Background technology
In the disease of harm humans health, tumour has become and has caused mankind's main causes of death.The number 10 million that affected by cancer, according to the < < world cancer report > > of the World Health Organization, expect the year two thousand twenty, annual morbidity will reach 1,500 ten thousand people, dead 1,000 ten thousand people.The mortality ratio of domestic tumour has risen to first in city.This means that the situation that oncotherapy faces is very severe.It is main at present for the treatment of cancer, being still to perform the operation clinically, and chemotherapy and radiation is auxiliary, but the specificity for the treatment of is not enough, and usually normal tissue and organ cause damage, and the operative treatment problem that still has postoperative height recurrence and easily shift.The immunotherapy of tumor vaccine can produce the specific reaction for tumour, even can remove remaining tumor focus, and produces immunological memory.Now become the 4th kind of oncotherapy mode after operation, chemotherapy and radiation, and had obvious complementarity with three large routine treatments.
Tumor vaccine is a kind of mode of immunotherapy.Wherein utilizing polypeptide to make vaccine is to utilize tumour specific antigen and other immunity regulatory cell is treated and prophylaxis of tumours.In anti tumor immune response process, first by antigen presenting cell (APC), tumour antigen processing treatment submission are excited to T cell.Dendritic cell (Dendritic Cell, DC) as the strongest full-time APC of function in human body, there is typical dendron shape form, film surface high expression level HLA I, HLA ll quasi-molecule, can absorb efficiently, processing and submission antigen, start the cell-mediated immune response of T, thereby become the key link of anti tumor immune response.The DC of application load tumour antigen, can excite in-vivo tumour specific T-cells, thus effective killing tumor cell, and can set up lasting antineoplastic specificity immunne response.
People's MUC-1 (MUC1) is a kind of height O-glycosylation and contains the high molecular glycoprotein that continuous repetition peptide sequence is feature, is present in multiple epithelium, has several functions.Saliva Orthana is unconventionality expression in cancerous tissue, and expression amount is abundant.People's MUC-1 (MUC1) is a kind of tumor associated antigen (Tumor associated antigen, TAA), is present in the molecule on 90% cancer cells surface.In healthy human body cell, also contain MUC-1, but quantity seldom, cannot trigger immune response.Immunity system meets with the high cancer cells of MUC-1 concentration may trigger immune response, and cancer cells can be attacked and kill to the toxic T lymphocyte of activation.
People's MUC-1 has signal peptide.Signal peptide Chang Zhixin synthesizes the aminoacid sequence of the N-end of the cross-film transfer that is used in reference to nest egg white matter in polypeptide chain.Generally by 15~30 amino acid, formed.Signal peptide comprises San Ge district: the N-terminal of a positively charged, is called alkaline N-terminal: hydrophobic sequence in the middle of, take neutral amino acids as main, and can form one section of β spirane structure, it is the major function district of signal peptide; Long electronegative C-terminal, containing small molecules amino acid, is signal sequence cleavage site, also claims processing district.
In immunotherapy of tumors, remove the immunization that tumour cell relies on T cell.The identification of T cell to antigen is not complete antigen molecule.In antigen presenting cell (APC), tumor associated antigen is resolved into polypeptide by enzyme, and then in the presence of peptide chain translocator, be transported to endoplasmic and new synthetic HLA molecule (human leukocyte antigen, human leucocyte antigen, comprise HLA I molecule or HLA II molecule) in conjunction with and move to APC surface, form HLA/ antigenic peptide complexes, after the HLA/ antigenic peptide complexes of T cell by special TCR identification antigen presenting cell surface, its surface, then receive costimulatory signal (B7 molecule and CD28 interaction of molecules).Under two signals model, T cell is activated and breeds, most of and then be divided into effector cell.CD wherein
8 +t cell has lethality, foreign cell is broken and death.CD
8 +the cytokines such as T emiocytosis interleukin make CD
8 +t cell,
and variously have the white corpuscle of phagocytic activity to concentrate on around tumour cell, tumour cell is eliminated.Wherein a part of T lymphocyte, becomes memory cell, and while again running into same antigen stimulation, it incites somebody to action more promptly proliferation and differentiation is effector cell.Minority memory cell is split into memory cell again, carries out enduringly specific immune function.
