CN1443778A - Monoclonal antibody of melanic related antigen, and its application and medicine composition - Google Patents
Monoclonal antibody of melanic related antigen, and its application and medicine composition Download PDFInfo
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Abstract
The present invention discloses a monoclonal antibody capable of identifying specific antigen of melanotumor. It also discloses the application of said monoclonal antibody in separation of melanotumor cell and detection of melanotumor. Said invention also relates to the medicine composite containing the monoclonal antibody and its application for curing melanotumor, said invention also relates to the recombinant protein of the monoclonal antibody.
Description
Technical field
The present invention relates to field of immunology, specifically, the present invention relates to monoclonal antibody of melanic related antigen, melanic related antigen and uses thereof.
Background technology
Malignant melanoma is the very high tumour of a kind of grade malignancy, and the person accounts for 20% of whole body to occur in the neck, mainly betides skin and chamber, hole mucous membrane.People's malignant melanoma starts from deleterious black mole usually, and these black moles are grown behind radiostimulation, forms destructive transfer melanoma.Melanoma very easily to the whole body diffusion, is difficult to control clinically.Chemotherapy and radiation treatment are inoperative to melanoma usually.
At present, to detect normally used antibody be HMB-45, anti-S-100 and NKI/C3 (referring to Smoller BR, PATHOLOGY:State of the Art Reviews, 2:371-383,1994) to the melanoma immunohistochemistry.
HMB-45 is a kind of monoclonal antibody of known anti-human melanoma cell, and it can discern the premelanosome sphaeroprotein, can react with melanic related antigen gp-100.HMB-45 can also act on melanoma, connect mole, cambiform cell and epithelial cell mole, nevus maternus etc.Yet,, when detecting, can leave over a certain amount of melanoma and be difficult for being detected (referring to Wick MR et al., J Cutan Pathol 1988 because HMB-45 lacks the susceptibility (its susceptibility is between 67%-93%) of height; 15:201-207; Ordonez NG et al., Am.J.Clin.Pathol.1988; 90:385-390).
The proteic antibody of S-100 has higher susceptibility than HMB-45, and (referring to Nakajima T et al., Cancer 1982; 50:912-918; Kindblom LG et al., Acta.Pathol.Macrobial.Immunol.Scand.1984; 92:219-230), but its specificity is very poor.Anti-S-100 antibody may be attached on the benign cell, as sialisterium and sweat gland, skeletal muscle and cardiac muscle, histocyte, Schwann cell, adipocyte, chondrocyte, astroglia cell, oligodendrocyte (referring to Stefansson K etal., Nature 1982,295:63-64; Stefansson K et al., BrainRes.1982,234:309-317) and the tumour that produces of these cells (referring to Tabuchi K et al., Ac taNeurochir Wien 1982,65:239-251).Anti-S-100 antibody can also be attached on the plain knurl malignant tumor tissues of many non-blacks (Drier JK et al., Arch.Pathol.Lab.Med., 1987,111:447-452).
NKI/C3 antibody also can act on many benign or virulent tumours, and this non-specific activity makes the use of NKI/C3 antibody on melanoma detects be subjected to very big restriction.
KBA.62 antibody is similar to HMB-45 for melanomatous susceptibility, but it can be attached on squamous cell carcinoma and the rodent cancer, lacks specificity (referring to Cohen Knafo Eet al., J Clin Pathol1984; 37:367-372).
This shows that though set up manyly at melanomatous monoclonal antibody at home and abroad, the specificity of these monoclonal antibodies and neutralising capacity are all not ideal enough.Therefore, need foundation can be used for the monoclonal antibody of clinical treatment and the monoclonal antibody of humanized high-affinity.
Summary of the invention
The object of the present invention is to provide the specific monoclonal antibody SM5-1 of a kind of melanoma, the antigen molecular of its correspondence is 220-240KD.Paraffin-embedded melanoma (elementary melanoma and metastatic tumor) and cutaneous nevus sample are carried out immunohistochemical staining, and the result shows that SM5-1 can act on all moles and malignant melanoma.On the other hand, SM5-1 not with inactive epidermal melanophore, non-melanocyte tumour cell, keratinocyte, endotheliocyte, smooth muscle cell and peripheral nerve cytosis.These results show that SM5-1 is extremely sensitive and specific for melanoma.
Monoclonal antibody of the present invention is carried out preservation in American type culture collection, and preserving number is ATCC No.HB-12588.
The invention still further relates to a kind of isolating, purifying or synthetic antigen, can specificity in conjunction with the antibody of SM5-1.Antigen of the present invention is present in the cytolemma and tenuigenin of everyone melanoma cell, includes but not limited to mole, elementary melanoma cell and shifts melanoma cell.In addition, detect discovery with SM5-1, this antigen is non-existent in normal inactive human melanoma cell, does not also exist in other tumour cell.
Antigen of the present invention can be used for preparing a kind of melanoma vaccines, can cause melanomatous specific immune response, thus prevention and treatment melanoma.Melanoma-associated antigen can be joined a kind of adjuvant and form pharmaceutical composition.The adjuvant that uses includes but not limited to that Fu Luoyideshi fully and Freund, mineral rubber, surface active substrate.
