CN1580247A - Esophagus carcinoma monoclonal antibody, and its radioactive marker and chemical cross-linked substance, and its use - Google Patents
Esophagus carcinoma monoclonal antibody, and its radioactive marker and chemical cross-linked substance, and its use Download PDFInfo
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Abstract
The invention relates to a kind of monoclonal antibody against carcinoma of esophagus. It aims at different kinds of esophagus cancer cell albumen and can secrete hybridoma cell against monoclonal antibody against carcinoma of Esophagus, which preservation number is CTCC-C200308,CCTCC-C200309, CCTCC-C200310, CCTCC-C200311. The invention also publishes radioactive marker and chemical crosslink material of the monoclonal antibody against carcinoma and their application for curing growth and therapeutic action.
Description
Technical field
The invention belongs to bioengineering field, what relate to is a class esophageal carcinoma monoclonal antibody and radioactively labelled substance and chemically crosslinked thing and their purposes.
Background technology
As everyone knows, although in decades, people, perform the operation to the existing many important breakthroughs of the understanding of tumour, radiotherapy, and chemotherapy is the modern treatment means of core, can only save the life of minority malignant tumor patient so far.Tumor treatment is made progress so difficultly, and its reason is many-sided.Many tumours are too dangerous owing to its position, or because it has been diffused into fatal position, lost the possibility of any operation when finding.
Radiotherapy is very effective to some circumscribed tumor focus, but is not all responsive to all malignant tumours, and in addition, to metastatic lesion, radiotherapy is invalid in general.And but the chemotherapy of today also is a kind of supplementary means of or mitigate the disease at the most.Even radiotherapy and chemotherapy can make progress on curative effect from now on, also they are class ideal methods of treatment hardly, selectively do not act on malignant cell and normal cell because these class methods have, the side effect that is caused often is impatient at by the patient.
Fritz Paul Ehrlich proposes the imagination of antineoplastic guide medicine at first in 20 beginnings of the century, the targeted therapy (Targeted Therapy) of so-called tumour is a kind ofly can selectivity to kill malignant cell and to the new ideas of the treatment tumour of normal cell toxicological harmless.Owing to there is not the ideal oriented carrier, for a long time, the research of targeted drug makes progress very slowly always.
1975, Koehler and Milstein have prepared first mouse resource monoclonal antibody in the world with hybridoma technology, for biotechnology has been opened a brand-new field, along with development of biology, monoclonal antibody technique has also had very big development, and is various whole molecules, micromolecular, cocktail, bifunctional so that all appearances successively of anti-unique monoclonal antibody; The appearance of monoclonal antibody technique and development have encouraged many people to go to explore the feasibility of targeted therapy again, go to seek the old dream at the beginning of last century again.Exploration and effort through more than ten years, as a kind of important content of biological respinse adjustment of treatment (Biological ResponseModulizer), be that the guiding antitumor drug of carrier has become over more than 100 year in the immunotherapy of tumors field one of the most noticeable progress with the monoclonal antibody.
As everyone knows, a kind of monoclonal antibody can only be discerned a kind of antigenic determinant, solve polymorphism (heterogeneicity) problem of tumour cell, also need develop a kind of energy identification compound monoclonal antibodies (being so-called cocktail monoclonal antibody) different tumor associated antigens, different antigenic determinants, that do not shelter mutually, although also come out without any a kind of antitumor compound monoclonal antibody that can be used as medicine up to now, its prospect is self-explantory.
According to the dead retrospective survey of 1974-1976 whole nation malignant tumour, China's esophageal carcinoma both sexes crude death rate is 16.7/10 ten thousand, and it is 23.4/10 ten thousand that the world adjusts human mortality.The neopathy number of world's esophageal carcinoma in 1980 is 31.04 ten thousand, accounts for 14.3% of all cancer morbidity numbers.
The esophageal carcinoma is some countries and regions common malignancy of the world.The statistics that the World Health Organization announced in 1977 shows that for the highest, the ratio that accounts for whole malignant tumour death is also than countries in the world height with China for world's adjustment mortality ratio of the esophageal carcinoma.Data according to China's national treatment and prevention of tumour research report in 1978.Male esophageal cancer is classified second of malignant tumour death as, is only second to cancer of the stomach; Women's esophageal carcinoma then accounts for the 3rd, inferior to cancer of the stomach and cervical cancer.As seen the esophageal carcinoma is one of tumour of serious threat China people's life health.
