biochem 57:443-78 (1988). The dimerization of these growth factor receptors after the binding of the growth factor is another important process in signal transmission. Conversion of an extracellular signal to an intracellular N.Z. PATcr.iT OFFICE" JUN 1993 JVED 24 4 23 ? signal by growth factor receptors with tyrosine kinase activity can be broken down into the following five steps: 1. Binding of the growth factor (also known as the ligand) to the extracellular domains of the receptor induces a conformational change in the ^^ receptor; 2. which, in turn, results in dimerization of the receptors with an altered conformation; 3. with the simultaneous induction of an allosteric change in the cytoplasmic domains by means of which, in mm, the kinase activity is induced; 4. transphosphorylation of tyrosine residues by the receptor dimer which, in turn, produces an activated receptor conformation and stabilizes it; and . phosphorylation of polypeptide substrates and their interaction 15 with cellular factors.

Hyperfunction of this signal transmission chain can lead to excessive divisional activity of the corresponding cell. In the extreme case it can lead to a degenerate cancer cell. A survey of growth factor receptors and their function in signal transmission from the extracellular level to the intracellular 20 level as well as the possible influence of mutated receptors on the development of cancer can be found in Ullrich et ai, Cell 67:203-212 (1990).

The epidermal growth factor receptor (EGF-R) is a 170 kD glycoprotein with tyrosine kinase activity (Ullrich et ai, Nature 309:418-425 (1984)). The molecular processes involved in binding its ligands and 25 stimulating its kinase activity are described in detail by Ullrich et ai, Cell 67:203-212 (1990). Although EGF normally induces a mitogenic response in fibroblasts, hyperfunction of the signal transmission mechanism by the EGF receptor due to overexpressed receptors leads to a ligand-dependent transformation of NIH 3T3 mouse cell (Riedel et ai, Proc. Natl. Acad Sci., 30 USA 55:1477-1481 (1988) and Di Fiore et ai, Cell 57:1063-1070 (1987)).

Intensive clinical studies have supported the function of this receptor in the an 60 » development of certain carcinomas such as breast cancer, ovarian cancer and lung cell cancer (Slamon et ai., Science 235:177-182 (1987); id.. Science 244:707-712 (1989); and Kern et al., Cancer Res. 50:5184-5191 (1990)).

It has now surprisingly been found that the five-step signal transmission chain described above can be blocked or inhibited by mutant receptor tyrosine kinases such as mutant growth factor receptors that no longer possess tyrosine kinase activity, or are otherwise rendered signalling incompetent, if the mutant receptors are expressed together with the wild-type receptors by a cell. Thus, mutant growth factor receptors are suitable for use as a medication for the treatment of diseases that occur in conjunction with an increased transmission of growth signals to the interior of the cell by their respective receptors.

III. Summary of the Invention The present invention provides genetically engineered signalling incompetent receptor tyrosine kinases, including growth factor receptors, which are capable of dimerizing with a signalling competent receptor, wherein the dimerization leads to inactivation of receptor tyrosine kinase, with the proviso that the receptors are not mutant epidermal growth factor receptors.

The present invention also provides pharmaceutical compositions comprising genetically engineered signalling incompetent receptor tyrosine kinases which are capable of dimerizing with a signalling competent receptor, wherein the dimerization leads to inactivation of the receptor tyrosine kinase activity, in combination with appropriate pharmaceutical excipients and vehicles.

IV. Brief description of the Figures Figure 1: Schematic diagram of the human EGF wild-type receptor (EGF-R wt) and mutant EGF receptors. The position of cysteine-rich domains (cys), the tyrosine kinase (TK) domains and the transmembrane (TM) domains 24 4 23 9 are indicated. The mutant HERK721A has a point mutation in position 721 (lysine is exchanged tor alanine) while the mutants HERCD-533 and HERCD-566 have C-terminal deletions of 533 and 566 amino acids, respectively. These mutants are described in detail by Livneh et al., J. Biol. Chem. 260:12490-12497 (1986) and Honegger et al., Cell 57:199-209 (1987).

Figure 2 (A): Tyrosine phosphorylation of the wild-type receptor and mutant EGF receptor. Cells that coexpress either the wild-type receptor alone, or the wild-type receptor and mutant receptors, were labeled overnight with [35S]-methionine and then were incubated for 10 minutes in the presence or absence of 2 ng/ml EGF. The cells were placed in solution and were precipitated withanti-EGF receptor antibody (mAb 108). They were separated by SDS-PAGE and were analyzed immunologically with anti-phosphotyrosine antibodies (5E2) followed by an ECL substrate reaction. In both Figures 2A and 2B, HERc means human "HGF Receptor complete". The other abbreviations have the same meaning as for Figure 1.