As used herein, what " isolating " limited is a kind of from the isolating or synthetic polypeptide (antigen or antibody) of native state (as cell, the outer material of born of the same parents), has broken away from from normal natural surroundings and has come.This isolated polypeptide is unique, is different from purifying, natural isolated polypeptide." purifying " limits be a kind of be not pure polypeptide fully, purer relatively than native state.
The invention still further relates to and a kind ofly can discern the antigenic affinity molecule of SM5-1 antibody recognition.These affinity molecule include but not limited to the polymkeric substance that antibody, peptide and other can the specificity conjugated antigens, and wherein preferred affinity molecule is a monoclonal antibody.Monoclonal antibodies different among the present invention can be discerned different antigenic determinants.Preferred monoclonal antibody is a purifying, isolating among the present invention.
The condition that possesses as a kind of preferred monoclonal antibody of the present invention is: (1) can be discerned mole, elementary melanoma cell and shift melanoma cell antigen; (2) can not discern normal disactivation melanoma cell antigen (promptly can not combine) with its any antigen; (3) can not discern other any tumour cell (promptly can not combine) with its any antigen.
Antibody of the present invention can be any immunoglobulin (Ig), such as IgA, IgG, IgD, IgE, IgM and subclass thereof, also can be complete antibody or antibody fragment in addition, includes but not limited to FAB, F (ab)
2', Fv.
The invention still further relates in addition with antibody of the present invention and have the specific antibody derivatives of identical combination, these derivatives are had some change in conjunction with non-critical areas at its antigen.
Monoclonal antibody of the present invention also can be connected with other molecule, and particularly marker and toxin include but not limited to enzyme (as peroxidase), toxin (as ricin or Toxins,exo-, cholera), fluorescein and radioactively labelled substance.
Monoclonal antibody can be obtained by the cell-fusion techniques of present technique field routine.Antibody secreting cell system also can be obtained by other technology beyond the integration technology, such as directly transforming bone-marrow-derived lymphocyte with carcinogenic DNA, virus.The antigen of different sources is used to excite normal bone-marrow-derived lymphocyte to be, changes immortal cell line afterwards into.Such as, can be used as immunogen with the antigen of SM5-1 antibody generation immunoprecipitation stimulates Mammals (as mouse, rat, hamster etc.).Collection is used technological incorporation well-known in the art to the infinite multiplication cell by the lymphocyte of antigenic stimulation, perhaps is transformed into immortal cell line.Can be by selecting to take place with antigen the colony screening melanoma cell antibody product of strong reaction; Also can be by selecting to combine melanoma cell and/or its antigenic clone isolating or purifying screens melanoma cell antibody product with SM5-1 competitiveness.
The clone that the present invention relates to is hybridoma cell line preferably, and another kind of optimal way is an immortal cell line.
Monoclonal antibody among the present invention can be used for separating melanoma-associated antigen (referring to Harlow and Lane, " immune purifying ", 511-552 page or leaf) with other affinity molecules.
Monoclonal antibody among the present invention also can be used for separating melanoma cell with other affinity molecules.Thereby the present invention also provides a kind of mass production to be rich in the method for the cell mass of human melanoma cell, comprise: select a kind of expectation to comprise the histocyte suspension of melanoma cell, cell suspending liquid is suspended altogether with discerning the antigenic monoclonal antibody of SM5-1 antibody recognition (or other affinity molecule), then from cell suspending liquid separation and combination the cell of antibody.
People's melanoma cell and other SM5-1 of expression discern antigenic cell and can use antibody of the present invention, separate from other cell by indirect immunological technique, used technology comprises that fluorescence activated cell separates (FACS) technology and other technology well-known in the art, carry out mark such as the antibody that will be attached to cell, with the markd cell of cell separation equipment decoupled band.
In addition, antimelanoma antibody or other affinity molecule can be attached on the solid support, and these solid supports are well-known in the art, include but not limited to sepharose 4B, magnetic bead, lustrex, hollow-fibre membrane and plastics petri dish.The cell attachment that can be incorporated on the antibody arrives solid support, thereby is separated from supernatant.
Monoclonal antibody of the present invention and other affinity molecule can be used for screening the melanoma sample in the tissue of patient, comprise biopsy samples, blood and other humoral sample.The melanoma that can use the immunohistochemical staining technology to carry out the living tissue sample detects.Tissue sample can be stored in the sanitas of formalin or other standard, dewaters and is embedded in paraffin, sample can be cut into slices, and is fixed on the slide.Sliced piece specimen also can directly prepare with frozen tissue.
Except biopsy samples, monoclonal antibody specificity melanoma-associated antigen of the present invention also can detect in tissue sample, the method that adopts is an immunological method well known in the art, prepares different samples to be tested according to the different sources of biological sample.The analytical technology that adopts can be referring to Harlowand Lane, immunoassay (471-510) and immunoblotting (552-612).
Available marker of the present invention includes but not limited to active nucleus, enzyme, fluorescein-quencher mixture, chemoluminescence element, magnetic particle, radiation opacity dyestuff.These markers can directly be attached on the monoclonal antibody by many covalent attachment linking groups or functional group.Use some known technologies that antigen-antibody complex is fixed on the solid support (as particulate or wall of container), also adopt ELISA, RIA, EIA technology such as (referring to Frye et al., 1987) to measure in addition.
