CN101654482B - High specific anti-tumor monoclonal antibody - Google Patents
High specific anti-tumor monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a high specific anti-tumor monoclonal antibody, relating to the fields of oncology and medical science, in particular relating to an anti-human tumor specific monoclonal antibody and an application thereof. The light chain variable region of the antibody is composed of 105 amino acids; and the heavy chain variable region is composed of 111 amino acids. The monoclonal antibody of the invention can significantly improve ADCC activity of NK cells, leads the kill rate of the NK cells to the tumor to increase by 50%. More importantly, the amino acid sequence of the variable region of the monoclonal antibody has self uniqueness, is totally different from that of other reported anti-HAAH monoclonal antibodies, which is fundamental for distinguishing different antibody characteristics.
Description
[technical field]
The present invention relates to oncology and medical field.The invention particularly relates to the monoclonal antibody specific and the application thereof of anti-people's tumour.
[background technology]
Tumour is the human second largest killer who is only second to cardiovascular diseases, and the early diagnosis and therapy of tumour is the important topic that life science faces.HAAH is intracellular a kind of enzyme, is detected in embryo and tumour, and in the normal human, the expression of haah gene expression is in closing condition, so do not produce HAAH.The expression of HAAH is obviously raised in the tumour cell.Because the generation of HAAH and tumour and develop close getting in touch all arranged, so HAAH is an important symbol of early diagnosis of tumor, also is an important target of oncotherapy.Immunodiagnosis and immunotherapy to tumour are the emphasis that people pay close attention to always, but breakthrough progress and few.People more and more pay attention to research and application to tumor associated antigen when constantly seeking tumour specific antigen.HAAH is a kind of special carcinomebryonic antigen, and its singularity is: (1) is relevant with nearly all tumour, and tumours known and more than 12 kinds are relevant, are a kind of carcinomebryonic antigen of wide spectrum.(2) HAAH and tumour have close dependency, and detection HAAH has very high specificity to the diagnosis of tumour, so have the meaning of making a definite diagnosis.(3) has the ADCC effect that mediates the NK cell at the antigenic specific antibody of HAAH, the effect of performance specific killing tumour cell.
(antibody-dependent cell-mediatedcytotoxicity, ADCC) effect is one of important channel of vivo immuning system specificity removing tumour and virus infected cell to the cell-mediated cell toxicant that antibody relies on.Film surface expressions such as NK cell, scavenger cell have IgG Fc acceptor, combine the effect that performance kills and wounds target cell by the Fc section with the IgG antibody that is combined in target cell surfaces such as virus infected cell or tumour cell.The NK cell is the main cell of performance ADCC effect.In the generating process of antibody-mediated ADCC effect, antibody at first combines with corresponding antigens epitope specificity on the target cell, and the NK cell kills and wounds the target cell with antibodies then, has special guiding lethal effect.
Effect that tumour is removed and ADCC effect strong and weak closely related, the power of ADCC effect is then closely related with the characteristics of antibody-antigenic action.Be not that all antibody all has mediation ADCC effect, different separately avidity and different mediation ADCC activity also arranged at the antibody of same antigen difference epi-positions.Therefore, screening has the monoclonal antibody specific of strong mediation ADCC effect, and is significant for tumor treatment, also has important use naturally and be worth on the specific diagnosis of tumour.This application need based on diagnosing tumor and treatment, the present invention has filtered out a strain and has had the monoclonal antibody of very strong mediation NK cell ADCC effect from the hybridoma of a large amount of fusions, in vivo with external experiment in, can make the NK cell that the killing activity of tumour is improved 50%-80%.Antibody is applied in the diagnosis of tumour very high specificity is arranged also, the Showed by immune group result of pathological section, with the diagnostic result of routine pathology section relatively, its positive coincidence rate is 100%.
[summary of the invention]
Purpose of the present invention provides a kind of specificity antineoplastic significant antigenic monoclonal antibody.