According to the dead data from retrospective survey of China to the esophageal carcinoma, this disease differs greatly at the sickness rate of various places, has its unique geography to distribute the district occurred frequently anomalous concentration.The Wild jujube in Taihang Mountain Area that Henan, Hebei, Shanxi San Sheng have a common boundary.With Lin County, Henan Province, Ci County, Hebei province and Yangcheng County, Shanxi Province is that the sickness rate of representative is the highest; The Yanting of the northwestward, the Sichuan Basin, Langzhong, North Jiangsu Area, the Da Bie Mountain area that Anhui, Hubei Province has a common boundary, border land, Guangdong, Fujian, Kazak ethnic population is lived in concentrated communities area etc. and is located all to have the esophageal carcinoma district occurred frequently of concentrating relatively.These regional esophageal carcinoma annual mortality ratio surpass 1,00/,100,000, and esophageal carcinoma death toll is occupied the 12%-20% of the total death toll of the people.In recent years according to the dead retrospective statistics of the various places esophageal carcinoma, this disease twenty or thirty over year incidence and mortality still do not have obvious decline.
The men and women's that this is sick ratio is about 2: 1, and the women of district occurred frequently falls ill and increases relatively, and men and women's ratio is about 1.5: 1.Age structure is mainly more than 40 years old, see at most in 65-69 year, but the sicken age of district occurred frequently is then generally on the low side, sends out the district 10 years in advance than low approximately.
Retrieve according to Medline, till 10 days July in 2003, the document of the whole world relevant human body esophageal carcinoma monoclonal antibody has 286 pieces, and what wherein relate to cocktail (cocktail) is 2 pieces (all on-radiation, non-therapeutic researchs), what relate to radioactivity (radioactive) is 0 piece, relates to
131Iodine (
131Iodineor 131I-labeled) be 0 piece, what relate to humanization (humanized) is 0 piece, what relate to chimeric antibody (chimeric) is 0 piece.
Summary of the invention
The inventor has studied esophageal carcinoma monoclonal antibody in order to address the above problem, especially about
131The preparation method of the esophageal carcinoma monoclonal antibody of iodine labeling (comprising its cocktail series), characteristic and problem such as possibility of its application in clinical diagnosis, treatment thereof.So one of purpose of the present invention provides a class esophageal carcinoma monoclonal antibody; Two of purpose provides its radioactively labelled substance and chemically crosslinked thing; Three of purpose is to disclose its application in clinical diagnosis, treatment.
The object of the present invention is achieved like this: a class esophageal carcinoma monoclonal antibody, it is characterized in that at antigen be different esophageal cancer cell membranins, the preserving number of secreting the hybridoma of these esophageal carcinoma monoclonal antibodies is CCTCC-C200308, CCTCC-C200309, CCTCC-C200310, CCTCC-C200311.
The form of wherein, such monoclonal antibody use separately, binary combination or polynary combination is used.
Such monoclonal antibody is the mouse source, the people source, or human mouse chimeric.
Such monoclonal antibody can be the antibody of whole molecule, or the fragment of antibody molecule, as the Fab fragment.
Such monoclonal antibody can be made protein chip (protein chip) and test kit (kit) with various forms.
And such monoclonal antibody can connect radioactively labelled substance, and this radioactively labelled substance comprises the isotropic substance of beta activity, as:
131Iodine,
125Iodine,
123Iodine,
188Rhenium,
90Yttrium,
32Phosphorus etc.; And alpha-active isotropic substance, as
212Bismuth etc.
Said
131The marking method of iodine labeling can adopt Iodogen method, 6-ammonia T method or other method; Its specific activity scope is 1.0 to 50.0; Its clinical using dosage scope in the 20mCi/ agent to the 500mCi/ agent.
In addition, this monoclonal antibody can also connect the chemically crosslinked thing, and this chemically crosslinked thing comprises toxalbumin cross-linking agent, pharmaceutical chemicals cross-linking agent and other chemical cross-linking agents; Described have ricin, toxalbumin, Chinese honey locust toxalbumin, Snakegourd Root toxalbumin, Pseudomonas aeruginosa toxalbumin, diphtheria corynebacterium toxalbumin etc. as crosslinked toxalbumin; Described have 5 FU 5 fluorouracil, methotrexate, vincristine(VCR), mitomycin etc. as crosslinked chemicals.
Monoclonal antibody of the present invention at different antigenic determinants can be glycoprotein character glycoprotein character or non-; Can be that one-level mechanism is special or tertiary structure is special; These all antigenic determinants can be used for preparing the monoclonal antibody of new anti esophageal cancer, can belong to same hypotype or subclass with original monoclonal antibody, also can be different subtype or subclass.
Described esophageal carcinoma monoclonal antibody has application in the pharmaceutical composition of esophagus cancer diagnosis and therapeutic action in preparation, and it comprises above-mentioned esophageal carcinoma monoclonal antibody, also comprises pharmaceutically acceptable carrier or vehicle.
The radioactively labelled substance of described esophageal carcinoma monoclonal antibody has application in the pharmaceutical composition of esophagus cancer diagnosis and therapeutic action in preparation, and it comprises above-mentioned radioactively labelled substance, also comprises pharmaceutically acceptable carrier or vehicle.
