CN1071586A

CN1071586A - The application of receptor of mutation growth factor of using as medicine and treatment cancer thereof

Info

Publication number: CN1071586A
Application number: CN 92111396
Authority: CN
Inventors: A·乌尔里克; N·H·雷德曼
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: 1992-09-05
Publication date: 1993-05-05
Also published as: AU2518592A; EP0667899A1; FI941053A; NZ244239A; NO940778L; AU669857B2; JPH07502884A; DE4129533A1; PT100844A; CA2117073A1; FI941053A0; WO1993005148A1; NO940778D0; MX9205084A

Abstract

The present invention relates to be suitable for doing the receptor of mutation growth factor of medicine.This class receptor of mutation growth factor is useful especially to the treatment cancer, especially that carcinoid that treatment growth factor receptors hyperfunctioning when the carcinogenesis is worked.The invention discloses as the EGF receptor mutation kind of controlling the cancer specific drug, wherein the tyrosine-kinase enzyme activity of wild-type receptor is eliminated by point mutation in the tyrosine kinase domain or disappearance.

Description

The application of receptor of mutation growth factor of using as medicine and treatment cancer thereof

The present invention relates to the medicine that has the receptor of mutation growth factor of curative properties, contain a kind of receptor of mutation growth factor at least, and the application of one or more these type of mutant receptors treatment cancers.

The cell growth is a kind of careful adjustment process that depends on the body special requirement.In young body, the cell division rate surpasses cell mortality, and this just causes body to increase.In full grown body, it is flat rare that cell regeneration and cell death reach, and forms " permanent attitude ".But under some rare situations, cell proliferation is out of control, even do not need this type of cell of higher number in the body especially, they but still begin growth and division.This cell out of control growth is the cause of cancer.Can cause the factor of cell growth out of control, usually be factor chemically, but also can be the factor of physics aspect, for example radioactive radiation.

The approach that two kinds of treatment cancers are arranged at present, basically.Or remove cancerous cell through surgical operation fully from the sufferer body, or manage to make the cell of body mesometamorphism not work the mischief, for example by administration or physical therapy method such as X-ray therapy.

In chemotherapy, usually use medicine, these medicines influence the DNS metabolism with certain form, and make the ramp cell that certainly leads to higher metabolic efficiency undermined, become and do not divide or only slow splitted cell.Yet a critical defect of many amic therapy methods is that used high effective substance specificity is very little, makes healthy cell also undermined in chemotherapy.In addition, the also requirement of this very little specificity of high effective substance, it decides dose at every turn all must be in kill cancer cell, and cell as few as possible impairs one's health.This does not usually accomplish, thereby cancer patient causes the necessary various function that earns a bare living because of dead in the later stage disappearance because cancerous cell constantly spreads.

Task of the present invention is, the high effective substance that provides another kind to have valuable curative properties, and a kind of medicine that contains this high effective substance is provided, this high effective substance or medicine are useful especially to the treatment cancer.

By the present invention, this task is made medicine with a kind of receptor of mutation growth factor and is solved with the medicine that contains a kind of receptor of mutation growth factor at least.

According to the present invention, can prove that receptor of mutation growth factor has valuable curative properties, they are particularly useful for treating cancer.

Growth factor receptors in the human cancer cell is taken place and bred, plays decisive role.Growth factor receptors participates in Normocellular Growth Control.Fissional real signal is a somatomedin, and the needs of body are depended in its formation.Receptor plays a part to transmit signal; Also promptly, it participates in the conversion of extracellular growth signals at cell interior in the cell division activity.With regard to many growth factor receptorses, they transfer to phosphate base that the ability on the tyrosine-based plays decisive role in the protein after somatomedin is attached to extracellular domain.These receptors are also referred to as receptor tyrosine kinase.Yarden, Y. and Ullrich, A. be at Rev.Biochem.1988, and 57, introduced the overview of receptor tyrosine kinase among the 443-78.After the somatomedin combination, the dimerization of this growth factor receptors is another significant process in signal transduction process.Under the intervention of the growth factor receptors with tyrosine kinase activity, the extracellular signal becomes the process of signal in the cell, can be decomposed into following 5 stages:

1, somatomedin (being also referred to as ligand) is attached on the extracellular domain of receptor, brings out conformation change; This causes

2, the receptor that changes of conformation carries out dimerization;

3, bring out the allostery variation of endochylema domain simultaneously, bring out the kinases vigor again with this;

4, in the receptor dimer, tyrosine-based generation transphosphorylation, this effect produces a kind of activated receptor conformation again, and makes it stable; And

5, peptide substrate generation phosphorylation, and with cytokine generation reciprocal action.

