CN1103404A - Novel monoclonal antibodies recognizing the epidermal growth factor-receptor, cells and methods for producing them and compositions containing them - Google Patents
Novel monoclonal antibodies recognizing the epidermal growth factor-receptor, cells and methods for producing them and compositions containing them Download PDFInfo
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Abstract
The present invention relates to a monoclonal antibodies capable of recognizing the EGF receptor in human placenta or cells with high affinity and capable of inhibiting EGF binding to the receptor. The monoclonal antibodies also has the ability to produce antibody dependent cellular cytotoxicity; to develop complement-mediated cytotoxic activity; to inhibit tumoral growth in certain cell lines and to show a synergetic effect to inhibit these cell lines proliferation when they are in combination with gangliosides or monoclonal antibodies against EGF.
Description
The present invention relates to the monoclonal antibody field and provide a kind of screening to produce to discern the method for heterozygote of the monoclonal antibody of EGF acceptor (a kind of be present in the normal cell in epithelium source and the antigen in the tumour cell).This antibody-like has superior performance, and can use it for diagnosis, treatment and the research of malignant tumour and other disease.
The invention still further relates to the treatment of having used this antibody uses and composition for diagnosis.
Urogastron (EGF) is a kind of polypeptide that to stimulate epidermic cell and mesenchymal cell propagation, molecular weight be 6KD, be made up of 53 amino acid, it is considered to one of somatomedin relevant with vicious transformation (Cohen S. always, Carpenter G., PNAS USA 72,1317,1975).This effect mainly is (a kind of by 1 by its membrane receptor, the glycoprotein of the heavy 170KD of 186 amino acid compositions) realize, EGF-R ELISA (EGF-R) more and more is subjected to the parent and looks at (Carpenter G. in cancer research, Cohen S., Recepter Regulation(B Serie) Vol 13,41, Lefkowitz, Ed.Chapman and Hall).
Malignant tumour in the face tissue source is as detecting high-caliber EGF-acceptor in mammary cancer, bladder cancer, ovarian cancer, carcinoma vulvae, colorectal carcinoma, lung cancer, cerebral tumor and the esophagus cancer.The effect in regulating tumor growth of EGF and acceptor thereof it be unclear that, but it is believed that the expression of EGF-acceptor in tumour cell provides a kind of autocrine (autocrine) growth-stimulating mechanism, this stimulation can cause unconfined propagation (Schlessinger J., Schreiber A.B., Levi A., Liberman T., Yarden Y.Crit.Rev.Biochem.1983,14 (2) 93-111).
The appearance of EGF-R has been proved to be the sign of human breast cancer prognosis mala in the tumour cell.But also think that this growth factor receptors also widened the notion of hormonal dependent in the mammary cancer (Perez R., Pascual M.R., Maclas A., Lage A., Breast Cancer Research and Treatment 4,189-193,1984).
The report that more existing monoclonal antibodies with anti-EGF acceptor (MAbs) are studied, these researchs particularly select to have the monoclonal antibody of blocking-up EGF with EGF-R bonded antigen recognition ability.Some preliminary study of carrying out with this antibody show that the EGF acceptor has preferentially cumulative characteristics in the tumour in face tissue source.Therefore in the immunodetection of this class tumour and immunotherapy, use this monoclonal antibody and have potential value (Mondelsohn) J., Masui H., Macleod C.Proc.Am.Assoc.Can.Res.26,287-294,1985; Gullick W.J., Marsden J.J., Whitle N., Ward B., Borrow, L.and Waterfield M.D.Cancer Research 46:285-292,1985).
Employing biological respinse effector designs the treatment plan at various disease, has constituted very tempting possibility.Monoclonal antibody comprises one group of biomolecules (Deush K. that is used for this kind purpose, Mauthe B., Reiter C., Endres N., Classen M., Riethmuller G., Proceeding of the International Congress of Immunollogy, Budapost Hungary, 11,1992).A lot of researchists have studied the immunotherapy method that adopts monoclonal antibody, to test its effect (Frodin J.E., Philstedt P. when the different position tumour of treatment, Lefvert A.K., Mellstedt H.Hybridoma, Vol 7, No.4,1988).Multiple treatment plan had been proposed, comprising: natural, that be used in combination with medicine, dual specific, antiidiotype and people's monoclonal antibody.The high expression level that breaks up EGF acceptor in the relatively poor epidermis tumour, provide with this natural antibody or with after medicine, toxin or active nucleus combine as the possibility that is used for the tomour specific methods of treatment.（Vollmar?A.M.,Banker?D.E.,Mendelsohn?J.,Herschman.H.R.J.Cell Physil.,1987,131,418-428）。Adopt the clinical trial of this mab treatment malignant tumour making progress among (Magdelenat H., Delarue J.Y., Mady E., Faillot T., Vega F., Poisson M., Proceedings of VI Conference of MESTOM International Journal of Tumor Markers, Nice, France, in November, 1991).
Be used to the antibody of the anti-EGF acceptor for the treatment of, at first will have selected its antigen recognition effect, produced the effect that suppresses tumor growth by this antigen recognition process.This restraining effect is (Mendolsohn J., Kawamoto T., Sato G., the Sato D. that takes place by the signal transduction mechanism that relates to the EGF acceptor; Schlesinger J.Civol D., Kris R., U. S. application number: 23988, EPO-0359282).
On the other hand, some ganglioside is regarded as EGF receptor phosphorylation function inhibitor and the cell signalling inhibitor that causes cell inhibitory effect.This shows that Sphingolipids,sialo can be used as the different sites tumour, especially have the tumour of EGF acceptor effective therapeutical agent (Daris R.J., J.Biol.Chem.1990,265:12059-12066).
In addition, also described in some tumour, comprised himself somatomedin to proliferation function, the possibility of autocrine growth control effect.This inspires us to adopt this mechanism, monoclonal antibody by the long factor of antibiosis and growth factor receptors suppresses tumor growth, and with its another feasible program (Todaro C.J.etal., Proc.Natl.Acad.Sci.USA 77:5258-5262,1980) as the treatment malignant tumour.And author of the present invention has obtained to exist in the mastadenoma experimental evidence (not delivering) of EGF and EGF acceptor.
Up to now, also nobody estimates the feasibility that screening can produce the hybridoma with following desired characteristics monoclonal antibody: the antigen recognition ability, cytotoxic effect complement-mediated or that produce by antibody, tumor growth restraining effect and combine the synergism that the back suppresses some cell line proliferation by monoclonal antibody with Sphingolipids,sialo or anti-EGF.Only to estimating to some extent by the possibility that suppresses to suppress tumor growth by the transduction signal that the EGF acceptor is produced.