(B): Expression of EGF receptor by NIH 3T3 cells. Cells that coexpress either the wild-type receptor alone, or the wild-type receptor and mutant receptors, were labeled overnight with [35S]-methionine and then were incubated in the presence or absence of 20 ng/ml EGF for 10 minutes. The cells were placed in solution and were precipitated with anti-EGF receptor antibody (mAb 108). They were separated with the help of SDS-PAGE and were analyzed immunologically with an anti-phosphotyrosine antibody (5E2) followed by an ECL substrate reaction. The ECL substrate was washed with PBS containing 0.2% Tween 20. The f35S]-methionine-labeled proteins were detected by autoradiography.

Figure 3: EGF-stimulated [3H]-thymidine incorporation. Cells that coexpress either the wild-type receptor alone (dotted line), or wild-type receptor and mutant receptors (solid line) were cultured in 12-hole Costar plates to the point of confluence and then were starved for 2 days in DEM, containing 0.5% FCS (Figure 3A, wild-type EGF receptor + K721A; Figure 3B, wild-type EGF receptor + CD-533; and Figure 3C, wild-type EGF 2- receptor + CD-566). Ten percent FCS or different EGF concentrations were added. Eighteen hours after adding EGF, [3H]-thymidine (0.5 /xCi/hole) was added for 4 hours, its incorporation into the DNA was determined. The mitogenic response was recorded in order to show the relationship between dose and response. The values were corrected by the amount of the basal thymidine incorporation and the maximum response to EGF observed was defined as 100%. The arrows indicate half the maximum incorporation of thymidine.

V. Definitions In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Any terms which are not specifically defined in this or other sections of this patent application have the ordinary meaning they would have when used by one of skill in the art to which this invention applies.

As used herein, "receptor tyrosine kinase" means any receptor which possesses tyrosine kinase activity. The term is meant to include, but is not limited to, growth factor receptors which possess tyrosine kinase activity as well as HER2 or the met receptors.

As used herein "signalling incompetent" means a mutant receptor which is no longer capable of translating an extracellular growth or other signal into an intracellular signal such that said signal is partially inhibited or completely blocked.

As used herein, "growth factor" means any mitogenic chemical, usually a polypeptide, which is secreted by normal and/or transformed mammalian cells which plays an essential role in the regulation of cell growth especially in stimulating the proliferation of the cells and in maintaining their viability. As used herein, the term "growth factor" is meant to include any compound generally recognized by those of skill in the art as being a "growth factor" and includes, but is not limited to. epidermal growth factor, platelet-derived growth factor and nerve growth factor.

As used herein, "growth factor receptor" means a membrane-spanning polypeptide that binds a growth or differentiation factor and contains an intrinsic or associated tyrosine kinase activity in its intracellular portion.

As used herein, "mutant receptor tyrosine kinase" means a tyrosine kinase receptor which has a change in its structure from the wild-type receptor so that it no longer has the tyrosine kinase activity of the wild-type receptor tyrosine kinase.

As used herein, "mutant growth factor receptor" means a growth factor receptor which has a change in its structure from the wild-type receptor so that it no longer has the tyrosine kinase activity of the wild-type growth factor receptor.

As used herein, a "wild-type" growth factor or other receptor means the naturally occurring growth factor or other receptor which has tyrosine kinase activity and is thus signalling-competent.

As used herein, the "extracellular domain" of the growth factor or other receptor means that portion of the growth factor or other receptor which normally protrudes out of the cell and into the extracellular fluid. The extracellular domain is meant to include, but is not limited to, the portion of the growth factor or other receptor to which a growth factor or other molecule binds.

As used herein, the "transmembrane region" of the growth factor or other receptor means the hydrophobic portion of the growth factor or other receptor which normally is situated in the cellular membrane of the cell which expresses the receptor.

As used herein, the "tyrosine kinase domain" or "cytoplasmic domain" of the growth factor or other receptor means that portion of the growth factor or other receptor which is normally located within the cell and which produces transphosphorylation of tyrosine residues. 24 4 25 9 As used herein, "an effective amount" means an amount of the composition which is capable of achieving the desired therapeutic effect.

As used herein, "platelet-derived growth factor" (PDGF) means a mitogenic polypeptide which is contained in platelets and which stimulates mesenchymally-derived cells to stimulate the intrinsic autophosphorylating protein tyrosine kinase activity when it binds to the wild-type PDGF receptor.

As used herein, "epidermal growth factor" (EGF) means a mitogenic polypeptide which normally generates a mitogenic response in fibroblasts and which stimulates the intrinsic autophosphorylating protein tyrosine kinase activity of the wild-type epidermal growth factor receptor.