The superfunction of signal transduction chain causes the mitotic activity of corresponding cell too high, and degenerates into cancerous cell under extreme case.Ullrich, A. and Schlessinger, J. at Cell(1990) 61, among the 203-212 brief growth factor receptors and in the effect of signal when extracellular environment is delivered to intracellular environment, and mutant receptors is to the issuable influence of the formation of cancerous cell.

Be surprised to find that now when the wild-type receptor co expression of mutant receptors and a kind of cell, the signal transduction chain in above-mentioned 5 stages can be receptor of mutation growth factor and blocks or suppress.Therefore, receptor of mutation growth factor is suitable to medicine, available it treat the long signal of syngenesis and aggravate to be delivered to cancer relevant in the cell by corresponding receptor.

The tyrosine that preferred receptor mutation kind no longer has wild-type receptor activates enzyme activity.After ligand formed, this mutant can not make the tyrosine-based generation phosphorylation in receptor dimer or the peptide substrate again.Therefore, the extracellular growth signals becomes in the cell that the transformation of signal is blocked or part suppresses.

Have a point mutation just can achieve the goal in the wild-type receptor because when wild-type receptor when this point mutation has lost the tyrosine-kinase enzyme activity, it just no longer has ability to function.As medicine, a kind of like this point mutation is preferred.

In addition, preferential what select is a kind ofly at tyrosine kinase domain the mutant receptors of disappearance to be arranged, and this disappearance makes the tyrosine kinase devitalization.

Preferably, in the cytoplasmic structure territory, lack the receptor that is suddenlyd change, still also contain and stride the film district.Have existing mutant receptors of striding the film district, can suppress the growth signals transmission very effectively, and thereby than there not being the receptor stride the film district, for example the mutant of being made up of extracellular domain demonstrates better therapeutical effect.

Especially being suitable as medicine, is the mutant of receptor tyrosine kinase, for example EGF receptor, pdgf receptor or NGF receptor.

The mutant receptors of epidermal growth factor (EGF) especially is particularly suitable for.

Under a kind of situation of particularly preferred EGF receptor mutation kind, on aminoacid 721 positions of wild-type receptor sequence a point mutation is arranged.In a kind of preferred mutant, 721 locational lysine bases are replaced by the alaninyl of mutant.This mutant is deposited with German microorganism and cell culture preservation company limited (DSM) according to budapest treaty with numbering DSM6678.In another preferred EGF receptor mutation kind, the 533C-end aminoacid of disappearance wild-type receptor.This mutant is deposited with numbering DMS6679.

The receptor mutation kind can be according to general gene technology method, prepare by wild-type receptor, these gene technology methods are Sambrook for example, and J. etc. are at (1989) Molecular Cloning(Cold Spring Harbor Laboratory Press) in existing the description.

Medicine of the present invention contains a kind of said mutation growth factor receptors and adjuvant drug and carrier mass commonly used at least.

Particularly preferred, be a kind of medicine that is encapsulated in one or more receptor mutation kinds in the liposome that contains.In order to make liposome arrive target tissue selectively, if liposome contains antibody in its film, and the defined epitope of these antibody capable identification target cells, and be selectively combined on these epi-positions, then be favourable.Therefore, the receptor mutation kind arrives target tissue selectively, and brings into play the effect of its expection there.