In the method that is adopted so far, filtered out crossbred according to antigen recognition, in addition, also obtained antibody, and it has been screened according to the antineoplastic activity that is produced by this antigen recognition effect by carrying out immunity with the antigen that tumor cell line produced.
The investigator of this area does not consider this monoclonal antibody by complement-mediated or the issuable antitumor action of antibody-dependant cell toxic action, does not consider yet the monoclonal antibody of this anti-EGF acceptor and the antibodies of other biomolecules such as Sphingolipids,sialo or the somatomedin or the long factor of other antibiosis are used to produce the synergism of anticancer system propagation.
The invention provides a kind of can the generation and have the screening method of the crossbred of the monoclonal antibody of superperformance quantitatively, can use it for development diagnositc system, treatment and research.
As mentioned above, main purpose of the present invention provides the monoclonal antibody with excellent characteristic, this antibody can high-affinity EGF-acceptor in identification Placenta Hominis or the A-431 cell, can suppress EGF and receptors bind, it also has the ability of the cytosis that produces the antibody dependence, produce the ability of the cytotoxicity of complement-mediated, it also has the feature that suppresses tumor growth in some clone, also shows the synergism that suppresses these cell line proliferations when its monoclonal antibody with Sphingolipids,sialo or anti-EGF is used in combination.
Another object of the present invention provides a kind of method of screening the new hybrid cell line that can produce said monoclonal antibody.
Further object of the present invention provides the hybridoma that can produce monoclonal antibody that meets screening criteria.
One of most important purpose of the present invention provides a kind of pharmaceutical composition that contains monoclonal antibody and the Sphingolipids,sialo or the anti-EGF monoclonal antibody of this anti-EGF acceptor, and it can produce cytostatic synergism.
The importance of this preparation is embodied in treatment lung cancer, face tissue, neural system any other tumour in source or unusual effect is all arranged when having any other tumour of EGF-acceptor.
Therefore, the invention provides the screening method that acquisition prepares monoclonal antibody by this way, and be used to diagnose and composition therapeutic purpose, that adopt this antibody.
The present invention includes with partially purified Placenta Hominis EGF-R ELISA composition and make mouse immune; Excise its spleen and be prepared into cell suspension; Splenocyte and myeloma cell are merged; Dilution is also cultivated these cells; Determine to contain the supernatant liquor of above-mentioned hybridoma; Screening and clone can produce the hybridoma of the antibody with following feature: can discern the EGF acceptor.Has high-affinity, have the ability of EGF that suppress with its receptors bind, ability with the cytotoxicity that produces cytotoxic effect (ADCC) that antibody relies on and complement-mediated, have growth of tumor in some clone of inhibition, and when the monoclonal antibody with Sphingolipids,sialo or anti-EGF is used in combination these cell line proliferations of inhibition, show the ability of synergism: from above-mentioned supernatant liquor, reclaim antibody and collect malignant ascite and the serum that contains required antibody.
Can understand the screening preparation and the feature of the antibody that hybridoma and it is produced better by the following description and embodiment.
The hybridoma preparation method generally includes following step, comprising the method for screening hybridoma.
A. the EGF-R composition with partially purified Placenta Hominis makes mouse immune.Although confirmed that female Balb/C mouse is best, the mouse of other strain also can be used.Immune programme for children and antigen concentration must conscientiously measure, and with preparation consumption arranged, suitably the splenocyte that stimulates.Confirmed that one of handy method is: at interval 0 day, 14 days, 21 days and 28 days, every mouse carries out immunity with the immunogen of 25 μ g in the physiological salt solution that is dissolved in the 0.2ml phosphoric acid buffer, used Freund's complete adjuvant in the time of 0 day, full adjuvant tood many or too much for use in the time of 14,21 and 28 days.Last inoculation is the immunogen of intravenous injection 50 μ g before cytogamy.
B. the spleen that excises every immune mouse is also with an amount of medium preparation splenocyte suspension.Is suitable according to each spleen of current experiments technology with about 50ml substratum.
C. under the effect of fusogen, with splenocyte suspension and the suitably rat bone marrow tumour cell fusion of clone.Preferred proportion is 10 splenocytes of each myeloma cell.Per 10
8The fusion substratum that individual cell adopts cumulative volume to be about 0.5-ml is appropriate.Multiple muroid myeloma cell line is also can obtaining from academic institution or specialty preservation of generally acknowledging.But used clone should be " anti-medicine " type, and like this, the myeloma cell of fusion can not be survived in selecting substratum, and hybrid cell then can survive.The most frequently used kind is the clone of anti-8-azaguanine, and it lacks xanthoglobulin-guanine-nucleotidyl transferase, therefore, can not survive in HAT substratum (xanthoglobulin-aminopterinum and thymidine).Also have generally and to adopt so-called " nonsecreting type " myeloma cell line, because itself does not produce antibody.But, if necessary, also can adopt the type of energy secretory antibody.
Although preferably merge promotor is the polyoxyethylene glycol that molecular-weight average is about 1000-4000, also can adopt the promotor of other type.
D. with the mixture diluted of non-fusion splenocyte, non-fusion myeloma cell and fused cell and in cultivating plate hole with non-fusions myeloma cell selective medium (the being HAT) cultivation of can not surviving.After one week, non-fusion splenocyte stops breeding.On the other hand, fused cell since malignant characteristics of inherit from the parental generation myelomatosis and parental generation splenocyte during selecting environment survival ability and can inherit breeding.
E. hybridoma screening method
1. detect the hybridoma supernatant liquor in each hole, to determine existing of antibody, this antibody capable is optionally discerned the EGF acceptor of Placenta Hominis and this acceptor in the A-431 tumor cell line with high avidity, and these monoclonal antibodies can also suppress EGF and receptors bind.This detection can be carried out according to preferred radioreceptor assay or immunoassay, and this acceptor in the Placenta Hominis of crossing with the clone that is rich in the EGF acceptor with according to following affinity purification method partial purification is an antigen.
2. detect the hybridoma supernatant liquor in every hole, to determine to have existing of the antibody that suppresses lung carcinoma cell tumor growth characteristic.The monoclonal antibody of different concns is added in the cell suspending liquid, and do not add antibody with one, another control group that adds uncorrelated antibody compares.The mensuration of protein concn is undertaken by the Lowry-Rosebrough method, but the method for any mensuration total protein concentration all can be used.
3. detect the hybridoma supernatant liquor in each hole, to determine to have existing of the antibody that produces the antibody-dependent cellular cytotoxicity feature, measure this activity with standard method of measurement, this method has adopted
51The target cell of Cr mark, the monoclonal antibody of effector cell and different concns.