1991, Rotzschke etc. disclosed with X ray crystalline diffraction, and the antigen peptide land that HLA I quasi-molecule top α 1 and α 2 chains form is groove shape structure, can hold the small peptide that 8~12 amino-acid residues form.Amino acid forms at other HLA I molecule camber of different shaped conservatively in groove, can form stable and powerful hydrogen bond with epitope peptide N end residue and C end residue, has guaranteed that HLA I molecule can identify special epi-position.In the groove of different genotype HLA I, amino acid can have various variations, and this is the basis that forms HLA I polymorphism.Although antigen peptide and HLA I quasi-molecule be combined with certain selectivity, but different with the mechanism that antibody and antigen are combined, as long as combined polypeptide has 2~3 crucial amino-acid residues that have similar chemical property (anchored site), just can be connected to rightly on the corresponding position of the polypeptide binding motif (binding motif) in groove.Polypeptide with it in conjunction with after, and be transported to cell surface submission to CD
8 +t cell.CD
8 +t cell is by after the HLA/ antigenic peptide complexes on its surperficial φt cell receptor (TCR) specific recognition antigen presenting cell surface, and being activated also bred, and then be divided into effector cell.This architecture basics just, so each HLA I molecular energy is combined with the not homopolypeptide of some amount.This sequence for other molecule binding peptide of different shaped is carried out Antigen Epitope Prediction provides theoretical basis, thereby the prediction that makes computer auxiliaring means carry out CTL epi-position becomes possibility.
The HLA II quasi-molecules such as HLA-DP, DQ, DR are comprised of the α chain of HLA II genoid coding and the glycoprotein of the non-covalent connection of β chain.α chain and β chain, after endoplasmic reticulum is synthetic respectively, is coiled into separately a α spiral and β lamella and forms groove.In groove, also have anchored site, groove is combined with antigen peptide and is formed HLA/ complex of polypeptides.Because its end is open, therefore can hold longer polypeptide (approximately 12~20 amino acid).When the groove of HLA II, be combined with antigen peptide and form MHC/ complex of polypeptides, and be transported to cell surface submission to CD
4 +t cell.CD
4 +t cell, by after the HLA/ antigenic peptide complexes on its surperficial TCR specific recognition antigen presenting cell surface, is activated and breeds, and then being divided into effector cell.CD
8 +t cell must have stimulation CD
4 +the signal of T cell, could produce effective immune response.The CD activating
4 +t cell can effectively promote CD
8 +t cell produces memory cell, thereby makes body produce long-term antineoplastic ctl response.In addition, the CD of activation
4 +t cell also can direct killing tumour cell.
The not structural difference between isoallele at HLA different genes seat or homogenic same seat, can form the structure of different HLA molecule grooves.Cause thus different HLA allelotrope coding molecules to there is selectivity to the combination of various antigen peptide.And, can with the antigen peptide of same class HLA molecule combination, its anchored site and anchor residues are often same or similar.This shows, specific HLA molecule can rely on optionally conjugated antigen peptide of needed concomitant motif, and in this sense, both combinations have certain selectivity.In fact, different HLA molecules are optionally in conjunction with the peptide section with different positioning of anchors and residue, therefore different HLA allelotrope products are likely offered the different epi-positions of same antigen molecule, cause Different Individual (with different MHC allelotrope) in intensity, to occur difference to replying of same antigen.On this, be that HLA participates in and regulate and control a kind of important mechanisms of immunne response with its polymorphism.
Further investigation is discovery also, and HLA molecule not presents strict one-one relationship to the identification of antigen peptide.This can flexibility can show different aspect: one, form the antigen peptide of concomitant basic sequence, its order and structurally variable; Two, the desired anchor residues of same HLA molecule (as HLA II molecule) is a more than seed amino acid often, result is that the quantity of peptide chains resulted of the specific common base sequence of correspondence can be considerably many, causes a kind of HLA molecule in conjunction with plurality of antigens peptide, to activate a plurality of special T cell clones; Three, the antigen peptide that different HLA molecules are received, can have similar common motif.For example, at least known A2, A3 ,B4 ,B44Si Ge family in HLA I quasi-molecule, the member in these families (various allelotrope product) optionally corecognition has the antigen peptide of same or similar anchor residues.This means the antigen peptide that can be identified and offer by a certain HLA molecule, also may be offered by other molecule in family under it.This is to application polypeptide vaccine, and external sensitization dendritic cell vaccine or T cell vaccine carry out immunoprophylaxis and immunotherapy is provided convenience.While is also for the polypeptide sequence of evaluation and transformation tumor vaccine provides foundation.