As used herein, "hyperplastic disorder" means a disorder of a tissue or organ, including but not limited to, skin epidermis, intestinal epithelium, hepatocytes, fibroblasts, bone marrow cells, other bone cells, cartilage and smooth muscle, which is characterized by an increase in the number of cells of the tissue or organ and includes, but is not limited to psoriasis and endometrial hyperplasia.

As used herein, "HER2" means a receptor tyrosine kinase with sequence homology to the epidermal growth factor receptor.

As used herein, "liposomes" means panicles in an aqueous medium which are formed by lipid bilayers enclosing on aqueous compartment.

As used herein, "hyperfunction" means excess uncontrolled activation of a given growth factor receptor signalling pathway which produces excessive cell divisional activity and other consequences, such as those which occur in some cancer cells, compared to those which occur in normal cells of similar cell type.

As used herein, "recombinant vectors" means vectors which have been genetically altered using recombinant DNA technology to incorporate nucleic acid fragments that code for normal and mutant receptor tyrosine kinase constructs. The recombinant vectors can infect target cells and cause the target cells to express these normal and mutant receptors.

As used herein, "recombinant retroviral vectors" are "recombinant vectors", as previously defined, which are retroviruses. -9 VI. Description of the Preferred Embodiments A preferred mutant receptor is a signalling-incompetent mutant receptor. An example of a signalling-incompetent mutant receptor is a mutant receptor tyrosine kinase which lacks the tyrosine kinase activity of its wild-type receptor. This mutant is no longer capable of phosphorylating tyrosine residues in the receptor dimer or in polypeptide substrates after the binding of its ligand to the mutated receptor. Thus, the translation of the extracellular growth signal into an intracellular signal is blocked or partially inhibited.

A point mutant is preferred as the mutation in the receptor. Even a point mutation in the wild-type receptor can be sufficient for the wild-type receptor to be rendered incapable of functioning if it has lost its tyrosine kinase activity due to the point mutation (see, for example. Fig. 1: HERK721A).

A mutant receptor which has a deletion in the tyrosine kinase domains leading to a loss of tyrosine kinase activity is also preferred.

It is preferable for the mutant receptor which has a deletion in the cytoplasmic domains to still have the transmembrane region (see, for example. Fig. 1: HERCD-533). Mutant receptors with the transmembrane region lead to more effective inhibition of the growth signal transmission and thus have a better therapeutic effect than receptors without the transmembrane region such as mutants, for example, which consist only of the extracellular domains (see, for example. Fig. 1: HERCD-566).

Mutants of the receptor tyrosine kinases such as EGF, PDGF or HER2 receptors are preferred for use as the medication.

A mutant receptor of EGF is most preferred.

With an especially preferred mutant of the EGF receptor, there is a point mutation in amino acid position 721 of the wild-type receptor sequence. With this preferred mutant, the lysine residue in position 721 is replaced by an alanine residue in the mutant. This mutant has been deposited with the German Collection of Microorganisms and Cell Cultures according to the Budapest Treaty under file code DSM 6678. In another preferred EGF receptor mutant, the 533 C-terminal amino acids of the wild-type receptor have been deleted. This mutant is on file as DSM 6679.

The receptor mutants can be produced starting from the wild-type receptors by the usual methods of genetic engineering, e.g., as described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

A medication containing the receptor mutant(s) packaged in liposomes is especially preferred. In order to bring the liposomes selectively to the target tissue, it is advantageous if the liposomes contain an antibody in their membrane which antibody recognizes the specific epitopes of the target cells and selectively binds to them. Thus, the receptor mutants selectively reach the target tissue where they can produce their desired effect. The administration of active ingredients packaged in liposomes is a common form of administration today.

A medication containing the receptor(s) in the form of one or more recombinant retroviral vectors converted into viruses is also preferred. The recombinant vectors contain nucleic acid fragments that code for the receptor(s). After administering the medication to the patients, the retroviruses infect the target cells where they lead to the expression of the receptor mutants.

An especially preferred medication contains the retroviral vectors pNTK-HERK721A and/or pNTK-HERCD-533 that code for EGF receptor mutants. These were deposited on August 22, 1991 with the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroden Weg 1 B, D-3300 Braunschweig, Federal Republic of Germany, under the Budapest Treaty and have been given deposit numbers DSM 6678 and DSM 6679, respectively.

Those of skill in the art will know how to incorporate these mutant receptors into medications using standard techniques and tests such as those described in Remington's Pharmaceutical Sciences (Osol, A.. Kd.) Mack 24*2 3 9 11- Publishing Company, Easton, PA, (1980) and subsequent volumes thereof, which are well known to those of skill in the an.