4. detect the hybridoma supernatant liquor in each hole, to determine whether to exist existing of antibody with the cytotoxicity feature that produces complement-mediated.The measuring method of employing standard is measured this activity, and this method has been utilized
51The monoclonal antibody of the target cell of Cr mark, the complement of rabbit and different concns.
5. detect the hybridoma supernatant liquor in each hole, have the antibody that when using jointly, produces the synergism feature that suppresses cell proliferation with Sphingolipids,sialo to determine whether to exist.
Obtain cell suspending liquid and with its resuspending in substratum, add Sphingolipids,sialo with the concentration of 0.5～50 μ M, the concentration with 10～100 μ g/ml adds monoclonal antibody (supernatant liquor) then; Measured total protein concentration at the 3rd and the 6th day, preferably adopt the Lowry-Rosebrough method, but any other method can adopt all.
6. detect the hybridoma supernatant liquor in each hole, have the antibody that when using jointly, produces the synergism feature that suppresses cell proliferation with the monoclonal antibody of anti-EGF to determine whether to exist.
Can obtain cell suspending liquid and resuspending in substratum, add the Mab of anti-EGF, and the concentration of this monoclonal antibody with 10～100 μ g/ml is added with proper concn.When the 3rd day and the 6th day, measure total protein concentration, adopt the Lowry-Rosebrough method better, still, measure any method of total protein concentration and all can use.
F. screening (that is: by restricted dilution) and clone produce the hybridoma of this antibody.
In case the ideal hybridoma is screened and clone after 2 times one of promptly available following two kinds of methods preparation antibody at least.
A) monoclonal antibody can by in appropriate culture medium with one fixed time of hybridoma vitro culture, from supernatant liquor, reclaim required antibody subsequently and be prepared.Suitable substratum and incubation time are known or are easy to determine.What this vitro culture technology was produced is single specific antibody basically, has exempted anti-human body specific immunoglobulin basically.Owing to contain a spot of foreign sera (foetal calf serum or new-born calf serum) in the substratum, also have a small amount of other immunoglobulin (Ig) to exist.This in vitro method can enlarge in bio-reactor pari passu, to obtain an amount of required monoclonal antibody.
B) another kind of production method comprises hybridoma is expelled in the isogenic mouse body.After cultivating a suitable period, can cause to produce the formation of the non-firm tumour of antibody.So just can in muroid host's blood and PE (ascites), produce the required antibody (5-20mg/ml) of high density.Though also have normal antibody in this host's blood and ascites, the concentration of monoclonal antibody is higher.In addition, because this normal antibody specificity of anti-human body not, resulting monoclonal antibody is not subjected to any anti-human immunoglobulin's pollution basically.Mixing the immunoglobulin (Ig) that is produced by the myelomatosis light chain is nonspecific irrelevant peptide class, and it only dilutes monoclonal antibody and does not influence basic specificity.
Consistent therewith is, the invention provides the hybridoma cell line that can produce the monoclonal antibody with following feature: this antibody can be discerned the EGF acceptor with high-affinity, can suppress the combination of EGF isoacceptor, can produce the cytotoxicity that antibody relies on, can produce the cytotoxicity of complement-mediated, the growth of tumor that can suppress some clone can produce the synergism that suppresses this tumor growth when using jointly with the monoclonal antibody of Sphingolipids,sialo or anti-EGF.
Above-mentioned antibody can be used to develop immunoassay technology, be used to develop the new treatment that adopts biomolecules, be used to study antigenic molecular topology structure, be used to develop the antibody of antiidiotype and peopleization, study its effect in the idiotype regulation and control in immunological network, and the effect on the antigenic structure signature analysis that can discern in immune purifying and it.
G. contain the monoclonal antibody that obtains to some extent, the acquisition that can produce the therapeutic composition of immunization.
In case obtain antibody purified, can develop any mab treatment composition that contains significant quantity, these monoclonal antibodies are to be produced by the hybridoma that obtains by the hybridoma screening method, and said composition has immunization.The antibody that screens according to these conditions has the cytotoxicity of complement-mediated and the features such as cytotoxicity that antibody relies on.These can adopt standard method to be confirmed; To use
51The A-431 clone of Cr mark is as target cell.The target cell of proper concn is moved on on little culture dish of monoclonal antibody with transfer pipet.The complement source can be human serum or the rabbit anteserum that adds in the substratum.The cultivation of H-125 clone and MAb shows that cytotoxicity is arranged when having complement.Realize with Balb/C mouse peritoneum scavenger cell action effect cell by antibody-mediated cytotoxicity, thereby obtain the cell destructive effect.
In said preparation, should use pharmaceutical carrier, these carriers can be any carriers of using in the conventional pharmacopeia, or maltose or trehalose, sucrose or polyoxyethylene glycol 800 or these carriers are used in combination with the Zn ionic agent with different concns.Said preparation can be by said antibody or its fragment (Fv, Fab, Fab ', F(ab ') 2) make, or make by the antibody of the peopleization, chimeric, " configuration again ", dual specific or with toxin, radionuclide or the above-mentioned any antibody of any other available medicine bonded, perhaps it can by obtained, at the antiidiotypic antibody (AB of said antibody
2, AB) make; To be used for the treatment of or to prevent disease before malignant tumour or the tumour, especially occur in the tumour of tumour, head and neck in the central nervous system, and mammary gland and lung's gland cancer.
H. make up the therapeutic composition that is obtained by gained monoclonal antibody and Sphingolipids,sialo.
In case obtain the monoclonal antibody of purifying, can send the therapeutic composition of any monoclonal antibody that contains significant quantity, these monoclonal antibodies are to be produced by the hybridoma that obtains by the hybridoma screening method, it also comprises the Sphingolipids,sialo of proper concn.
Shown by this monoclonal antibody and different Sphingolipids,sialo are mixed and used the synergism that is produced.The Sphingolipids,sialo of different concns are in that it can produce the growth-inhibiting effect that suppresses cell proliferation when using with the monoclonal antibody of anti-EGF acceptor.
Obtain cell suspending liquid from H-125 and U-1752 clone respectively, and with its resuspending in substratum.Add Sphingolipids,sialo and add the monoclonal antibody of anti-EGF-acceptor with the concentration of 10-100 μ g/ml with the concentration of 0.5-50 μ M.
Measure total protein concentration the 3rd day and the 6th day with Lowry-Rosebrough method (Loury D.H.1951 J.Biol.Chem.193:265), but, other any method all can adopt.Compare with used contrast in the test, observed the synergism of composition aspect inhibition lung cancer cell line propagation of Sphingolipids,sialo-anti-EGF acceptor.
Compared with the control, inhibition of proliferation is about 100%, the simple addition of effect when using separately greater than two kinds of molecules in conjunction with the back effect that is produced.