Not only each HLA I molecular energy is combined with the not homopolypeptide of some amount, and T cell also has cross reaction phenomenon, and single T cell can be identified the protein complex of two or more different polypeptide antigens and MHC.This provides again the foundation of identifying and transform the peptide sequence of tumor vaccine in another aspect.
Research shows, overcomes the obstacle of HLA polymorphism, and finding the effectively most of crowd's of covering tumour epiposition vaccine has become the important prerequisite that the treatment of tumour-specific polypeptides vaccine immunity is studied.
According to statistics, only two of HLA A2 (comprising A*0201, A*0202, A*0204, A*0205, A*0206, A*0207 and A*0208) and HLA A3 (comprising A*0301, A*1101, A*3101 and A*6801) are large-scale surpasses 85% in Chinese overall average distribution frequency.The peptide sequence of same large-scale identification is closely similar.
This area also needs the most of crowd's of more effective covering tumor vaccine.
Summary of the invention
The invention provides and can and and then excite the antigenic determinant of people's mucoid I of Tumor-assaciated T cell by HLA I and HLA II identification.These antigenic determinants, Aisa people, have particularly accounted for great majority (being greater than 50%) in Chinese, the frequency occurring is higher.The present invention also provides polypeptide and the related neoplasms polypeptide vaccine preparing according to the antigenic determinant of these people's mucoids I.These tumour polypeptide vaccines that prepare have good therapeutic action in larger crowd.
Concrete, the invention provides a kind of isolated polypeptide or its variant, described polypeptide comprises the aminoacid sequence identical or substantially the same with following (a) or aminoacid sequence (b):
(a) aminoacid sequence shown in SEQ ID NO:1;
(b) fragment of the aminoacid sequence of described (a).
Aminoacid sequence shown in SEQ ID NO:1 is MTPGTQSPFFLLLLLTVLTVVTGS.
Polypeptide of the present invention or its variant can be in conjunction with HLA I or HLA II molecules and by CD
8 +t cell or CD
4 +t cell recognition.
In the present invention, term " separation " refers to non-natural form.
In the present invention, term " variant " or " variant of polypeptide " refer to described polypeptide at protein-active, for example, in antigenicity, are equal to or have the variant of better activity in epi-position or in immunology.In some embodiments, those skilled in the art can not destroy its active part by changing on described polypeptide, for example, replace, lack, insert, increase the one or more amino acid in the aminoacid sequence of this polypeptide and obtain described variant.In some embodiments, can be tested and appraised the residue of molecule conservative between similar polypeptide and partly described polypeptide be carried out the replacement of related amino acid and obtains described variant.Those skilled in the art can also analyze three-dimensional structure and the aminoacid sequence relevant to structure in similar polypeptide.According to such information, those skilled in the art can predict the aminoacid sequence comparison of antigen three-dimensional structure, prepare thus the test variant that comprises single amino acid metalepsy at the amino-acid residue place of each expectation.Then can apply and well known to a person skilled in the art that determination of activity screens these variants.
In the present invention, the implication that comprises " comprising " " having " in statement " polypeptide comprises certain aminoacid sequence ".
Polypeptide provided by the invention comprises the fragment (immunogenic fragments) of the polypeptide with the aminoacid sequence shown in SEQ ID NO:1, a sequential portion with the aminoacid sequence shown in described SEQ ID NO:1, this part can produce the immunne response of the polypeptide of the aminoacid sequence shown in identification SEQ ID NO:1.Preferred fragment comprises, for example, has the brachymemma polypeptide of the continuous aminoacid sequence of a part of SEQ ID NO:1.
In one aspect of the invention, provide aforementioned polypeptides, wherein fragment described in (b) is comprise or have in the aminoacid sequence shown in SEQ ID NO:1 continuous 8,9 or 10 amino acid whose fragments.Of the present invention, aspect one of them, provide aforementioned polypeptides, wherein fragment described in (b) is comprise or have continuous 10 amino acid whose fragments in the aminoacid sequence shown in SEQ ID NO:1.