The chemically crosslinked thing of described esophageal carcinoma monoclonal antibody has application in the pharmaceutical composition of esophagus cancer diagnosis and therapeutic action in preparation, comprises above-mentioned chemically crosslinked thing, also comprises pharmaceutically acceptable carrier or vehicle.
Monoclonal antibody name of the present invention is called:
A. mouse source esophageal carcinoma monoclonal antibody SG5B7, corresponding human monoclonal antibody RSG5B7, corresponding people mouse chimeric mAb CSG5B7 and corresponding small molecular antibody series.
B. mouse source esophageal carcinoma monoclonal antibody SG5B8, corresponding human monoclonal antibody RSG5B8, corresponding people mouse chimeric mAb CSG5B8 and corresponding small molecular antibody series.
C. mouse source esophageal carcinoma monoclonal antibody SG5B11, corresponding human monoclonal antibody RSG5B11, corresponding people mouse chimeric mAb CSG5B11 and corresponding small molecular antibody series.
D. mouse source esophageal carcinoma monoclonal antibody SGD5, corresponding human monoclonal antibody RSGD5, corresponding people mouse chimeric mAb CSGD5 and corresponding small molecular antibody series.
The source cell of above series product is that hybridoma (monoclonal antibody mouse-anti human cell line) SG5B8, SG5B11, SGD5 and SG5B7 have been Chinese typical culture collection center (CCTCC) preservation in the school of Wuhan City, Hubei Province Wuhan University in the address on July 11st, 2003, and the acquisition preserving number is respectively: CCTCC-C200308, CCTCC-C200309, CCTC-C200310 and CCTCC-C200311.
Description of drawings
Fig. 1 is the positive colour developing photo of esophageal carcinoma monoclonal antibody SG5B7 in the human esophageal carcinoma section.
Fig. 2 is the feminine gender colour developing photo of esophageal carcinoma monoclonal antibody SG5B7 in lung tissue section.
Fig. 3 is that esophageal carcinoma monoclonal antibody SG5B7 is at pancreatic tissue section feminine gender colour developing photo generally.
Fig. 4 is the positive colour developing photo of esophageal carcinoma monoclonal antibody SG5B8 in the human esophageal carcinoma section.
Fig. 5 is the feminine gender colour developing photo of esophageal carcinoma monoclonal antibody SG5B8 in the spleen tissue section.
Embodiment
The preparation method of embodiment 1 mouse source esophageal carcinoma monoclonal antibody (comprising its cocktail type):
1). antigen and immunity
We are the antigen immune BALB/C mice with EC-109 esophageal cancer cell (or other esophageal cancer cell), get 2 * 10
5-9Individual cell injects mouse peritoneal, and immune 3-5 time altogether, face with reinforcement in preceding 3-5 days 1 time, get spleen after 3-5 days and merge.
2). cytogamy
The spleen chopping that to extract in the mouse body, the centrifugation splenocyte is suspended in RPMI 1640 substratum and cleans.In kind in the RPMI 1640 that contains foetal calf serum (or new-born calf serum), cultivate murine myeloma cell strain Sp2/0-Ag14 (or any murine myeloma cell strain) and obtain the myeloma cell, and in above-mentioned RPMI 1640 substratum, clean, then with myeloma cell and above-mentioned splenocyte with 1: the ratio of 5-10 is put into centrifuge tube and is mixed, with (Clin.Exp.Immunol. according to a conventional method under the Macrogol 4000 effect, 42,458-462,1980) carry out cytogamy.Then, resulting fused cell is scattered in the 96 hole flat boards of RPMI 1640 substratum that contain HAT, and at 37 ℃ and 5%CO
2In insulation can, cultivate it under the condition.Change the liquid several times with the HAT nutrient solution, continue and change liquid, form until the clone with the HT nutrient solution.
3). screening
With the EC-109 cell is target cell, filters out the purpose clone.Its method is:
A. centrifugal and reclaim target cell, the cell precipitation thing is suspended in the 100 μ l hybridoma culture supernatant and 37 ℃ of reactions 1 hour down.
B. after cleaning 1 time with PBS, in cell, add the sheep anti-mouse igg antibody of FITC mark and be incubated 1 hour.
C. after cell suspension being washed 1 time, analyze it with fluorescence microscope.Pick out esophageal cancer cell positive and, obtain SG5B7, SG5B8, SG5B11 and SGD5 etc. altogether to the fused cell (being so-called hybridoma) of other cell feminine gender.
D. use limiting dilution assay then, routinely program repeat clone (at least 6 times) resulting fused cell.
4). the initiation of ascites
With quantity is 3-5 * 10
7Individual hybridoma (CCTCC-C200308, CCTCC-C200309, CCTCC-C200310 or CCTCC-C200311) injects the Balb/c mouse peritoneal that excites through pristane.2-3 collects its ascites after week.
