CN102443060B - Anti-HPV (human papilloma virus) antibody and preparation method and application thereof - Google Patents
Anti-HPV (human papilloma virus) antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an antibody used for treatment of diseases, in particular to cervical cancer, related to human papilloma virus infection, and a preparation method and an application thereof.
Description
Technical field
The present invention relates to immunotherapy field.More specifically, the present invention relates to a kind of for example, antibody for prevention or treatment human papillomavirus (human papillomavirus, HPV) infection relative disease (cervical lesions, particularly cervical cancer).The invention still further relates to the preparation method and application of described antibody.
Background technology
Current research shows, HPV infects the obvious dependency that has with uterine neck systemic disease---particularly cervical cancer---.Nineteen ninety-five, (the International Agency for Research of Cancer of IARC, IARC) result of study of announcing confirms, the uterine neck such as HPV and cervical cancer systemic disease all has close cause-effect relationship: in 99.8% cervical cancer patient, can detect HPV and infect, and cervical cancer can occur HPV negative patient hardly; In addition, in more than 98% cervical disease patient, all can detect HPV.
HPV virus can be divided into different types and hypotype according to the sequence homology of L1 gene, and homology is a type more than 90%, and the HPV of homology between 90～98% can be divided into different hypotypes.According to the relation of the type of HPV or hypotype and female genital tract malignant tumour, HPV is divided into low risk and high-risk-type.Low risk HPV, the types such as such as HPV 6,11,34,40,42 are more common in optimum cervical lesions, as uterine neck condyloma and the slight Atypical hyperplasia pathology of epithelium of cervix uteri; More common with HPV 16,18,31,33,35,39,45 types infection in epithelium of cervix uteri severe atypical hyperplasia and cervical cancer, these types are high-risk HPV.
For a series of researchs of different crowd, confirmed that HPV 16,18 types of reproductive tract infect and the generation height correlation of epithelium of cervix uteri severe pathology (CIN I/CINII) cervical cancer, closer compared with other Hazard Factor.In the < < world wide of announcing according to the World Health Organization (WHO), HPV and cervical cancer 2 007 are reported > > result, and in China women, 58.7% cervical cancer is directly related with HPV16 type.
HPV16E7 gene is sustainable existence high expression level in cervical cancer, in the maintaining of the formation of cervical cancer and pernicious proterties, plays an important role.HPV16E7 albumen is totally 98 amino acid, and its sequence is as follows: MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVT FCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP.This albumen has three conserved regions (conserved region, CR): CR1 to be positioned at N end, and CR2 contains LXCXE structural domain, and CR3 is containing two zinc-finger structure territories.The center function of E7 is exactly by one of three conservative regions and Rb protein family (as PRb, P107, P130) knot merga pass ubiquitin-26S proteasome pathway degraded Rb family protein.The key link of Rb protein family in the cell cycle G1/S outpost of the tax office.Rb protein binding transcription factor E2f albumen under dephosphorylation state, stops E2f albumen to activate numerous protein factors relevant with DNA replication dna, thereby suppress DNA, synthesizes and cell cycle progress, makes cell in the G0/G1 phase and carries out cytodifferentiation.E7 protein binding the Rb albumen of degrading, discharge E2f albumen, thereby activate many relevant DNA synthetic proteins factors, accelerates the division growth of DNA replication dna and cell, promotes the differentiation of cell.In addition, E7 albumen can also be in conjunction with other albumen of kind more than 10 such as cyclin A, cyclin E, P48, improve exogenous DNA integration and cytogene mutagenicity etc., accelerate transformation.
Therefore, HPV16E7 albumen is the antigen of Cervical Tumor cell specific expression, is that tumour cell is different from the antigen target of normal cell at intracellular expression.Owing to being the antigen of expressing in born of the same parents, must be by the process of angtigen presentation to cell surface.Antigen presenting cell is (such as dendritic cell, scavenger cell) picked-up corresponding antigens, after processing treatment, antigen is degraded into 8-10 amino acid whose small peptide, these small peptides in golgi body and the combination of MHC-I quasi-molecule, then by after birth circularly exhibiting at cell surface.This process is just called angtigen presentation.The MHC-I molecule of offering at cell surface and small peptide (being called t cell epitope), by T cell recognition, thus the immune response in activated T cell downstream.In early days the research of HPV16 HPV-16 E7 is shown to HPV16E7 has the t cell epitope of three high-affinities, respectively 11-20 amino acids residue: YMLDLQPETT, 49-57 amino acids residue RAHYNIVTF (SEQ ID NO:5 calls " 49-57 peptide " in the following text) and 86-93 amino acids residue: TLGIVCPI (calling " 86-93 peptide " in the following text).