Of the present invention aspect another, aforementioned polypeptides is provided, it has FLLLLLTVLT(SEQ ID NO:2), LLLLLTVLTV(SEQ ID NO:3), LLLLTVLTVV(SEQ ID NO:4), FFLLLLLTVL(SEQ ID NO:5) aminoacid sequence, GTQSPFFLLL(SEQ ID NO:6), TQSPFFLLLL(SEQ ID NO:7).
In one aspect of the invention, aforementioned polypeptides can be in conjunction with HLA I molecule and by CD
8 +t cell recognition.CD
8 +t cell is also referred to as cytotoxic T cell (CTL).Of the present invention, aspect another, wherein said HLA I is HLA A2 or HLA A3 type.
Herein, " substantially the same aminoacid sequence " refers in aminoacid sequence a, disappearance for example, to several (2,3,4 or 5) amino acid replaced, adds or insert, its compare with this aminoacid sequence have same or similar or better active.In the present invention, described activity can refer to, for example, and by HLA I and the identification of HLA II and and then excite the activity of Tumor-assaciated T cell.
Can be according to the synthetic polypeptide of the present invention of the method for using in conventional chemistry of peptides.These currently known methodss comprise the method for for example describing in following document: Peptide Synthesis, Interscience, New York, 1966.
Also can pass through conventional preparation polypeptide of the present invention.For example, can prepare described polypeptide by Nucleotide synthetic with conventional DNA and coding said polypeptide prepared by gene engineering method.By following method, prepare described polypeptide: above-mentioned polynucleotide are inserted to conventional expression vector; With the recombinant expression vector transformed host cell obtaining; The transformant that cultivation obtains; With from culture, collect described polypeptide.Can reference example carry out as the method to describe in Publication about Document: Molecular Cloning, the people such as T.Maniatis, CSH Laboratory (1983).
The present invention also provides the separated nucleic acid that forms of base sequence by coding aforementioned polypeptides of the present invention.Nucleic acid of the present invention can be cDNA, mRNA or DNA/RNA mosaic, preferably DNA.Described nucleic acid can be two strands or strand.When described nucleic acid is double-stranded, it can be double-stranded DNA, double-stranded RNA or DNA:RNA heterozygote.When polynucleotide of the present invention are two strands, can be inserted into expression vector to produce for expressing the recombinant expression vector of peptide of the present invention.Therefore, nucleic acid of the present invention comprises by double-stranded polynucleotide of the present invention is inserted to the recombinant expression vector that expression vector obtains.
The base sequence of above-mentioned signal peptide of the present invention of encoding is not particularly limited, as long as it produces any one of aminoacid sequence of aforementioned polypeptides of the present invention after translation.The base sequence of encode such amino acid sequences can be usingd genomic dna or the RNA that contains Saliva Orthana sequence by PCR method etc. and be obtained as template.On the other hand, consider the expression efficiency in host cell, conventionally preferably select very through being usually used in the codon of host cell used.In various biological varieties, the frequency of utilization data of codon can obtain from genetic code frequency of utilization database.Nucleic acid of the present invention also can carry out chemosynthesis by DNA/RNA automatic DNA synthesizer DNA.
In one aspect of the invention, the polypeptide that contains the invention described above or vaccine and the pharmaceutical composition of nucleic acid are provided.At described vaccine provided by the invention and pharmaceutical composition, can be used for treatment or preventing cancer.The polypeptide of the invention described above can be used as vaccine and the pharmaceutical composition for the treatment of or preventing cancer.The nucleic acid of the invention described above also can be used as vaccine and the pharmaceutical composition for the treatment of or preventing cancer.Thereby nucleic acid of the present invention can be expressed and be obtained polypeptide of the present invention by habitual mode, and then for such use.
Polypeptide of the present invention can be the HLA antigen that is handed to antigen presenting cell, the tumour that therefore can treat or prevent patient.Polypeptide of the present invention can be the HLA that is handed to antigen presenting cell, the T cell, particularly CD of specificity excitation energy identification HLA antigen and the mixture of the combination of the polypeptide of presenting
8 +t cell, kills tumour cell thereby can breed, and therefore can be used to treatment or prevention patient's tumour.
Comprise polypeptide of the present invention as activeconstituents for inducing T cell, CD particularly
8 +the reagent of T cell can with pharmaceutically acceptable carrier for example suitable adjuvant mix or combined administration, thereby effectively set up cellular immunization.The example of spendable adjuvant is included in document Clin.Microbiol.Rev., 7:277-289, it is incorporated herein by reference 1994(herein) middle those that describe.