In addition, it is preferred that a kind of medicine is arranged, and this medicine contains above-mentioned one or more receptors of one or more recombinant retroviral vector forms.Recombinant vector contains the nucleic acid fragment that above-mentioned one or more receptors of coding are used.To after the patient dispensing, retrovirus is with regard to target cell infection, and the receptor mutation kind is expressed therein.Giving to be encapsulated in the high effective substance in the liposome, is a kind of dosing mode commonly used at present.

A kind of particularly preferred medicine is arranged, and it contains the retroviral vector pNTK-HER-K721A and/or the pNTK-HERCD-533 of coding EGF receptor mutation kind, and they are deposited with DSM with numbering DSM6678 and DSM6679 respectively.

Said mutation receptor or contain the medicine of these mutant receptors is specially adapted to treat cancer.Wherein, can treat especially effectively because of growth factor receptor function those cancer classes due to hyperfunction.Belong to these cancer classes, especially mastocarcinoma, ovarian cancer and pulmonary carcinoma.Slamon, D.J. etc. are at Science(1987), 235,177-182 and Science(1989), 244,707-712, and Kern, J.A. wait at Cancer Res.(1990), 50, discussed surface receptor role in these cancers among the 5184-5191 in detail.

Be not subjected to the limitation of certain particular theory, people can think why described receptor mutation kind can bring into play its effect in target cell, are because except that wild-type receptor, mutant also is embedded in the target cell membrane, so the receptor mutation kind just makes the hypofunction of wild-type receptor.

Mutant with the EGF receptor is that model elaborates the present invention below.

Ullrich, people such as A. are at Nature(1984), 309, the EGF-R ELISA of recording and narrating among the 418-425 (EGF receptor) is the 170kD glycoprotein with tyrosine-kinase enzyme activity.Ullrich, A. and Schlessinger, J. is at Cell(1990), 81, described in detail among the 203-212 from conjunction with ligand up to the molecular level process that stimulates the kinases vigor.As Riedel, H. wait at Proc.Natl.Acad.Sci., USA is among (1988) 85, the 1477-1481 and Di Fiore, P.P. wait the people at Cell(1987), 51, described in the 1063-1070, when EGF under normal circumstances causes mitogenesis when reaction in the fibroblast, EGF receptor signal propagation function due to the overexpression receptor is hyperfunction, has caused the ligand dependency of DIH 3T3 mouse cell to transform.Clinical examination completely, supported this function of this receptor in the forming process of various cancers such as mastocarcinoma, ovarian cancer and pulmonary carcinoma, this is as Slamon, and people such as D.J. are at Science(1987), 235,177-182 and Science(1989), 244,707-712, and Kern, J.A. wait the people at Cancer Res.(1990), 50, described in the 5184-5191.

Now be surprised to find that no longer have the EGF receptor mutation kind of tyrosine-kinase enzyme activity,, can cancel the phenotype that has transformed expressing the expression that transforms in the cancerous cell to be carried out of EGF wild-type receptor.

Material and method

The preparation of recombinant retrovirus

Keller, people such as G. be at Nature, (1985), 318,149-154; Stewart, people such as C.L. be at EMBO J., (1987), 6,383-388; Von Ruden, T. and Wagner, E.F. be at EMBO J., and (1988), 7, retrovirus expression vector pN2, pNTK2 and pNTK-HERC have been described in detail in detail among the 2749-2756.PNTK-HER-k 721A prepares by the BgL II fragment of CMVHER-k721A among the clone pNTK-HERc.By as Sambrook, J. wait the people at Molecular cloning(Cold Spring Habour Laboratory Press, 1989) the general cloning process described in, pass through Liven, E. wait the people at J.Biol.Chem., 260, method described in the 12490-12491, on the both sides of the big fragment XBa I/Xho of the 2kb of pLSXNA 8 I, form a CLa I site and prepare pNTK-HERCD-533, then with making the cracked pNTK2 of CLaI connect 2kb CLaI fragment.By with the ClaI fragment cloning of CVNHERXCD to the CLaI position of pNTK2, prepare the NTK-HERCD-566 construct.This construct is deposited with numbering DSM6680.Eco Battalion (ecotrope) recombinant retrovirus of nourishing one's nature, at J.Virol., the non-auxiliary virus production described in (1988) 62, the 1120-1124 is that GP+E-86 prepares with Markowitz D..Stable GP+E-86 produces system, and by means of as Miller, A.D. and Buttimore, C. be at Mol.Cell.Biol., a kind of improvement infection writing-method (Infektionsprotkoll described in (1986) 6, the 2895-2902 ₁) prepare.Low liter both sexes trophism (amphotrophes) virus, by being to prepare among the PA317 to helper free packaging cell (to this with retrovirus expression plasmid transient transfection, Miller, A.D. wait the people at Mol.Cell.Biol., 1985,5, given explanation among the 431-437), and then at G418(1mg/ML) in to select production be after the clone of GP+E-86, infect secondary package cell GP+E-86 with it.The retrovirus dilution series that contains the liquid that disappears on the acellular GP+E-86 by usefulness infects NIH 3T3 cell, and measures the resistive colony number of G418, measures virus titer.Blashand, A.J. have discussed the retrovirus (ψ 2TGF α) that contains tumor growth factor α (TGF α) gene at Oncogene among (1990) 5, the 1213-1221.