Generally, the t cell epitope of antigen only has 8-10 amino acid.And there is three CDR (complementary determinant the variable region of monoclonal antibody, complementary-determining region) district, about each CDR can identify in district 10 amino acid left and right, monoclonal antibody is except the specific t cell epitope of affine combination, also to identify other epi-position, therefore the monoclonal antibody of, only identifying the specific T cells epi-position of antigen is difficult to obtain.Be embodied in using in classical Mice Body in monoclonal antibody screening, go separately immune animal with 8-10 amino acid whose small peptide, be difficult to generation antibody.
For this situation, screening t cell epitope antibody adopts two kinds of methods to solve at present.A kind of is to screen with external display technique of bacteriophage, and with the polypeptide of t cell epitope, as affine former screening, this method and technology platform requires high, and the avidity of antibody to be generally all not so good as the antibody that filters out in body high.Second method is first with other albumen and epitope polypeptide, to form mixture, to improve its immunogenicity, then carry out monoclonal antibody screening and epi-position evaluation with this mixture immune mouse, other albumen that use in this method mostly are MHC (ajor histocompatibility mixture) molecule or KLH (keyhole-limpet hemocyanin) molecule.Adopt the method for MHC molecule need to express in vitro MHC molecule and by external change renaturation, MHC molecule and antigen peptide be formed to mixture, the method process is loaded down with trivial details, and it is low to prepare the efficiency of mixture.Adopt the macromole couplings such as KLH to be faced with equally the inefficient problem of preparing mixture.And, do not form the MHC of mixture or KLH molecule owing to thering is equally immunogenicity, in immunity, after in animal body, can produce a lot of unwanted interference antibody, bring very large interference to the screening of target monoclonal antibody and epi-position evaluation subsequently.
Therefore, in this area, need monoclonal antibody and the production method thereof of a species specificity for the t cell epitope polypeptide of HPV16E7 albumen.
Summary of the invention
For solving the problems of the technologies described above, in a first aspect of the present invention, the antibody of one species specificity for the 49-57 peptide epitopes (as shown in SEQ ID NO:5) of HPV16E7 albumen is provided, and described antibody has light chain CDR1, CDR2 and the CDR3 region shown in heavy chain CDR1, CDR2 and CDR3 region and the SEQ ID NOs:9-11 as shown in SEQ ID NOs:6-8.
In one embodiment, antibody of the present invention has the weight chain variabl area sequence as shown in SEQ ID NO:1 and the light chain variable region sequence as shown in SEQ ID NO:2.
The present invention also provides a kind of method of preparing antibody of the present invention, and it comprises:
Polypeptide shown in a kind of double-stranded RNA adjuvant and SEQ ID NO:5 is mixed to form to mixture;
Polypeptide shown in a kind of double-stranded RNA adjuvant and SEQ ID NO:5 is mixed to form to mixture; By described mixture immunization host; With
From host's body fluid, tissue or cell, obtain antibody.
The present invention provides a kind of method of preparing antibody of the present invention especially, and it comprises:
Polypeptide shown in a kind of double-stranded RNA adjuvant and SEQ ID NO:5 is mixed to form to mixture; By described mixture immunization host;
Results host's splenocyte also merges itself and myeloma cell, and
The antibody that the cell that collection is merged produces.
In the present invention, prepare in the method for described antibody, the mixture that has adopted double-stranded RNA and epitope polypeptide to form carries out host's (for example, mouse) immunity, and then screens monoclonal antibody.For example, because double-stranded RNA is different from other protein molecular (MHC and KLH) that is generally used for forming with antigen mixture, itself does not produce interference antibody, and the antibody therefore producing is the monospecific antibody for epitope polypeptide entirely.This method Dispersal risk is easy fast, and without special technology platform requirement, and the antibodies specific filtering out is high.
In another aspect of this invention, antibody of the present invention is also provided, containing composition or the test kit of this antibody, for the preparation of prevention or treatment HPV16, has caught, the particularly purposes in the medicine of cervical lesions or cervical cancer.
Accompanying drawing explanation
Fig. 1 has shown the electrophoresis detection result of 6C10 monoclonal antibody of the present invention.
