CN102443060A - 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 the immunotherapy field.More specifically, the present invention relates to a kind of prevention or treatment human papillomavirus (human papillomavirus, HPV) antibody of infection relative disease (for example cervical lesions, particularly cervical cancer) of being used for.The invention still further relates to the preparation method and the application of said antibody.
Background technology
Present research shows, HPV infects and the uterine neck systemic disease---particularly cervical cancer---has tangible dependency.Nineteen ninety-five; (the International Agency for Research of Cancer of IARC; IARC) result of study of announcing confirms; 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 take place the HPV negative patient hardly; In addition, all can detect HPV among the cervical disease patient more than 98%.
HPV virus can be divided into different types and hypotype according to the sequence homology of L1 gene, and homology more than 90% is being a type, and the HPV of homology between 90～98% can be divided into different hypotypes.According to the type of HPV or the relation of hypotype and female genital tract malignant tumour, HPV is divided into low risk and high-risk-type.Low risk HPV, for example type such as HPV 6,11,34,40,42 is more common in optimum cervical lesions, like uterine neck condyloma and the slight atypism hyperplasia of epithelium of cervix uteri pathology; Then see that so that the infection of HPV 16,18,31,33,35,39,45 types more more these types are high-risk HPV in epithelium of cervix uteri severe atypical hyperplasia and the cervical cancer.
A series of researchs to different crowd have 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, and are closer than the Other Dangers factor.According to " HPV and cervical cancer 2 007 report in the world wide " result that The World Health Organization (WHO) announces, 58.7% cervical cancer is directly related with the HPV16 type among China women.
The HPV16E7 gene continue to exist in cervical cancer and high expression level, in the keeping of the formation of cervical cancer and malignant, plays an important role.HPV16E7 albumen is totally 98 amino acid, and its sequence is following: MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVT FCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP.This albumen has three conserved regions, and (conserved region, CR): CR1 is positioned at the N end, and CR2 contains the LXCXE structural domain, and CR3 contains two zinc-finger structure territories.The center function of E7 is exactly to combine with Rb protein family (like PRb, P107, P130) and through ubiquitin-26S proteasome pathway Rb family protein of degrading through one of three conservative regions.The Rb protein family is in the key link at the cell cycle G1/S outpost of the tax office.Rb protein binding transcription factor E2f albumen under the dephosphorylation state stops E2f albumen to activate numerous and the dna replication dna proteins associated factor, thus suppress DNA synthetic with the cell cycle progress, make cell be in the G0/G1 phase and carry out cytodifferentiation.The E7 protein binding and the Rb albumen of degrading discharge E2f albumen, thereby activate many relevant DNA synthetic proteins factors, accelerate the division growth of dna replication dna and cell, promote the differentiation of cell.In addition, E7 albumen can also combine other albumen of kind more than 10 such as cyclin A, cyclin E, P48, improves exogenous DNA integration and cytogene mutagenicity or the like, quickens transformation.
Therefore, HPV16E7 albumen is the antigen that the cervical cancer tumor cell specific is expressed, and is that tumour cell is different from the antigen target that normal cell is expressed in born of the same parents.Since be the antigen of in born of the same parents, expressing, must be to cell surface through the process of angtigen presentation.Antigen presenting cell is (such as BMDC; Scavenger cell) picked-up corresponding antigens after processing treatment, is degraded into 8-10 amino acid whose small peptide with antigen; These small peptides combine with the MHC-I quasi-molecule in golgi body, pass through the after birth circularly exhibiting then at cell surface.This process just is called angtigen presentation.MHC-I molecule of offering and small peptide (being called t cell epitope) at cell surface, by the T cell recognition, thus the immunoreation in activated T cell downstream.It is early stage that antigenic research shows that HPV16E7 has the t cell epitope of three high-affinities to HPV16 E7; Be 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, antigenic t cell epitope has only 8-10 amino acid.And there is three CDR (complementary determinant the variable region of monoclonal antibody; Complementary-determining region) district; Approximately each CDR district can discern about 10 amino acid, and monoclonal antibody also will be discerned other epi-position except the specific t cell epitope of affine combination; Therefore, the monoclonal antibody of only discerning antigenic specific T cells epi-position is difficult to obtain.Be embodied in using classical mouse body and in the monoclonal antibody screening, go immune animal separately, be difficult to produce antibody with 8-10 amino acid whose small peptide.