The invention still further relates to the mensuration test kit, can use and measure the quantitative and qualitative analysis that test kit comprises the melanic related antigen of monoclonal antibody or other affinity molecule.
Before surgical operation, chemotherapy, the radiotherapy, among and afterwards, can use antibody of the present invention and/or other affinity molecules to detect melanoma-associated antigen level in the body fluid (as serum), to determine tumour sign and the position that occurs in the body.
It is very high (referring to Swerdlow AJ et al.Int.J.Cancer 1995 suffering from the possibility that melanomatous patient suffers from secondary tumour (such as lung cancer); Therefore 61:773-779), for melanomatous analysis with identify extremely important.Because SM5-1 can discern the transfer melanoma, its analysis and evaluation for melanoma and metastatic tumor is very useful.And by using SM5-1 can correctly detect melanomatous origin.
The invention still further relates to the recombinant protein of SM5-1 monoclonal antibody, they contain through humanization modified aminoacid sequence, are suitable for high expression level in human body.
Reorganization SM5-1 of the present invention can get with the several different methods preparation, both can have been expressed by reorganization SM5-1 coding nucleic acid as described herein and get, and also can obtain by full chemosynthesis.How on the books the preparation of protein involved or peptide is in existing document, and these methods all are applicable to the recombinate preparation of SM5-1 of the present invention.In addition, proteinic evaluation also is the ordinary skill in the art, and is how on the books in existing document.Therefore, the given aminoacid sequence according to the present invention, those skilled in the art are not difficult to obtain reorganization SM5-1 of the present invention in conjunction with prior art.
The recombinant nucleic acid of coding SM5-1 recombinant protein can obtain with several different methods, comprising, be that template is carried out site-directed mutagenesis with disclosed native sequences; Or according to the present invention given sequence is carried out complete synthesis.These all have been the ordinary skill in the art.And the evaluation of nucleotide sequence also is the ordinary skill in the art, and is how on the books in existing document.Therefore, the given nucleotide sequence according to the present invention, those skilled in the art's be not difficult to obtain recombinant nucleic acid of coding SM5-1 of the present invention in conjunction with prior art for preparing.
The present invention also provides a kind of carrier, wherein contains recombinant nucleic acid of the present invention.In the art, by suitable clone technology, the purpose nucleic acid fragment being inserted suitable carriers to realize purposes such as preservation, amplification and/or expression, has been general routine techniques.Clone's purpose is depended in the selection of suitable carrier, and carrier is taken in, with the consistency of host cell, and multiple factor such as restriction enzyme site, those skilled in the art are not difficult to make as the case may be suitable selection.Can be in order to carrier of the present invention such as but not limited to pUC18, pUC19, pUC57, pBluescript sk+/sk-.
The present invention also provides the host cell that contains reorganization SM5-1 coding nucleic acid, for example intestinal bacteria.Operate in this area quite ripe for colibacillary genetically engineered.For example, according to standard technique, all purpose nucleic acid can be imported host cell by several different methods such as conventional transfection, conversion, microinjections, and, can obtain good reproducibility in conjunction with conventional screening method.
Based on the lethal effect of SM5-1 to K-1735, monoclonal antibody of the present invention and other affinity molecule or recombinant protein can be used for passive immunization melanoma patient.Therefore, the present invention also provides a kind of pharmaceutical composition that comprises one or more antibody of the present invention or affinity molecule.Pharmaceutical composition is optionally combined with pharmaceutical carrier, adjuvant etc., put on melanoma patients by suitable route of administration.
As used herein, " pharmaceutical carrier " are meant that they can not produce disadvantageous, hypersensitive or other untoward reaction when molecule body and composition suitably give the animal or human." the pharmaceutically acceptable carrier " of indication of the present invention should be compatible with monoclonal antibody of the present invention, can not reduce the effect of pharmaceutical composition with its blend under normal conditions significantly.The object lesson that can be used as some materials of pharmaceutically acceptable carrier or its component is a carbohydrate, as lactose, dextrose plus saccharose; Starch is as W-Gum and potato starch; Mierocrystalline cellulose and derivative thereof are as Xylo-Mucine, ethyl cellulose and methylcellulose gum; The tragakanta powder; Fructus Hordei Germinatus; Gelatin; Talcum; Solid lubricant is as stearic acid and Magnesium Stearate; Calcium sulfate; Vegetables oil is as peanut oil, Oleum Gossypii semen, sesame oil, sweet oil, Semen Maydis oil and theobroma oil; Polyvalent alcohol is as propylene glycol, glycerine, Sorbitol Powder, mannitol and polyoxyethylene glycol; Lalgine; Emulsifying agent is as Tween ; Wetting agent is as Sodium Lauryl Sulphate BP/USP; Tinting material; Seasonings; Tablet agent, stablizer; Antioxidant; Sanitas; Apirogen water; Deng oozing salts solution; With phosphate buffered saline buffer etc.
Pharmaceutical composition of the present invention can be made various formulations as required, and can by the doctor according to patient's kind, age, body weight and roughly factor such as disease condition, administering mode determine the useful dosage of patient is used.Administering mode for example can adopt perfusion and other therapeutic modality.