5) separation and purifying
A. liquid phase chromatography (as cationic exchange coloum) purifying routinely;
B. column chromatography (as Protein G post) purifying routinely.
E.. intersect inhibition test: with uranine four kinds of monoclonal antibodies such as direct mark SG5B7, SG5B8, SG5B11 and SGD5 respectively, replace second antibody, prove that four kinds of former determinants of antigen are independently of one another, do not shield mutually with the anti-clonal antibody of direct mark.
6). the prescription of esophageal carcinoma cocktail monoclonal antibody:
A. binary prescription (for example: the ratio between each monoclonal antibody is: 1: 1)
B. ternary prescription (for example: the ratio between each monoclonal antibody is: 1: 1: 1)
C. (for example: the ratio between each monoclonal antibody is the quaternary prescription: 1: 1: 1: 1)
7). flow cytometry analysis: with the indirect fluorescent method target cell is carried out mark, determine monoclonal antibody or cocktail monoclonal antibody recognition rate to target cell.
Embodiment 2 humanization esophageal carcinoma MONOCLONAL ANTIBODIES SPECIFIC FOR methods:
1). corresponding mouse source esophageal carcinoma monoclonal antibody: SG5B7, SG5B8, SG5B11, SGD5
2). cell cultures:
Esophageal cancer cell is that EC-109 cell (or other esophageal cancer cell) and Chinese hamster ovary line CHO-K1 (ATCC CCL 61) use RPMI 1640 (Gibco) and F12 Ham ' s nutrient solution to cultivate respectively, two kinds of nutrient solutions all add 10% foetal calf serum (Gibco), 5% microbiotic and antimycotic solution (Gibco) and 2mM/L glutamine (Gibco), at 37 degree, 5%CO
2, aseptic condition is cultivated down.
3). the antibody humanization:
Based on the sequence similarity of mouse, people's framework organizes kappa chain (VK) the variable region subgroup I of subgroup and variable region of heavy chain (VH) subgroup II to be selected to the antibody humanization.Do not keep the low danger of kappa chain site 42,83 and 106, but keep corresponding mouse source monoclonal antibody be combined with the framework that closes the position and organize residue and humanization.6 SHD-1 CDRs are arrived the rGD-A variable region by grafting, the most frequently used codon in expressing according to antibody gene, and the complete aminoacid sequence of rSHD-1 variable region is transformed into dna sequence dna.The NheI site is positioned at 3 ' terminal (GCTAGC) of VH, another restriction site, and hindIII then is introduced into 5 ' end, so that be cloned into the expression vector of mammalian cell.In addition, by changing the usage of codon, the restriction site that some are inner also is introduced in the humanized framework dna sequence dna as KpnI (GGTACC) and XbaI (TCTAGA).
4). the structure of expression vector
Synthesizing of the dna segment of the different zones of people source kappa chain and heavy chain: use six segment lengths' overlapping synthetic DNA oligonucleotide, get final product by overlapping PCR method.Six sections eclipsed oligonucleotides are set up in two steps and are formed.All annealing and the extensions in the reaction mixture of 50 μ l of the first step, each section of six sections oligonucleotides (5pmole).In second step, the oligonucleotide primer (50pmole) that designs each section is hybridized in difference pulsating 5 ' and 3 ' end.The human permanent cDNA segment that becomes the zone can use the primer of sex change to make the permanent district that becomes of heavy chain by PCR method clone.The structure of heavy chain carrier (pHeavy): the variable region dna segment and the people's heavy chain constant region cDNA segment that at first digest the people respectively with hindIII/NheI and NheI/XhoI, connect into the carrier of a hindIII/XhoI digestion then, pSectag2B/Hygro (Invitrogen); The structure of kappa chain carrier (pKappa): with the humanized variable region of hindIII enzymic digestion dna segment, people's constant region cDNA segment is 5 ' phosphorylation and use the XhoI enzymic digestion earlier, connect into the carrier of a hindIII/XhoI digestion then, pSectag2B (Invitrogen).The plasmid DNA of purifying is carried out transfection and order-checking, the hHP-1 dna sequence dna of the sequencing result of variable region and design relatively, cotransfection to the mammalian cell, between the different clones by exchange DNA restriction segment restitution point sudden change and disappearance.
5). antibody is expressed in CHO-K1 clone
5 μ l lipofectamine transfections are to Chinese hamster ovary CHO-K1 cell (ATCC) among two kinds of expression vectors (0.75 μ g pHeavy and 0.75 μ g pKappa) and the hybridoma-SFM (Gibco), in order to select stable transformant, the selection nutrient solution that contains 250 μ g/ml hygromycin B is added in transfection after 48 hours, select the fine and close colony and the sandwich ELISA method screening of passing through to secrete the supernatant of the resistance clone of monoclonal antibody, select secretory antibody to reach the resistance clone of highest level, with serum-free CHO-S-SFM nutrient solution (Gibco) preparation antibody, cultivate and collect supernatant after 3 days, with dawn white A affinity chromatography monoclonal antibody purification.The about 500ml blended of centrifuging cell conditioned medium, with 1MTris-HCL (pH8.0) balance, sample on the supernatant of handling, with white A column purification of dawn, antibody purified is kept at-20 ℃ in PBS solution, or adds white and 0.02%NaN3 (Sigma) of pure dawn of 1% ox blood and be kept at 4 ℃.People's IgG antibody 1 (or IG with concentration known
2a) kappa (Sigma) is standard, test by ELISA, antibody purified with the twice serial dilution is measured concentration, with the normal data mapping, the slope of the calibration tropic and Y-intercept are no less than 0.99 with relation conefficient to be calculated, and the data point of monoclonal antibody sample is in standard curve range, be used for calculating antibody concentration, the size of the purity of antibody sample and subunit (heavy chain and kappa chain) under reduction and non-reduced condition, is analyzed with SDS-PAGE respectively.