The mutant receptors described above and the medications containing them are especially suitable for the treatment of cancer. Forms of cancer that are the result of hyperfunction of growth factor receptors are especially suitable for this form of treatment. Such types of cancer include, but are not limited to, breast cancer, ovarian cancer and lung cancer. The role of surface receptors in these types of cancer has been described in detail (Slamon et al.. Science 255:177-182 (1987); Id.. Science 244:707-712 (1989); and Kern et al., Cancer Res. 50:5184-5191 (1990)).

As would be understood by one of ordinary skill in the an, such therapeutic compositions may contain salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the mutated growth receptors.

Compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Examples of non-aqueous solvents are propylene glycol, polyethylene gylcol, vegetable oils, such as olive oil, and injectable organic esters such as ethyl oleate.

Carriers or occlusive dressings can be used to increase skin permeability and enhance dermal absorption of the medication.

Liquid dosage forms may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents which are commonly used in the an, such as purified water.

Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or perfuming agents. Examples of other materials which are suitable for use in the pharmaceutical compositions of the present invention are provided in Remington's Pharmaceutical Sciences (Osol, A., Ed.), Mack Publishing Co., Easton, PA (1980)) and subsequent volumes thereof.

Treatment of an individual who has a tumor comprises administering an effective amount of the mutant receptors or recombinant vectors which produce the mutant receptors, in a single dose, multiple doses or an infusion to a patient or other animal.

According to the present invention, an "effective amount" of a therapeutic composition is one which is sufficient to achieve the desired biological effect. Generally, the dosage needed to provide an effective amount of the composition, and which can be adjusted by one of ordinary skill in the art, will vary depending upon such factors as the individual mutant receptor used, the presence and nature of any other therapeutic agent used, the animal's or patient's age, condition, sex, and clinical status, including extent of disease and other variables.

The preferred human doses for the pharmaceutical compositions of this invention are doses greater than 109 plaque forming units per person and will depend on the type of cancer and the extent of receptor hyperfunction.

The preferred routes of administration for the administration of the pharmaceutical compositions of this invention are parenteral routes. The most preferred routes are: intravenous; intraperitoneal; topical; and directly into the brain, spinal cord or tumor itself.

Without being limited to a certain theory, it is assumed that the receptor mutants described herein develop their effects in the target cells by the fact that the mutants are incorporated into the membrane of the target cells and the receptor mutant then impairs the function of the wild-type receptor in the target cells. 24 4 2 3 9 -4. The Production of Recombinant Retroviruses The retroviral expression vectors pN2, pNTK2 and pNTK-HERc have been described in detail (Keller et al., Nature J7S: 149-154 (1985); Stewart fet ai, EMBOJ. <5:383-388 (1987); von Ruden et al., EMBOJ. 7:2749-2756 (1988)). Vector pNTK-HERK72lA was produced by cloning a Bgl II fragment of pCMV-HERK721A in pNTK-HERc. The vector pNTK-HERCD-533 was produced by creating a Clal site on both sides of the Xbal/Xhol 2KB fragment of pLSXNA 8, described by Livneh et al. (J. Biol. Chem. 260:12490-12497 (1986)) by means of the usual cloning methods 10 (Sambrook et al.. Molecular Cloning, Cold Spring Harbour Laboratory Press, Cold Spring Harbor, NY (1989)). Then the 2KB Clal fragment was ligated with pNTK2 cleaved with Clal. The pNTK-HERCD-566 construct was produced by cloning a Clal fragment of pCVNHERXCD in the Clal site of pNTK2. This construct was deposited on August 22, 1991 with the German 15 Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1 B, D-3300 Braunschweig, Federal Republic of Germany, under the Budapest Treaty was given the deposit number DSM 6680. Ecotrophic recombinant retroviruses were produced from the helper virus-free producer cell line GP+ E-86 described by D. Markowitz (J. Virol. 62:1120-1124 (1988)). Stable 20 GP + E-86 producer lines were produced by means of a modified infection protocol as described by Miller et al. (Mol. Cell. Biol. 6:2895-2902 (1986)). Amphotrophic virus with a low titer was produced by transient transfection of retroviral expression plasmids in the helper virus-free packaging cell line PA317, described by Miller et al. (Mol. Cell. Biol. 5:431-437 (1985)) and was 25 used to infect secondary packaging cells GP+E-86 followed by selection of clones of the producer line GP +E-86 in G418 (1 mg/ml). The virus titer was determined by infecting NIH 3T3 cells with a dilution series of retrovirus containing the cell-free GP + E-86 supernatants and determining the number of G418 resistant colonies. A retrovirus (-^2TGFa) containing the gene for N.Z. PATENT OFFICE -4 JUN 1393 RECEIVED 24423 9 tumor growth factor a (TGFa) has been described by A.J. Blasband (Oncogene 5:1213-1221 (1990)).