A kind of anti-tumor monoclonal antibody of high specific, the variable region of light chain of this antibody is made up of 105 amino acid, and its sequence is as follows:
Gln?Phe?Val?Leu?Thr?Glu?Ser?Gly?Ala?Ile?Met?Ser?Thr?Ala?Met?Gly?GlyArg?Val?Thr?Ile?Ala?Cys?Ala?Thr?Ser?Gly?Gly?Ser?Asp?Thr?Arg?Asn?LeuHis?Trp?Tyr?Ala?Thr?Val?Thr?Arg?Leu?Pro?Gly?Arg?Val?Thr?Ser?Lys?ArgPro?Ser?Phe?Ala?Cys?Leu?Glu?Ser?Arg?Thr?Asn?Gly?Asp?Ala?Lys?Val?LeuPro?Gly?Phe?Phe?Asn?Gly?Gln?Thr?Phe?Ala?Ser?Pro?Phe?Val?Ala?Thr?ProAsn?Ala?His?Val?Pro?Ala?Asp?Gly?Leu?Leu?Gly?His?Ala?Gly?Ser?Lys?AlaPhe?Gly?Ser
Variable region of heavy chain is made up of 111 amino acid, and its sequence is as follows:
Ala?Val?Ser?Pro?Asn?Gly?Lys?Tyr?Lys?Gly?Asn?Ser?Arg?Pro?Thr?Cys?ProAsp?Cys?Phe?Pro?Ala?Thr?Ser?Leu?Glu?Gly?Leu?Phe?Leu?Glu?Leu?Ser?ArgArg?Ala?Lys?Cys?Phe?Pro?Thr?Val?Val?Arg?Gly?Phe?Glu?His?Thr?Cys?AsnThr?Arg?Glu?Leu?Pro?Asp?Leu?Gly?Arg?Ile?His?Ser?Ile?Lys?Ala?Gly?IleHis?Arg?Ash?Gly?Gly?Ile?Gln?Phe?Ile?Thr?Phe?Gly?Leu?Cys?Gly?Asp?ArgIle?Phe?Pro?Glu?Val?Asp?Asn?Arg?Val?Lys?Phe?Thr?Cys?Ser?Asn?Asp?SerVal?Asn?Gly?Ser?Ala?Phe?Ile?Arg?Cys
In one embodiment of the invention, the specificity combination takes place in above-mentioned antibody and antigen HAAH (Human asparaginylbeta-hydroxylase, human asparagine hydroxylase).
In another embodiment of the present invention, the constant region of this antibody is a mouse IgG2a subclass.
In a second aspect of the present invention, provide the application of said monoclonal antibody in preparation medicine for treating tumor thing.
In another embodiment of the present invention, said monoclonal antibody application in cancer, kidney, esophagus cancer, the bladder cancer medicine before preparation treatment liver cancer, lung cancer, mammary cancer, cancer of the stomach, cervical cancer, ovarian cancer, colorectal carcinoma, prostatitis.
Monoclonal anti physical efficiency of the present invention significantly improves the ADCC activity of NK cell, makes the NK cell improve at least 50% to the kill rate of tumour.Its amino acid sequences of what is more important has the uniqueness of himself, and the anti-HAAH monoclonal antibody that its variable region amino acid sequence and other reported is different fully, and this is distinguish the different antibodies characteristic basic.
[embodiment]
One, haah gene expression is recombinant expressed
Derive from the human breast carcinoma tissue of clinical excision, adopt the RNA extracting solution to extract total RNA from the tumor tissues that grinds, oligo (dt) primer reverse transcription obtains the cDNA of haah gene expression.The HAAH cDNA that adopts PCR method to amplify subsequently to be used to clone.The HAAH cDNA order-checking of amplification is confirmed rear clone on the pPIC9k Yeast expression carrier, behind the conversion GS115 yeast, and the transformed yeast bacterium of screening 3mg/ml G418 resistance.Utilize methyl alcohol that yeast is fermented and induced expression, purifying expression product from fermented liquid supernatant finally obtains purity and is 95% HAAH recombinant expression protein.