Should adopt the medicine carrier in said preparation, it can be any used carrier in the conventional pharmacopeia, or maltose or trehalose, sucrose or polyoxyethylene glycol-800 or above-mentioned carrier are with different concns and Zn ionic mixture.This preparation can be by above-mentioned antibody or its fragment (Fv, Fab, Fab ', F(ab ') 2) make, or make, or by the antiidiotypic antibody (AB of anti-above-mentioned antibody by the peopleization, chimeric, " configuration again ", bi-specific antibody or with toxin, radionuclide or the above-mentioned any antibody of any available medicine bonded
2, AB) make; Be used for the treatment of or prevent disease, particularly central nerve neuroma before malignant tumour or the tumour, head and tumor colli, and mammary gland and lung's gland cancer.
I. by the resulting therapeutic composition of the monoclonal antibody combination of gained monoclonal antibody and anti-EGF.
In case obtain antibody purified, just can develop the therapeutic composition that contains by the resulting hybridoma of hybridoma screening method any monoclonal antibody that produce, significant quantity, the monoclonal antibody that comprises the anti-EGF of proper concn, and pharmaceutical carrier, these carriers can be to adopt in the conventional pharmacopeia, or maltose or trehalose, sucrose or polyoxyethylene glycol-800 or above-mentioned substance are with different concns and Zn ionic mixture.Said preparation can be by above-mentioned antibody or its fragment (Fv, Fab, Fab ', F(ab ') 2) make, or make by the peopleization, chimeric, " configuration again ", bi-specific antibody, or by the antiidiotypic antibody (AB of anti-above-mentioned antibody
2, AB) make; Be used for the treatment of or prevent disease, particularly central nerve neuroma before malignant tumour or the tumour, tumor of head and neck, and mammary gland and lung's gland cancer.
The hybridoma screening method
A) according to antigen recognition method screening hybridoma.
Hybridoma supernatant liquor in each hole is measured, and to determine whether to exist a kind of like this antibody: it can optionally discern tissue-derived Placenta Hominis EGF acceptor of epidermis and A-431 clone EGF acceptor with high-affinity, also can suppress the combination of EGF isoacceptor.
After cytogamy, with cell in 37 ℃, 5%CO
2Wet condition under in selectivity HAT substratum (xanthoglobulin, aminopterinum and thymidine), cultivate.
After 3 weeks, take out 10 μ l and contain the supernatant liquor of hybridoma and it is added in the aperture on the polyvinyl chloride plate that scribbles the placental membrane extract that contains the EGF acceptor or A-431 clone extract in advance from culture, the preserving number of A-431 clone is ATCC-CRL-1555.By solid phase, indirect immunoenzymology assay method detect can with the antigen reactive antibody of in the film extract, expressing.By solidifying antigen with partially purified, solvable EGF acceptor application of polyvinyl plate.Adopt 10mM HEPES, the damping fluid of PH7.5.As the peroxidase that is combined with anti-mouse antibodies (Amersham) in the probe of second antibody.
Screening contain can with the hybridoma supernatant liquor of the antibody of the film extract reaction that contains acceptor, and according to the limiting dilution method under the condition that the conditioning cell exists with twice of its clone.
This method has the feature of screening simultaneously at following two kinds of antigenic antibody: from the acceptor of Placenta Hominis, it has normal antigenic determinant; Acceptor from A-431 clone, its antigenic determinant occurs during malignant development, this means, the antigenic determinant of selecting in antibody by screening method is present in the acceptor molecule in the healthy tissues, but also appears in the most receptors molecule in the tumor tissues in epithelium source.
B) select hybridoma according to anti-tumor activity
Detect the hybridoma supernatant liquor in each hole, can suppress lung cancer cell line U-1752 and H-125(NCI to determine whether to exist, Bethersda, the antibody of tumor growth Maryland).The propagation of these clones shows the dependency to EGF.Used substratum is the RPMI-1640(Gibco that replenishes the 5-10% bovine serum, USA).With above-mentioned cell in thermostat container, in 37 ℃ of CO in humidity
2Cultivate in the air (95%-5%).
Measure monoclonal antibody to the cell in trypsinase suspension and with 2.5 * 10
4The retarding effect of concentration resuspending in the cell of substratum of individual cell/ml wherein, adds the monoclonal antibody of different concns: 10,30 and 100 μ g/ml.Control group is studied with irrelevant antibody with the concentration of 100 μ g/ml equally.Cell suspending liquid is placed on the culture plate in 24 holes (every hole 1ml), and to each sample test 3 times.For each clone prepares culture plate and analyzes in the different treatment time (1,3,5 and 7 day).Quantitative determination process (D: H: Lowry) measure total cell count according to gross protein.
Compared with the control, H-125 and the effect of U-1752 lung cancer cell line inhibition of proliferation are reached 50%, this result can go out from Figure 12.
This part of this system of selection has such feature: the monoclonal antibody that the hybridoma that is screened produces has antigenic specific recognition power, has the ability that suppresses tumor growth simultaneously.
C) according to the ability screening hybridoma that produces antibody dependent cellular cytotoxicity.
Detect the hybridoma supernatant liquor in each hole, with existing of the antibody of determining to produce antibody dependent cellular cytotoxicity.Adopt and measure this active standard technique; To use
51Cr(100 μ Ci) the A-431 cell of mark is a target cell.
The effector cell is the hemocyte that adopts the normal adult of Ficoll-Hypaque gradient preparation, wherein, isolates monocyte from peripheral blood, cleans 3 times and be adjusted to the ratio of effector cell/target cell of 200:1.With concentration is 10
4Target cell move liquid to 96 well culture plates that contain 25 μ l monoclonal antibodies (ascites fluid dilution 1/10).To there be the plate of target cell and antibody to cultivate 30 minutes at 4 ℃.Effector cell (100 μ l) is added on the plate and centrifugal immediately 100 * g, 2 minutes.Then with its 37 ℃, contain 5-95% CO
2Cultivated 4 hours in the moist thermostat container of air.Can confirm 22% antibody dependent restraining effect by this experiment.
In this part of this screening method, obtained to produce the hybridoma of monoclonal antibody, it has three features simultaneously: the antigen recognition ability, the ability of inhibition tumor growth also has antibody dependent cellular cytotoxicity simultaneously.
D) select hybridoma according to the cytotoxicity that produces complement-mediated.
Adopt standard method of measurement to detect supernatant liquor in each hole, with existing of the Cytotoxic antibody of determining to produce complement-mediated: will use
51Cr(100 μ Ci) the A-431 clone of mark is as target cell.
With concentration is 10
4Target cell move liquid to 96 well culture plates that contain 25 μ l monoclonal antibodies (ascites fluid dilution 1/10 times).