B. Gene Transfer by Means of Retroviruses Subconfluent NIH 3T3 ceils (105 cells/6 cm plate) were incubated with supernatants of GP+E-86 cells that release a high titer of NTK-HERc virus (5 x 105 G418R colony forming units per ml). They were incubated for 4 to 12 hours in the presence of 4 jig/ml Polybrene (Aldrich) and then were incubated in a supernatant of GP+E-86 cells that release a hip,h titer of either N2, NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566 viruses. The expression level of the receptors was increased by multiple infection rounds as described by Bordignon et al. (Proc. Natl. Acad. Sd., USA 56:6748-6752 (1989)). In these experiments, infection was performed once with 1 ml of a diluted supernatant (1.25 x 10s 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 that release high titers of either N2, NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566 viruses.

C. Receptor Phosphorylation in Intact Cells Cells infected as described above were cultured in 10 cm plates to 90% confluence, were washed and cultured for 16 hours in methionine-free DMEM (Gibco), supplemented with 1% FCS containing 50 /xCi/ml [35S]-methionine (Amersham). The cells were stimulated for 10 minutes with 20 ng/ml EGF (Amgen Corp.) and were lysed at 4°C in 0.5 ml lysis buffer (50mM HEPES, pH 7.2, 150mM NaCl, 1.5mM MgCI2, ImM EGTA, 10% glycerol, \% Triton X-100, ImM PMSF, 10 mg/ml aprotinin, and 100 fiM sodium onhovanadate). The lysates were centrifuge*) for 10 minutes at 4°C in an Eppendorf centrifuge at about 12,000 g's. The supernatants were then incubated for 4 hours at 4°C with an excess of mAb 108.1 (Honegger et al.. 24 4 23 9 Proc. Natl. Acad. Sci., USA 86:925-929 (1989)) and protein A-Sepharose. The immune precipitates were washed twice with HNTG (20 mM HEPES, pH 7.3, 150mM NaCl, 0.1% Triton X-100 and 10% glycerol). The pellet was resuspended in sample buffer, was boiled for 5 minutes and then was analyzed on the basis of SDS-PAGE (7.5%). The proteins were transferred by electrophoresis to nitrocellulose and then were incubated with a monoclonal mouse antibody tophosphotyrosine (5E2) (Fendly et al., Cancer Res. 50.1550-1558 (1990)). The nitrocellulose filter was incubated with a peroxidase-coupled goat anti-mouse antibody followed by an ECL substrate reaction (Amersham) for detection. After detection of the ECL substrate reaction with Kodak X-Omat film, the nitrocellulose filter was washed with PBS containing 0.2% Tween 20. Then the [35S]-methionine-labeled proteins were detected by autoradiography. The density of the bands was determined by densitometry.

D. Incorporation of ^HJ-Thymidine Subconfluent NIH 3T3 cells (105 cells/6 cm plate) were coinfected as described above with NTK-HERc followed by 4 infection rounds with either N2 (wild-type control vims), NTK-HERK721A, NTK-HERCD-533 or NTK-HERCD-566. The cells were divided on 12-hole Costar plates. After reaching confluence, the cell monolayer was starved for 24 hours in 0.5 ml DMEM, 0.5% FCS. Eighteen hours after adding EGF, the cells were labeled with a 0.5 /xCi methyl-[3H]-thymidine (Amersham) for 4 hours. The cells were washed twice with PBS and then were precipitated with 10% TCA for one hour on ice. The precipitate was washed with 10% TCA and was dissolved again in 200 y\ 0.2N NaOH/O.2% SDS. The lysates were neutralized and the incorporated radioactivity was determined quantitatively by scintillation countings. 24 42 3 9 E. Transformation Tests In order to investigate the ability of NIH 3T3 cells to form colonies in soft agar, confluent NIH 3T3 cells (105 cells/6 cm plate) were infected with NTK-HERc followed by 4 rounds of infection with either N2, NTK-5 HERK72IA, NTK-HERCD-533 or NTK-HERCD-566. In those cases when autocrine stimulation was to be induced, the cells were infected with ^2TGFa virus (5 x 104 G418R colony forming units per ml). NIH 3T3 cells (105) were plated out on 6 cm plates in the presence or absence of 10 ng/ml EGF in 3 ml medium added to the top layer of MEM containing 10% FCS and 0.2% agar 10 (Gibco). The bottom layer contained MEM, 10% FCS and 0.4% agar.