Fig. 2 has shown the result of identifying 6C10 monoclonal antibody avidity of the present invention by ELISA method.
Fig. 3 has shown the result of carrying out the epi-position evaluation of 6C10 monoclonal antibody of the present invention by the experiment of T2 load peptide.Fig. 3 A and Fig. 3 B show respectively T2 cell streaming cell detection results and the fluorescent microscope detected result that the 49-57 peptide load of E7 is crossed; Fig. 3 C and Fig. 3 D show respectively T2 cell streaming cell detection results and the fluorescent microscope detected result that E786-93 peptide load is crossed; Fig. 3 E and Fig. 3 F show respectively T2 cell streaming cell detection results and the fluorescent microscope detected result without any peptide load, crossed.
Fig. 4 has shown and uses 6C10 monoclonal antibody of the present invention to suppress the tumor formation rate of tumor research.
Fig. 5 has shown and uses 6C10 monoclonal antibody of the present invention to suppress the tumour inhibiting rate of tumor research.
Fig. 6 has shown aminoacid sequence and the nucleotide sequence of the variable region of heavy chain of 6C10 monoclonal antibody of the present invention.
Fig. 7 has shown aminoacid sequence and the nucleotide sequence of the variable region of light chain of 6C10 monoclonal antibody of the present invention.
Sequence explanation
SEQ ID NO:1 is antibody heavy chain variable region of the present invention aminoacid sequence.
SEQ ID NO:2 is antibody chain variable region of the present invention aminoacid sequence.
SEQ ID NO:3 is the nucleotide sequence of code book invention antibody heavy chain variable region.
SEQ ID NO:4 is the nucleotide sequence of code book invention antibody chain variable region.
SEQ ID NO:5 is the 49-57 amino acids sequence of HPV 16E7 albumen.
The aminoacid sequence of the CDR 1-3 that SEQ ID NOs:6-8 is heavy chain of antibody of the present invention.
The aminoacid sequence of the CDR 1-3 that SEQ ID NOs:9-11 is light chain of antibody of the present invention.
Embodiment
Although described embodiment represents the preferred embodiments of the invention, it should be understood that those skilled in the art can make any conversion without departing from the spirit of the invention.
Preferably, antibody of the present invention is to prepare by hybridoma technology the monoclonal antibody obtaining.Particularly preferably, antibody of the present invention is by preserving number, to be the monoclonal antibody of the 6C10 hybridoma generation of CGMCC 4144.
The present invention provides a kind of pharmaceutical composition that comprises antibody of the present invention or test kit in embodiments.In one embodiment, pharmaceutical composition of the present invention can further contain other treatment agent, wherein antibody of the present invention can be with described other treatment agent with form independently separately, or the form of mixing mutually each other exists, then or can form each other coupling combination.Described therapeutical agent can be antitumor or antiviral activity therapeutical agent or biotoxin, such as 5'-FU, fludarabine, CldAdo, cytosine arabinoside, gemcitabine, capecitabine, troxacitabine, lamivudine, emtricitabine, zalcitabine, zidovudine, Abacavir, didanosine, the two isopropyl acyloxy methyl esters of tynofovir fumaric acid, stavudine, Abacavir, cis-platinum, ifosfamide, carboplatin, bleomycin or PTX etc.
At an antibody of the present invention and other treatment agent, form in the embodiment of conjugate, antibody of the present invention can play the effect in the therapeutical agent target therapeutic interest region of its coupling.
In another embodiment, test kit of the present invention can further contain therapeutical agent described above, and the operation instruction of this test kit.
" mixture " of the present invention is by the polypeptide shown in a kind of double-stranded RNA adjuvant and SEQ ID NO:5 is mixed and formed.Preferably, the double-stranded RNA that is used to form described mixture is polyinosinic acid (PolyIC, Polyinosinic acid-polycytidylic acid) or PolyICLC.
In an embodiment of preparation method for antibody of the present invention, double-stranded RNA fully to be mixed with E7 (49-57) peptide, its ratio of mixture can be between 1: 0.5 to 1: 4 (w/w).
In another embodiment, described host is mouse, rat, rabbit.
In another embodiment, described myeloma cell is SP2/0 oncocyte.
In preparation method for antibody of the present invention, the described cell clone through fusion is preferably the mono-clonal that still produces described antibody after more than three times subclone.Particularly preferably, described cell clone is 6C10 cell clone.