The complement source is that 100 μ l concentration in substratum is 25% rabbit anteserum.Place it in moist, have a 5-95%CO
2Cultivated 4 hours in thermostat containers air, 37 ℃.Then that culture plate is centrifugal, on γ-counter, measure 100 μ l supernatant liquor per minutes countings (cpm).
Several working fluids of antibody have been tested, to check its maximum toxicity value.
In this experiment, confirmed to have the restraining effect of 60% complement-mediated, as shown in figure 14.
E) basis and N-acetyl GM
3Share the ability screening hybridoma that the back produces synergism.
To express 10
4-10
5Individual EGF receptor binding site also is categorized as the experimental model of the H-125 clone of adenocarcinoma of lung as screening.Obtaining concentration is 10
4-10
5The cell suspending liquid of individual cell/ml, and with its resuspending in substratum.Concentration with 5 μ M adds N-acetyl GM then
3, add the monoclonal antibody of anti-EGF acceptor with the concentration of 30 μ g/ml.
At the 3rd day and the 6th Lowry-Rosebrough method measurement total protein concentration of using in the sky.
Compare with contrast used in this test, can find out N-acetyl GM
3The monoclonal antibody of-anti-EGF acceptor is share the synergism aspect inhibition lung cancer cell line propagation.Compared with the control, inhibition of proliferation is almost reached 100%.Therefore, share the effect that produced the simple addition of role is much bigger alone than these two kinds of molecules by this.
This result can be as seen from Figure 15.
F) according to share the ability screening hybridoma that the back produces synergism with the monoclonal antibody of anti-EGF.
To express 10
4-10
5Individual EGF receptor binding site also is categorized as the experimental model of the H-125 clone of adenocarcinoma of lung as screening.Obtaining concentration is 10
4-10
5The cell suspending liquid of individual cell/ml, and with its resuspending in substratum.Add the MAbs of anti-EGF with the concentration of 5 μ g/ml, add the monoclonal antibody of anti-EGF acceptor with the concentration of 30 μ g/ml.Adopted the Lowry-Rosebrough method to measure total protein concentration at the 3rd day and the 6th day.
Compare with the contrast that this test is adopted, the monoclonal antibody of the anti-EGF acceptor of the MAbs-of anti-EGF is share the synergism aspect inhibition lung cancer cell line propagation as can be seen.
The antibody that produces according to this screening method gained hybridoma has superior characteristic, and makes it have following distinguishing characteristics: with high-affinity identification EGF receptor antigen; The cytotoxicity that antibody relies on; The cytotoxicity of complement-mediated; Suppress the clone tumor growth ability and with N-acetyl GM
3Or the MAbs of anti-EGF share the ability of the synergism that produces.
Biochemical character by monoclonal antibody that this screening method obtains.
The gained monoclonal antibody is an IgG2a type immunoglobulin (Ig), and it is by SP
2/ Ag14 rat bone marrow tumour is secreted with the fused cell of and Balb/C mouse splenic lymphocyte excessively immune with partially purified Placenta Hominis EGF acceptor.The purifying of dissolving EGF acceptor is by the affinity chromatography purifying.In brief: at room temperature in 1 hour the rough film fraction of placenta is dissolved in the Tris-HCL 20mM damping fluid, PH7.4 contains glycerine 10%, Triton X-1001%.Homogenate is centrifugal with 10,000 * g, and supernatant liquor is added on the EGF affinity matrix.Be rich in the fraction PH9.7 of EGF, contain the 5mM thanomin buffer solution elution of 10% glycerine and 0.1% Triton X-100 solution.This cut that is rich in soluble EGF receptor has and 125 I-EGF bonded ability, and occurs the master tape of 170kd when the SDS disk electrophoresis.Each 100 grams of handling.Every average yield that restrains wet tissue is the partially purified EGF acceptor of 15-25 μ g.
This monoclonal antibody has following feature:
Calculating the KD value according to resulting semi-saturation concentration curve is 10
-9The high-affinity of M to acceptor, as shown in Figure 1, the similar receptor affinity of this avidity with EGF.
It can suppress 125 I-EGF ' S and combine (Fig. 2 and 4) with the membrane receptor that adopts Placenta Hominis microsome cut, from its 10
-8The MKi value can be reasoned out the different of its binding site and Urogastron, and its restraining effect excites by configuration effect or steric effect.This monoclonal antibody can suppress the effect of EGF to A-431 clone, also is (Fig. 6 and 7) like this when having synergistic agent such as γ-IFN to exist.This MAbs can also suppress to depend on the asexual growth of the NRK cell of EGF, and can suppress to be regarded as the autophosphorylation that depends on the EGF acceptor (Fig. 8) of the committed step of cell proliferation signal transduction, this has confirmed the EGF-antagonistic action that this monoclonal antibody rose.
The research that confirms this effect is carried out (Fig. 9 and 10) by Mab and by the Fab fragment of papain digestion.This effect is proved to be dose-dependently.This fragment keeps the same capabilities that suppresses EGF cell proliferation.
The ability that this monoclonal antibody suppresses different cell line proliferations is confirmed equally.Find that in the lung cancer cell line of being studied (M-125 and U-1752) when the application concentration of this antibody was 10 μ g/, growth slightly weakened, compared with the control no significant difference; Then, when working concentration is 30 μ g/ml, realized inhibition, when cultivating the 3rd day, kept stable, in the time of the 5th day, shown 50% inhibition (Figure 12 and Figure 13) compared with the control growth.
This monoclonal antibody also has the cytotoxicity of complement-mediated and the cytotoxicity that antibody relies on.
Confirm that by cultivate H-125 clone with monoclonal antibody under the situation that has the rabbit complement, when antibody concentration was 12.5 μ g/ml, cytotoxic effect was 60%(Figure 14).Antibody-mediated cell toxicity test is performed such: with Balb/C mouse abdomen scavenger cell is the effector cell, the effector cell: target cell ratio is 110:1, produces 22% cell Decomposition.
The Sphingolipids,sialo of this monoclonal antibody and different concns share the synergism that is produced and also are confirmed.The propagation of this bound energy pair cell produces the growth-inhibiting effect.N-acetyl GM
3Concentration with 5 μ M is added, and adds Mab with the concentration of 30 μ g/ml.
Test used contrast with this and compare, observed N-acetyl GM
3The monoclonal antibody of-anti-EGF acceptor is share the synergism aspect inhibition lung cancer cell line propagation.
Compared with the control, to inhibition of proliferation near 100%, this result (Figure 15) who share the effect that produced greater than the simple addition that two kinds of molecules are acted on alone.
