Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• 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.
• the cell division rate outweighs the death rate of cells, which leads to an increase in the size of the organism.
• the formation of new cells and cell death are balanced so that a "steady state" arises.
• 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.
• 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.
• 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.
• 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.
• Deective 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.
• 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.
• 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.
• 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.
• Platinum-derived factor 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.
• EGF Extracellular growth factor
• 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.
• Retroviral vectors are understood to mean recombinant vectors that are retroviruses.
• 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
• receptor tyrosine kinases 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.
• 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:
• EGF-R epidermal growth factor receptor
• 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.
• 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.
• a point mutant e.g. HERK721A is particularly preferred.
• a mutated receptor that carries a deletion in the tyrosine kinase domain that leads to a loss of tyrosine kinase activity.
• 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.
• EGF receptor is located at amino acid position 721 of the wild-type receptor sequence, a point mutation.
• 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.
• 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.
• 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.
• 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.
• 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.
• these compositions can also contain additives, wetting agents, emulsifying and suspending agents or fragrances.
• 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.
• an "effective amount" of a pharmaceutical composition is an amount sufficient to achieve the desired biological effect.
• 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.
• 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.
• 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.
• 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 ⁇ g / 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.
• 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 ⁇ Ci / 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 ⁇ M 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 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.
• E2 phosphotyrosine
• 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.
• 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 ⁇ Ci 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.
• NIH 3T3 cells 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 "_, 2TGF ⁇ - virus (5x10 G418 R colony forming units per mL).
• NIH 3T3 cells (10) were dissolved in 6 cm plates plated in the Anoch ⁇ 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.
• 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.
• 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.
• 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.
• 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.
• mAb 108 anti-EGF receptor antibody
• 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 (mAblO ⁇ ), 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.
• 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 ⁇ Ci / 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.
• 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.
• EGF receptor 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. - -
• 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.2TGF ⁇ ) carrying TGF- ⁇ DNA to produce an autocrine activation system (Table; 1 average values from four experiments are shown).
• NIH 3T3 cells formed approximately 250 colonies in soft agar in the presence of 10 ng / ml EGF.
• Coinfection with _2TGF ⁇ virus resulted in the formation of 148 colonies under otherwise identical conditions (Table 1).
• 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.
• 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
• EGF receptor mutants have both a clear antiproliferative and anioncogenic potential and are therefore outstandingly suitable for the treatment of cancer.
• NTK-HERK721A 0 0 NTK-HERCD-533 0 0 NTK-HERCD-566 0 0
• CFU colony-forming units
• NTK-HERK721A 0 0 NTK-HERCD-533 0 0 NTK-HERCD-566 0 0
• CFU colony-forming units

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