To 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 as affine former the screening, this method and technology platform requires high with the polypeptide of t cell epitope, and the avidity of antibody generally all be not so good as the antibody height that filters out in the body.Second method is to form mixture with other albumen and epitope polypeptide earlier; To improve its immunogenicity; Carry out monoclonal antibody screening and epi-position evaluation then with this mixture immune mouse, mostly other albumen that use in this method are MHC (main histocompatibility complex) molecule or KLH (KLH) molecule.Adopt the MHC molecule method need vivoexpression MHC molecule and through external change renaturation with MHC molecule and antigen peptide composition mixture, this procedure is loaded down with trivial details, and it is low to prepare the efficient of mixture.Macromole couplings such as employing KLH are faced with the inefficient problem of preparing mixture equally.And, do not form MHC or the KLH molecule of mixture owing to have immunogenicity equally, after immunity is in the animal body, can produce a lot of unwanted interference antibody, evaluation brings very big interference with epi-position to give subsequently target monoclonal antibody screening.
Therefore, need monoclonal antibody and the working method thereof of a specific specificity in this area to the proteic t cell epitope polypeptide of HPV16E7.
Summary of the invention
For solving the problems of the technologies described above; In first aspect of the present invention; The antibody of one specific specificity to the proteic 49-57 peptide epitopes of HPV16E7 (shown in SEQ ID NO:5) is provided, and said antibody has heavy chain CDR1, CDR2 and CDR3 zone and the light chain CDR1 shown in the SEQ ID NOs:9-11, CDR2 and the CDR3 zone shown in SEQ ID NOs:6-8.
In one embodiment, antibody of the present invention has weight chain variabl area sequence and the light chain variable region sequence shown in SEQ ID NO:2 shown in SEQ ID NO:1.
The present invention also provides a kind of method for preparing antibody of the present invention, and it comprises:
Polypeptide shown in a kind of double-stranded RNA adjuvant and the SEQ ID NO:5 is mixed the formation mixture;
Polypeptide shown in a kind of double-stranded RNA adjuvant and the SEQ ID NO:5 is mixed the formation mixture; With said mixture immunization host; With
Obtain antibody from host's body fluid, tissue or cell.
The present invention provides a kind of method for preparing antibody of the present invention especially, and it comprises:
Polypeptide shown in a kind of double-stranded RNA adjuvant and the SEQ ID NO:5 is mixed the formation mixture; With said mixture immunization host;
Results host's splenocyte and with its with myeloma cell fusion and
Collect the antibody that cell produced that merges.
Prepare in the method for said antibody in the present invention, the mixture that has adopted double-stranded RNA and epitope polypeptide to form carries out host's (for example, mouse) immunity, and screens monoclonal antibody then.Because double-stranded RNA is different from other protein molecular (for example MHC and KLH) that is generally used for forming with antigen mixture, itself does not produce interference antibody, and the antibody that therefore produces is the monospecific antibody to epitope polypeptide entirely.It is easy fast that this legal system is equipped with antibody, the requirement of no special technique platform, and the antibodies specific that filters out is high.
In another aspect of this invention, compsn or the test kit that antibody of the present invention also is provided, contains this antibody is used for the preparation prevention or treatment HPV16 catches, particularly the purposes in the medicine of cervical lesions or cervical cancer.
Description of drawings
Fig. 1 has shown the electrophoresis detection result of 6C10 monoclonal antibody of the present invention.
Fig. 2 has shown the result who identifies 6C10 monoclonal antibody avidity of the present invention through the ELISA method.
Fig. 3 has shown the result who carries out the epi-position evaluation of 6C10 monoclonal antibody of the present invention through the experiment of T2 load peptide.Fig. 3 A and Fig. 3 B show T2 cell streaming cell detection results and the fluorescent microscope detected result that the 49-57 peptide load of E7 is crossed respectively; Fig. 3 C and Fig. 3 D show T2 cell streaming cell detection results and the fluorescent microscope detected result that E786-93 peptide load is crossed respectively; Fig. 3 E and Fig. 3 F show T2 cell streaming cell detection results and the fluorescent microscope detected result that no any peptide load is crossed respectively.