By the retrovirus metastatic gene

In the presence of 4 μ g/L polybrenes (Aldrich), with the release height NTK-HERc virus (5 * 10 of tiring ⁵G418 ^RThe every mL of colony forming unit) GP+E-86 cell conditioned medium liquid is cultivated inferior junction NIH 3T3 cell (10 ⁵Individual cell/6cm culture plate) 4-12 hour, then in the GP+E-86 cell conditioned medium liquid that discharges high titre N2, NTK-HERK 721A, NTK-HERCD-533 virus or NTK-HERCD-566 virus, cultivates.As Bordignon, people such as C. described in the 6748-6752, improve receptor expression level by repeatedly infecting at Proc.Natl.Acad.Sci.USA86.In above-mentioned test, with 1mL GP+E-86 cell dilution supernatant (1.25 * 10 ⁵Colony forming unit) infect once, or with the undiluted GP+E-86 cell conditioned medium liquid (5 * 10 of equal volume ⁵Colony forming unit) infect 1 to 4 time, described GP+E-86 cell discharges high titre N2, NTK-HERK721A, NTK-HERCD-533 virus or NTK-HERCD-566 virus.

Receptor phosphorylation in the intact cell

With the cell that is as above infected, on the foster flat board of 10cm a word used in place name, cultivate 90% and join, wash, and at the DMEM(Gibco that does not contain methionine) in, add the 50 μ Ci/mL that contain 1%FCS ³⁵S-methionine (Amersham) was cultivated 16 hours.With 20ng/mL EGF(Amgen Corp.) irritation cell 10 minutes, and under 4 ℃, cell is dissolved in 0.5mL dissolving buffer (50mM HEPES pH7.2,150mMNaCl, 1.5mM MgCl, 1mM EGTA, 10% glycerol (Glyerzin), 1%Triton X-100,1mM PMSF, 10mg/mL aprotinin, 100 μ M sodium vanadates).Under 4 ℃, with lysate in Eppendorf type centrifugal separator centrifugal 10 minutes with about 12000g.Then, with supernatant with excessive monoclonal antibody 108.1(Honegger, A.M., Ullrich, A. and Schlessinger, J. is at Proc.Natl.Acad.Sci., USA addresses this among (1989) 86, the 925-929) and protein A-agarose cultivated 4 hours down at 4 ℃.Immunoprecipitate HNTG(20mM Hepes pH7.3,150mM NaCl, 0.1% Triton X-100 and 10% glycerol) wash twice.Pellet is resuspended in the test buffer, boils 5 minutes, and use SDS-PAGE(7.5%) analyze.With electrophoresis method protein transduction is moved on on the nitrocellulose, then (see Fendly, people such as B.M., 1990, Cancer Research, 50,1550-1558) cultivation with the monoclonal mouse antibody (5E2) of anti-phosphorus tyrosine.For the purpose of checking, in ECL-substrate-reaction (Amersham) afterwards, cultivate the nitrocellulose filter with the goat anti-mouse antibody that connects peroxidase.After detecting, the nitrocellulose filter is washed with the PBS that contains 0.2%Tween 20 with the X-Omat of Kodak (Kodak) egative film.Then, verify by the radioautogram art ³⁵The protein of S-methionine labelling.Strip-like developing pipe density is determined with the developing densitometry.