Method of the present invention can further comprise that the monoclonal cell that screening is obtained sets up cell strain.This cell strain can be stablized and goes down to posterity, and secreting specificity antibody.Therefore, method of the present invention can comprise the cell monoclonal that obtains continuous release specific antibody from cell strain.
By mentioned earlier, the present invention also provides a kind of method of producing the cell that can produce antibody of the present invention, and the method that produces 6C10 cell clone is provided especially.
The method of Dispersal risk of the present invention can further comprise carries out purifying by the antibody of collection.
The present invention also provides the cell that produces a kind of antibody of the present invention, and in one embodiment, it is (CGMCC), the 6C10 cell that preserving number is CGMCC 4144 that is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
The conventional purification process that the purifying of antibody of the present invention can be known by those of ordinary skills carries out, for example, by affinity chromatography, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography etc.
Antibody of the present invention can be used for prevention or treatment HPV16 catches, and comprises the antibody of the present invention that gives significant quantity to experimenter, and particularly preferred antibody is that preserving number is the monoclonal antibody of the 6C10 cell hybridization oncocyte generation of CGMCC 4144.In one embodiment, antibody of the present invention can be used for the multiple cervical lesions of preventing/treating, particularly CIN I or CIN II, and cervical carcinogenesis.
Those of ordinary skills by following examples, describe the present invention in detail, so that can understand the present invention better.Following embodiment, only for exemplary purpose, is not intended to limit scope of the present invention.Endeavoured to ensure the accuracy about numeral (as quantity, temperature etc.), but should consider that meeting exists some errors and deviation.Except as otherwise noted, temperature is take ℃ as unit or as envrionment temperature, and pressure approaches or equals normal atmosphere.Should be understood that, except as otherwise noted, in following each embodiment, plant and instrument used is the conventional equipment of this area.Except as otherwise noted, the substratum using is commercially available conventional medium, and those skilled in the art all know composition and content wherein.For for simplicity, likely use in this article various general abbreviations, those skilled in the art can understand its implication completely.
Embodiment
Preparation and the immunization of the immunocomplex of embodiment 1:E7 albumen 49-57 peptide
The 49-57 peptide of HPV16 virus E7 albumen, aminoacid sequence: RAHYNIVTF; Molecular weight 1119Da, theoretical iso-electric point 9.04.This peptide is synthesized by gill biochemical corp, and purity is greater than 95%.Take the double-stranded RNA synthesizing---PolyIC is adjuvant, and it is purchased from Sigma company (catalog number (Cat.No.): P9582).
Take PolyIC adjuvant dry powder and E7 (49-57) peptide dry powder, mix at 5: 2 in mass ratio, add pure water to dissolve, fully mix, form macroscopic white granular mixture, within 5-10 minute that dissolves, this mixture is passed through to subcutaneous abdomen immunity BALB/c mouse, every mouse immune 100 μ g PolyIC+40 μ g 49-57 peptide mixts.After initial immunity the 14th day and the 28th day, with same immunizing dose twice of booster immunization again.After initial immunity, about the 35th day, collection mice serum can detect that the associated antibodies of corresponding E7 albumen produces (detection method is as described in following examples 2).
Embodiment 2: the detection of serum antibody titer
For the mouse by carrying out immunity described in embodiment 1, after initial immunity, from tail vein, take a small amount of serum the 35th day time, with direct enzyme-linked immunosorbent assay (ELISA) detection serum antibody titer.
It is as follows that ELISA detects serum antibody titer step:
1. (preparation method of this albumen is: synthetic total length HPV 16E7 gene first to use HPV16E7 albumen, then in pET32a carrier and BL21 (DE3) host, give expression to the E7 fusion rotein with Trx label, with enteropeptidase, Trx label is cut away and can obtain HPV16E7 albumen again), with coated 96 orifice plates of amount of every hole 1 μ g.Coated plate is hatched 16 hours at 4 ℃, washs each hole 6 times with lavation buffer solution (containing the PBS solution of 0.05%Tween-20), and every hole adds confining liquid (containing the PBS solution of 5% skim-milk) 100 μ l to seal 2 hours.
2. every hole adds the serum sample of 50 times of 100 μ l PBS solution dilutions, and 37 ℃ are reacted one hour.Then with lavation buffer solution, wash each hole 3 times, each 5 minutes.