The sequential analysis of the immunoglobulin heavy chain variable region that obtains is undertaken by polymerase chain reaction (PCR): at least 2 the independently PCR samples that increase, adopt at least 2 independently PUC19 clone to determine sequence.In case the variable region of light chain and heavy chain obtains amplification, and it is cloned in the PUC 19 carrier sequences, the sequence of heavy chain is determined.According to Kabat Database, this sequence is classified into different groups.
The rearrangement of variable region of heavy chain defines the antigenic determinant of EGF acceptor, and this position can be discerned by this monoclonal antibody.
Reactive characteristics by monoclonal antibody that this screening method obtains.
Select flesh tissue to be used for research, these tissues comprise heart, prostate gland, liver, lung, kidney, spleen, ovary, pancreas, brain, testis, suprarenal gland, hypophysis, lymphatic node, medullary substance, the remorse gland of first, tonsilla, stomach, small intestine, colon, skin, submaxillary gland, placenta and cerebellum.Several nervous tissues, hemopoietic tissue tumour and sarcoma have also been studied.
Be organized in the liquid nitrogen these aquatic foods freezing and-20 ℃ of preservations.Adopt the painted section of hematoxylin-eosin to carry out histopathology and detect research.To being used for the partial continuous section employing Immunoperoxidase technology dyeing that histopathology detects.
Adopt vitamin H-suis-avidin-superoxide enzyme complex (Amersham, U.K.) reactivity of checking tissue.This research comprises the contrast of the part (Tris buffered salts solution) of male part (epidermis part) and feminine gender.With generation brown precipitation after the enzyme reaction, and can be divided into following type:
* reactionless (-);
* mild reaction (+);
* moderate reaction (++);
* reaction by force (+++).
Take fresh healthy tissues from the corpse of firm dissection, according to the pattern of setting up with pancreas and testis, the reactivity of this tissue is an intensity.
Take fresh healthy tissues from the corpse that firm aquatic foods cut open, according to the pattern of being set up with placenta, skin, prostate gland, liver, kidney, ovary, Tiroidina, small intestine and non-lymph tonsilla tissue, the reactivity of tissue is medium tenacity (seeing Table 1).
According to the pattern of being set up, epidermoma and central nervous system knurl such as neurospongioma, meningioma, malignant histiocytoma, neurofibrosarcoma, and mastadenoma and rhinopharyngocele show strong reaction, and with the healthy tissues phase specific concentration higher (table 2) of taking from identical source.
Detect and positively to only occur in the tissue that contains acceptor, this has confirmed that the identification to the antigenic determinant of molecule is specific, with and unique and in other antigen, lack relevant antigenic determinant.
Can produce the exploitation of the treatment of immunization with compound.
Developed a kind of treatment compound that contains the monoclonal antibody of significant quantity, this monoclonal antibody is to be produced by the hybridoma that is obtained by the hybridoma screening method, can produce antibody dependent cellular cytotoxicity.For this reason, we have adopted and have measured this active canonical measure technology, will use
51The A-431 clone that (100 μ Ci) mark of Cr is crossed is as target cell.The effector cell is a hemocyte, takes from normal adult, adopts the preparation of Ficell-Hypaque gradient, isolates monocyte by it from peripheral blood, washs 3 times, and is adjusted to effector cell/target cell ratio of 200:1.
To there be the culture plate of target cell and antibody under 4 ℃ of temperature, to cultivate 30 minutes.Effector cell (100 μ l) is added on this plate and centrifugal 2 minutes immediately with the speed of 100Xg.Place it in then and have 5-95%CO
2Cultivated 4 hours in 37 ℃ the moist the Huanshui River incubator of air.On γ-counter, measure the counting (cpm) of 100 μ l supernatant liquor per minutes.
The measuring method of employing standard is to use
51The A-431 cell of Cr radioactivity (100 μ Ci) mark is a target cell, measures the cytotoxicity of complement-mediated.
With concentration is 10
4Target cell move liquid to 96 well culture plates that contain the diluted 1/10 times monoclonal antibody (ascites fluid) of 25 μ l.
Complement source is to cultivate the rabbit anteserum that concentration wherein is 25% 100 μ l.It had the CO of 5-95%
2Cultivated 4 hours at 37 ℃ in the wet thermostat container of air.Subsequently that culture plate is centrifugal, and on γ-counter, measure the counting of 100 μ l supernatant liquor per minutes.Several antibody working fluids have been tested, to find its maximum toxicity.
These experiments show that under the condition that the rabbit complement is arranged, cellular cytoxicity activity was 59% when concentration was 12.5 μ g/ml, and produce 22% cell decomposition effect by the antibody-mediated cell toxicant performance that peritoneal macrophages carried out.
Said preparation comprises a kind of pharmaceutical carrier, and it contains the alkali valency sodium phosphate of 2.55mg, 9.00mg bibasic sodium phosphate, 43.00mg sodium-chlor, 1mg Spheron MD 30/70-80 and 5.00 injection waters.
Contain N-acetyl GM
3Treatment with the exploitation of compound.
Developed a kind of contain significant quantity, by the monoclonal antibody and the N-acetyl GM that produce by hybridoma screening method institute hybridoma
3The treatment compound.
Observe N-acetyl GM
3Share the back in the synergism aspect the inhibition lung cancer cell line propagation with monoclonal antibody.
In order to implement this experiment, obtained cell suspending liquid and with its resuspending in substratum, add N-acetyl GM with the concentration of 5 μ M
3, add Mab with the concentration of 30 μ g/ml.Measure total protein the 3rd day and the 6th day with the Lowry method.
Compared with the control, be 100% to inhibition of proliferation, and the effect that is produced is greater than simple additive effect.
Said preparation comprises a kind of pharmaceutical carrier, and it contains the alkali valency sodium phosphate of 2.55mg, 9.00mg bibasic sodium phosphate, 43.00mg sodium-chlor, 1mg Spheron MD 30/70-80 and 5.00ml injection water.
The treatment exploitation of compound that contains the MAB of anti-EGF.
Developed a kind of contain significant quantity, by the Mab of monoclonal antibody of producing and anti-EGF and a kind of therapeutic composition of pharmaceutical carrier by hybridoma hybridoma that screening method obtains, this carrier contains 2.55mg-alkali valency sodium phosphate, 9.00mg bibasic sodium phosphate, 43.00mg sodium-chlor, 1mg Spheron MD 30/70-80 and 5.00ml injection water.
Fig. 1 represents resulting saturation curve during the solid phase ELISA of placental membrane and A-431 cell cut.The abscissa axis is represented the logarithm of antibody concentration.A kind of irrelevant antibody is done as negative control (antibody concentration is 0.5-7500ng/ml).
Fig. 2 represents the displacement curve by the radioreceptor assay gained.Competition experiments is carried out with the microsome cut of Placenta Hominis and the antibody and the resistive contrast (anti-cell Keratin sulfate monoclonal antibody) of different concns.The EGF displacement curve is as positive control.