[ ³H]-thymidine mixes

After carrying out 4 subinfections, infect inferior junction HIN 3T3 cell (10 with NTK-HERc is auxiliary as described with N2, NTK-HERK 721A, NTK-HERCD-533 or with NTK-HERCD-566 ⁵Individual cell/6cm culture plate).This cell is all assigned on the 12 hole Coase tower flat boards (costarplatten).After reaching junction, make cell monolayer at 0.5mL DMEM, among 0.5% FCS hungry 24 hours, and after adding EGF18 hour, with this cell with 0.5 μ Ci methyl-[ ³H] thymidine (Amersham) labelling 4 hours.Cell is washed twice with PBS, and then precipitating 1 hour with 10%TCA on ice.Use the 10%TCA washing precipitate, and be dissolved in 200 μ L 0.2N NaOH/0.2%SDS again.In and lysate, and the radioactivity of coming quantitative assay to mix with scintillation counting technique.

Conversion test

In order to check the colony of NIH 3T3 cell in soft agar to form ability, using N2, NTK-HERK 721A, NTK-HERCD-533, or with after the NTK-HERCD-566 infection 4 times, with the NIH3T3 cell (10 after the NTK-HERc infection junction ⁵Individual cell/6cm culture plate).Can produce under those situations of autocrine stimulation, with ψ 2TGF α virus (5 * 10 ⁴G418 ^RThe every mL of colony forming unit) infection cell.On the 6cm culture plate, with NIH 3T3 cell (10 ⁵Individual) in the 3mL culture medium, contain or do not contain under the situation of 10ng/mL EGF, be coated onto fully by MEM and form and contain on the surface layer of 10%FCS and 0.2% agar (Gibco).Bottom contains MEM, 10%FCS and 0.4% agar.The four stars after date is counted visible colony.

Form test for carrying out plaque (Foci), using N2, NTK-HERK 721A, NTK-HERCD-533 virus, or with behind the NTK-HERCD-566 viral infection four times, with NTK-HERc(1 * 10 ⁴G418 ^RThe every mL of colony forming unit) comes the auxiliary inferior junction NIH 3T3 cell (10 that infects ⁵Individual cell/6cm culture plate).In some experiment, with ψ 2TGF α virus (1 * 10 ³G418 ^RThe every mL of colony forming unit) this cell of repeated infection.Metainfective cell with containing the DMEM of 4%FCS, is cultivated being with or without in the presence of the 10ng/mL EGF on the 6cm culture plate.Changed a subculture every 3 days.The culture plate violet staining was counted plaque at the 18th day.

Description of drawings

Fig. 1: illustrative people's EGF wild-type receptor and sudden change EGF receptor.Pointed out the position of rich cysteine structure territory (CYS), tyrosine kinase (TK) domain and transmembrane domain (TM) among the figure.Mutant HERK 721A has a point mutation (replacing lysine with alanine) on 712, HERCD-533 and HERCD-566 then are loaded with 533 and 566 amino acid whose C terminal deficiencies respectively.Livneh, people such as E. be at J.Biol.Chem., and (1986), 260,12490-12497, and Honegger, people such as A.M. are at Cell(1987), 51, these mutants have been described in detail in detail among the 199-209.

Fig. 2 A: the tyrosine phosphorylation of wild type EGF receptor and sudden change EGF receptor.To only express wild-type receptor, or the cell of while co expression wild-type receptor and mutant receptors, with [ ³⁵S]-the methionine labelling spends the night, and then is being with or without in the presence of the 2ng/mL EGF, cultivated 10 minutes.With cytolysis, and with anti-EGF receptor antibody (mAb108) make it the precipitation, separated with SDS-PAGE, and carried out immune analysis with anti-phosphotyrosine receptor (5E2).