6). the evaluation of humanization esophageal carcinoma monoclonal antibody RSG5B7
RSG5B7 combines with the specificity of EC-109 cell with immunofluorescence dyeing test confirmer source helping digestion pipe carcinoma monoclonal antibody, the EC-109 cell reacts with humanization esophageal carcinoma monoclonal antibody RSG5B7 and esophageal carcinoma monoclonal antibody SG5B7 earlier, resist reaction with two of FITC coupling subsequently, under fluorescent microscope, observe stained.By indirect elisa method measure RSG5B7 (or other) in conjunction with active, in 96 hole elisa plates (Coring), cultivate EC-109 cell and fixing with 2% Paraformaldehyde 96 (Sigma), normal human tissue sample (Clontech) with 5 μ g/ml is coated on the elisa plate, after the 1%BSA sealing, the RSG5B7-A and the non-specific human IgG kappa antibody (Sigma) that add various concentration (0-20 μ g/ml), mouse-anti human IgG monoclonal antibody (Pharmingen) with the HRP mark detects bonded antibody, and the successful bag of people's tissue sample is to use mouse-anti people's white I monoclonal antibody of myocardium calcium dawn (HyTest) at heart tissue's sample.To the EC-109 cell, RSG5B7-A is arranged, non-specific human IgG1 kappa antibody (Sigma) and three kinds of antibody of cold RSG5B7-A antibody and biotin labeled RSG-5B7-A (biotin-RSG5B7-A) competition, inoculation EC-109 cell, with 2% formaldehyde fixed and 1%BSA sealing, the competition antibody sample that adds various concentration (0-20 μ g/ml), vitamin H-the RSG-5B7 that adds 5 μ g/ml subsequently, usefulness white streptavidin-HRP of antibiosis dawn (Pharmingen) detection of biological element-RSG-5B7 combines with the EC-109 cell.
Test for the HAMA sero-reaction, three kinds of liquid phase antibody sample (SG5B7, RSG5B7 and human IgG kappa) and the combining of solid phase SG5B7 competition and human antimouse antibody, bag is added then by the RSG5B7 of 5 μ g/ml and contains HAMA serum (Type2SQ earlier on elisa plate, Boehringer, Mannheim) and the serum/antibody mixed solution of the antibody sample of various concentration (0-0.5 μ g/ml), add the crosslinked mouse-anti human IgG1 monoclonal antibody of 2.5 μ g/ml vitamin Hs and white streptavidin-HRP of antibiosis dawn subsequently, people's anti-mouse IgG antibody in the detection HAMA serum and the combination of solid phase SHD-1.Above-mentioned three kinds of tests mentioning, all use 0.1M carbonate buffer solution (pH9.2) bag white by the dawn, in conjunction with and the incubation step of detection all be every hole 50 μ l, room temperature 1 hour, dilute all serum and antibody sample with confining liquid (the pure dawn of 1% ox blood is white among the PBS), wash plate six times with PBS between per step, after the detection, add OPO substrate solution (Sigma) colour developing, absorb light at 450nm.
Embodiment 3 esophageal carcinoma monoclonal antibodies
131-Iodine labeling (is example with the Iodogen method)
1). methylene dichloride is coated with pipe.
2). esophageal carcinoma monoclonal antibody is moved into methylene dichloride be coated with pipe, make it to react 3-5 minute.
3). will with mechanical manipulator
131-Sodium iodide is added to above-mentioned being coated with in the pipe, reacts more than half an hour in ice bath.
4). its mark dosage can be 20mCi, also can be more than the 20mCi,
5). with mechanical manipulator the above-mentioned pipe that is coated with is shifted out again.
6). the above-mentioned mixed solution that is coated with in the pipe is separated in the Sephadex chromatography column.
7). analyze with gamma counter, obtain radioactivity cocktail esophageal carcinoma monoclonal antibody.
The marker of embodiment 4. other radio isotope and esophageal carcinoma monoclonal antibody
The present invention also with some other labelled with radioisotope that cancer cells is had lethality in esophageal carcinoma monoclonal antibody, made a series of markers, as
125-Iodine,
123-Iodine,
188-Rhenium,
90-Yttrium,
32-Phosphorus,
212-The marker of bismuth etc.Its marking method is all undertaken by techniques well known.