3. every hole adds the sheep anti-mouse igg antibody (purchased from Calbiochem company, numbering 401215) of the horseradish peroxidase of 100 μ l, hatches 0.5 hour for 37 ℃.Then with lavation buffer solution, wash each hole 6 times, to remove unconjugated enzyme labelled antibody.
4. every hole adds 100 μ l TMB nitrite ions, and room temperature is carried out color reaction 20 minutes.
5. every hole adds stop buffer (2M sulphuric acid soln) the color development stopping reaction of 50 μ l.After 5 minutes, by microplate reader, measure the photoabsorption at 450nm wavelength place, and record result.There is situation in the antibody of judging serum by absorbance value.
Measured altogether four and carried out immune mouse and the serum without immune negative control mouse according to method described in embodiment 1, four are respectively by immune serum OD reading: 0.323,1.805,0.347,0.709, and negative serum reading 0.055.Infer thus the immunity system that can activate mouse by the mixture immune mouse of PolyIC and antigen peptide, and produce the specific antibody of corresponding E7 albumen.
Embodiment 3: the screening of authentic monoclonal antibody
1. cytogamy
Select a highest mouse of serum titer, the mixture of abdominal injection 200 μ g PolyIC+80 μ g49-57 peptides carries out booster immunization, within after booster immunization the 4th day, gets spleen and carries out cytogamy.Concrete steps are as follows: mouse is plucked eyeball and gets blood, by blood collecting to 1.5ml centrifuge tube, until dead mouse.The blood separation serum of collecting ,-20 ℃ of preservations.Positive colony is used as positive control when detecting.The mouse that has gathered blood carries out the aseptic spleen of adopting.In 200 order steel screen clothes, grind spleen, collect splenocyte.
The splenocyte of collecting is resuspended with 10ml RM1640 basic medium, and splenocyte and myeloma cell (SP2/0 cell) mix according to the ratio of 10: 1 to 1: 1, mix centrifugal 10 minutes of 1000rpm.Supernatant discarded, adds the PEG 15001ml of 37 ℃ of preheatings in 1 minute, 37 ℃ of water-baths 1 minute slowly added 20ml RM1640 basic medium (Gibico company) in 5 minutes, mixed centrifugal 10 minutes of 960rpm.Supernatant discarded, adds 120ml to contain the RM1640 substratum of 1%HAT, mixes, and is inoculated in 6 96 orifice plates, and every hole 200 μ l, at 37 ℃, 5%CO
2under condition, cultivate.After 5 to 7 days, treat that Kong Zhongyou clones clearly visible, each clone's cell count reaches 10
3above, by the substratum sucking-off in hole, add the fresh RM1640 substratum containing 1%HT, every hole 200 μ l, at 37 ℃, 5%CO
2under condition, continue to cultivate.Whether the substratum supernatant of sucking-off detects and has specific antibody to produce by ELISA method as above.The clone of picking ELISA tests positive continues subclone screening subsequently.
2. subclone screening
The preparation of feeder cell: need to have the existence of feeder layer cells when adopting limiting dilution assay to carry out subclone.Carry and prepare feeder layer cells the day before yesterday.Get 1 above Kunming mouse collection in female 8 week age peritoneal macrophage, and be inoculated in 96 orifice plates.
Limiting dilution assay screening subclone: above-mentioned selected clone is made to cell suspension with liquid-transfering gun piping and druming, use blood cell counting plate numeration.According to numeration result, sucking-off adds 20ml to contain in the RM1640 substratum of 10% foetal calf serum containing the suspension of 100 cells, is seeded in 1 96 orifice plate, and 200 μ l/ holes, are placed in 37 ℃, 5%CO
2under condition, cultivate, after 7 to 10 days, treat that clone in 96 orifice plates grows to 1/6th to three of whole hole/for the moment, get cell conditioned medium and carry out the detection of specific antibody, then select higher, the well-grown clone of OD value, carry out subclone screening.
If continuous three subclones of mono-clonal, when all clones that obtain are all positive, can assert that this monoclonal cell can stably excreting specific antibody, by this cell strain built, enlarged culturing, the frozen cell seed bank of setting up.
The clone who goes out to be numbered 6C10 according to above-mentioned standard picking, sets up cell seed bank.The about 800ml of collecting cell culture supernatant, with Protein A medium purification antibody, obtains 6C10 monoclonal antibody, as further identifying.The 6C10 hybridoma cell strain of above-mentioned foundation is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on September 3rd, 2010, and preserving number is CGMCC 4144.