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 the 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 the aminoacid sequence and the nucleotide sequence of the variable region of light chain of 6C10 monoclonal antibody of the present invention.
The sequence explanation
SEQ ID NO:1 is an antibody heavy chain variable region aminoacid sequence of the present invention.
SEQ ID NO:2 is an antibody chain variable region aminoacid sequence of the present invention.
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 proteic 49-57 amino acids of a HPV 16E7 sequence.
SEQ ID NOs:6-8 is the aminoacid sequence of the CDR 1-3 of heavy chain of antibody of the present invention.
SEQ ID NOs:9-11 is the aminoacid sequence of the CDR 1-3 of light chain of antibody of the present invention.
Embodiment
Though described embodiment is represented the preferred embodiments of the invention, it should be understood that those skilled in the art can make any conversion under the situation that does not deviate from spirit of the present invention.
Preferably, the monoclonal antibody of antibody of the present invention for getting through the hybridoma technology preparation.Particularly preferably, antibody of the present invention is for by preserving number being the monoclonal antibody of the 6C10 hybridoma generation of CGMCC 4144.
The present invention provides a kind of pharmaceutical composition or test kit that comprises antibody of the present invention in embodiments.In one embodiment; Pharmaceutical composition of the present invention can further contain the other treatment agent; Antibody wherein of the present invention can with said other treatment agent with form independently separately, perhaps phase blended form exists each other, can form coupling again or each other and combine.Said therapeutical agent can be antitumor or antiviral activity therapeutical agent or biotoxin, for example 5 '-Fluracil, fludarabine, CldAdo, cytosine arabinoside, gemcitabine, capecitabine, troxacitabine, lamivudine, emtricitabine, zalcitabine, zidovudine, Abacavir, didanosine, the two different propionyloxy methyl esters of tynofovir fumaric acid, stavudine, Abacavir, cis-platinum, ifosfamide, carboplatin, bleomycin or safe element etc.
Form in the embodiment of conjugate at antibody of the present invention and other treatment agent, antibody of the present invention can play the effect with its link coupled therapeutical agent target therapeutic interest zone.
In another embodiment, test kit of the present invention can further contain therapeutical agent as stated, and the operation instruction of this test kit.
" mixture " according to the invention is to form through the polypeptide shown in a kind of double-stranded RNA adjuvant and the SEQ ID NO:5 is mixed.Preferably, the double-stranded RNA that is used to form said mixture is Polyribocytidylic acid-polyriboinosinic acid complex (PolyIC, Polyinosinic acid-polycytidylic acid) or PolyICLC.
In an embodiment of preparation method for antibody of the present invention, double-stranded RNA and E7 (49-57) peptide are carried out thorough mixing, its ratio of mixture can be between 1: 0.5 to 1: 4 (w/w).
In another embodiment, said host is mouse, rat, rabbit.
In another embodiment, said myeloma cell is the SP2/0 oncocyte.
In preparation method for antibody of the present invention, said cell clone through fusion is preferably the mono-clonal that behind subclone more than three times, still produces said antibody.Particularly preferably, said cell clone is the 6C10 cell clone.
Method of the present invention can comprise further that the monoclonal cell that screening is obtained sets up cell strain.This cell strain can be stablized and goes down to posterity the justacrine specific antibody.Therefore, method of the present invention can comprise the cell monoclonal that obtains the 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 the 6C10 cell clone is provided especially.
The method for preparing antibody of the present invention can further comprise carries out purifying with the antibody of collecting.
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 common micro-organisms center.
The conventional purification process that purifying antibody of the present invention can be known by one of ordinary skill in the art carries out, for example through 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 the experimenter, and special preferred antibodies 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 describe the present invention in detail through following examples, so that can understand the present invention better.Following embodiment is property purpose presented for purpose of illustration only, is not intended to limit scope of the present invention.Endeavoured to ensure the accuracy of relevant numeral (like quantity, temperature etc.), but should consider that there are some sum of errors deviations in meeting.Except as otherwise noted, temperature to be ℃ being unit or being envrionment temperature, pressure near or equal normal atmosphere.Should be understood that except as otherwise noted, used plant and instrument is the conventional equipment of this area among following each embodiment.Except as otherwise noted, employed substratum is the commercially available conventional substratum that gets, and those skilled in the art all know composition and content wherein.For for simplicity, might use various general abbreviations in this article, those skilled in the art can understand its implication fully.
