CN1151843C - Novel anti-tumor medicine and its screening method and use - Google Patents
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Abstract
The present invention aims to provide a method for sieving an antitumor drug. The present invention aims to provide an antitumor drug capable of powerfully and specifically inhibiting the growth of a tumor cell and a method for powerfully and specifically inhibiting the growth of a tumor cell.
Description
The present invention relates to immunology.More particularly, relate to the enzyme with ErbB, especially the ErbB2/ErbB3 heterodimer is as the method for effect target inhibition growth of tumour cell, the method for screening antitumor drug, and the antitumor drug that obtains thus.
Tumor disease is a kind of human dead principal disease that causes.It results from physiology cell proliferation out of control has influenced the body normal physiological condition, thereby produces serious pathological reaction, often causes death.Though spending great effort aspect research of tumor disease and the processing, tumor disease remains human main causes of death at present.The method of multiple processing tumor disease is arranged, comprise operation, radiotherapy and chemotherapy.Operation can not fully be driven away tumor cell among the patient body with radiotherapy, and chemotherapy combines the growth that generally is used to control tumor cell separately or with additive method.The common target of antitumoral compounds among the patient body is to prevent tumor cell proliferation or kill somatoblast.When chemical compound during to the tumor cell toxigenicity, they also seriously influence those usually to the teleorganic proper splitting cell of people.Therefore, be exactly to seek a single-minded blocking-up of energy or kill tumor cell and the method that do not influence normal cell propagation to a main direction in the tumor disease research.Currently be starved of a kind of like this method of handling tumor disease patient.
ErbBS is a receptoroid protein tyrosine kinase, and the cell signal of ErbB mediation plays an important role in the cardiac function of fetal development or adult.On cellular level, signal (Gassmann and Lemke, 1997 that the propagation of the receptor-mediated cell of ErbB, differentiation, migration and cellularity are reset; The ErbB member that four kinds of similar Tzahar and Yarden, 1988), are arranged: ErbB-1, ErbB2, ErbB3 and ErbB-4.EGF can discern and in conjunction with one of part of ErbB1.ErbB3 and ErbB4 also can be discerned by several bodies of accompanying, comprising Neuregulin-1 (NRG-1).So far, also do not find the part of ErbB2.Yet ErbB2 can form heterodimer with ErbB3 or ErbB-4, and the formation of this heterodimer is conclusive to the cell signal effect of NRG-1.
The discovery of studying in the body of doing with gene mapping experiment etc. proves that ErbB2, ErbB3 and ErbB-4 be (Meyer and Birchmeier, 1996 in the activated signal chains of NRG-1; Gassmann etc., 1996; Lee etc., 1996; Erickson etc., 1997).
Except developmental role, be amplified in people's adenoma of people's ErbB2 through being everlasting dissimilar and overexpression.ErbB2 is considered to work as the part of heterodimer always.And this process originates in combining of NRG-1 and ErbB3 or ErbB4.This part has been considered to two fully independently receptors bind points: one has high-affinity to ErbB3 or ErbB-4, and another all has less but nonselective affinity to all ErbB members.Like this, when NRG-1 existed, the ErbB3 of cell surface or ErbB4 just formed heterodimer with ErbB2.ErbB3 more special because: 1) ErbB3 easily with ErbB2 formation heterodimer; 2) when ErbB2 and ErbB3 while transformant, can obtain higher conversion ratio; 3) ErbB3 is in tumor cell, often is accompanied by the expression of ErbB2 and expresses; 4) in the ErbB2 transgenic mouse, ErbB3 is overexpression also.
The present inventor's systematic study ErbB member and the effect between them, disclosed the formation mechanism of heterodimer, and their effects in the tumor cell breeding, and potential purposes.
The object of the present invention is to provide a kind of method that suppresses the growth of tumor cell stronger, more single-mindedly.
The present invention also aims to provide a kind of method of screening antitumor drug.
The present invention also aims to provide a kind of antitumor drug, it can suppress the growth of tumor cell stronger, more single-mindedly.