Polypeptide of the present invention can especially effectively be presented and inducing specific cytotoxic T cell (CTL) in two of HLA A2 and HLA A3 are large-scale.HLA A2 type comprises A*0201, A*0202, A*0204, A*0205, A*0206, A*0207 and A*0208.HLA A3 comprises A*0301, A*1101, A*3101 and A*6801.These two large-scale surpasses 85% in Chinese overall average distribution frequency.Therefore, vaccine or the pharmaceutical composition of polypeptide of the present invention and the prevention based on polypeptide of the present invention and treatment tumour can effectively cover most of crowd.Preferably, polypeptide of the present invention can effectively be presented and inducing specific cytotoxic T cell (CTL) in HLA A2 type.Preferred, polypeptide of the present invention can effectively be presented and inducing specific cytotoxic T cell (CTL) in A*0201, A*0202 type.
Comprise polypeptide of the present invention or derivative as activeconstituents for induce prevention or the vaccine of therapeutic immune response can with pharmaceutically acceptable carrier for example suitable adjuvant mix or combined administration, thereby more effectively set up immune response.
The example of spendable adjuvant comprises, for example, microbe-derived composition or derivatives thereof, cytokine, the composition or derivatives thereof of plant origin, the composition or derivatives thereof in marine organisms source, mineral substance gel is aluminium hydroxide, lysolecithin for example, and tensio-active agent is polyvalent alcohol for example, polyanion, peptide, oil emulsifier (emulsifying agent preparation) etc.Also can consider Liposomal formulation in addition, with the Nanoparticulate formulations that the pearl with several micron diameters is connected, there is the preparation of the fat of connection, microball preparation, microcapsule formulation etc.
The method of using of above-mentioned vaccine of the present invention or pharmaceutical composition comprises intracutaneous, subcutaneous, intramuscular, intravenous administration etc.The dosage of the peptide of the present invention in preparation can be according to disease to be treated, and patient's age and body weight etc. are suitably adjusted.Conventionally, the dosage of peptide of the present invention in preparation is 0.0001 to 1000mg, and preferably 0.001 to 1000mg, and more preferably 0.1 to 10mg, preferably within every several days or 1, uses 1 time to some months.
The vaccine of the invention described above and pharmaceutical composition also can be used for the treatment of tumour patient by vitro method.In other words, polypeptide of the present invention or nucleic acid can contact with antigen presenting cell and/or immune effector cell in vitro, and generation can be identified the antigen presenting cell of antigen of the present invention or antigenic complex, thus inducing specific T cell, particularly CD
8 +t cell, then returns in patient body for prevention or treatment cancer.The invention provides by vitro the peripheral blood lymphocyte that derives from tumour patient being contacted to the T cell, particularly CD of inducing with polypeptide of the present invention or nucleic acid
8 +t cell, and the method that produces above-mentioned cell.
In one aspect of the invention, provide the method that produces antigen presenting cell.Of the present invention, aspect one of them, described method comprises the polypeptide of the invention described above and the step with the cells contacting of antigen presentation ability.Of the present invention, aspect one of them, described method comprises the nucleic acid of the invention described above and the step with the cells contacting of antigen presentation ability.Polypeptide of the present invention as above and nucleic acid can with the cells contacting with antigen presentation ability, can produce antigen presenting cell.By polypeptide of the present invention or nucleic acid and there is the cells contacting of antigen presentation ability, can produce antigen presenting cell.Polypeptide of the present invention and nucleic acid can be used in vitro, for prevention or treatment tumour.For example, by vitro, by polypeptide of the present invention or nucleic acid and there is the cells contacting of antigen presentation ability, can produce antigen presenting cell.Polypeptide of the present invention and nucleic acid also can be used in vivo, for prevention or treatment tumour.
In the present invention, the cell that has an antigen presentation ability is at described cell surface expression, to present the cell of the HLA antigen of polypeptide.Wherein a kind of cell with antigen presentation ability is dendritic cell.
The invention provides a kind of antigen presenting cell, at described cell surface expression, present the cell of the HLA antigen of polypeptide of the present invention.Wherein a kind of cell with antigen presentation ability is dendritic cell.Antigen presenting cell of the present invention can be by adding the above-mentioned cell with antigen presentation ability to prepare polypeptide of the present invention or nucleic acid.