Embodiment
The preparation and the immunization of the immunocomplex of embodiment 1:E7 albumen 49-57 peptide
The proteic 49-57 peptide of HPV16 virus E7, aminoacid sequence: RAHYNIVTF; Molecular weight 1119Da, theoretical iso-electric point 9.04.This peptide is synthetic by the gill biochemical corp, and purity is greater than 95%.With the synthetic double-stranded RNA---PolyIC is an adjuvant, and it is available from Sigma company (catalog number (Cat.No.): P9582).
Take by weighing PolyIC adjuvant dry powder and E7 (49-57) peptide dry powder; Press mass ratio and mix at 5: 2, add the pure water dissolving, fully mixing; Form macroscopic white granular mixture; Within dissolved 5-10 minute, this mixture is passed through subcutaneous abdomen immunity BALB/c mouse, every mouse immune 100 μ g PolyIC+40 μ g 49-57 peptide mixts.Behind initial immunity the 14th day and the 28th day is with same immunizing dose twice of booster immunization again.Behind the initial immunity about the 35th day, gather mice serum and can detect the proteic associated antibodies of corresponding E7 and produce (detection method such as following examples 2 are said).
Embodiment 2: the detection of serum antibody titer
For by the embodiment 1 said mice immunized of carrying out, behind initial immunity, take a small amount of serum from the tail vein the 35th day the time, detect serum antibody titer with direct enzyme-linked immunosorbent assay (ELISA).
It is following that ELISA detects the serum antibody titer step:
1. (this proteic preparation method is: synthetic total length HPV 16E7 gene with HPV16E7 albumen earlier; In pET32a carrier and BL21 (DE3) host, give expression to the E7 fusion rotein of band Trx label then; With enteropeptidase the Trx label is cut away again and can obtain HPV16E7 albumen), encapsulate 96 orifice plates with the amount of every hole 1 μ g.Encapsulate plate and hatched 16 hours at 4 ℃, wash each hole 6 times with lavation buffer solution (the PBS solution that contains 0.05%Tween-20), every hole adds confining liquid (the PBS solution that contains 5% skim-milk) 100 μ l sealing 2 hours.
2. every hole adds the serum sample of 100 μ l with 50 times of PBS solution dilutions, and 37 ℃ were reacted one hour.Wash each hole 3 times with lavation buffer solution then, each 5 minutes.
3. every hole adds the sheep anti-mouse igg antibody (available from Calbiochem company, numbering 401215) of the horseradish peroxidase of 100 μ l, hatches 0.5 hour for 37 ℃.Wash each hole 6 times with lavation buffer solution then, to remove unconjugated enzyme labelled antibody.
4. every hole adds 100 μ l TMB colour developing liquid, and room temperature was carried out coupling reaction 20 minutes.
5. every hole adds stop buffer (2M sulphuric acid soln) the color development stopping reaction of 50 μ l.Measure the photoabsorption of 450nm wavelength after 5 minutes with ELIASA, and the record result.There is situation in the antibody of judging serum through absorbance value.
Measured four altogether and carried out mice immunized and the serum without the negative control mouse of immunity according to embodiment 1 said method, four are respectively by immune serum OD reading: 0.323,1.805,0.347,0.709, and negative serum reading 0.055.Infer the immunity system that can activate mouse through the mixture immune mouse of PolyIC and antigen peptide thus, and produce the proteic specific antibody of corresponding E7.
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, and getting spleen on the 4th day behind the booster immunization carries out cytogamy.Concrete steps are following: mouse is plucked eyeball and gets blood, with blood collecting to the 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 of having 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) be according to 10: 1 to 1: 1 mixed, mixing, 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), mixing, centrifugal 10 minutes of 960rpm in 5 minutes.Supernatant discarded adds the RM1640 substratum that 120ml contains 1%HAT, and mixing is inoculated in 6 96 orifice plates, and every hole 200 μ l are at 37 ℃, 5%CO
2Cultivate under the condition.After 5 to 7 days, it is visible to treat that Kong Zhongyou clones clearly, and each clone's cell count reaches 10
3More than, with the substratum sucking-off in the hole, adding the fresh RM1640 substratum that contains 1%HT, every hole 200 μ l are at 37 ℃, 5%CO
2Continue under the condition to cultivate.Whether the supernatant of culture medium of sucking-off detects with aforesaid ELISA method has specific antibody to produce.The clone of picking ELISA tests positive continues subclone screening subsequently.