As a kind of preferred mode be, the unitary dose of the antibody of passive immunization is between the 1-200mg scope, and this unitary dose also can repeatedly impose on melanoma patients.Monoclonal antibody can directly put on the melanoma position, and (referring to Irie et al., 1986 Proc.Natl.Acad.Sci.USA 83:8694-8698), also can integral body impose on patient's (transfer has especially taken place).Antibody of the present invention can be applied to the patient separately, also can use jointly with toxin, radiological agent or other treatment means.
Melanoma-associated antigen can be coated on the antigen presenting cell (APC), also can transfection or pulse enter APC, preparation can stimulate the melanoma cellullar immunologic response maybe can prevent melanomatous cell vaccine.The APC here includes but not limited to dendritic cell, B cell and scavenger cell.Also isolating melanoma cell and APC cytogamy can be prepared cell vaccine.Can handle with cytokine, with the amplifying cells vaccine, this technology can be the patent of WO 97/16238 referring to the PCT publication number.
The antibody of melanoma-associated antigen can self produce the specific antibody of mono-clonal Id as antigen, whether anti-idiotype polyclone or monoclonal antibody can be used for detecting existence that the melanoma-associated antigen host goes up HMB45, and wherein the antibody of melanoma-associated antigen and analyzed body fluid (as blood, serum, urine) must derive from same individuality.The anti-idiotype monoclonal antibody can be used for measuring melanoma-associated antigen level in people's body fluid.The anti-idiotype monoclonal antibody can be produced such as rodent, preferably rat or mouse by any host.The anti-idiotype monoclonal antibody also can be humanized or human antibodies.
Monoclonal antibody of the present invention also can be used for screening melanoma-associated antigen.Can use the cDNA library and the rotaring redyeing COS cell of mammalian cell transient expression vector construction melanoma A2058 cell strain, with melanoma monoclonal antibody SM5-1 is that probe carries out multi-turns screen to above-mentioned expression library, and the positive colony that screening obtains carried out sequential analysis and evaluation, obtain corresponding melanoma-associated antigen.
Description of drawingsFig. 1 is the melanoma immunohistochemical staining figure that combines monoclonal antibody SM5-1.Wherein: Figure 1A is a mole cell homology colored graph, and inactive basic melanophore is not colored.
Figure 1B is a melanoma original position colored graph.
Fig. 1 C is melanomatous surface arrangement colored graph.
Fig. 1 D is the colored graph that melanoma is transferred to lymphoglandula.
Embodiment
1.SM5-1 the preparation of antibody
From patient's elementary melanoma, gather K-1735 SMMU-1 (referring to Guo et al., 1994, Cancer Res.54:561-1565), with it immune mouse.Then mouse is handled with endoxan and removes activatory B cell.The transfer K-1735 SMMU-2 that mouse immune is gathered in the same patient body, splenocyte with mouse prepares hybridoma, the related technology of the preparation of hybridoma is that the routine techniques in present technique field is (referring to Kohler G et al., Eur.J.Immunol.1976; 6:511-519).
2. the immuning tissue of tissue sample dyeing
The SM5-1 sample can be freezing or be embedded in paraffin.Collect the supernatant of SM5-1 hybridoma, use the method for chain enzyme antibiotin-biotin labeling (LSAB) to carry out immunohistochemical staining.In brief, sample and main antibody were cultivated 10 minutes altogether, then cultivated altogether with biotinylated chain enzyme antibiotin and the substrate chromogen solution that is connected antibody, peroxidase labelling.The SM5-1 supernatant that uses in steps through diluting (1: 50).
The melanoma sample is also used HMB-45 (dilution in 1: 50) and the proteic monoclonal antibody of S-100 (dilution in 1: 20) dyeing.Also select the sample of many Humanmachine tumours to test, comprised elementary melanoma and melanoma metastatic tumor (skin, lymphoglandula etc.).
Prepare related tissue, after buffered 10% formaldehyde fixed, use paraffin embedding, study with conventional histological techniques then.
SM5-1 is listed in table 2 and the table 3 for the reactive behavior of plain knurl sample of non-black and benign tissue.
The staining method of HMB-45 is cytoplasmic granule dyeing, and the dyeing of SM5-1 can be tenuigenin, also can be cytolemma, and therefore two kinds of antibody recognition is antigen different on the cell.The result shows that for melanomatous reaction, SM5-1 has higher susceptibility than HMB-45, has higher specificity than anti-S-100 antibody.
3.SM5-1 can discern the antigen of malignant melanoma and mole
Shown in the table 1 that 226 melanomas and 16 moles are directed to the immunohistochemical analysis of SM5-1, HMB-45 and anti-S-100.No matter be what types of organization, all tested elementary melanomas and mole are male for the reaction of SM5-1, and Color is very strong, and relate to most tumour cells.Adopt the staining method of tenuigenin and cytolemma.The staining power of elementary melanoma and metastatic tumor is identical with staining method.
HMB-45 can detect all elementary melanoma and mole, yet 83% transfer melanoma HMB-45 can not be colored.The negative melanoma of all HMB-45 can be dyeed by SM5-1.Also find in addition, in by SM5-1 and the common painted tissue sample of HMB-45, the antigenic determinant of SM5-1 and HMB-45 is present in the different types of tumors, this antigenic determinant that shows SM5-1 and HMB-45 is different, and they are expressed in the different groups of melanoma tissue sample.