The present application people finds that the expression of ErbB2 in cell can not promote the breeding of cell, and the cell that can suppress oncogene ras mediation on the contrary is to cancerous cell transformation.The inventor finds that also ErbB2/ErbB3 can form the heterodimer that does not rely on part NRG-1, and this dimerization physical ability stimulates the growth of tumor cell.Particularly, the inventor has found the effect of ErbB3 in tumor forms first.Based on above discovery, the inventor has imagined following technical scheme to realize purpose of the present invention.
The invention provides a kind of method of screening antitumor drug.Promptly cell and the candidate compound with coexpression ErbB2 and ErbB3 interacts.Described cell can be, for example use ErbB2 and the permanent cotransfection of ErbB3 expression vector and to the NIH3T3 of cancerous cell transformation cell, human tumor cells is BT-474 or SK-BR-3 human breast cancer cell.And not transfected and the NIH3T3 cell independent transfection of ErbB2, or the MCF-7 cell then is used to do negative control.The screening object is various possible organic or inorganic molecules.Usually should be water-soluble or lipophilic molecule.Solvent can be normal saline, PBS, water etc.Lipophilic molecule can be dissolved in 100% ethanol or DMSO earlier, then is dissolved in the cell culture fluid again.Usually when ethanol or DMSO were added in the cell culture fluid processing cell, its dilution factor was more than 1: 1000.And there is the growth of situation and/or cell and differentiation situation a index as the screening cancer therapy drug with the ErbB2/3 heterodimer.Can suppress the cell of ErbB2, ErbB3 cotransfection when any medicine of discovery and grow, and can not suppress NIH3T3 cell non-transfection or the single transfection of ErbB2, these medicines will be taken as and the interactional candidate of ErbB2/3 heterodimer.Can also be further with the direct effect of these medicines and ErbB2/3 heterodimer, and detect, thereby verified with the method for aforesaid co-immunoprecipitation of the application or covalent coupling.In a typical example, the object that is screened is the antibody of anti-ErbB3.In another kind of example, the object that screens is the various possible molecules that ErbB3 had affinity.
The present invention also provides the antitumor drug that obtains with screening technique of the present invention.In a typical case, described antitumor drug is the ErbB3 antagonist, especially ErbB3 antibody.In another typical case, described antitumor drug also comprises ErbB2 antibody.Beat allly be, when two kinds of antibody couplings, antagonistic effect do not take place not only, obtained ideal stack or cooperative effect on the contrary.Than typical case be, described ErbB3 antibody is commercially available H3.105.5, described ErbB2 antibody is commercially available N12 (available from NEO MARKER company).
The invention provides a kind of method that suppresses growth of tumour cell, it comprises the formation that suppresses the ErbB2/ErbB3 heterodimer.One of mode that realizes described inhibition is to handle cell with the ErbB2/ErbB3 heterodimer antagonist of effective dose.Described antagonist is selected from the molecule that affinity is arranged with ErbB2 or ErbB3, and they can block, disturb or change the formation or the structure of ErbB2/ErbB3 heterodimer, thereby suppresses the growth of cell.Described antagonist is antibody for example, antibody fragment.Wherein be typically, described antagonist is the antibody in anti-ErbB3 extracellular protein zone or the antibody in anti-ErbB extracellular protein zone.Special character of the present invention is that the present invention finds that first ErbB3 antibody has the effect that suppresses growth of tumour cell by the formation that suppresses the ErbB2/ErbB3 heterodimer.Described effective dose depends on target cell, multiple factor such as used antagonist, and those skilled in the art can determine this amount by routine test.
Tumor cell of the present invention can be people's a breast cancer cell.
Past thinks that always the independent high expressed of ErbB2 just can be carcinogenic.But the present invention confirms that ErbB2 does not have the function of stimulating cellular growth separately, and ErbB2 and ErbB3 form heterodimer in non-ligand-dependent mode, and this heterodimer is relevant with growth of tumour cell.Thus, the inventor draws the mechanism that a kind of new antitumor cell is grown.Particularly, the present invention first ErbB3 be tumor suppression target molecule new except that ErbB2.Thus, the method for a series of new inhibition growth of tumour cell and new method for screening anticancer medicine have been drawn.