Visible colonies were counted after 4 weeks.

For foci forming tests, subconfluent NIH 3T3 cells (105 cells/6 cm plate) were coinfected with NTK-HERc (1 x 104 G418R colony forming units per ml) followed by 4 founds of infection with either N2, NTK-HERK721 A, 15 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 cultured on 6 cm plates with DMEM containing 4% FCS in the presence or absence of lOng/ml EGF. The medium was changed every 3 days. The plates were stained with crystal violet and the 20^ foci were counted on day 18.

Having now generally described the invention, the same will be more readily understood through reference to the following methods and examples, which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

Example 1 Tyrosine Phosphorylation by Cells LU O , O / Cells that express the wild-type receptor alone or together with the mutant receptors were labeled with [35S]-methionine. They were incubated in ' '< hi 1 Ul o Ul 17- 24 4 2 the presence or absence of EGF for 10 minutes, were lysed and were subjected to immunoprecipitation with a mouse antibody to human EGF receptor (mAb 108). The precipitated proteins were separated by SDS-PAGE, were transferred to a nitrocellulose filter and the phosphorylation of tyrosine was detected with the help of the phosphotyrosine-specific mouse antibody 5E2 (Figure 2A). The amount of receptor present in the immune precipitate was detected by autoradiography of the same nitrocellulose filter (Figure 2B).

As shown in Figure 2A, lanes b and c, the addition of EGF to intact NIH 3T3 cells infected with the virus containing the wild-type EGF receptor induced a strong phosphorylation of tyrosine of the 170KD EGF receptor band. Due to this phosphorylation, the rate of migration of the EGF receptor in the SDS-PAGE dropped in comparison with that of the unphosphoryiated EGF receptor (Figure 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 domains of the EGF receptor as coded by the NTK HERCD-566 virus genome, even when the extracellular domain was expressed in a 4-fold excess relative to the wild-type receptor (Figure 2A, lanes d to f; Figure 2B, lanes d to f).

On the contrary, in a similar experiment using a virus that expressed the EGF receptor deletion mutant HERCD-533 (Figure 1), there was a strong dose-dependent inhibition of the EGF-induced phosphorylation of the wild-type EGF receptor (Figure 2A, lanes g to i) although the level of the 170 KD EGF receptor protein remained constant (Figure 2B, lanes g to i). The intensity of the tyrosine-phosphorylated bands decreased from 100% to 71% or 30%. Under these conditions, the EGF receptor had the same electrophoretic properties as an unphosphoryiated receptor (Figure 2B, lane i), which is in agreement with its state of tyrosine phosphorylation as detected by mAb 5E2 (Figure 2A, lane i).

When the wild-type receptor was coexpressed with the kinase-negative mutant HERK721A, an increased tyrosine phosphorylation of the 170 KD band was detected (Figure 2A, lanes k to m). The intensity of the signal for 244 23 9 tyrosine phosphorylation of the receptor increased from 251% to 337% and 450% according to densitometry analysis of the autoradiographs. Since the wild-type receptor and the kinase-negative mutant were the same size, the increased 170 KD signal in Figure 2B, lanes k to m, represents (he sum of the phosphorylation of the two receptors because the mutant receptor can be transphosphorylated by the wild-type receptor.

Example 11 Inhibition of EGF-Induced Cell Division Rate EGF stimulates cell division in NIH 3T3 fibroblasts that express the EGF receptor (Riedel etal., Proc. Natl. Acad. Sci., USA 55:1477-1481 (1988); and Prywes et al., EMBOJ. 5:2179-2190 (1986)). The influence of mutant receptors on the cell division regulated by the EGF wild-type receptor was determined on the basis of induction of DNA synthesis.

DNA synthesis was determined as the incorporation of I3H]-thymidine in cells infected with the NTK-HERc virus and the N2 virus control. Synthesis was stimulated maximally at 2 ng/ml EGF with half-maximum stimulation (ED50) at 0.66 ng/ml (Figure 3). Like previous results (Honegger etal., EMBOJ. 7:3045-3052 (1988); and Riedel etal., Proc. Natl. Acad. Sci., USA 55:1477-1481 (1988)), higher EGF concentrations led to lower levels of incorporation of [3H]-thymidine as indicated in Figure 3. The coexpression of the EGF receptor with HERCD-533 and HERK721A led to a definite shift in the dose-dependent curve toward higher EGF concentrations after 4 rounds of infection with the respective viruses (Figures 3A and B). This indicates that the cells had become less sensitive to the growth factor in comparison with HERc/N2 cells. The deletion mutant HERCD-533 as well as the point mutant HERK721A (Figure 1) had similar effects on the cell division signal mediated by the EGF wild-type receptor and caused a ten-fold increase in ED50 to 6.6 ng/ml EGF. In contrast with this, superinfection with NTK-HERCD-566 virus had no significant effect on the DNA synthesis stimulated by the wild-type receptor by EGF (Figure 3C).