Implementation process is as follows:
1.HAAH the clone of cDNA:
At first synthetic following primer
Upstream primer: 5 ' at gaattc atg gtg att gca ttg ctg ggc 3 '
Downstream primer: 5 ' at gcggccgc cta aat tgc tgg aag gct gcg tc 3 '
Upstream primer has been introduced EcoR I restriction enzyme site gaattc, and downstream primer is introduced Not I restriction endonuclease sites; Clone's detailed process is as follows:
Total RNA in the extracting tumor tissues: get clinically through about 0.1 gram of the breast cancer tissue that pathology is made a definite diagnosis, the Trizol RNA extracting solution (Invitrogen company product), the chloroform 200 μ l extractings that add 1ml after freeze thawing-homogenate, centrifugal 5 minutes of 12000rpm, draw water 500 μ l, add Virahol 500 μ l, centrifugal 15 minutes of 12000rpm obtains the RNA precipitation, and 75% ethanol is washed precipitation one time.Sedimentary total RNA dries up in super clean bench, is dissolved in the 50 μ l DEPC treated waters.Get the total RNA of 5 μ l, as reverse transcriptase primer, AMV is the first chain cDNA that reversed transcriptive enzyme obtains HAAH with OligodT 16.With 8 μ l reverse transcription products is template, is amplimer with above-mentioned upstream and downstream primer, with the haah gene expression that increases in the PCR method.Amplification condition is: 94 ℃ of 2min, 94 ℃ 50s-55 ℃ 50s-72 ℃ of 1min30s totally 30 circulations.Amplified production has an amplified band clearly at about 2.3kb size place after 1% agarose electrophoresis.From sepharose, reclaim this DNA band, be connected with the T carrier subsequently.Connect product and transform DH5 α intestinal bacteria (competence), select 8 white colonies on the penicillin flat board of IPTG and X-gal containing, extract plasmid respectively, carry out double digestion with EcoR I and Not I and identify that 5 clones' plasmid can both cut out the fragment of 2.3kb size as a result.Get one of them clone and deliver the order-checking of order-checking company, the result shows that the HAAH cDNA sequence of institute's cloned genes and announcement is in full accord.
2. the expression vector of construction expression HAAH:
Behind EcoR I and Not I double digestion T carrier, reclaim the haah gene expression of 2.3kb, be connected with the pPIC9K carrier of same double digestion, transform DH5 α competence bacteria, get 4 clones and identify that with EcoR I and Not I double digestion the result can both cut out two fragments (fragment is the carrier of 9.3kb, and another is the haah gene expression of 2.3kb), get one of them clone and carry out sequencing, turn out to be haah gene expression and be cloned in the pPIC9K carrier.The clone who confirms extracts plasmid, and after Sal I enzyme was cut into linearity, purifying reclaimed.The about 0.1 μ g of linearizing cloned plasmids (in 0.2 μ l pure water) mixes with the competence GS115 Pichia yeast of 50 μ l, and at 25 μ F, electroporation under the 2.5kV condition changes cloned plasmids in the Pichia yeast over to.GS115 Pichia yeast after electricity is worn is coated on the MD flat board.Behind 30 ℃ of cultivation 5d, collect all pichia spp bacterium colonies, be coated on again after mixing on the YNB flat board that contains 3mg/ml G418, behind 30 ℃ of cultivation 5d, obtain the Yeast engineering bacteria clone of 2 anti-4mg/ml G418.Get one of them clone and carry out small-scale abduction delivering experiment: after in the BMGY substratum, expanding bacterium, in containing the BMMY substratum of methyl alcohol, carry out abduction delivering, express supernatant, determine to see that at the 90kd place obvious expressed proteins is arranged through the SDS-PAGE electrophoresis.Adopt the anti-HAAH polyclonal antibody of rabbit (source is stated as follows) that it is carried out western-blot dyeing, the albumen that proves the 90kd place is HAAH.This result shows the Yeast engineering bacteria that successfully constructs expression HAAH.
3. the polyclonal antibody for preparing the anti-HAAH of rabbit:
For recombinant expressed HAAH albumen is identified, we are before this according to the 86th to 110 aminoacid sequence among the HAAH, chemosynthesis 25 amino acid whose small peptides, the synthetic small peptide mixes the back immunizing rabbit with Freund's complete adjuvant, obtained the polyclonal antibody of the anti-HAAH of rabbit.
4.HAAH fermentation, expression and purification in yeast:
(1) fermentation of Yeast engineering bacteria with induce: express the Yeast engineering bacteria 10ml of HAAH, in the 500mlBMGY substratum, cultivated 48 hours, forward in the 30L fermentor tank that contains the 15L basic culture solution.Temperature, oxygen dissolved and pH value are set in 30 ℃, 60%, 5.0 respectively.Carrying out batch feeding with glycerine cultivated 72 hours.After stopping glycerine stream and adding, begin stream and add methyl alcohol and carry out abduction delivering, induction time is 72h.
(2) separation of fermented liquid: the fermented liquid of collecting in the fermentor tank is total to 30L, centrifugal collection supernatant liquor 20L.The supernatant that takes a morsel is identified expression product with the SDS-PAGE electrophoretic method.The result shows that a tangible protein expression is arranged at about 90kd place, with the standard content human albumin comparison of 0.5mg/ml, determines that tentatively the HAAH expressing quantity of this engineering bacteria can reach 0.5mg/ml.