4.SM5-1 lethal effect to K-1735
In the PRIM1640 substratum, cultivate melanoma A2058 cell strain to 106 cells/ml.The SM5-1 monoclonal antibody of adding above-mentioned cell suspending liquid of 20ul and different amounts in each hole of 96 orifice plates (0,1,5,10,20,50,100ul; Monoclonal antibody is dissolved in FCS PRIM1640 substratum, and concentration is 1ug/ul), every hole adds the PRIM1640 substratum to 500ul.Each is handled 3 times and repeats.Carry out the MTT reaction after in 37 degree 5%CO2 incubators, cultivating 24 hours, react the 570nm place is read in the back on microplate reader photoabsorption at test under microscope survivaling cell number or through MTT then.
(1) cell counting.From above-mentioned sample, take out 0.2 milliliter and carry out cell counting, the result represents that with survivaling cell percentage ratio concrete data are as follows: SM5-1 add-on 015 10 20 50 100 (ul) cell survivaling number 100.0 87.8 68.8 53.2 42.6 32.6 22.6
The The above results explanation, SM5-1 has the effect that significantly kills and wounds melanoma cell.(2) MTT detects: 0.2 milliliter in the cell that the SM5-1 that learnt from else's experience handles, and adding 1/10 volumetric concentration is the MTT solution of 5mg/ml, 37 degree are cultivated the photoabsorption of reading the 570nm place after 30 minutes on microplate reader.Found that the cell of handling through SM5-1 obviously reduces than undressed contrast photoabsorption.This explanation SM5-1 handles the mass mortality that can cause melanoma cell.SM5-1 add-on 015 10 20 50 100 (ul) photoabsorption 100.0 96.3 84.9 66.2 47.7 33.6 20.1
From above-mentioned two detected results as can be seen, SM5-1 has the effect that significantly kills and wounds melanoma cell, therefore might be used for the treatment of melanoma clinically.
5.SM5-1 effect with the melanoma frozen section
36 melanoma samples (comprising 17 elementary melanomas and 19 metastatic tumors) are divided into 2 groups, use paraffin embedding for one group, another group is carried out moment embedding (tissue is put in the liquid nitrogen, with microtome (5 μ m) cutting), uses SM5-1 (dilution in 1: 51) dyeing then at low temperatures.The result shows that all frozen sections are similar to the SM5-1 Color that paraffin-embedded mode obtains.
6.SM5-1 can not dye in plain malignant tumour of non-black and people's healthy tissues
For detect SM5-1 whether with other malignant melanoma generation cross reaction, the present invention also carries out paraffin embedding to the plain knurl samples of many non-blacks, detects their dyeing situations for SM5-1.Tested tumor tissues comprises skin, brain, stomach and intestine etc., the results are shown in Table 2, and all testing sample coloration results all are negative.
In people's healthy tissues, except can taking place the faint reaction with dendritic cell around plasma cell, exocrine gland secretion thing, some myofibroblasts and the blood vessel, most normal cell can not react with SM5-1.
7.SM5-1 the preparation of recombinant protein
1) antibody variable gene of screening SM5-1 from antibody library
According to people J.Mol.Biol. such as Marks, 222,581-597; Hoogenboom and Winte, J.Mol.Biol., 227,381-388; Haidaris CG etc., J Immunol Methods.2001 Nov1; 257 (1-2): 185-202; Griffiths, EMBO J. such as A.D., 13,3245-3260 (1994); Nissim, EMBO J. such as A., 13, the method for describing among the 692-698 (1994) makes up human antibody library.
The antibody library bacterial strain of recovery is added 14 milliliters of fresh LB substratum for 1 milliliter, in 50 milliliters of triangular flasks, cultivated 16 hours for 37 ℃.
12000rpm high speed centrifugation 10 minutes shifts in supernatant to one 50 milliliters of aseptic centrifuge tubes, preserves standby.Its titre should be more than 2 * 1011.Melanoma-associated antigen with purifying is an antigen, and bag is by 25 ml cells culturing bottles.Bag by after the cell bottle in add and to be no less than 3 * 1010 phage particles, 37 ℃ of incubations 1 hour.Then, outwell the liquid in the bottle, wash culturing bottle 10 times with 10 milliliters of PBS that added 1%Tween-20.The TG1 cell that adds 1 milliliter of logarithmic phase in culturing bottle, 37 ℃ of incubation concussions were cultivated 16 hours.
Repeat the described step of epimere totally 4 times.
With cell dilution to 100000 cells/ml of above-mentioned acquisition, on 1.5% agar plate that adds 0.1% penbritin, cultivate to obtain mono-clonal then.Get being cloned on the 96 hole depth orifice plates on the above-mentioned flat board and cultivate, the clone in every hole does 960 clones (10 96 orifice plates) altogether.With above-mentioned deep-well plates centrifugal 20 minutes of 5000RPM on 96 orifice plate whizzers, supernatant is transferred to new aseptic deep-well plates, it is standby to be preserved in 4 degree after sealing.
Get 10 of 96 orifice plates, add in every hole the conventional bag of SM5-1 (10 mcg/ml) 10 microlitres by after, add supernatant 10 microlitres of above-mentioned preservation respectively, 37 ℃ of incubations after 1 hour with the PBS washing that contains 1%Tween-20 20 times.The goat-anti M13 monoclonal antibody that adds 1 microlitre HRP mark, 37 ℃ of incubations use the PBS of 1%Tween-20 to wash 10 times after 30 minutes.