Example III Antineoplastic Activity of the EGF Receptor Mutants It is known that overexpression of the EGF receptor causes an EGF-dependent cell transformation of NIH 3T3 cells (Di Fiore et al.. Cell 57:1063-1070(1987); VeLu« ai. Science237:1408-1410 (1987); and Riedel etal., Proc. Natl. Acad. Sci., USA 55:1477-1481 (1988)). In order to investigate the transformative potential of overexpressed EGF receptor mutants, EGF receptor was coexpressed with receptor mutants and then its ability to produce colonies in soft agar or foci in a monolayer cell culture was investigated. Stimulation of overexpressed EGF receptor was achieved either by adding EGF to the medium or by infection with a virus (^2TGFcr) having a TGF-a cDNA in order to produce an autocrinic activation system (Table 1; average values of four experiments are shown).

After infection with the NTK-HERc virus and the N2 controls, NIH 3T3 cells produced approximately 250 colonies in soft agar in the presence of 10 ng/ml EGF. By coinfection with ^2TGFa virus the formation of 148 colonies was achieved under otherwise identical conditions (Table 1). However, when the cells infected with EGF receptor were superinfected with either NTK-HER-K721A or NTK-HERCD-533 viruses, the colony forming capacity was suppressed almost completely. Coexpression of the EGF receptor with the extracellular domain HERCD-566 reduced the colony forming ability by about 50% due to stimulation with 10 ng/ml EGF in the agar layer, and by about 33% with stimulation by the autocrinic TGFa after infection with i/^TGFa virus.

Similarly, the focus-forming potential of the NTK-HERc virus in NIH 3T3 monolayer cultures was determined either in the presence of 10 ng/ml I:GF, which led to 920 foci per 10'' viruses, or after coinfection with 239 l/^TGFor virus, which led to 480. foci per 106 NTK-HERc viruses. Superi nfection with NTK-H ERK721A or NTK-HERCD-533 viruses suppressed the number of foci by more than 90% when stimulation was performed with EGF or with the ^2TGFa virus (Table 2), respectively.

Cells that coexpressed the EGF wild-type receptor and HERCD-566 had 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 stimulation with EGF and also with the ^2TGFa virus.

These data indicate that EGF receptor mutants have a definite antiproliferative potential as well as an antineoplastic potential and are thus excellent for the treatment of cancer. 24 4 23 9 1 Table 1 Formation of Colonies in Soft Agar Infection Number of C 10® CF + 10 ng/ml EGF Colonies/ U *2TGF N2 0 0 NTK-HERK721A 0 0 NTK-HERCD-533 0 0 NTK-HERCD-566 0 0 NTK-H ERc/N2 246 148 NTK-HERc/riTK-HERK721A 8 2 NTK-HERc/NTK-HERCD-533 6 4 NTK-HERc/NTK-HERCD-566 128 100 These colonies were counted after 4 weeks. The values represent the averages of four independent experiments. CFU stands for colony forming units.

Table 2 NIH 3T3 FOCUS FORMATION Cell Line Number ol 106CF +10 ng/ml EGF Foci/ U *2TGF N2 0 0 NTK-HERK721A 0 0 NTK-HERCD-533 0 0 NTK-HERCD-566 0 0 NTK-HERC/N2 920 480 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 the averages of four independent experiments. CFU stands for colony forming units. 244239 *22*

Claims (35)

WHAT WE CLAIM IS:

1. A genetically engineered signalling incompetent receptor tyrosine kinase which is capable of dimerizing with a signalling competent receptor, wherein said dimerization leads to inactivation of receptor tyrosine kinase activity, with the proviso that the receptor is not a mutant epidermal growth factor receptor.

2. The signalling-incompetent receptor of claim 1, wherein said receptor lacks the tyrosine kinase activity of its corresponding wild-type receptor.

3. The signalling-incompetent receptor of claim 2, wherein said receptor has a deletion in its tyrosine kinase domain.

4. The signalling-incompetent receptor of claim 2, wherein said receptor has a point mutation in its tyrosine kinase domain.

5. The signalling-incompetent receptor of claim 2, wherein said receptor comprises an extracellular domain and transmembrane region.

6. The signalling-incompetent receptor of claim 2, wherein said receptor comprises an extracellular domain.

7. The signalling-incompetent receptor of claim 5, wherein said extracellular domain and said transmembrane region are both of the wild-type.