2. subclone screening
The preparation of feeder cell: when adopting limiting dilution assay to carry out subclone, need the existence of feeder layer cells.Carry and prepare feeder layer cells previous day.Get 1 female above Kunming mouse collection in 8 ages in week peritoneal macrophage, and be inoculated in 96 orifice plates.
Limiting dilution assay screening subclone: above-mentioned selected clone is processed cell suspension with liquid-transfering gun piping and druming, use the blood cell counting plate numeration.The suspension that contains 100 cells according to numeration sucking-off as a result adds 20ml and contains in the RM1640 substratum of 10% foetal calf serum, is seeded in 1 96 orifice plate, and 200 μ l/ holes place 37 ℃, 5%CO
2Cultivate under the condition, after 7 to 10 days, treat that clone in 96 orifice plates grows to the sixth to three in whole hole/for the moment, get the detection that cell conditioned medium carries out specific antibody, select higher, the well-grown clone of OD value again, carry out subclone and screen.
If continuous three subclones of mono-clonal, when all clones that obtain are all positive, can assert that this monoclonal cell can the stably excreting specific antibody, with this cell strain built, enlarged culturing, the frozen cell seed bank of setting up.
Go out to be numbered the clone of 6C10 according to above-mentioned standard picking, set up the cell seed bank.The about 800ml of collecting cell culture supernatant liquid with Protein A medium purification antibody, obtains the 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 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 to behind the purifying is carried out the SDS-PAGE electrophoresis detection.The result is as shown in Figure 1, in WR 34678 reduction electrophoretogram, and the heavy chain band of about 52KD of visible this monoclonal antibody and the light chain band of 25KD.
The evaluation of embodiment 5:6C10 monoclonal antibody avidity
Respectively as envelope antigen, the 6C10 monoclonal antibody carries out ELISA and detects as detecting antibody with E7 full-length proteins, Trx-E7 (polypeptide that the 30-67 amino acids residue of E7 is formed) and BSA (bovine serum albumin).The result is shown in Fig. 2.
Can find out from the ELISA result of Fig. 2, contain the albumen of E749-57 peptide, for example E7 full-length proteins and Trx-E7 (30-67) albumen can have very strong combination with the 6C10 monoclonal antibody, and the OD value is greater than 1.5 in the ELISA experiment.The albumen that does not contain the E749-57 peptide does not almost combine such as BSA albumen and 6C10 monoclonal antibody, and the OD value is less than 0.2 in the ELISA experiment.
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 (available from U.S. ATCC storehouse, preserving number CRL-1992) is the cell of a kind of endogenous TAP defective, surperficial unloaded HLA-A2 molecule.The antigen Toplink that external source adds is loaded on the HLA-A2 molecule of T2 cell surface.
With the 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, hatched 4 hours.After the washing once,, hatched 30 minutes for 37 ℃ with the 6C10 monoclonal antibody of the resuspended back adding of dyeing damping fluid (PBS+0.5%BSA+2mM EDTA) 100 μ l purifying.After the washing once; Resuspended with dyeing damping fluid (PBS+0.5%BSA+2mM EDTA); The sheep anti-mouse igg antibody (available from BD company, catalog number (Cat.No.) 349031) that adds 2 μ l FITC marks then detects cell green fluorescence situation with flow cytometer and fluorescent microscope after room temperature kept in Dark Place 20 minutes.The result is as shown in Figure 3.
Fig. 3 A and Fig. 3 B show T2 cell streaming cell detection results and the fluorescent microscope detected result that E749-57 peptide load is crossed respectively; Fig. 3 C and Fig. 3 D show T2 cell streaming cell detection results and the fluorescent microscope detected result that E786-93 peptide load is crossed respectively; Fig. 3 E and Fig. 3 F show T2 cell streaming cell detection results and the fluorescent microscope detected result that no any peptide load is crossed respectively.