Add and contain PBS 200 microlitres of 0.025%DAB developer and the H2O2 of 1 microlitre 1%, the 37 ℃ of incubation colour developing was read 595 nanometers after 20 minutes on plate reading machine photoabsorption.Determine the hole that color reaction is strong according to the photoabsorption reading, the corresponding clone in these holes is the stronger antibody variable region clone of avidity.
Filter out 415 positive colonies altogether by said process, determine wherein 5 the strongest clones of avidity according to reading.These 5 clones are seeded in respectively in 100 milliliters of LB substratum, after concussion under 37 ℃ of 260rpm conditions is cultivated 9 hours, add final concentration and be isopropylthiogalactoside (IPTG) inducing culture 10 hours of 1mM.Separation and purification reorganization SM5-1 antibody protein then.The reorganization SM5-1 protein of extracting and purifying is used for avidity research, finds out the positive colony with high expression level amount and high-affinity, is used for follow-up research.
The bacterial strain of above-mentioned positive colony is increased in 100 milliliters of LB substratum, with the plasmid DNA extracting and purifying test kit plasmid DNA purification of Promega company.
With XbaI with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after the NheI enzyme is cut above-mentioned plasmid DNA, get band about 350bp and carry out glue and reclaim, the gained fragment is the variable region of heavy chain encoding sequence.
With HindIII with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after Bsi WI enzyme is cut above-mentioned plasmid DNA, get band about 320bp and carry out glue and reclaim, the gained fragment is the variable region of light chain encoding sequence.
At first above-mentioned variable region of heavy chain encoding sequence is inserted into expression vector pMG18-3K then (referring to DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORING BASED ON INCP-9PLASMIDS SEQUENCES.A.Greated, R.Krasowiak, M.Titok, C.M.ThomasSchool of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK and Faculty of Biology, Dept of Microbiology, Belarus State University Scorina Av.4, Minsk 220080 Belarus) in the Xba I/NheI site, with Hind III and Bsi WI above-mentioned antibody chain variable region encoding sequence is inserted in the HindIII/Bsi WI site of the pMG18-3K that is inserted with the variable region of heavy chain encoding sequence again, is built into the expression vector of the anti-SM5-1 antibody gene of humanization.
2) screening of the transfection of Chinese hamster ovary celI and recombinant clone
Above-mentioned steps 3) expression vector that has antibody gene that makes up in is inoculated in 100 milliliters of LB substratum at e.colistraindh5 and increases, with ultrapure plasmid DNA purification kit (Ultrapure Plasmid DNA Purification Kit) the extracting and purifying plasmid DNA of Qiagen company.The plasmid DNA of above-mentioned purifying is adopted the liposome method test kit transfection CHO cell of Invitrogen company, and operation is carried out with reference to the specification sheets of producer.
The Chinese hamster ovary celI that transforms carries out the extreme dilution at last and cultivates in the selection of selecting to carry out on the substratum continuous 9 weeks on 96 orifice plates, carry out continuously 3 times, carry out mono-clonalization.
The monoclonal cell of choosing ties up on RPMI 1641 substratum and cultivates, and supernatant is carried out the Western Blot experiment, judges expression intensity according to staining reaction, picks out the stronger clone of 10 expression as candidate cell strain (seeing Table 1).
The purifying of above-mentioned monoclonal antibody adopts the directly separation and purification from cells and supernatant of Protein A (Pharmacia company) affinity column, and proves that with the SDA-PAGE electrophoresis products therefrom purity is greater than 90%.The product of above affinity chromatography passes through sieve chromatography once more, has obtained the sample of purity＞98%.These samples are used for following further analysis and research.
3) research of antibody gene expression intensity in Chinese hamster ovary celI
The high expression level candidate clone that above-mentioned screening is obtained is incubated in the tissue culture ware of 10cm, adopt the ELISA method to measure the expression amount of antibody: goat anti-human igg (Fc) bag quilt is in elisa plate, 4 ℃ are spent the night, and in 37 ℃ of sealings 2 hours, add culture supernatant to be measured and standard substance (human IgG1) through 2%BSA, hatched 2 hours for 37 ℃, add HRP-goat anti-human igg (κ) and carry out association reaction, hatched 1 hour for 37 ℃, add TMB in 37 ℃ of effects 10 minutes, use the H2SO4 termination reaction at last, survey the A450 value.The expression amount that records above-mentioned 10 candidate clones is as follows: table 1 antibody gene expression intensity in Chinese hamster ovary celI
Cell strain is compiled E9 D7 C6 H8 A6 B4 D8 F6 G7 G2 354563445 No. 6
Expression amount 1133433121 (ug/ milliliter) 16.7 28.9 66.7 43.2 08.6 72.1 32.1 76.4 31.4 46.9
As can be seen from the above table, the expression level that is numbered the cell strain of 4C6,5H8,6A6 and 3B4 has very high expression level (340-410 mg/ml), head and shoulders above the expression level of domestic and international monoclonal antibody class (50-100mg/ milliliter).
8. the screening of melanoma-associated antigen
(1) the cDNA library of structure melanoma cell strain A2058.