(3) the proteic purifying of HAAH: the supernatant liquor that fermentation inducement is expressed carries out ultrafiltration and concentration with the ultrafiltration plate of 50K, obtains ultrafiltration and concentration liquid 1L.Ultrafiltration and concentration liquid adopts the method purifying target protein wherein of ion exchange chromatography subsequently at first by sephadex G50 removal salt ion wherein.The iso-electric point of HAAH is 4.8, thus adopt DEAE-sepharose-FF anion-exchange chromatography post, under the PBS (pH7.0) of 0.1-2M concentration tonsure NaCl wash-out, purifying HAAH albumen.HAAH albumen behind the purifying is through SDS-PAGE electrophoresis and gray scale scanning, and proteic purity can reach 99% behind the confirmation purifying.The final HAAH albumen 3g that obtains behind the purifying.
Two, anti-HAAH Monoclonal Antibody
Immune BALB/c mouse behind the reorganization HAAH of purifying and the Freund mixed grinding, the dosage of mouse immune is: the HAAH of every each subcutaneous immune 0.5mg of mouse, immunity is 4 times altogether, each 10 days at interval.Get the spleen of immune mouse, screen cloth grinds the back and merges with sp2/0 myeloma cell, the preparation hybridoma.Screening has the mono-clonal hybridoma of secretion in conjunction with HAAH antibody from the hybridoma of fusion growth, and preparation contains the mouse ascites of monoclonal antibody.Obtain altogether 3 strains can with HAAH bonded monoclonal antibody, respectively called after 2C6,2D5,2H8.
Specific implementation process is as follows:
1. immune Balb/c mouse
Freund's complete adjuvant (the sigma company product) mixing of recombinant expressed HAAH albumen 0.5ml (containing 1.5mg HAAH albumen) and 1ml, every subcutaneous 3 point of mouse back, 2 of inguinal regions carry out immunization, every some injection 0.1ml, immunity 3 BALB/c mouse (BALB/c mouse is available from The Fourth Military Medical University's Experimental Animal Center) altogether.Carry out the immunity second time after 10 days, the reorganization HAAH albumen of abdominal injection 0.5mg.Carried out immunity for the third time on the 20th day, immunizing dose and immunization route are with for the second time.A week adopt a spot of blood after the immunity for the third time and carry out titration from the tail vein.ELISA indirect method result confirms that the anti-HAAH antibody titer of mice serum has reached 1: 10000.The 30th day mouse peritoneal booster immunization injection 1mgHAAH albumen was got mouse spleen, and was used to prepare hybridoma on the 35th day.
2, the fusion of hybridoma
(1) main agents: the cell culture medium that uses in the hybridoma technology is RPMI-1640 (Gibco BRL).With RPMI-1640 is basic culture solution, according to the composition difference of adding, is used for different purposes in hybridoma fusion preparation process.
Incomplete RPMI-1640 substratum: every 100ml contains RPMI-1640 substratum stoste 96ml, 100 * glutamine solution 1ml (0.2mol/L), two anti-solution 1ml (containing penicillin 10,000 units/ml, Streptomycin sulphate 10mg/ml), 7.5%NaHCO
3Solution 2ml, HEPES solution 1ml (1mol/L).Aforesaid liquid is all by filtration sterilization.
Complete RPMI-1640 substratum: every 100ml contains incomplete RPMI-1640 substratum 80ml, foetal calf serum 20ml.Complete 1640 cultivations that are used for myeloma cell SP2/0 and build the hybridoma after the strain.
HT substratum: complete RPMI-1640 substratum 99ml, 1ml xanthoglobulin and thymidine (HT).
HAT substratum: complete RPMI-1640 substratum 98ml, 1ml HT, 1ml aminopterin-induced syndrome (A).
(2) fusion of myeloma cell and splenocyte
Myeloma cell: collect the myeloma cell (SP2/0) of logarithmic growth before merging, be suspended in the incomplete substratum.