Can find out that from result shown in Figure 3 6C10 monoclonal antibody of the present invention can specific recognition be positioned at the E749-57 peptide of T2 cell surface load.Confirmed the epitope specificity of 6C10 monoclonal antibody thus.
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), presses test kit specification sheets operation extracting RNA.Total RNA that about 3 μ g extractings are gone out is used to carry out single stage method RT-PCR (using the OneStep RT-PCR test kit of Qiagen company).
According to antibody variable gene FR1 district and the synthetic weight strand primer of FR4 district conserved regions sequences Design, the heavy chain primer is following:
VH forward primer: 5 '-GA GGTGCA GCTGCA GGA GTC-3 ';
VH reverse primer: 5 '-TGAGGA GACGGTGACCG TGGTCCCTTGGCCC-3 ';
The light chain primer is following:
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 into pMD18-T carrier (Takara company) respectively, picks out and deliver to Shanghai behind the positive colony and give birth to worker company and carry out dna sequencing.Sequencing result shows that the light/heavy chain variable region gene that is obtained contains 4 framework regions and 3 antigen complementary determining regions.
The weight chain variabl area sequence of this monoclonal antibody is shown in SEQ ID NO:1.
6C10 monoclonal antibody VH full length gene 348bp, 116 amino acid of encoding.The amino acid sequence analysis result shows; VH contains clear and definite 4 framework regions (FR) and 3 the complementary district of antigenic determinant (CDR) (they have sequence shown in the following SEQ ID NOs:6-8 respectively); At the 22nd and the 95th is halfcystine, is to form two kinds of relevant characteristic amino acid with the 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 shown in SEQ ID NO:2.
6C10 monoclonal antibody VL full length gene 324bp, 108 amino acid of encoding.The amino acid sequence analysis result shows; VL contains clear and definite 4 framework regions (FR) and 3 the complementary district of antigenic determinant (CDR) (they have sequence shown in the following SEQ ID NOs:9-11 respectively); At the 92nd is halfcystine, and this light chain is unusual 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 (available from U.S. ATCC storehouse, deposit number CRL-2785) inoculation to set up the cervical cancer tumor model of E7 protein expression.Express the antigenic TC-1 cell of E7 in the lung primary cell that derives from the C57BL/6 mouse, through E7 gene that imports HPV16 and the people C-Ha-ras gene that is activated, this primary cell strain is transformed and is obtained unlimited prolificacy.
To this tumor model injected in mice 6C10 monoclonal antibody and various reference protein, observe the tumor growth situation.
The outer good TC-1 cell of incubation growth of collection body, PBS washes 3 times, and the adjustment 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 the inoculation mouse is divided into 4 groups at random, 8 every group, carries out administration by following four kinds of modes.Negative control PBS administration group: abdomen is subcutaneous to be administered twice respectively at inoculation in back 2 days, 16 days; Positive control VR111 vaccine administration group: abdomen is subcutaneous to be administered twice respectively at inoculation in back 2 days, 16 days; 6C10 monoclonal antibody administration group: nape is subcutaneous to be administered four times respectively at inoculation in back 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 in back 0 day, 7 days, 14 days, 21 days.Wherein, positive control VR111 vaccine is based on the proteic therapeutic vaccine of HPV16E7, and good restraining tumor growth effect is arranged in the mouse tumor animal model.The preparation of this vaccine and sign can be referring to International Application No. WO 2008/154867A1, and the full content of this application is included this paper in through the mode of quoting.Homotype contrast 16X1 monoclonal antibody is the antibody that specificity is directed against the coat protein L1 of HPV16 virus, does not have any compatible reaction with HPV16E7 albumen.The 16X1 monoclonal antibody through with the virus-like particle of HPV 16L1 as the antigen immune BALB/c mouse, adopt traditional hybridoma technology screening to obtain then.
Observe the animal tumor growing state every day, the number of animals (length diameter all＞=3mm just be calculated as tumer positive) of tumour appears in record inoculation back different time, is calculated to be ratio of outflow, the ratio of tumour number of animals and every group of actual number of animals promptly occurs.After tumour can be touched, 2 times with the vernier caliper measurement tumour line of apsides weekly, calculated 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.When observing 60 days, kill whole mouse.