The electrophoresis detection of embodiment 4:6C10 monoclonal antibody
6C10 monoclonal antibody after purifying is carried out to SDS-PAGE electrophoresis detection.Result as shown in Figure 1, is reduced in electrophoretogram in dithiothreitol (DTT), the heavy chain band of the approximately 52KD of this monoclonal antibody and the light chain band of 25KD as seen.
The evaluation of embodiment 5:6C10 monoclonal antibody avidity
Using E7 full-length proteins, Trx-E7 (polypeptide of the 30-67 amino acids residue composition of E7) and BSA (bovine serum albumin), respectively as envelope antigen, 6C10 monoclonal antibody, as detecting antibody, carries out ELISA detection.The results are shown in Fig. 2.
From the ELISA result of Fig. 2, can find out, the albumen that contains E749-57 peptide, for example E7 full-length proteins and Trx-E7 (30-67) albumen can have very strong combination with 6C10 monoclonal antibody, and in ELISA experiment, OD value is greater than 1.5.Do not contain the albumen of E749-57 peptide, such as BSA albumen, and 6C10 monoclonal antibody almost do not have combination, and in ELISA experiment, OD value is less than 0.2.
The evaluation of embodiment 6:6C10 monoclonal antibody epi-position
The epi-position of using the experiment of T2 load peptide to carry out monoclonal antibody of the present invention is identified.T2 cell (purchased from U.S. ATCC storehouse, preserving number CRL-1992) is the cell of a kind of endogenous TAP defect, surperficial unloaded HLA-A2 molecule.The antigen Toplink that external source adds is loaded on the HLA-A2 molecule of T2 cell surface.
By T2 cell in substratum with E749-57 peptide or the E786-93 peptide of final concentration 20 μ g/ml or do not add any peptide, hatch 4 hours.After washing once, the 6C10 monoclonal antibody that adds 100 μ l to purify after resuspended with the damping fluid (PBS+0.5%BSA+2mM EDTA) that dyes, hatches 30 minutes for 37 ℃.After washing once, resuspended with dyeing damping fluid (PBS+0.5%BSA+2mM EDTA), then add the sheep anti-mouse igg antibody of 2 μ l FITC marks (purchased from BD company, catalog number (Cat.No.) 349031), after keeping in Dark Place 20 minutes, room temperature detects cell green fluorescence situation with flow cytometer and fluorescent microscope.Result as shown in Figure 3.
Fig. 3 A and Fig. 3 B show respectively T2 cell streaming cell detection results and the fluorescent microscope detected result that E749-57 peptide load is crossed; Fig. 3 C and Fig. 3 D show respectively T2 cell streaming cell detection results and the fluorescent microscope detected result that E786-93 peptide load is crossed; Fig. 3 E and Fig. 3 F show respectively T2 cell streaming cell detection results and the fluorescent microscope detected result without any peptide load, crossed.
From the result shown in Fig. 3, can find out, 6C10 monoclonal antibody of the present invention can specific recognition be positioned at the E749-57 peptide of T2 cell surface load.Confirmed thus the epitope specificity of 6C10 monoclonal antibody.
Embodiment 7: variable region of mab (CDR) order-checking and sequential analysis
Get 1 × 10
7individual 6C10 hybridoma, adopts the total RNA purification kit of PureLink (Invitrogen company), by test kit specification sheets operation extracting RNA.Total RNA that approximately 3 μ g are extracted is for carrying out single stage method RT-PCR (using the OneStep RT-PCR test kit of Qiagen company).
According to the synthetic weight strand primer of antibody variable gene FR1 district HeFR4 district conserved regions sequences Design, heavy chain primer is as follows:
VH forward primer: 5 '-GA GGTGCA GCTGCA GGA GTC-3 ';
VH reverse primer: 5 '-TGAGGA GACGGTGACCG TGGTCCCTTGGCCC-3 ';
Light chain primer is as follows:
VL forward primer: 5 '-GACA TTGTGA TGACCCA GTCT-3 ';
VL reverse primer: 5 '-CCGTTTTA TTTCCA GCTTGGTCCC-3 '.
The pcr amplification product of heavy chain and light chain is connected respectively into pMD18-T carrier (Takara company), picks out and deliver to Shanghai Sheng Gong company after positive colony and carry out DNA sequencing.Sequencing result demonstration, the light/heavy chain variable region gene obtaining contains 4 framework regions and 3 antigen complementary determining regions.