Anti-ErbB3 antibody (H3.105.5) past that is used to study has been found the not influence (NeoMarks Catalogue, 1999) of growth of tumour cell to expressing ErbB3.Be different from research in the past, we have tested 5 people's breast tumor cell line: MCF-7, T47D, SK-BR-3, MDA-MB-453 and BT474.The cell of latter two cell strain and ErbB3 antibody are done the time spent separately, and the speed of growth is suppressed.What is interesting is that SK-BR-3 cell and ErbB3 antibody effect separately are not remarkable, and demonstrate ErbB2 and the big 2.5 times reactivity of ErbB3 antibody conjugates comparison ErbB2 antibody.When two kinds of antibody use simultaneously, anti-ErbB antibody can be reduced by at least five times, but still can obtain the effect that antitumor cell is grown, its threshold is suitable with the independent maximum effect value of anti-ErbB antibody of using, and because of ErbB2 is expressed in the myocardial cell, anti-ErbB antibody is applied in and causes 28% patient that heart failure takes place among the patient.And ErbB3 there is no expression in cardiac muscle, therefore the use of anti-ErbB3 antibody or and being used in combination of anti-ErbB antibody, make the use amount of anti-ErbB antibody reduce, can both reduce the side effect that produces heart failure in the tumour patient.
Fig. 1: during no part NRG-1, the Western trace of ErbB2, ERB3 immunoprecipitation separately or behind the cotransfection NIH 3T3.Among the figure, transfection: the independent transfection of 2 expression ErbB2 expression plasmid pRC/CMV-ErbB2 coding total length people ErbB2 cDNA, the independent transfection of 3 expression ErbB3 expression plasmid PCMVneo-HER3,2/3 expression pRC/CMV-ErbB2 and pCMVneo-HER3 cotransfection, 2+3 are represented the mixture of cell pyrolysis liquid after the above-mentioned independent transfection; NRG-1: "-" expression NRG-1 does not exist, and "+" expression exists; Immunoprecipitation: 2 expressions precipitate with ErbB2 antibody (NCL-CB11), 3 expressions ErbB3 antibody (NCL-PC11) precipitation; Up is the Western trace that carries out with anti-phosphotyrosine antibody (anti-PTry) (reorganization RC20:HRPO is available from Transduction Laboratories); Middle row is the Western trace that carries out with ErbB2 antibody (NCL-CB11); Descending is the Western trace that carries out with ErbB3 antibody (NCL-PC11).
Fig. 2: the Western trace that detects homodimer or heterodimer with e.Among the figure, covalent coupling: covalent coupling has been carried out in "+" expression, and covalent coupling is not carried out in "-" expression.
Fig. 3: detect interior ErbB2 of tumor cell and ErbB3 form heterodimer in non-ligand dependent mode Western trace.
Fig. 4: a) the ErbB2/ErbB3 heterodimer and the bonded Western trace of signaling molecule Shc of non-ligand dependent in the detection ErbB2/ErbB3 cotransfection NIH3T3 cell; B) the ErbB2/ErbB3 heterodimer of non-ligand dependent and the bonded Western trace of signaling molecule Shc in the NIH3T3 tumor cell of ErbB2/ErbB3 coexpression.Anti--SHC represent with anti--Shc (C20) carry out the Western trace.
Fig. 5: a) common multi-resistance IgG of mice and ErbB3 antibody H3.105.5 suppress the microscope inspection comparison of non-adhere-wall culture BT-474 human breast cancer cell growth.B) amount effect curve of common multi-resistance IgG of mice and ErbB3 antibody H3.105.5 inhibition adhere-wall culture BT-474 human breast cancer cell growth relatively.Wherein, abscissa is represented antibody concentration, and vertical coordinate is for suppressing percentage ratio.