The chemically crosslinked thing of embodiment 5. esophageal carcinoma monoclonal antibodies
Esophageal carcinoma monoclonal antibody can connect the chemically crosslinked thing, and the chemically crosslinked thing comprises toxalbumin cross-linking agent, pharmaceutical chemicals cross-linking agent and other chemical cross-linking agents.As crosslinked toxalbumin ricin, toxalbumin, Chinese honey locust toxalbumin, Snakegourd Root toxalbumin, Pseudomonas aeruginosa toxalbumin, diphtheria corynebacterium toxalbumin etc. are arranged; As crosslinked chemicals 5 FU 5 fluorouracil, methotrexate, vincristine(VCR), mitomycin etc. are arranged.
The present invention has also made these esophageal carcinoma monoclonal antibodies various forms of protein chips or reagent as clinical diagnosis or vitro detection.
These monoclonal antibodies at antigen be different esophageal cancer cell membranins, at different antigenic determinants can be glycoprotein character, also can be non-glycoprotein character, can be primary structure special, also can be that tertiary structure is special.
The specificity analysis (shown in accompanying drawing 1～5) of Application Example 1 esophageal carcinoma monoclonal antibody
Indirect immunofluorescence
Organization name N1 N2 P1 P2 SG5B7 SG5B8 SG5b11 SGd5
Brain--++----
Cerebellum--++----
Sciatic nerve--++----
Lung--++----
Liver--++ ±---
Kidney--++--±-
Bladder--++----
Spleen--++----
Stomach--++----
Intestines--++----
Pancreatico--++-±-±
Tiroidina--++----
Suprarenal gland--++----
The heart--++----
Voluntary muscle--++----
Testiculo--++----
Skin--++----
The esophageal carcinoma--++++++
N1, N2: be respectively first and second negative controls
N1: the human IgG of same concentrations is as first antibody, to get rid of because of the false positive due to the non-specific adsorption.
N2: damping fluid replaces first antibody, with the false positive due to the eliminating solvent composition.
P1, P2: be respectively first and second positive controls
P1: mouse anti human esophageal cancer cell film whole serum (dilution in 1: 5) is as first antibody, to check antigenic activity.
P2: human esophagus cancer EC-109 cell is as antigen, to check the activity of antibody to be checked.
The biochemical characteristics of Application Example 2 esophageal carcinoma monoclonal antibodies
[proterties] colourless clear liquid.
[inspection] pH value 6.5-7.5 (two appendix VI of Chinese Pharmacopoeia nineteen ninety-five version H).
[aseptic] this product should be tested by biological products rules (nineteen ninety-five version one one).
[bacterial endotoxin] this product is checked pyrogen with bacterial endotoxin test.Bacterial endotoxin interference test by trial-product shows that this product is not disturbed bacterial endotoxin test after diluting 10 times, thereby available bacterial endotoxin test is checked the pyrogen of this product.In the once maximum consumption 10ml of monoclonal antibody, consider that again other reagent also can carry bacterial endotoxin in the labeling process, so the bacterial endotoxin of regulation this product be every milliliter less than 5Eu.
[mycoplasma inspection] any biological products all do not allow mycoplasma contamination.In view of mycoplasma hominis, mycoplasma pneumoniae and molten urea urea substance are most representative three mycoplasma species, whether the existence of selected this three mycoplasma species of this standard is the discrimination standard whether this product has mycoplasma contamination.Mycoplasma can be grown on specific biphasic culture, forms colony; And make the substratum variable color.Mycoplasma pneumoniae is sour owing to the energy decomposition glucose produces, thereby makes substratum by pink flavescence; Mycoplasma hominis be owing to can decompose arginine and discharge ammonia, thereby makes substratum turn green by yellowish green; Molten urea urea substance is owing to the energy decomposing urea and discharge ammonia and carbonic acid gas, thereby makes substratum by purple stain indigo plant, and therefore, we can be by directly cultivating the pollution of mycoplasma with the detection mycoplasma on specific biphasic culture.The detected result of this product all should be negative.
[monoclonal antibody purity] is in view of advantages such as simple and direct, reliable, sensitivities, the selected SDS-polyacrylamide gel electrophoresis of this standard is as the measuring method of monoclonal antibody purity, and require this product through the SDS-polyacrylamide gel electrophoresis continue with after Coomassie blue R-250 (the Coomassie Blue R-250) dyeing, under the reductive condition two clear and definite colour developing bands (laying respectively at the position that molecular weight is 55KD and 25KD), be one or two clear and definite colour developing bands under the non-reduced condition, be positioned at the position that molecular weight is 160KD or 160KD and 320KD.Through running gel scanning integral and calculating, the monoclonal antibody of this product (monomer and dimer) purity should be not less than 95%.