Fig. 2 (B): the expression of EGF receptor on NIH 3T3 cell.To only express wild-type receptor, or the cell of while co expression wild-type receptor and mutant receptors, with [ ³⁵S]-the methionine labelling spends the night, is connected on to be with or without 20ng/mL EGF and to exist down and cultivated 10 minutes.With cytolysis, and, separate with SDS-PAGE, and carry out immunology detection, wherein carry out the ECL substrate reactions with anti-phosphotyrosine receptor (5E2) with anti-EGF receptor antibody (mAb108) precipitation.With the PBS flush away ECL substrate that contains 0.2% Tween 20, and with autoradiography detect [ ³⁵S]-protein that the methionine labelling is crossed.

Fig. 3: simulation EGF [ ³H]-the thymidine insertion.Only expressing wild-type receptor (dotted line), or in the cell of co expression wild-type receptor and mutant receptors, for example among the A wild type EGF receptor+K721A, among the B wild type EGF receptor+CD-533, among the C wild type EGF receptor+CD-566, in 12 hole Coase tower culture plates, cultivate junction, and in containing the EMEM of 0.5%FCS hungry 2 days.Add the EGF of 10%FCS or variable concentrations, and interpolation in 18 hours after adding EGF [ ³H]-thymidine (0.5 μ Ci/ hole), last 4 hours, and measure its mixing in DNS.The reaction of record mitogenesis is to disclose the relation between dosage and the reaction.Institute's measured value mixes with the substrate thymidine and proofreaies and correct, and the maximum observing response of EGF is decided to be 100%.Shared triangle indicates the half value that maximum thymidine inserts.

The result

To only express the cell of wild-type receptor or co expression wild-type receptor and mutant receptors, with [ ³⁵S]-the methionine labelling, cultivated 10 minutes being with or without in the presence of the EGF, dissolving, and with mouse antibodies (mAb108) immunoprecipitation of anti-human body EGF receptor.Sample separates with SDS-PAGE, transfers in the nitrocellulose filter, and with phosphotyrosine specificity mouse antibodies 5E2(Fig. 2 A) detect tyrosine phosphorylation.Be present in the acceptor quantity in the immunoprecipitate, carry out autoradiography with same nitrocellulose filter and detect (Fig. 2 B).

Shown in mottling b and c among Fig. 2 A, EGF is added with the normal NIH 3T3 cell that virus infected that contains wild type EGF receptor, induced the strong tyrosine phosphorylation of 170kDEGF receptor strip-like developing pipe.Compare with unphosphorylated EGF receptor, the migration velocity of EGF receptor in SDS-PAGE reduces because of phosphorylation, as being seen by speckle b and c among Fig. 2 B.Wild-type receptor stimulates the degree of EGF phosphorylation, just as passing through NTK-HERCD-566 viral gene group coding, the co expression owing to the solubility ectodomain of EGF receptor does not reduce, even the expression of ectodomain is than wild-type receptor excessive 4 times of also right (Fig. 2 A, speckle d to f; Fig. 2 B, speckle d to f).

On the contrary, in a kind of similar test, use a kind of receptor (Fig. 1) that film is stablized EGF receptor deletion mutation HERCD-533 of expressing, EGF to wild type EGF receptor induces phosphorylation to produce strong dose-dependent inhibition effect (Fig. 2 A, speckle g to i), although 170kD EGF receptor protein content remains unchanged (Fig. 2 B, speckle g to i).The intensity of tyrosine phosphorylation strip-like developing pipe reduces to 71% and 30% from 100% respectively.With this understanding, the EGF receptor demonstrates with electrophoretic characteristic (Fig. 2 B, speckle i) that the phosphorylation receptor is not the same, this with mAb 5E2(Fig. 2 A, speckle i) situation of its tyrosine phosphorylation of being detected is consistent.

When wild-type receptor and negative kinase mutant kind HERK721A co expression, detect the tyrosine phosphorylation aggravation (Fig. 2 A, speckle k to m) of 120kD strip-like developing pipe.According to the autoradiography analysis of density measurement, receptor tyrosine phosphorylation signal intensity brings up to 337% and 450% respectively from 251%.Because wild-type receptor size the same with negative kinase mutant kind, thus among the speckle k to m of Fig. 2 B, the enhancing of 170kD signal, the phosphorylation summation of two kinds of receptors of expression, in such cases, mutant receptors can be changeed phosphorylation by wild-type receptor.