Fig. 6: do not have under the part NRG-1 condition, anti-ErbB antibody and anti-ErbB3 antibody list usefulness and coupling are to the inhibition of human breast carcinoma growth of tumour cell.Abscissa is that used antibody: IgG represents the common multi-resistance IgG of mice (5 μ g/ml), ErbB2 represents the single usefulness of anti-ErbB antibody N12, ErbB3 represents that ErbB3 antibody H3.105.5 is single with (5 μ g/ml), and ErbB2/3 represents N12 and H3.105.5 coupling (concentration of two kinds of antibody all is 2.5 μ g/ml).Vertical coordinate is the absorbance at 495nm place.Above the Nogata post is the inhibition percentage ratio of the common multi-resistance IgG of relative mice, and the display error line.
Fig. 7: anti-ErbB antibody and the coupling of anti-ErbB3 antibody are to the inhibition of human breast carcinoma growth of tumour cell.Abscissa is represented used antibody, and anti-ErbB antibody is N12 (50ng/ml); Anti-ErbB3 antibody is H3.105.5 (2.5 μ g/ml).Vertical coordinate is cell growth indexes (absorbance at 495nm place).
Embodiment
Material
NIH 3T3 cell and breast tumor cell strain SK-BR-3, MDA-MB-453 and BT-474 are available from AmericanType Culture Collection.The LipofectAMINETM transfection reagent is available from Life Technologies, Inc..ErbB2 expression plasmid pRC/CMV-ErbB2 coding total length people ErbB2 cDNA, ErbB3 expression plasmid pCMVneo-HER3 coding total length people ErbB3 cDNA, from Drs Rodney Fiddes and Roger Daly (The Garvan Institute ofMedical Research, Darlinghurst, NSW 2010, Australia).NRG1 and be used for the ErbB2 antibody N12 of antitumor cell growth and ErbB3 antibody H3.105.5 available from NEO MARKER company.Connect reagent BS
3Obtain from PIERCE Chemical Company.The antibody (NCL-CB11) of anti-ErbB that is used for the test of immunoprecipitation and Western trace is available from Novocastra Laboratories Ltd..Anti--ErbB3 (NCL-PC11) is available from Santa CruzBiotechnology.Anti--Shc antibody (C-20) is available from Santa Cruz Biotechnology, and anti--phosphorylated tyrosine (reorganization RC20:HRPO) is available from Transduction Laboratories.The protease inhibitor completely that is used for lysis and immunoprecipitation is available from Boehringer Mannheim.Chemiluminescence agent (ECL) agent of second antibody that horseradish peroxidase-a flat iron plate for making cakes closes and reinforcement is available from NEN Life Science Products.
ErbB2 and ErbB3 form heterodimer in non-ligand dependent mode
In order to study under the condition that does not have part NRG-1, the interaction of ErbB2 and ErbB3, the inventor carries out the transient expression of ErbB2 and/or ErbB3 with the low NIH3 cell (available from American Type Culture Collection) of expressing and not expressing ErbB3 of ErbB2.
37 ℃ in NIH 3T3 cell, 5%CO
2Maintain under the condition in the improved Eagle culture medium of Dulbecco (DMEM) (add 10% calf serum (FBS) and select antibiotic (100 μ g/ml penicillin and streptomycin)).With reference to illustrating with LipofectAMINETM reagent (available from Life Technologies, Inc.) with ErbB2 expression plasmid pRC/CMV-ErbB2 and ErbB3 expression plasmid pCMVneo-HER3 difference or cotransfection NIH 3T3.The NRG-1 activity that may exist in the serum, cell is then got a cotransfection cell sample and was kept in the DMEM that adds 0.1%FBS hungry 18 hours being cultured in serum-free DMEM culture medium 24 hours after the transfection, uses 10 then
-9M NRG-1 (available from NEO MARKER company) handled 10 minutes.
Gather in the crops whole cells transfected (1-2 * 10
6) with cold PBS washing and be dissolved in the 1ml ice crack and separate in the buffer (50mMTris[pH7.4], 5mM EGTA, 150mM NaCl, 1%Triton X-100, the 2mM sodium orthovanadate, the 50mM sodium fluoride, 2mM phenyl methyl sulfuryl fluoride, mixing protease suppresses sheet).The cytolysis thing was hatched 60 minutes at 4 ℃ with anti-ErbB antibody (NCL-CB11) or ErbB3 antibody (NCL-PC11).Immune complex is collected and is washed four times with lysis buffer with protein A or Protein G Sepharose.