Melanoma A2058 cell extracts total RNA with Trizol reagent, with Oligo (T) fibre column chromatography method separating mRNA, is cDNA with random primer with the mRNA reverse transcription of extracting, mending flat back links to each other with the joint of BstXI restriction enzyme site, cut rear clone in Mammals transient expression carrier pCDM8 (Invitrogen) through the BstXI enzyme, transformed into escherichia coli MC1061/P3 constitutes the cDNA library.
(2) expression in library and screening.
The COS-7 cell is taped against in the culture dish of 20 10cm, transfection is carried out by the lipofectin method in cDNA library with above structure, cell is laid in the new culture dish after with trysinization again after 12 hours, transfection 72 hours, collecting cell is resuspended in the PBS/10mM EDTA/5%FBS liquid that contains monoclonal antibody SM5-1, ice bath 1 hour, cell is resuspended in the PBS/EDTA/0.5%FBS liquid after washing, cell is taped against respectively in 10 sub-sieve wares with sheep anti-mouse igg two anti-bag quilts, room temperature leaves standstill after 2 hours with PBS/EDTA/5%FBS liquid and washs (will be not removing with the cell of antibodies) gently, collect the cell that is adsorbed on the sub-sieve ware then, adopt the plasmid DNA in the Hirt method recovery cell, transformed into escherichia coli MC1061/p3 increases and reconstitutes a new cDNA library.
The COS-7 cell is taped against in the plastic culture dish of 6 10cm, carries out transfection, carry out second according to the method in the first round and take turns screening, the new cDNA storehouse of getting back then with the new cDNA storehouse lipofectin method of above structure.
Taking turns transfection, expression, sub-sieve and plasmid through 4 reclaims; with bed board behind the plasmid transformation escherichia coli MC1061/P3 that uses the Hirt method to reclaim for the last time; the a plurality of mono-clonal amplifications of picking at random, extracting plasmid; and use Lipofectin method transfection COS-7 cell respectively; transfection was carried out 12 hours; cell is laid in the new plastic ware after with trysinization again; transfection adds monoclonal antibody SM5-1 after 72 hours; and with the anti-dyeing of the goat anti-human igg two of FITC mark; under fluorescent microscope, observe at last; identify positive colony, the COS-7 cell with the empty carrier transfection compares simultaneously.(3) evaluation of sequential analysis and positive colony
The positive colony that obtains is checked order, and compare, be defined as new gene with international gene pool data.Derive its functional area of antigenic amino acid sequence analysis with the cDNA sequence.
Carry out the evaluation of positive colony with Western Blot method.The COS-7 cell (obtaining through the empty carrier plasmid transfection) of COS-7 cell, non-antigen expressed gene, the COS-7 cell (obtaining through the positive colony plasmid transfection) and the melanoma cell strain A2058 of antigen expressed gene are extracted above epicyte protein respectively with high density SDS, carry out the SDS-PAGE electrophoresis, then albumen is transferred on the nitrocellulose filter, nitrocellulose filter adds the sheep anti-mouse igg of SM5-1 monoclonal antibody, HRP mark successively after the BSA sealing, with the DAB colour developing, analyze the expression and the antigen molecular size of antigen gene at last.
With the expression of flow cytometry analysis antigen gene in mammalian cell.The COS-7 cell is taped against in the plastic ware, carries out transfection by the Lipofectin method, use the empty carrier transfectional cell simultaneously in contrast with containing 10 μ g plasmids.Transfection is in the time of 12 hours, transfer in the new plastic ware with trysinization COS-7 cell and with it, when transfection is carried out 72 hours, collecting cell, be resuspended in the PBS/EDTA/5%FBS liquid that contains melanoma monoclonal antibody SM5-1, A103 and T311 ice bath 1 hour after the washing respectively.Collecting cell is resuspended in after the washing and contains in the anti-PBS/EDTA/5%FBS liquid of FITC-sheep anti-mouse igg two, and ice bath 1 hour is analyzed with flow cytometer behind the PBS/EDTA washing transfectional cell.The anti-S-100 of reaction immunohistochemical staining melanoma SM5-1 NMB-45 of table 1:SM5-1, HMB-45 and anti-S-100 and optimum and malignant melanoma
(positive/testing sample) (positive/testing sample) (positive/testing sample) mole
Melanoma
The reactive behavior tumorous type SM5-1 immunohistochemical staining of the plain knurl of table 2:SM5-1 and non-black (specimens paraffin embedding slices)
| Compound mole | 6/6 | 6/6 | 6/6 |
| The dysplastic type mole | 5/5 | 5/5 | 5/5 |
| Blue nevus | 2/2 | 2/2 | 2/2 |
| Combined mole | 3/3 | 3/3 | 3/3 |
| Sum total | 16/16(100％) | 16/16(100％) | 16/16(100％) |
| Elementary melanoma: | |||
| Melanoma on the original position | 9/9 | 9/9 | 9/9 |
| Clark standard I I | 7/7 | 7/7 | 7/7 |
| Clark standard I II | 19/19 | 19/19 | 19/19 |
| Clark standard I V | 47/47 | 47/47 | 47/47 |