Splenic lymphocyte: get the above-mentioned BALB/c mouse of immunity, in 75% alcohol, soaked 1 minute after extracing eyeball bloodletting and execution, in super clean bench, cut off peritonaeum, take out spleen and place plate, respectively with the incomplete substratum washing of 20ml 2 times with aseptic operation.Peel off around the spleen behind the reticular tissue, spleen is moved on the 200 order copper mesh (placing plate), with 1ml glass syringe nook closing member crush and grind spleen gently, with the incomplete substratum flushing of 10ml screen cloth.The results splenocyte suspension, centrifugal 10 minutes of 1000rpm, not exclusively the substratum centrifuge washing is 3 times, at last cell is resuspended in the incomplete substratum of 10ml.
The preparation of feeder cell: get a normal BALB/c mouse (not passing through immunity), prepare feeder cell according to the method identical with preparing splenic lymphocyte.96 porocyte culture plates, it is standby that every hole adding 0.1ml contains 106 feeder cell.
The fusion of myeloma cell and splenic lymphocyte:
With 1 * 108 splenocyte with after 1 * 107 myeloma cell SP2/0 mixes, centrifugal 5 minutes of 1000rpm, exhaustion supernatant.Light finger is flicked and is hit at the bottom of the fusion pipe, makes sedimentation cell loose evenly, puts 37 ℃ of preheatings.Add PEG (sigma) 1ml of 37 ℃ of preheatings in 1 minute, the limit edged stirs gently.Add the incomplete substratum of 20ml preheating in 90 seconds, left standstill 10 minutes.Centrifugal 5 minutes of 1000rpm, supernatant discarded.Add 5ml HAT substratum, the pressure-vaccum sedimentation cell suspends and mixing it gently, adds the HAT substratum then to 50ml.The above-mentioned 96 porocyte culture plates that contain feeder cell, every hole 0.1ml, culture plate put 37 ℃, 5%CO
2Cultivate in the incubator.After 5 days with HAT substratum 1/2 substratum that swaps out.After 10 days with the HT substratum HAT substratum that swaps out; Often observe the hybridoma growing state, treat that it grows to hole floorage 1/10 sucking-off supernatant detection antibody when above.
(3) hybridoma screening
The reorganization HAAH albumen bag of purifying is by elisa plate, 20 μ g/ holes.Get the culture supernatant 0.1ml in hybridoma growth hole, add and wrapped by in the good elisa plate, negative control hole adds complete 1640 substratum of 0.1ml, 37 ℃ 1 hour.Wash and add sheep anti mouse enzyme labelled antibody 0.1ml behind the plate, wash plate after 1 hour, add the colour developing of OPD substrate for 37 ℃.The hybridoma of positive strong hole correspondence goes to and continues in 24 orifice plates to cultivate, and carries out three times cloning.This fusion has obtained three strain positive colonies.Difference called after: 2C6,2D5,2H8.This three strains monoclonal antibody detects through the antibody subclass and confirms to be IgG2a.
(4) monoclonal antibody ascites preparation
15 of BALB/c mouse, every abdominal injection whiteruss 0.2ml, 7 days above-mentioned three strain of hybridoma that filter out of pneumoretroperitoneum injection.Every kind of hybridoma injected 5 BALB/c mouse, 106 hybridomas of every injection.Extract ascites after 13 days ,-20 ℃ of preservations are standby after the centrifugal collection.
Three, the ADCC activity of anti-HAAH mediated monoclonal antibody NK cell
With A549 lung carcinoma cell, 7721 liver cancer cells, MCF breast cancer cell is target cell, and it is the effector cell that magnetic bead separates the NK cells of human beings that obtains, and cultivates in 96 orifice plates after two kinds of cytomixis, and the ratio of effector cell and target cell quantity is 5: 1.Control group has only tumour cell and NK cytomixis to cultivate, and experimental group also adds the monoclonal antibody ascites of 10 μ l except tumour cell and NK extracellular.In order to calculate the needs of kill rate, also established the control wells of having only independent NK cell and having only independent tumour cell in addition.Each organizes cell on same 96 well culture plate, cultivates after 4 hours for 37 ℃, uses the amount of Cell Countingkit-8 kit measurement viable cell respectively, calculates kill rate.Kill rate (%)=(1-(Ae+t-Ae)/At) * 100%; Ae: simple effector cell hole is the A value (light absorption value) of NK cell hole; At: simple target cell hole is the A value of K562 cell hole; Ae+t: the effector cell adds the A value in target cell hole.The kill rate result such as the following table of each group:
Table 1.3 strain mediated monoclonal antibody ADCC exercising result
| The average kill rate of tumour cell+NK cell | The average kill rate of tumour cell+NK cell+2C6 monoclonal antibody | The average kill rate of tumour cell+NK cell+2D5 monoclonal antibody | The average kill rate of tumour cell+NK cell+2H8 monoclonal antibody | |
| The A549 lung carcinoma cell | 25％±6％ | 26％±8％ | 38％±8％ | ?24％±9％ |
| 7721 liver cancer cells | 33％±5％ | 36％±7％ | 50％±9％ | ?35％±6％ |
| The MCF breast cancer cell | 38％±5％ | 32％±6％ | 72％±10％ | ?34％±9％ |
From the experimental result of last table as can be seen this strain monoclonal antibody of 2D5 have tangible mediation NK cell ADCC effect, other two strain monoclonal antibody 2C6 and 2H8 have only very weak or do not have the ADCC effect.