Carry out the Western trace test of immunoprecipitation then.The albumen of immunoprecipitation is with the sample loading buffer dissolving of boiling and do SDS-PAGE (6%) electrophoresis.The albumen electricity consumption is transferred to pvdf membrane.With the PBS that contains 5% skim milk add 0.02%Tween 20 4 ℃ spend the night blocking-up after, film at room temperature uses specificity first antibody (NCL-CB11, NCL-PC11 or reorganization RC20:HRPO) to survey 60 minutes.Albumen manifests by the second antibody that the chemical illuminating reagent (NEN LifeScience Products) with a kind of reinforcement is connected with horseradish peroxidase.
The result as shown in Figure 1.Row the 3rd row have the ErbB2 antigen active among Fig. 1 in the precipitation of anti-ErbB3 Antibody Preparation.This is not an antibody cross reaction, because middle row the 5th row show that anti-ErbB3 antibody can not deposit E rbB2, perhaps, and shown in end ranks 1, can not be in conjunction with ErbB2.In like manner, anti-ErbB antibody can not be in conjunction with ErbB3 (middle ranks 2 and row 5).
For the complex that proves ErbB2 and ErbB3 is a heterodimer, the cell of independent or cotransfection is carried out the covalent coupling reaction.Cell after the transfection was cultivated 12 hours in serum-free DMEM culture medium.Control cells was handled 10 minutes with the serum-free DMEM culture medium that contains 50nMNRG-1 (Neo Markers) earlier.With PBS washed cell three times, at room temperature use coupling agent BS then
3At 2mM/PBS, (PIERCE) the middle processing 30 minutes adds 10mM then, the Tris of pH7.5, and cessation reaction is 15 minutes under 0.9%NaCl and the 0.1 lysine solution room temperature.Gather in the crops as mentioned above then, cracking, immunoprecipitation.Immunoprecipitation carries out the test of Western trace behind the 4%SDS-PAGE electrophoresis.
In the ErbB2/ErbB3 of non-ligand dependent heterodimer, the tyrosine residue of ErbB3 is by phosphorylation
Among Fig. 1, comparison array 3 and row 4 as seen, the existence of part NRG-1 strengthens the formation of ErbB2/ErbB3 heterodimer.Up, shown in row 3 and the row 4, in the cell of ErbB2/ErbB3 coexpression, the residue of two kinds of proteic tyrosine is all by phosphorylation.Because the tyrosine kinase activity of ErbB3 disappearance, therefore, ErbB3 should be by the ErbB2 phosphorylation.
ErbB2 and ErbB3 form heterodimer in non-ligand dependent mode in the tumor cell
In the tumor cell of ErbB2 high expressed, whether also exist the above-mentioned dimer that does not rely on part to form in order to test, personnel selection breast tumor cell line SK-BR-3, MDA-MB-453 and BT474 replace the NIH3T3 cell, in serum-free DMEM culture medium, cultivated 24 hours earlier, carry out the test of immunoprecipitation and Western trace then respectively as previously mentioned.The result as shown in Figure 3, and is similar to row 4 to the row 3 among Fig. 1, has the ErbB2/ErbB3 heterodimer of non-ligand-dependent to form in the tumor cell, and the existence of part NRG-1 then significantly strengthens the formation of heterodimer.
The ErbB2/ErbB3 heterodimer of non-ligand dependent combines with signaling molecule Shc
The dependent ErbB2/ErbB3 heterodimer of known ligand is known to be combined with cell signal molecule Shc, and whether the ErbB2/ErbB3 heterodimer that the inventor has detected non-ligand-dependent also combines with Shc.Rotaring copolymering NIH 3 T 3 cell was as previously mentioned cultivated in serum-free DMEM culture medium after 12 hours, results, cracking and immunoprecipitation as previously mentioned, and carry out the Western trace with anti-ErbB3 antibody and anti-Shc antibody and test.