The inhibition of EGF inducing cell division rate

As Riedel, people such as H. are at Proc.Natl.Acad.Sci.USA, (1988) 85,1477-1481, and Prgwes, people such as R. are at EMBO J.(1986), described in the 2179-2190, EGF can stimulate the NIH3T3 fibroblast division of expressing the EGF receptor.The influence that the cell division that mutant receptors is controlled the EGF wild-type receptor produces is synthesized by means of inducing DNA and to be measured.

This DNA is synthetic, in the cell that infects with NTK-HERc virus and N2 virus contrast groups, be chosen to be [ ³H]-thymidine mixes, and this synthesize when 2ng/mL EGF, stimulate maximum, at 0.66ng/mL(Fig. 3) time, be half maximal stimulus (ED ₅₀).Be similar to previously as Honegger, people such as A.M. are at EMBO J., and among (1988) 7, the 3045-3052 and Riedel, people such as H. are at Proc.Natl.Acad.Sci., USA, that the result described in (1988) 85, the 1477-1481, higher EGF concentration cause is lower [ ³H]-the thymidine incorporation, as shown in Figure 3.After with corresponding viral infection 4 times, the co expression of EGF receptor and HERCD-533 and HERR721A makes the dose dependent curve move (Fig. 3 A and B) to high EGF concentration direction significantly.This shows, compares with the HERc/N2 cell, and this cell is not quite responsive to somatomedin.No matter deletion mutation kind HERCD-533, or point mutation kind HERK721A(is Fig. 1), all the cell division signal that the EGF wild-type receptor is facilitated shows similar influence, and makes ED ₅₀Become 10 times of ground to be increased to 6.6ng/mL EGF.In contrast, synthetic with the repeated infection that NTK-HERCD-566 virus is carried out for stimulate the DNA of wild type with EGF, not appreciable impact (Fig. 3 C).

The carcinogenesis activity of EGF receptor mutation kind

As everyone knows, the repeating of EGF receptor expressed, and causes that the EGF dependent cell of NIT 3T3 cell transforms, this is as Di Fiore, and people such as P.P. are at Cell, and (1987) 51, among the 1063-1070, Velu, people such as T.J. are at Science, among (1987) 237, the 1408-1410, and Riecel, H. wait the people at Proc.Natl.Acad.Sci., USA is described in (1988) 85, the 1477-1481.Whether can be suppressed in order to check the EGF receptor that repeats to express to transform potentiality, make EGF receptor and receptor mutation kind co expression, and check that then it forms colony in soft agar, or in cell monolayer is cultivated, form the ability of plaque by EGF receptor mutation kind.By adding EGF to culture medium, or by infecting with the virus (ψ 2TGF α) that has TGF-α DNA, reach stimulation, so that produce a kind of autocrine (Autocrin) activation system (table 1 illustrates the meansigma methods of 4 tests) to overexpression EGF receptor.

After infecting with NTK-HERc virus and N2 matched group, NIH 3T3 cell forms about 250 colonies in the soft agar that has 10ng/mL EGF to exist.Under the identical situation of other conditionally complete,, then form 148 colonies (seeing Table 1) by with the coinfection of ψ 2TGF α virus.Yet when the cell that infected with the EGF receptor during with the viral repeated infection of NTK-HER-K721A or NTK-HERCD-533, colony formation ability is almost constrained fully.The co expression of EGF receptor and extracellular domain HERCD-566 when stimulating with 10ng/mL EGF in agar layer, makes colony form ability and reduces by 50% approximately, and after with ψ 2TGF α viral infection, when stimulating with autocrine TGF, then reduces by 33% approximately.