Four, 2D5 monoclonal antibody subclass and weight, light chain variable region amino acid sequence determines
The Hybridoma Cell Culture supernatant of secretion 2D5 monoclonal antibody adopts mouse antibodies classification identification kit to identify.Confirm that the 2D5 monoclonal antibody belongs to mouse IgG2a subclass.Extract total RNA of 2D5 hybridoma, adopt 5 groups of primers of mouse variable region cDNA gene amplification, counterweight, chain variable region gene increase respectively.As a result, respectively there is a pair of primer to amplify variable region of heavy chain cDNA and variable region of light chain cDNA respectively.Heavy, variable region of light chain cDNA carries out gene sequencing after being cloned into the T carrier respectively, carries out sequence alignment in gene pool, does not find the gene identical with this sequence.Confirm that through sequence alignment the structure of 2D5 variable region of mab has the typical skeleton structure feature of murine antibody with this gene order deduced amino acid, the aminoacid sequence of hypervariable region also has particular structure in the variable region, and existing antibody variable region amino acid sequence does not have repeating sequences with it.
As follows according to the corresponding aminoacid sequence that 2D2 monoclonal antibody variable region of light chain cDNA sequence is derived:
Gln?Phe?Val?Leu?Thr?Glu?Ser?Gly?Ala?Ile?Met?Ser?Thr?Ala?Met?Gly?GlyArg?Val?Thr?Ile?Ala?Cys?Ala?Thr?Ser?Gly?Gly?Ser?Asp?Thr?Arg?Asn?LeuHis?Trp?Tyr?Ala?Thr?Val?Thr?Arg?Leu?Pro?Gly?Arg?Val?Thr?Ser?Lys?ArgPro?Ser?Phe?Ala?Cys?Leu?Glu?Ser?Arg?Thr?Asn?Gly?Asp?Ala?Lys?Val?LeuPro?Gly?Phe?Phe?Asn?Gly?Gln?Thr?Phe?Ala?Ser?Pro?Phe?Val?Ala?Thr?ProAsn?Ala?His?Val?Pro?Ala?Asp?Gly?Leu?Leu?Gly?His?Ala?Gly?Ser?Lys?AlaPhe?Gly?Ser
As follows according to the corresponding aminoacid sequence that 2D2 monoclonal antibody variable region of heavy chain cDNA sequence is derived:
Ala?Val?Ser?Pro?Asn?Gly?Lys?Tyr?Lys?Gly?Asn?Ser?Arg?Pro?Thr?Cys?ProAsp?Cys?Phe?Pro?Ala?Thr?Ser?Leu?Glu?Gly?Leu?Phe?Leu?Glu?Leu?Ser?ArgArg?Ala?Lys?Cys?Phe?Pro?Thr?Val?Val?Arg?Gly?Phe?Glu?His?Thr?Cys?AsnThr?Arg?Glu?Leu?Pro?Asp?Leu?Gly?Arg?Ile?His?Ser?Ile?Lys?Ala?Gly?IleHis?Arg?Asn?Gly?Gly?Ile?Gln?Phe?Ile?Thr?Phe?Gly?Leu?Cys?Gly?Asp?ArgIle?Phe?Pro?Glu?Val?Asp?Asn?Arg?Val?Lys?Phe?Thr?Cys?Ser?Asn?Asp?SerVal?Asn?Gly?Ser?Ala?Phe?Ile?Arg?Cys
Five, the anti-tumor experiment of 2D5 monoclonal antibody on SCID-Hu mouse tumor animal model
With SCID mouse (T nucleus B cell combined immunodeficient mouse) is prototype, the mosaic type mouse with human immune feature that makes up behind the introducing human peripheral blood single nucleus cell is set up the solid tumor models of A549 lung carcinoma cell, 7721 liver cancer cells, MCF breast cancer cell respectively as animal model for tumour.36 tumor-bearing mices are divided into 9 groups, 4 every group.The control group tumor-bearing mice is not injected NK cell and 2D5 monoclonal antibody.Tumor-bearing mice+NK group, every tumor-bearing mice is injected 107 NK cells of human beings.Tumor-bearing mice+NK group+2D5 monoclonal antibody group, every tumor-bearing mice is injected the 2D5 monoclonal antibody of 107 NK cells of human beings and 1mg purifying.Parallel laboratory test, the relatively size of knurl body between each group.The results are shown in Table 2.