The results are shown in Figure 4, the 1 descending demonstration of Fig. 4 a row, the Shc subspecies of 52kDa are not in conjunction with the Erb3 homodimer, and among this figure, the Shc subspecies of 46kDa are because of being hidden and can not record by a kind of non-differential protein; Fig. 4 a row 2 and row 3 show that no matter whether NRG1 exists, Shc combines with the ErbB2/ErbB3 heterodimer.Fig. 4 b shows that in the tumor cell of ErbB2/ErbB3 coexpression, Shc also interacts with ErbB3, and this effect does not rely on part.
By suppressing the growth that the ErbB2/ErbB3 heterodimer suppresses tumor cell
With ErbB3 is the growth that target molecule suppresses tumor cell
BT474 human breast cancer cell with anti-ErbB3 antibody treatment ErbB2/ErbB3 high expressed.Used antibody is H3.105.5 (available from NEO MARKER company), and this antibody never is in the news and suppresses the effect of growth of tumour cell.That contrast is used is the common multi-resistance IgG of mice) (available from Sigma).In soft agar, carry out non-adherent growth inhibition test respectively, on 96 porocyte culture plates, carried out the adherent growth inhibition test.
In non-adherent growth inhibition test, detailed process: by the density of 5 * 104/ml the BT474 human breast cancer cell is inoculated into and contains in the 4% soft agar DMEM culture medium.37 ℃, 5%CO
2Cultivate 24hr under the condition.The H3.105.5 and the common multi-resistance of mice that in test and control sample, add 5 μ g/ml respectively.Cultivated again 30 days.Carry out microscope inspection then.With cell proliferation reagent box (Cell Titre Aqueous Promega).The result is shown in Fig. 5 a: after the antibody treatment 30 days, the cell of handling with the common multi-resistance IgG of mice grew up to very significantly cell mass; And the cell of handling with H3.105.5, its growth is significantly suppressed.
In the adherent growth inhibition test, the BT474 human breast cancer cell is inoculated on the 96 porocyte culture plates of the DMEM culture medium that contains 10%PBS with the density of 3000-5000 cells/well.37 ℃, 5%CO
2Cultivate 16hr under the condition, make it adherent.The H3.105.5 and the common multi-resistance IgG of mice that add 1-5 μ g/ml in test and control sample respectively continue to cultivate 5-14 days.By specification then, with the cell number of corresponding each concentration of Cell Titre Aqueous enumeration of Promega company, and curve plotting.The result is shown in Fig. 5 b: H3.105.5 also has the obvious suppression effect to the BT474 human breast cancer cell of adherent growth, and presents positively related dose dependent.
This shows, be the growth that target molecule can suppress tumor cell by the formation that suppresses the ErbB2/ErbB3 heterodimer with ErbB 3.
Unite and use ErbB2 and ErbB3 antibody suppressing the effect of growth of tumour cell
The inventor has also tested to unite and has used ErbB2 and ErbB3 antibody to suppressing the effect of growth of tumour cell.Used tumor cell is BT474, SK-BR-3 and MDA-MB-453.As previously mentioned with the density of 2000 cells/well with cell inoculation on 96 porocyte culture plates of the DMEM culture medium that contains 10%PBS, 37 ℃, 5%CO
2Cultivate about 16 hours under the condition to adherent, add antibody: the common multi-resistance IgG:5 of mice μ g/ml, ErbB3 antibody H3.105.55 μ g/ml, ErbB2 antibody N12 (available from NEO MARKER company) 5 μ g/ml, each 2.5 μ g/ml of two antibody, the same terms was cultivated 14 days down.During this time, changed to contain the fresh culture of antibody in per 48 hours.Measure cell quantity with nonspecific cell proliferation reagent box (Promega).The result as shown in Figure 6, the coupling of two kinds of antibody all has the obvious suppression effect to 3 kinds of cells that tried.Wherein, the antibody coupling has the synergistic effect that suppresses growth to BT474 and MDA-MB-453; SK-BR-3 then had the synergism that suppresses growth.