Fig. 3 represents the immunoreactivity mensuration (spot is inhaled seal) of the EGF-R of monoclonal antibody and A-431 cell purification.With purifying EGF-R(30ng/ml) and 3.0 μ l, 1.0 μ l, 0.1 μ l sample are added on the nitrocellulose and with the antibody incubation of different concns.
Fig. 4 represents with the competition experiments between the different monoclonal antibodies of the anti-EGF acceptor of 125-I mark.
The competition experiments that Fig. 5 represents to adopt the monoclonal antibody of Placenta Hominis microsome cut and the anti-EGF-R of different concns to do.To be used as radiotracer with the EGF of 125-I mark.
Fig. 6 represents the effect of monoclonal antibody to the A-431 cell.With cell and EGF(1 μ g/ml); Gamma-IFN(100 μ g/ml); EGF+G-IFN; And the monoclonal antibody of different concns is hatched separately or with the mixture of Gamma-IFN.The protein of measuring in the monolayer cell at the 7th day that cultivates.
Fig. 7 has reflected the bioactive antagonistic action of monoclonal antibody to EGF.With cell and EGF(1 μ g/ml); Gamma-IFN(100 μ g/ml); EGF+G-IFN; And share with the monoclonal antibody of Gamma-IFN and EGF and hatch together.The protein of measuring in the monolayer cell at the 7th day that cultivates.
Fig. 8 represent because of in the placental membrane tissue by the inhibition of the caused autophosphorylation to EGF excitation of monoclonal antibody.Film with
32The ATP of P mark is together in no EGF(1 road) or the EGF(2.8 road is arranged) situation under cultivate, and analyze by polyacrylamide gel electrophoresis and radioactive automatic developing.Observed the effect that Mab concentration increases by in EGF-R, integrating P-32.2 roads and 3 roads do not have Mab; 4 roads: 1.8 μ g/ml Mab; 5 roads: 6 μ g/ml Mab; 6 roads: 18 μ g/ml Mab; 7 roads: 60 μ g/ml Mab; 8 roads: 180 μ g/ml Mab.
Fig. 9 is illustrated in that the Fab fragment has the same ability that can suppress EGF with its receptors bind with monoclonal antibody in the A-431 cell.Adopt the Mab of the microsome of Placenta Hominis and different concns and Fab thereof the test that is at war with.The EGF of I-125 mark is used as tracer.
The antagonistic effect aspect Fab fragment that Figure 10 is illustrated in the EGF of A-431 cell has identical ability with natural monoclonal antibody.The Mab of cell and different concns and Fab thereof cultivate.Cultivating the protein of measuring monolayer cell in former the 7th day.
Figure 11 represents the immunocytochemical assay result of mammary cancer.Invasive duct carcinoma epithelial cell is painted heavier.
Figure 12 represents the effect of Mab to H-125 clone; Can find out the growth quickening (its speed of growth has been turned over several times) of control group.Its growth of group of handling with minimum concentration (10 μ g/ml) only is slightly slower than control group.The group of handling with higher concentration shows at the 1st, 2 day cell some growths.Beginning in the 3rd day, culture entered the stationary phase of not having growth, and the 5th day compared with the control, is 50% to the inhibition of growing.
Figure 13 represents the effect of Mab to U-1752 clone.Compared with the control, the group of handling with minimum concentration (10 μ g/ml) does not have the growth difference.The group of handling with higher concentration showed some growths at preceding 1.2 days, beginning in the 3rd day, culture entered the stationary phase of not having growth, and the 5th day compared with the control, is 50% to the inhibition of growing.
Figure 14 represents Mab cytotoxic effect to H-125 clone when the rabbit complement exists.
Figure 15 represents compared with the control, the Mab of fixed concentration and two kinds of different concns X-acetyl-GM
3The effect of share.
Control group 1: the irrelevant antibody identical with the MAb concentration of anti-EGF-R.
Control group 4: only contain the monoclonal antibody that concentration is 30 μ g/ml.
It is the N-acetyl GM that the concentration of monoclonal antibody of the anti-EGF acceptor of 30 μ g/ml is respectively 2.5 and 5 μ g/ml that control group 5 and 6 contains concentration
3
Table 1 and 2Mab are to the fresh healthy tissues of having carried out freezing microtome section and the identification situation of tumor tissues.
The recognition capability of table 1 Mab in normal organ
The organ reactivity
- -/+ + ++ +++
Note
Reactionless (-);
Weak reaction (+);
Moderate reaction (++);
Strong reaction (+++).
Cryo-etching
Table 2 Mab is to the recognition capability of tumour
The organ reactivity
- -/+ ++ +++
Note
Reactionless (-);
Weak reaction (+);
Moderate reaction (++);
Claims (39)
1, the monoclonal antibody of a kind of identification GG hEGF (EGF) acceptor, it can suppress EGF and receptors bind and can suppress to rely on the growth of the tumour cell of EGF, in addition, also has at least a in the following feature:
A) have antibody dependent cellular cytotoxicity,
B) have the cytotoxicity of complement-mediated,
C) if share with Sphingolipids,sialo, can produce the synergism that suppresses tumor cell proliferation,
D), can produce the synergism that suppresses tumor cell proliferation if share with the monoclonal antibody of anti-EGF.
Or the derivative of the said monoclonal antibody of from its form peopleization, chimeric, configuration, dual specific, bonded and antiidiotype again, electing,
Or the fragment of said monoclonal antibody or the derivative from its Fv, Fab, Fab ' and F (ab ') 2 fragments, selected.
2, monoclonal antibody according to claim 1, it has the cytotoxicity of antibody dependent cellular cytotoxicity and complement-mediated.
3, monoclonal antibody according to claim 2 if it share with Sphingolipids,sialo, can produce the inhibition tumour cell and draw the synergism of growing.
4,,, can produce the synergism that suppresses tumor cell proliferation if it share with the monoclonal antibody of anti-EGF according to claim 2 or 3 described monoclonal antibodies.
5, according to each described monoclonal antibody in the claim 1～4, it can discern the EGF acceptor in human normal cell and the tumour cell.
6, according to claim 5 a described monoclonal antibody, it can discern the EGF acceptor of Placenta Hominis EGF acceptor and A-431 tumor cell line.
7, according to each described monoclonal antibody in the claim 1～6, it can suppress the growth of EGF-dependency lung cancer cell line U-1752 and/or H-125.
8, according to each described monoclonal antibody in the claim 1～7, it has antibody dependent cellular cytotoxicity in the test that with the A-431 cell is target cell.
9, according to each described monoclonal antibody in the claim 1～8, it has the cytotoxicity of complement-mediated in the test that with the A-431 cell is target cell.