Table .22D5 monoclonal antibody is to the effect of SCID-Hu tumor-bearing mice
| The 14th day average knurl body diameter (major diameter) of tumor-bearing mice | 14 days average knurl body diameters (major diameter) behind tumor-bearing mice+NK cell | 14 days average knurl body diameters (major diameter) after tumor-bearing mice+NK cell+2D5 monoclonal antibody | |
| The A549 lung carcinoma cell | 0.8cm±0.15cm | ?0.5cm±0.18cm | 0.2cm±0.09cm |
| 7721 liver cancer cells | 0.7cm±0.2cm | ?0.55cm±0.16cm | 0.35cm±0.11cm |
| The MCF breast cancer cell | 0.75cm±0.22cm | ?0.5cm±0.11cm | 0.15cm±0.06cm |
In the body of last table experimental result as can be seen, the 2D5 monoclonal antibody all has very strong raising NK cell killing to these three kinds of tumours, can obviously improve NK cell anti-tumor activity in vivo.
Six, anti-HAAH monoclonal antibody is used for the immunohistochemical staining of tumor tissue section
2D5 monoclonal antibody ascites is used for the immunohistochemical staining of tumour pathological section after the dilution in 1: 500.11 tumor tissue sections of having made a definite diagnosis, the healthy tissues section of 11 corresponding organs combines with the 2D5 monoclonal antibody respectively, again with sheep anti mouse ELIAS secondary antibody and substrate colour developing.The result shows that these 11 tumor tissue sections of having made a definite diagnosis are all positive.And the section of the healthy tissues of 11 corresponding organs is all negative.Shown the good specificity of 2D5 monoclonal antibody.11 tumour pathological sections of having made a definite diagnosis are respectively: cancer, kidney, esophagus cancer, bladder cancer before liver cancer, lung cancer, mammary cancer, cancer of the stomach, cervical cancer, ovarian cancer, colorectal carcinoma, the prostatitis.The negative control section is the healthy tissues section of 11 kinds of corresponding organs.
Sequence table
＜110〉Guangzhou Rosson Bioscience Co., Ltd.
＜120〉a kind of anti-tumor monoclonal antibody of high specific
<160>4
<210>1
<211>105
<212>PRT
＜213〉artificial sequence
<400>1
Gln?Phe?Val?Leu?Thr?Glu?Ser?Gly?Ala?Ile?Met?Ser?Thr?Ala?Met
1 5 10 15
Gly?Gly?Arg?Val?Thr?Ile?Ala?Cys?Ala?Thr?Ser?Gly?Gly?Ser?Asp
20 25 30
Thr?Arg?Asn?Leu?His?Tyr?Tyr?Ala?Thr?Val?Thr?Arg?Leu?Pro?Gly
35 40 45
Arg?Val?Thr?Ser?Lys?Arg?Pro?Ser?Phe?Ala?Cys?Leu?Glu?Ser?Arg
50 55 60
Thr?Asn?Gly?Asp?Ala?Lys?Val?Leu?Pro?Gly?Phe?Phe?Asn?Gly?Gln
65 70 75
Thr?Phe?Ala?Ser?Pro?Phe?Val?Ala?Thr?Pro?Asn?Ala?His?Val?Pro
80 85 90
Ala?Asp?Gly?Leu?Leu?Gly?His?Ala?Gly?Ser?Lys?Ala?Phe?Gly?Ser
95 100 105
<210>2
<211>111
<212>PRT
＜213〉artificial sequence
<400>2
Ala?Val?Ser?Pro?Asn?Gly?Lys?Tyr?Lys?Gly?Asn?Ser?Arg?Pro?Thr