Based on above result, inventor's imagination: if ErbB2 antibody and the coupling of ErbB3 antibody have cooperative effect.Then be low to moderate cell to inferior concentration when insensitive when the concentration of ErbB2 antibody, the stack of double antibody or cooperative effect still may show significantly overall inhibitory action.For this reason, the inventor has carried out foregoing adhere-wall culture, antibody treatment and detection with the SK-BR-3 human tumor cells.Different is, coupling be 50ng/ml ErbB2 antibody (N12) and the ErbB3 antibody H3.105.5 of 2.5 μ g/ml.The result is as shown in Figure 7: cell suppresses all not obvious, but then be subjected to remarkable inhibition when the double antibody coupling when single two kinds of antibody with experimental concentration.This shows, because the short growth of tumour cell effect of ErbB2/ErbB3 heterodimer, except known ErbB2, ErbB3 also can be as the target molecule of known cancer cell growth.Can obtain the stack or the cooperative effect of short growth of tumour cell during at both antibody in coupling.
Anticarcinogen screening technique based on the ErbB2/ErbB3 heterodimer
Based on above result of the test, the inventor also provides a kind of new method of screening cancer therapy drug, and has obtained a series of new cancer therapy drugs really.
Screening to ErbB3 antibody
Material therefor in this screening system for ErbB2 and the permanent cotransfection of ErbB3 expression vector and to the NIH3T3 of cancerous cell transformation cell.Not transfectedly then be used to do negative control with the NIH3T3 cell independent transfection of ErbB2.The object that is screened is the antibody of anti-ErbB3, and various antibody dissolves among the PBS, then is dissolved in the cell that is used in the culture medium handling in the cultivation.Cell should be cultivated in 96 porocyte culture plates, and cell density is 4000 cells in every hole.Cell is placed in 96 orifice plates and cultivated 24 hours earlier, makes it adherent, then adds the medicine of suffered screening, or solvent itself.Can not suppress the cell of ErbB2 cells transfected or untransfected if certain ErbB3 antibody capable suppresses the cell growth of ErbB2/3 cotransfection, then this antibody just might become the candidate of the antagonist of ERBB2/3 heterodimer.
Can also be further with the direct effect of these medicines and ErbB2/3 heterodimer, the method for aforesaid co-immunoprecipitation of available the application or covalent coupling detects.Nature ErbB3 antibody to the affinity of ErbB3 and specificity all the biochemical method of available routine identify.
Found that ErbB3 antibody H3.105.5 can be used as the active component of cancer therapy drug.
To the screening of the molecule of affinity being arranged with ErbB3
Determine the affinity of these molecules to ErbB3, we can use biochemical method.Such as the affinity column made from ErbB3 albumen, any ErbB3 has the molecule of affinity can be attached on this post and is detected.Another kind method be if this molecular energy by isotopic labeling.So just can be directly and the cell combination of ErbB2 transfection or ErbB2/3 cotransfection the molecule of this labelling.Bonded intensity is measured with the isotope exit dose of unit cell.If this molecular energy can not just illustrate that this molecule tool is to the specific affinity of ErbB3 in conjunction with the single cells transfected of ErbB2 in conjunction with the cell of ErbB2/3 cotransfection.These molecules just can be used to do aforesaid cell growth inhibiting test.Usually the medicine that goes out with above-mentioned screening system also will be used for doing the inhibition growth of human tumor cells.The typical human tumor cells relevant with ErbB3 is BT-474 and SK-BR-3 human breast cancer cell.As negative control, the MCF-7 cell also should be used for doing the cell growth inhibiting test.Method of being done experiment and aforementioned NIH3T3 test cell line are just the same.
Claims (27)
1. can stop or reduce the application that material that non-ligand-dependent ErbB2-ErbB3 heterodimer forms is used for preparing the cell growth inhibiting medicine.
2. according to the application of claim 1, wherein said cell is a cancerous cell.
3. according to the application of claim 2, wherein said cancerous cell is a human breast cancer cell.
4. according to the application of claim 1, wherein said material be selected from the chemical compound that causes ErbB2 high expressed in the cell, with the bonded chemical compound in zone, ErbB2 extracellular, prevention reduce chemical compound, the prevention that ErbB3 expresses in the cell or reduce ErbB3 and the interactional chemical compound of ErbB2 in the cell, to comprise coding adjustable or improve the DNA expression vector of protein that the ErbB2 homodimer forms or polypeptide cDNA and comprise to encode and prevent or reduce the DNA expression vector of the cDNA of ErbB3 and ErbB2 interacting proteins or polypeptide.