24 days tumor formation rate is as shown in Figure 4 behind the inoculated tumour cell, and the result of the tumour inhibiting rate in 60 days is as shown in Figure 5.
Can find out that from Fig. 4 and Fig. 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.Explain after the administration of 6C10 monoclonal antibody the effect that suppresses the tumour generation and suppress tumor growth is arranged.
Claims (18)
1. a specific specificity is to the antibody of epitope shown in SEQ ID:5 of human papillomavirus HPV16, and said antibody has heavy chain CDR1, CDR2 and CDR3 zone and the light chain CDR1 shown in the SEQ ID NOs:9-11, CDR2 and the CDR3 zone shown in SEQ ID NOs:6-8.
2. a specific specificity is to the antibody of epitope shown in SEQ ID:5 of human papillomavirus HPV16, and said antibody has weight chain variabl area sequence and the light chain variable region sequence shown in SEQ ID NO:2 shown in SEQ ID NO:1.
3. according to claim 1 or claim 2 antibody is characterized in that, said antibody is to merge the monoclonal antibody that the hybridoma cell line that forms produces by host's splenocyte and myeloma cell.
4. according to claim 1 or claim 2 antibody is characterized in that, said antibody is that preserving number is the monoclonal antibody that the 6C10 cell of CGMCC 4144 produces.
5. a pharmaceutical composition is characterized in that, said pharmaceutical composition comprises each antibody of claim 1-4.
6. pharmaceutical composition as claimed in claim 5 is characterized in that, said pharmaceutical composition comprises another kind of antiviral or antitumor drug.
7. pharmaceutical composition as claimed in claim 6 is characterized in that, said another kind antiviral or antitumor drug and said antibody coupling.
8. a test kit is characterized in that, said test kit comprises each antibody of claim 1-4.
9. the cell of a generation such as each described antibody of claim 1-4 is characterized in that said cell is that preserving number is the 6C10 cell of CGMCC 4144.
10. one kind prepares each the method for antibody of claim 1-4, it is characterized in that said method comprises:
It is compound that polypeptide shown in a kind of double-stranded RNA adjuvant and the SEQ ID NO:5 is mixed formation
Thing; With said mixture immunization host; With
Obtain antibody from host's body fluid, tissue or cell.
11. method as claimed in claim 10 is characterized in that, said method comprises:
Polypeptide shown in a kind of double-stranded RNA adjuvant and the SEQ ID NO:5 is mixed the formation mixture; With said mixture immunization host;
Results host's splenocyte and with its with myeloma cell fusion and
Collect the antibody that cell produced that merges.
12. method as claimed in claim 11 is characterized in that, said fused cell is for still producing the cell of said antibody behind subclone more than three times.
13., it is characterized in that said host is mouse, rabbit or rat like each described method of claim 10-12.
14., it is characterized in that said myeloma cell is the SP2/0 oncocyte like each described method of claim 10-13.
15., it is characterized in that said adjuvant and said polypeptide blended weight ratio are 1: 0.5 to 1: 4 like each described method of claim 10-14.
16., it is characterized in that said double-stranded RNA is PolyIC or PolyICLC like each described method of claim 10-15.
17., it is characterized in that said fused cell is the 6C10 cell like each described method of claim 10-16.
18. like each described antibody of claim 1-4, like each pharmaceutical composition of claim 5-7, test kit perhaps as claimed in claim 8 is used to prepare prevention or treatment HPV16 catches, the purposes of the medicine of cervical lesions or cervical cancer.
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| WO2016045563A1 (en) * | 2014-09-26 | 2016-03-31 | 艾托金生物医药（苏州）有限公司 | Cervial cancer-related hpv e7 protein monoclonal antibody and use thereof |
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| CN106366186A (en) * | 2015-07-21 | 2017-02-01 | 艾托金生物医药（苏州）有限公司 | Monoclonal antibody for recognizing HPV16 positive tumor cells, and applications thereof |
| 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 |
| JP2020529835A (en) * | 2017-06-28 | 2020-10-15 | リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． | Anti-human papillomavirus (HPV) antigen-binding protein and how to use it |
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| 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 |
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