[monoclonal anti body burden] adopts the Bradford method to measure the content of monoclonal antibody.The ultimate principle system of this method can form the stable bond thing with the arginic residue of protein molecule because of Coomassie Blue G 250 dyestuffs, dyestuff mainly exists with cationic form in acid reagent, its maximum light absorption (λ max) is 470nm, and dyestuff and combination of proteins thing mainly exist with anionic form in acid reagent, its maximum light absorption (λ max) is 595nm, in view of the above, the available standards product are made typical curve, measure the photoabsorption of trial-product 595nm under the same conditions, thereby obtain the content of monoclonal antibody.
[immunocompetence] adopts indirect immunofluorescence, is antigen with the EC-109 esophageal cancer cell, and the immunocompetence titre of this product (1mg/ml) (promptly can be diluted to 1 mcg/ml) more than 1: 1000 at least at least.
[specification] every milliliter contains monoclonal antibody 10mg.
To meet the aseptic pyrogen-free medicinal glass bottle of national standard, medicinal rubber bottle cover is sealed, and seals with aluminium lid [storage], preserves down at 2-8 ℃.
Application Example 3
131The biochemical characteristics of the radioactivity esophageal carcinoma monoclonal antibody of iodine labeling
This strain iodine [
131I] the aseptic virus-free no thermal source solution of liver cancer cell film monoclonal antibody Hepama-l of mark.By the time of putting down in writing on its label, contain iodine [
131I] radioactive activity should be the 90.0-110.0% of labelled amount.
This product contains 5% human serum albumin as stablizer.
[proterties] this product is flaxen clear liquid.
[discriminating] 1). it is an amount of to get this product, measures according to gamma spectrometer method (two appendix XIII of Chinese Pharmacopoeia nineteen ninety-five version), and the sub-energy of its key light is 0.364Mev.Or it is an amount of to get this product, measures according to transformation period assay method (two appendix XIII of Chinese Pharmacopoeia nineteen ninety-five version), should be up to specification.
2). measure according to the radiochemical purity assay method, being about 0 place in the Rf value has the radioactivity main peak.
[inspection] pH value should be 6.5-7.5 (two attached VI H of Chinese Pharmacopoeia nineteen ninety-five version).
It is an amount of that [bacterial endotoxin] gets this product, behind 30 times of bacterial endotoxin inspection dilute with waters, checks (two attached XI E of Chinese Pharmacopoeia nineteen ninety-five version) in accordance with the law.Bacteria endotoxin content in per 1 milliliter of stoste should be less than 15Eu.
[other] should meet every regulation relevant under the injection item (two appendix IB of Chinese Pharmacopoeia nineteen ninety-five version).
It is an amount of that [radiochemical purity] gets this product, is developping agent with acetone-0.9% sodium chloride solution (1: 1), measures according to radiochemical purity assay method (two appendix XIII one methods of Chinese Pharmacopoeia nineteen ninety-five version), and the radiochemical purity of this product should be not less than 95%.
It is an amount of that [immunocompetence] gets this product, measures the immunocompetence of this product with direct radioimmunoprecipitation.
[radioactive concentration] gets this product, measures according to radioactive concentration method of masurement (two appendix X of Chinese Pharmacopoeia nineteen ninety-five version III), and the radioactive concentration that contains iodine [131I] should be not less than 370MBq/ml.
The medication of [classification] radiation treatment.Be used for the treatment of the esophageal carcinoma.
The once about 925-1850MBq of [dosage] intravenous injection, maximum injection rate must not surpass 10mL.
[specification] (1) 1850MBq (2) 2775MBq
[storage] puts in the lead container, 2-8 ℃ of airtight preservation.Lead container surface emissivity level should be up to specification.
The present invention is a class
131The radioactivity esophageal carcinoma monoclonal antibody of iodine labeling (comprising its cocktail combination), and other radioactively labelled substance, chemically crosslinked thing and connecting method thereof can be released according to this, so above embodiment is in order further to illustrate the present invention, but does not limit the scope of the invention.
Claims (12)
1, a class esophageal carcinoma monoclonal antibody, it is characterized in that at antigen be different esophageal cancer cell membranins, the preserving number of secreting the hybridoma of these esophageal carcinoma monoclonal antibodies is CCTCC-C200308, CCTCC-C200309, CCTCC-C200310, CCTCC-C200311.
2, esophageal carcinoma monoclonal antibody as claimed in claim 1 is characterized in that the form of the use separately of this monoclonal antibody, binary combination or polynary combination is used.
3, esophageal carcinoma monoclonal antibody as claimed in claim 1 is characterized in that this monoclonal antibody is the mouse source, the people source, or human mouse chimeric.
4, esophageal carcinoma monoclonal antibody as claimed in claim 1 is characterized in that this monoclonal antibody can be the antibody of whole molecule, or the fragment of antibody molecule, as the Fab fragment.