10, according to each described monoclonal antibody in the claim 1～9, if with Sphingolipids,sialo N-acetyl GM
3Share, it can produce the synergism that suppresses tumor cell proliferation.
11, monoclonal antibody according to claim 10, wherein said tumour cell H-125 cell.
12, according to each described monoclonal antibody in the claim 1～11, if share with the monoclonal antibody of anti-EGF, it can produce the synergism that suppresses tumor cell proliferation.
13, monoclonal antibody according to claim 12, wherein said tumour cell are the H-125 cells.
14, according to each described monoclonal antibody in the claim 1～13, it is an IgG2a type immunoglobulin (Ig).
15, a kind of composition comprises according to each described monoclonal antibody in the claim 1～14, and carrier, thinner or its vehicle.
16, a kind of be used for the treatment of or prophylactic treatment malignant tumour and tumour before the pharmaceutical composition of disease, comprise can play the treatment and the amount of prophylactic effect, according to each described monoclonal antibody in the claim 1～14, and carrier, thinner or its vehicle.
17, pharmaceutical composition according to claim 16, it contains a kind of Sphingolipids,sialo.
18, according to claim 16 or 17 described pharmaceutical compositions, it contains the monoclonal antibody of anti-EGF.
19, a kind of monoclonal antibody method for preparing identification human body EGF-R comprises: make the laboratory animal immunity with human body EGF-R or its antigenic derivant or its fragment; In the animal body of immunity, separate the cell that produces antibody; Preserve and produce this antibody cell; Screening can produce the cell of monoclonal antibody from preserved, and the EGF-R of this monoclonal antibody identification human body can suppress the growth of EGF dependent tumors cell, has at least a in the following feature in addition:
A) have antibody dependent cellular cytotoxicity,
B) have the cytotoxicity of complement-mediated,
C) if share with Sphingolipids,sialo, can produce the synergism that suppresses tumor cell proliferation,
D) if share with the monoclonal antibody of anti-EGF, can produce the synergism that suppresses tumor cell proliferation,
And separation is by the monoclonal antibody of the cell generation of being screened.
20, method according to claim 19, the preservation step that it is characterized in that preserving isolated generation antibody cell in the immune animal body realizes by this cell and myeloma cell are merged to produce hybridoma.
21,, it is characterized in that the used laboratory animal of immune step is a rodent, preferably mouse or rat according to claim 19 and 20 described methods.
22,, it is characterized in that laboratory animal usefulness Placenta Hominis EGF-R immunity according to each described method in the claim 19～21.
23,, it is characterized in that the monoclonal antibody that screens has the cytotoxicity of antibody dependent cellular cytotoxicity and complement-mediated according to each described method in the claim 19～22.
24, method according to claim 23 is share the synergism that can produce the inhibition tumor cell proliferation if it is characterized in that the monoclonal antibody that screens with Sphingolipids,sialo.
25,, can produce the synergism that suppresses tumor cell proliferation if it is characterized in that the monoclonal antibody of the anti-EGF of monoclonal antibody that screens is share according to claim 23 or 24 described methods.
26,, it is characterized in that the monoclonal antibody that filters out can discern the EGF-R that is present in human normal cell and the tumour cell according to each described method in the claim 19～25.
27,, it is characterized in that the monoclonal antibody that filters out can discern the EGF-R of Placenta Hominis EGF-R and A-431 tumor cell line according to each described method in the claim 19～26.
28,, it is characterized in that the monoclonal antibody of being screened can suppress the growth of EGF-dependency lung cancer cell line U-1752 and/or H-125 according to each described method in the claim 19～27.
29,, it is characterized in that the monoclonal antibody that filters out has antibody dependent cellular cytotoxicity in the test that with A-431 is target cell according to each described method in the claim 19～28.
30,, it is characterized in that the monoclonal antibody that filters out has the cytotoxicity of complement-mediated in the test that with A-431 is target cell according to each described method in the claim 19～29.
31, according to each described method in the claim 19～30, if it is characterized in that the monoclonal antibody and Sphingolipids,sialo N-acetyl GM that filter out
3Share and to produce the synergism that suppresses tumor cell proliferation.
32, method according to claim 31, wherein said tumour cell are the H-125 cells.
33,, share the synergism that can produce the inhibition tumor proliferation with the monoclonal antibody of anti-EGF if it is characterized in that the monoclonal antibody that filters out according to each described method in the claim 19～32.
34, method according to claim 33, wherein said tumour cell H-125 cell.
35,, it is characterized in that the monoclonal antibody that filters out is an IgG2a type immunoglobulin (Ig) according to each described method in the claim 19～34.
36, according to each described method in the claim 19～35, it is characterized in that cell to the selected monoclonal antibody of generation of being preserved can adopt carries out vitro culture in the substratum of suitable tissue, or in the body fluid of tissue compatible, carry out culturing in vivo, or in fermentor tank such as product matrix, cultivate, from above-mentioned matrix, separate the cell of preserving subsequently.
37,, it is characterized in that the monoclonal antibody that is produced is purified and derivatize or fragmentation selectively according to each described method in the claim 19～36.
38, a kind of preservation cell that can produce according to each described monoclonal antibody in the claim 1～14.
39, according to the purposes of each described monoclonal antibody in the claim 1～14.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CU1993017 | 1993-03-01 | ||
| CA17/93 | 1993-03-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1103404A true CN1103404A (en) | 1995-06-07 |
Family
ID=5459200
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 94104094 Pending CN1103404A (en) | 1993-03-01 | 1994-02-26 | Novel monoclonal antibodies recognizing the epidermal growth factor-receptor, cells and methods for producing them and compositions containing them |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1103404A (en) |
| CA (1) | CA2116753C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103998098A (en) * | 2011-12-27 | 2014-08-20 | 分子免疫中心 | Pharmaceutical compositions for the treatment of tumours that express EGFR and ganglioside N-glycolyl GM3 (NeuGcGM3) |
-
1994
- 1994-02-26 CN CN 94104094 patent/CN1103404A/en active Pending
- 1994-03-01 CA CA 2116753 patent/CA2116753C/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103998098A (en) * | 2011-12-27 | 2014-08-20 | 分子免疫中心 | Pharmaceutical compositions for the treatment of tumours that express EGFR and ganglioside N-glycolyl GM3 (NeuGcGM3) |
| CN103998098B (en) * | 2011-12-27 | 2017-08-04 | 分子免疫中心 | For the pharmaceutical composition for the tumour for treating expression EGFR and N hydroxyl acetyl GM3 gangliosides (NeuGcGM3) |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2116753C (en) | 2001-09-04 |
| CA2116753A1 (en) | 1994-09-02 |
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