8. The signalling-incompetent receptor of claim 6, wherein said extracellular domain is of the wild-type. 3 o MAR 1395 24 h 139

9. The signalling-incompetent receptor of any one of claims 1-8, wherein said receptor is a signalling-incompetent growth factor receptor.

10. The signalling-incompetent receptor of claim 9, wherein said receptor is a mutant platelet-derived growth factor receptor.

11. The signalling-incompetent receptor of any one of claims 1-8, wherein said receptor is a mutant HER2 receptor.

12. The signalling-incompetent receptor of any one of claims 1-8, wherein said receptor is a met receptor.

13. A pharmaceutical composition comprising: a genetically engineered, signalling incompetent receptor tyrosine kinase which is capable of dimerizing with a signalling competent receptor, wherein said dimerization leads to inactivation of receptor tyrosine kinase activity; appropriate pharmaceutical excipient(s); and appropriate pharmaceutical vehicles).

14. The pharmaceutical composition of claim 13, wherein said receptor is a mutant epidermal growth factor receptor.

15. The pharmaceutical composition of claim 14, wherein said receptor has a point mutation in amino acid position 721 of the wild-type epidermal growth factor receptor sequence. 244239

16. The pharmaceutical composition of claim 15, wherein said point mutation comprises an alanine residue in amino acid position 721 and for which producer microbcs have been deposited under file code.number DSM 6678 with the German Collection of Microorganisms and Cell Cultures.

17. The pharmaceutical composition of claim 14, wherein the 533 C-terminal amino acids of the wild-type epidermal growth factor receptor are deleted.

18. The pharmaceutical composition of claim 14, wherein the 566 C-terminal amino acids of the wild-type epidermal growth factor receptor are deleted and for which producer microbes have been deposited under file code number DSM 6679 with the German Collection of Microorganisms and Cell Cultures.

19. A pharmaceutical composition comprising: the genetically engineered signalling-incompetent receptor of any one of claims 1-10; appropriate pharmaceutical excipient(s); and appropriate pharmaceutical vehicle (s).

20. A pharmaceutical composition comprising: the signalling-incompetent receptor of claim 11; appropriate pharmaceutical excipients; and appropriate pharmaceutical vehicle (s).

21. A pharmaceutical composition comprising: the signalling-incompetent receptor of claim 12; appropriate pharmaceutical excipients; and -25- 244239 appropriate pharmaceutical vehicle(s).

22. The pharmaceutical composition of claim 13 or 19, wherein said receptor is associated with liposomes.

23. The pharmaceutical composition of claim 20, wherein said receptor is associated with liposomes.

24. The pharmaceutical composition of claim 21, wherein said receptor is associated with liposomes.

25. The pharmaceutical composition of claim 13 or 19, wherein said composition contains said receptor in the form of one or more recombinant vectors having nucleic acid fragments that code for said receptor.

26. The pharmaceutical composition of claim 20, wherein said composition contains said receptor in the form of one or more recombinant vectors having nucleic acid fragments that code for said receptor. ,

27. The pharmaceutical composition of claim 21, wherein said composition contains said receptor in the form of one or more recombinant vectors having nucleic acid fragments that code for said receptor.

28. The pharmaceutical composition of claim 25, wherein said vector is a recombinant retroviral vector. 744239 -26-

29. The pharmaceutical composition of claim 26, wherein said vector is a recombinant retroviral vector.

30. The pharmaceutical composition of claim 27, wherein said vector is a recombinant retroviral vector.

31. The pharmaceutical composition of claim 28, wherein said retroviral vector is selected from the group consisting of pNTK-HER-K721A and pNTK-HERCD-533, and for which microbes containing said vectors have been deposited under file codes DSM 6678 and DSM 6679, respectively, with the German Collection of Microorganisms and Cell Cultures.

32. The pharmaceutical composition of claim 29, wherein said retroviral vector is selected from the group consisting of pNTK-HER-K721A and pNTK-HERCD-533, and for which microbes containing said vectors have been deposited under file codes DSM 6678 and DSM 6679, respectively, with the German Collection of Microorganisms and Cell Cultures.

33. The pharmaceutical composition of claim 30, wherein said retroviral vector is selected from the group consisting of pNTK-HER-K721A and pNTK-HERCD-533, and for which microbes containing said vectors have been deposited under file codes DSM 6678 and DSM 6679, respectively, with the German Collection of Microorganisms and Cell Cultures.

34. A pharmaceutical composition as claimed in claim 13, substantially as herein described with reference to the examples. r 244239

35. A genetically engineered signalling incompetent receptor tyrosine kinase as claimed in claim 1, substantially as herein described with reference to the examples. ' authorised agents j r'ARK & SON P