5. according to the application of claim 1, material wherein comprises with ErbB2 or ErbB3 and combining, and blocking-up, stops or disturb the ErbB2-ErbB3 heterodimer to form or change the chemical compound of ErbB2, ErbB3 and/or ErbB2-ErbB3 allos two dimeric structures.
6. according to the application of claim 1, material wherein comprises anti-ErbB extracellular zone antibody and anti-ErbB3 antibody.
7. according to the application of claim 6, material wherein can be worked in coordination with and be suppressed or stop the cell growth.
8. according to the application of claim 6, material wherein is anti-ErbB antibody N12 and anti-ErbB3 antibody H3.105.5.
9. according to the application of claim 6, wherein anti-ErbB extracellular zone antibody and anti-ErbB3 antibody are monoclonal antibody.
10. according to the application of claim 9, wherein said monoclonal antibody is a Humanized monoclonal antibodies.
11. according to the application of claim 1, wherein said part is NRG-1.
12. comprising, a blocking-up, cytostatic compositions, said composition prevent, reduce the medicine that non-ligand-dependent ErbB2-ErbB3 heterodimer forms in the described cell.
13. according to the compositions of claim 12, wherein said compositions comprises the chemical compound that can make ErbB2 high expressed in the cell, with the bonded chemical compound in zone, ErbB2 extracellular, prevention or reduce ErbB3 and the interactional chemical compound of ErbB2 in the cell, comprise coding and regulating or improve the protein of ErbB2 homodimer formation or the DNA expression vector of the cDNA of polypeptide and comprise the DNA expression vector that prevents or reduce the cDNA of ErbB3 and ErbB2 interacting protein or polypeptide.
14. according to the compositions of claim 12, wherein said compositions comprises with ErbB2 or ErbB3 and combining, and blocking-up or disturb the ErbB2-ErbB3 heterodimer to form or change the material of ErbB2, ErbB3 and/or ErbB2-ErbB3 heterodimer structure.
15. according to the compositions of claim 12, wherein said compositions comprises anti-ErbB extracellular zone antibody and anti-ErbB3 antibody.
16. according to the compositions of claim 15, wherein said compositions can be worked in coordination with and be suppressed or stop the cell growth.
17. according to the compositions of claim 15, wherein said compositions comprises anti-ErbB antibody N12 and anti-ErbB3 antibody H3.105.5.
18. according to the compositions of claim 15, wherein said anti-ErbB extracellular zone antibody and anti-ErbB3 antibody are monoclonal antibody.
19. according to the compositions of claim 18, wherein said monoclonal antibody is a Humanized monoclonal antibodies.
20. according to the compositions of claim 12, wherein said part is NRG-1.
21. a method of screening antitumor drug, this method comprises:
A), determine that the ErbB2-ErbB3 heterodimer forms situation in this cell with the cell and the test substance effect of ErbB2 and ErbB3 coexpression;
B) do not exist under the situation at test substance, determine that the ErbB2-ErbB3 heterodimer forms situation;
C) relatively a) and b) the ErbB2-ErbB3 heterodimer forms in the step difference,
Here the tester that makes the ErbB2-ErbB3 heterodimer form minimizing and/or inhibition cell transformation is potential cancer therapy drug.
22. can stop or reduce the application that the anti-ErbB3 antibody of non-ligand-dependent ErbB2-ErbB3 heterodimer formation is used for preparing the cell growth inhibiting medicine.
23. according to the application of claim 22, wherein said cell is a cancerous cell.
24. according to the application of claim 23, wherein said cancerous cell is a human breast cancer cell.
25. according to the application of claim 22, wherein said anti-ErbB3 antibody is monoclonal antibody.
26. according to the application of claim 25, wherein said monoclonal antibody is a Humanized monoclonal antibodies.
27. according to the application of claim 22, wherein said anti-ErbB3 antibody is H3.105.5.
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