1 5 10 15
Cys?Pro?Asp?Cys?Phe?Pro?Ala?Thr?Ser?Leu?Glu?Gly?Leu?Phe?Leu
20 25 30
Glu?Leu?Ser?Arg?Arg?Ala?Lys?Cys?Phe?Pro?Thr?Val?Val?Arg?Gly
35 40 45
Phe?Glu?His?Thr?Cys?Asn?Thr?Arg?Glu?Leu?Pro?Asp?Leu?Gly?Arg
50 55 60
Ile?His?Ser?Ile?Lys?Ala?Gly?Ile?His?Arg?Asn?Gly?Gly?Ile?Gln
65 70 75
Phe?Ile?Thr?Phe?Gly?Leu?Cys?Gly?Asp?Arg?Ile?Phe?Pro?Glu?Val
80 85 90
Asp?Asn?Arg?Val?Lys?Phe?Thr?Cys?Ser?Asn?Asp?Ser?Val?Asn?Gly
95 100 105
Ser?Ala?Phe?Ile?Arg?Cys
110
<210>3
<211>29
<212>DNA
＜213〉artificial sequence
<400>3
atgaattcat?ggtgattgca?ttgctgggc?29
<210>3
<211>33
<212>DNA
＜213〉artificial sequence
<400>4
atgcggccgc?ctaaattgct?ggaaggctgc?gtc?33
Claims (3)
1. the anti-tumor monoclonal antibody of a high specific is characterized in that the variable region of light chain of this antibody is made up of 105 amino acid, and its sequence is as follows:
Gln?Phe?Val?Leu?Thr?Glu?Ser?Gly?Ala?Ile?Met?Ser?Thr?Ala?Met?Gly?GlyArg?Val?Thr?Ile?Ala?Cys?Ala?Thr?Ser?Gly?Gly?Ser?Asp?Thr?Arg?Asn?LeuHis?Trp?Tyr?Ala?Thr?Val?Thr?Arg?Leu?Pro?Gly?Arg?Val?Thr?Ser?Lys?ArgPro?Ser?Phe?Ala?Cys?Leu?Glu?Ser?Arg?Thr?Asn?Gly?Asp?Ala?Lys?Val?LeuPro?Gly?Phe?Phe?Asn?Gly?Gln?Thr?Phe?Ala?Ser?Pro?Phe?Val?Ala?Thr?ProAsn?Ala?His?Val?Pro?Ala?Asp?Gly?Leu?Leu?Gly?His?Ala?Gly?Ser?Lys?AlaPhe?Gly?Ser
Variable region of heavy chain is made up of 111 amino acid, and its sequence is as follows:
Ala?Val?Ser?Pro?Asn?Gly?Lys?Tyr?Lys?Gly?Asn?Ser?Arg?Pro?Thr?Cys?ProAsp?Cys?Phe?Pro?Ala?Thr?Ser?Leu?Glu?Gly?Leu?Phe?Leu?Glu?Leu?Ser?ArgArg?Ala?Lys?Cys?Phe?Pro?Thr?Val?Val?Arg?Gly?Phe?Glu?His?Thr?Cys?AsnThr?Arg?Glu?Leu?Pro?Asp?Leu?Gly?Arg?Ile?His?Ser?Ile?Lys?Ala?Gly?IleHis?Arg?Asn?Gly?Gly?Ile?Gln?Phe?Ile?Thr?Phe?Gly?Leu?Cys?Gly?Asp?ArgIle?Phe?Pro?Glu?Val?Asp?Asn?Arg?Val?Lys?Phe?Thr?Cys?Ser?Asn?Asp?SerVal?Asn?Gly?Ser?Ala?Phe?Ile?Arg?Cys
The specificity combination takes place in described monoclonal antibody and antigen HAAH (Human asparaginyl beta-hydroxylase, human asparagine hydroxylase); The constant region that it is characterized in that this antibody is a mouse IgG2a subclass.
2. the application of monoclonal antibody as claimed in claim 1 in preparation medicine for treating tumor thing.
3. monoclonal antibody as claimed in claim 1 is the application in cancer, kidney, esophagus cancer, the bladder cancer medicine before preparation treatment liver cancer, lung cancer, mammary cancer, cancer of the stomach, cervical cancer, ovarian cancer, colorectal carcinoma, prostatitis.
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| CN101307302A (en) * | 2007-05-17 | 2008-11-19 | 陕西北美基因股份有限公司 | Process for preparing hybridoma cell for secreting anti-human asparagine hydroxylase monoclonal antibodies |
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