The plaque of measuring NTK-HERc virus in the NIH 3T3 monolayer culture with similar approach forms potentiality, when 10ng/mL EGF exists, and per 10 ⁶Individual virus forms 920 plaques, or after using the coinfection of ψ 2TGF α virus, per 10 ⁶Individual NTK-HERc virus forms 430 plaques.With the repeated infection of NTK-HERK721A or NTK-HERCD-533 virus, when stimulating with EGF, the plaque number reduces by 100% or 90% approximately, when stimulating with ψ 2TGF α virus, the plaque number reduce about 75% or 71%(table 2).

The cell of co expression EGF wild-type receptor and HERCD-566 shows with the same plaque number of cell of expressing the EGF receptor and infecting with contrast virus N 2.Not only when stimulating, and when stimulating, all observe this result with ψ 2TGF α virus with EGF.

From the above mentioned as seen, EGF receptor mutation kind not only has clear and definite anti-hypertrophy potentiality, and has the potentiality of carcinogenesis, therefore is applicable to the treatment cancer unsurpassedly.

The formation of colony in table 1 soft agar

Colony number/10 ⁶CFU

Infect

+10ng/mL EGF+2TGF

N2 0 0

NTK-HERK721A 0 0

NTK-HERCD-533 0 0

NTK-HERCD-566 0 0

NTK-HERc/N2 246 148

NTK-HERc/NTK-HERK721A 8 2

NTK-HERc/NTK-HERCD-533 6 4

NTK-HERc/NTK-HERCD-566 128 100

The colony of counting after 4 weeks.Institute's indicating value is the meansigma methods of 4 parallel tests.CFU represents colony forming unit.

Table 2 NIH 3T3 plaque forms

Plaque number/10 ⁶CFU

Cell line

+10ng/mL EGF+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 plaque of counting after 14-16 days.Institute's indicating value is the meansigma methods of 4 parallel tests.CFU represents colony forming unit.

Claims

1, is used as the receptor of mutation growth factor of medicine.

2, the described receptor of mutation growth factor of claim 1 is characterized in that, it no longer has the tyrosine-kinase enzyme activity of wild-type receptor.

3, each described mutant receptors in the claim 1 or 2 is characterized in that, it is a point mutation kind of wild-type receptor.

4, each described mutant receptors in the claim 1 or 2 is characterized in that, it has a disappearance in tyrosine kinase domain.

5, each described mutant receptors in the claim 1,2 or 4 is characterized in that, the film district is striden but exist in its disappearance cytoplasmic structure territory.

6, each described mutant receptors in the claim 1 to 5 is characterized in that, it is a kind of receptor tyrosine kinase of sudden change.

7, the described mutant receptors of claim 6 is characterized in that, it is a kind of sudden change EGF receptor.

8, the described mutant receptors of claim 7 is characterized in that, it has a point mutation on the amino acid position 721 of wild-type receptor sequence.

9, the described mutant receptors of claim 8 is characterized in that, it has an alaninyl on 721 positions.

10, the described mutant receptors of claim 7 is characterized in that, the 533C-end aminoacid deletion of wild-type receptor.

11, the medicine that contains each described receptor of mutation growth factor and adjuvant drug commonly used and carrier mass in a kind of claim 1 to 10 at least.

12, the described medicine of claim 11 is characterized in that, it contains one or more and is encapsulated in described receptor in the liposome.

13, claim 11 or 12 described medicines is characterized in that, it contains one or more described receptors of one or more recombinant retroviral carrier formats, and described carrier carries the nucleic acid fragment of coding receptor.

14, the described medicine of claim 13 is characterized in that, it contains retroviral vector pNTK-HER-K721A and/or pNTK-HERCD-533, and these two kinds of carriers are deposited with German microbial preservation chamber with numbering DSM6678 and DSM6679 respectively.

15, at least one described, as to be used for the treatment of cancer medicine in the claim 11 to 14.

16, each described mutant receptors is used for the treatment of the application of cancer in the claim 1 to 10.

17, the described application that is used for the treatment of the cancer class of claim 16, described cancer system by growth factor receptor function hyperfunction due to.

18, the described application that is used for the treatment of mastocarcinoma, ovarian cancer and/or pulmonary carcinoma of claim 17.