5, esophageal carcinoma monoclonal antibody as claimed in claim 1 is characterized in that this monoclonal antibody makes protein chip (protein chip) and test kit (kit) with various forms.
6, esophageal carcinoma monoclonal antibody as claimed in claim 1 is characterized in that this monoclonal antibody can connect radioactively labelled substance, and this radioactively labelled substance comprises the isotropic substance of beta activity, as:
131Iodine,
125Iodine,
123Iodine,
188Rhenium,
90Yttrium,
32Phosphorus etc.; And alpha-active isotropic substance, as
212Bismuth etc.
7, esophageal carcinoma monoclonal antibody as claimed in claim 6 is characterized in that this
131The marking method of iodine labeling adopts Iodogen method, 6-ammonia T method or other method; Its specific activity scope is 1.0 to 50.0; Its clinical using dosage scope in the 20mCi/ agent to the 500mCi/ agent.
8, esophageal carcinoma monoclonal antibody as claimed in claim 1 is characterized in that this monoclonal antibody can connect the chemically crosslinked thing, and this chemically crosslinked thing comprises toxalbumin cross-linking agent, pharmaceutical chemicals cross-linking agent and other chemical cross-linking agents; Described have ricin, toxalbumin, Chinese honey locust toxalbumin, Snakegourd Root toxalbumin, Pseudomonas aeruginosa toxalbumin, diphtheria corynebacterium toxalbumin etc. as crosslinked toxalbumin; Described have 5 FU 5 fluorouracil, methotrexate, vincristine(VCR), mitomycin etc. as crosslinked chemicals.
9, esophageal carcinoma monoclonal antibody as claimed in claim 1, it is characterized in that this monoclonal antibody at different antigenic determinants be glycoprotein character or non-glycoprotein character; Be that one-level mechanism is special or tertiary structure is special; These all antigenic determinants can be used for preparing the monoclonal antibody of new anti esophageal cancer, can belong to same hypotype or subclass with original monoclonal antibody, or different subtype or subclass.
10, esophageal carcinoma monoclonal antibody as claimed in claim 1 has application in the pharmaceutical composition of esophagus cancer diagnosis and therapeutic action in preparation, it is characterized in that described pharmaceutical composition comprises the described esophageal carcinoma monoclonal antibody of claim 1, also comprises pharmaceutically acceptable carrier or vehicle.
11, radioactively labelled substance as claimed in claim 6 has application in the pharmaceutical composition of esophagus cancer diagnosis and therapeutic action in preparation, it is characterized in that described pharmaceutical composition comprises the described radioactively labelled substance of claim 6, also comprises pharmaceutically acceptable carrier or vehicle.
12, chemically crosslinked thing as claimed in claim 8 has application in the pharmaceutical composition of esophagus cancer diagnosis and therapeutic action in preparation, it is characterized in that described pharmaceutical composition comprises the described chemically crosslinked thing of claim 8, also comprises pharmaceutically acceptable carrier or vehicle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031422969A CN1580247A (en) | 2003-08-15 | 2003-08-15 | Esophagus carcinoma monoclonal antibody, and its radioactive marker and chemical cross-linked substance, and its use |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031422969A CN1580247A (en) | 2003-08-15 | 2003-08-15 | Esophagus carcinoma monoclonal antibody, and its radioactive marker and chemical cross-linked substance, and its use |
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|---|---|
| CN1580247A true CN1580247A (en) | 2005-02-16 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA031422969A Pending CN1580247A (en) | 2003-08-15 | 2003-08-15 | Esophagus carcinoma monoclonal antibody, and its radioactive marker and chemical cross-linked substance, and its use |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139394B (en) * | 2006-09-08 | 2011-10-12 | 中国医学科学院肿瘤研究所 | Cytoplasma membrane surface antigen monoclonal antibody and uses thereof |
| CN101134778B (en) * | 2006-09-01 | 2011-11-23 | 中国医学科学院肿瘤研究所 | Monoclonal antibody of esophagus cancer resistant blood vessel endothelium cell |
| CN1873410B (en) * | 2005-06-16 | 2011-11-30 | 中国医学科学院肿瘤研究所 | Method for detecting id marks related to tumor in blood serum from patient of oesophagus cancer |
-
2003
- 2003-08-15 CN CNA031422969A patent/CN1580247A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1873410B (en) * | 2005-06-16 | 2011-11-30 | 中国医学科学院肿瘤研究所 | Method for detecting id marks related to tumor in blood serum from patient of oesophagus cancer |
| CN101134778B (en) * | 2006-09-01 | 2011-11-23 | 中国医学科学院肿瘤研究所 | Monoclonal antibody of esophagus cancer resistant blood vessel endothelium cell |
| CN101139394B (en) * | 2006-09-08 | 2011-10-12 | 中国医学科学院肿瘤研究所 | Cytoplasma membrane surface antigen monoclonal antibody and uses thereof |
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