The weight chain variabl area sequence of this monoclonal antibody is as shown in SEQ ID NO:1.
6C10 monoclonal antibody VH full length gene 348bp, 116 amino acid of encoding.Amino acid sequence analysis result shows, VH contains clear and definite 4 framework regions (FR) and 3 complementary districts (CDR) of antigenic determinant (it has respectively sequence shown in following SEQ ID NOs:6-8), at the 22nd and the 95th, being halfcystine, is to form two kinds of relevant characteristic amino acid with antibody disulfide linkage.
SEQ?ID?NO：6：
Asn?Tyr?Gly?Met?Ser
SEQ?ID?NO：7：
Thr?Ile?Asn?SerAsn?Gly?Asn?Thr?Tyr?Tyr?Pro?Asp?Ser?Val?Gln?Gly
SEQ?ID?NO：8：
Glu?Tyr?Gly?Lys?Ala?Phe?Ala?Tyr
The light chain variable region sequence of this monoclonal antibody is as shown in SEQ ID NO:2.
6C10 monoclonal antibody VL full length gene 324bp, 108 amino acid of encoding.Amino acid sequence analysis result shows, VL contains clear and definite 4 framework regions (FR) and 3 complementary districts (CDR) of antigenic determinant (it has respectively sequence shown in following SEQ ID NOs:9-11), at the 92nd, it is halfcystine, this light chain is abnormal transcription product, has carried out premature termination.
SEQ?ID?NO：9：
Arg?Ala?Ser?Lys?Ser?Val?Ser?Thr?Ser?Gly?Tyr?Ser?Tyr?Met?His
SEQ?ID?NO：10：
Leu?Val?Ser?Asn?Leu?Glu?Ser
SEQ?ID?NO：11：
Gln?His?Ile?Arg?Glu?Leu?Thr
The effect research that the inhibition tumour of embodiment 8:6C10 monoclonal antibody generates or grows
Adopt classical mouse source TC-1 cell (purchased from U.S. ATCC storehouse, deposit number CRL-2785) inoculation to set up the Cervical Tumor model of E7 protein expression.Express the TC-1 cell of HPV-16 E7 in the lung primary cell that derives from C57BL/6 mouse, by importing the E7 gene of HPV16 and the people C-Ha-ras gene being activated, this primary cell strain is converted and obtains unlimited fecundity.
To this tumor model injected in mice 6C10 monoclonal antibody and various reference protein, observe tumor growth situation.
The outer good TC-1 cell of incubation growth of collection body, PBS washes 3 times, adjusts cell density, gives the armpit subcutaneous injection of C57BL/6 mouse left side, and every inoculation 0.2ml cell suspension, is about 1 × 10
5cell.After inoculation, mouse is divided into 4 groups at random, 8 every group, by following four kinds of modes, carries out administration.Negative control PBS administration group: abdomen is subcutaneous to be administered twice respectively at inoculation for latter 2 days, 16 days; Positive control VR111 vaccine administration group: abdomen is subcutaneous to be administered twice respectively at inoculation for latter 2 days, 16 days; 6C10 monoclonal antibody administration group: nape is subcutaneous to be administered four times respectively at inoculation for latter 0 day, 7 days, 14 days, 21 days; 16X1 homotype contrast monoclonal antibody administration group: nape is subcutaneous to be administered four times respectively at inoculation for latter 0 day, 7 days, 14 days, 21 days.Wherein, positive control VR111 vaccine is the therapeutic vaccine based on HPV16E7 albumen, has good inhibition tumor growth effect in mouse tumor animal model.The Preparation and characterization of this vaccine can be referring to International Application No. WO 2008/154867A1, and the full content of this application is included in herein by the mode of quoting.Homotype contrast 16X1 monoclonal antibody is the antibody of specificity for the coat protein L1 of HPV16 virus, with HPV16E7 albumen without any compatible reaction.16X1 monoclonal antibody as antigen immune BALB/c mouse, then adopts traditional hybridoma technology screening to obtain by the virus-like particle using HPV 16L1.