| Clark standard V | 6/6 | 6/6 | 6/6 |
| Sum total | 88/88(100％) | 88/88(98％) | 88/88(100％) |
| Shift melanoma | 135/138(100％) | 114/138(83％) | 137/138(100％) |
| The melanoma sum total | 223/226(98％) | 201/226(88％) | 225/226(99％) |
(positive/testing sample) skin
Prominent property dermatofibrosarcoma 0/2 is fallen
Merkel's cell knurl 0/1
Sweat gland carcinoma 0/3
Sqnamous glucagonoma 0/4 brain
Neurospongioma 0/1
Astrocytoma 0/4
Oligodendrocyte knurl 0/1
Meningioma 0/3
Glioblastoma 0/2
Neu knurl 0/1 gi tract
Esophagus cancer 0/2
Cancer of the stomach 0/2
Colorectal carcinoma 0/2
Carcinoma of the pancreas 0/2 other
Prop up organ cancer 0/1
Thyroid carcinoma 0/1
Thymic carcinoma 0/1
Prostate cancer 0/1
Ovarian cancer 0/1
Kidney 0/1
Leiomyosarcoma 0/2
M.paget???????????????????????????????0/1
Angioleiomyoma 0/1
Angiosarcoma 0/3
porocarcinoma?????????????????????????0/1
Urothelial carcinoma 0/1
Osteosarcoma 0/2
The reactive behavior tissue of sum total 0/46 table 3:SM5-1 and healthy tissues, normal cell, innocent tumor or the dyeing of knurl SM5-1 immunochemistry
Negative glomerulus-----cilliated epithelium tissue-----Beaker cell-----Sebacious gland-----hepatic tissue-----eumelanin cell-----keratinocyte-----islet cells-----peripheral nerve-----neutrophil cell-----smooth muscle cell-----stomach lining-----ependymocytoma-----the cheloid-----melanocyte of activation-----
Faint positive activatory scavenger cell+myofibroblast+plasma cell+exocrine gland sweat gland secretion thing+thyroid secretion+
Claims (14)
- One kind can specificity in conjunction with the antigenic monoclonal antibody of human melanoma cell, wherein antigen: (a) antibodies that can be produced by the hybridoma of ATCC preserving number HB-12588; (b) be present in melanocytic film of people and the tenuigenin; (c) be not present in the plain cell tumour cell of normal inactive human melanoma cell and non-black.
- 2. monoclonal antibody as claimed in claim 1, this antibody is similar to the monoclonal antibody that the hybridoma of ATCC preserving number HB-12588 produces.
- 3. monoclonal antibody as claimed in claim 1, this antibody are to be produced by the hybridoma of ATCC preserving number HB-12588.
- 4. hybridoma, monoclonal antibody that can production claim 1.
- 5. hybridoma, monoclonal antibody that can production claim 2.
- 6. hybridoma, monoclonal antibody that can production claim 3.
- 7. the antigen of isolating a, purifying, it is characterized in that: (a) can specificity be attached on the monoclonal antibody that the hybridoma of ATCC preserving number HB-12588 produces, (b) be present in the cytolemma and tenuigenin of human melanoma cell, (c) be not present in normal inactive people's melanophore and non-melanophore tumour cell.
- 8. one kind is detected the method whether melanoma exists, and relates to:(1) sample is combined with antigen-specific antibodies, antigen wherein (a) can be by the monoclonal antibody combination of ATCC preserving number HB-12588 hybridoma generation, (b) be present in melanocytic film of people and tenuigenin, (c) be not present in normal inactive people's melanophore and non-melanophore tumour cell;(2) detect the immunogenic compound that forms, judge that whether melanoma cell exists.
- 9. method according to Claim 8, tissue sample wherein is paraffin-embedded or freezing sample.
- 10. according to the method for claim 9, the monoclonal antibody wherein or the second antibody of monoclonal antibody are attached on the detectable signal marker.
- 11. method of utilizing the monoclonal antibody screening antigen gene of claim 1 or 2 or 3, it is characterized in that, cDNA library and transfection host cell with the strain of mammalian cell transient expression vector construction melanoma cell, with the melanoma monoclonal antibody is that probe screens expression library, obtains positive colony.
- 12. the method as claim 11 is characterized in that, with the cDNA library and the transfection COS-7 cell of mammalian cell transient expression vector construction melanoma A2058 cell strain.
- 13. the recombinant protein of the monoclonal antibody of a claim 1 is characterized in that, contains through humanization modified aminoacid sequence, can be in human body high expression level.
- 14. the melanomatous pharmaceutical composition of treatment is characterized in that it contains pharmaceutically claim 1 or 2 or 3 described human monoclonal antibodies or the described recombinant protein of claim 13 and the pharmaceutically acceptable carrier of significant quantity.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617734A (en) * | 2011-12-28 | 2012-08-01 | 暨南大学 | Antibody Dab-2 against FGF-2 and application thereof |
| CN105861437A (en) * | 2011-10-20 | 2016-08-17 | 加利福尼亚干细胞公司 | Antigen presenting cancer vaccine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861437A (en) * | 2011-10-20 | 2016-08-17 | 加利福尼亚干细胞公司 | Antigen presenting cancer vaccine |
| CN102617734A (en) * | 2011-12-28 | 2012-08-01 | 暨南大学 | Antibody Dab-2 against FGF-2 and application thereof |
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