Observe animal tumor growing state every day, after record inoculation there is the number of animals (the equal >=3mm of length diameter is just calculated as tumer positive) of tumour in different time, be calculated to be ratio of outflow, occur the ratio of tumour number of animals and every group of actual number of animals.After tumour can be touched, 2 times with the vernier caliper measurement tumour line of apsides weekly, calculates gross tumor volume, i.e. gross tumor volume=(major diameter × minor axis
2)/2, tumour inhibiting rate calculation formula: tumour inhibiting rate=100%-administration group tumour mean size/PBS control group tumour mean size.While observing 60 days, kill whole mouse.
After inoculated tumour cell, as shown in Figure 4, the result of the tumour inhibiting rate in 60 days as shown in Figure 5 for the tumor formation rate of 24 days.
As can be seen from Figure 4 and Figure 5,6C10 monoclonal antibody and VR111 vaccine are different from PBS control group and homotype 16X1 monoclonal antibody group in the effect remarkable (P < 0.001) that suppresses tumour generation and inhibition tumor growth.Illustrate after the administration of 6C10 monoclonal antibody and have the effect that suppresses tumour generation and suppress tumor growth.
Claims (10)
1. a species specificity is for the antibody of epitope as shown in SEQ ID:5 of human papillomavirus HPV16, and described antibody has light chain CDR1, CDR2 and the CDR3 region shown in heavy chain CDR1, CDR2 and CDR3 region and the SEQ ID NOs:9-11 as shown in SEQ ID NOs:6-8.
2. a species specificity is for the antibody of epitope as shown in SEQ ID:5 of human papillomavirus HPV16, and described antibody has the weight chain variabl area sequence as shown in SEQ ID NO:1 and the light chain variable region sequence as shown in SEQ ID NO:2.
3. antibody as claimed in claim 1 or 2, is characterized in that, described antibody is the monoclonal antibody that is merged the hybridoma cell line generation forming by host's splenocyte and myeloma cell.
4. antibody as claimed in claim 1 or 2, is characterized in that, described antibody is that preserving number is the monoclonal antibody of the 6C10 cell generation of CGMCC4144.
5. a pharmaceutical composition, is characterized in that, the antibody that described pharmaceutical composition comprises claim 1-4 any one.
6. pharmaceutical composition as claimed in claim 5, is characterized in that, described pharmaceutical composition comprises another kind of antiviral or antitumor drug.
7. pharmaceutical composition as claimed in claim 6, is characterized in that, described another kind antiviral or antitumor drug and described antibody coupling.
8. a test kit, is characterized in that, the antibody that described test kit comprises claim 1-4 any one.
9. a cell for the antibody of generation as described in claim 1-4 any one, is characterized in that, described cell is that preserving number is the 6C10 cell of CGMCC4144.
Antibody as described in claim 1-4 any one, as the pharmaceutical composition of claim 5-7 any one, or test kit as claimed in claim 8 for the preparation of prevention or treatment HPV16 catch, the purposes of the medicine of cervical lesions or cervical cancer.
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| CN105504049B (en) * | 2014-09-26 | 2019-07-05 | 艾托金生物医药（苏州）有限公司 | The relevant HPV E7 protein monoclonal antibody of cervical carcinoma and its application |
| CN106366186B (en) * | 2015-07-21 | 2020-03-24 | 艾托金生物医药（苏州）有限公司 | Monoclonal antibody for identifying HPV16 positive tumor cells and application thereof |
| AU2018290856A1 (en) * | 2017-06-28 | 2020-01-02 | Regeneron Pharmaceuticals, Inc. | Anti-human papillomavirus (HPV) antigen-binding proteins and methods of use thereof |
| CN108837150A (en) * | 2018-06-01 | 2018-11-20 | Wnl生物医学科技有限公司 | Polypeptide composition with specific immunity function, vaccine and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101487010A (en) * | 2008-01-15 | 2009-07-22 | 上海泽润生物科技有限公司 | Method for preparing vaccine for anti-HPV 18 infection by pichia yeast expression system |
| CN101487009A (en) * | 2008-01-15 | 2009-07-22 | 上海泽润生物科技有限公司 | Method for preparing vaccine for anti-HPV 16 infection by pichia yeast expression system |
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| CN101487010A (en) * | 2008-01-15 | 2009-07-22 | 上海泽润生物科技有限公司 | Method for preparing vaccine for anti-HPV 18 infection by pichia yeast expression system |
| CN101487009A (en) * | 2008-01-15 | 2009-07-22 | 上海泽润生物科技有限公司 | Method for preparing vaccine for anti-HPV 16 infection by pichia yeast expression system |
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