Antigen presenting cell of the present invention can obtain by following method: the separated cell with antigen presentation ability from tumour patient, with polypeptide of the present invention irritation cell in vitro, and allows antigen presenting cell to present the mixture of HLA antigen and polypeptide.When using dendritic cell, can be from the peripheral blood of tumour patient separated lymphocyte, remove and can not adhere to the cell of culture dish, under the existence of GM-CSF and IL-4, cultivate adherent cell with induction dendritic cell and cultivate and use peptide stimulation dendritic cell of the present invention and prepare antigen presenting cell of the present invention.
In being joined to the cell with antigen presentation ability, nucleic acid of the present invention preparing antigen presenting cell of the present invention.Described nucleic acid can be DNA or rna form.Described nucleic acid can be at the polypeptide of the present invention of the cells with antigen presentation ability.
The invention provides a kind of immune effector cell, described cell can have been presented the delivery cell that is of polypeptide of the present invention and HLA antigenic compound by specific recognition surface expression, and is activated and breeds, and is divided into effector cell.Effector cell comprises various T cells, for example CD
8 +t cell and CD
4 +t cell.In one aspect of the invention, the invention provides a kind of immune effector cell, it is CD
8 +t cell, has lethality to cancer cell, and cancer cell is broken and death, also becomes memory cell, and while again running into same antigen stimulation, it incites somebody to action more promptly proliferation and differentiation is effector cell.
Immune effector cell of the present invention can, by polypeptide of the present invention or nucleic acid are added to the cell with antigen presentation ability, then be prepared the delivery cell that is of having presented polypeptide of the present invention and HLA antigenic compound with having the cells contacting of immunological effect ability.
The antigen presenting cell of the invention described above provided by the invention can be used as vaccine or the pharmaceutical composition for the treatment of or preventing cancer.Antigen presenting cell of the present invention has immune induction activity, can be used for preparing inducing antigen-specific effector cell's reagent.Derivative CD
8 +t cell can be by the generation performance antitumor action of cytotoxic effect and lymphokine.Therefore, antigen presenting cell of the present invention can be and is used for the treatment of or the vaccine of prophylaxis of tumours or the activeconstituents of pharmaceutical composition.Comprise antigen presenting cell as activeconstituents for inducing the vaccine of CTL can comprise salt solution, phosphate buffered saline buffer (PBS), substratum etc. stably to maintain antigen presenting cell.The method of using comprises that intravenously uses.Can using comprise antigen presenting cell as activeconstituents for inducing CD
8 +the reagent of T cell returns in patient's body, therefore can be in the patient body that polypeptide of the present invention is responded effective inducing specific CD
8 +t cell, result can be treated or prophylaxis of tumours.
Immune effector cell of the present invention can be used as vaccine or the pharmaceutical composition for the treatment of or preventing cancer.Effector cell of the present invention comprises various T cells, for example CD
8 +t cell.CD of the present invention
8 +t cell can be by the generation performance antitumor action of cytotoxic effect and lymphokine.Therefore, immune effector cell of the present invention can be and is used for the treatment of or the vaccine of prophylaxis of tumours or the activeconstituents of pharmaceutical composition.Comprise antigen presenting cell and can comprise salt solution, phosphate buffered saline buffer (PBS), substratum etc. stably to maintain immune effector cell as the vaccine of the immune effector cell of activeconstituents.The method of using comprises that intravenously uses.Can return in patient's body comprising the reagent of immune effector cell as activeconstituents, result can be treated or prophylaxis of tumours.
In one aspect of the invention, the antigen presenting cell that contains the invention described above or vaccine and the pharmaceutical composition of immune effector cell are provided.At described vaccine provided by the invention and pharmaceutical composition, can be used for treatment or preventing cancer.The antigen presenting cell of the invention described above or immune effector cell can be used as vaccine and the pharmaceutical composition for the treatment of or preventing cancer.
The vaccine that contains antigen presenting cell or immune effector cell and the pharmaceutical composition of the invention described above can be used for the treatment of tumour patient by vitro method.Polypeptide of the present invention or nucleic acid can contact with antigen presenting cell and/or immune effector cell in vitro, and generation can be identified the antigen presenting cell of antigen of the present invention or antigenic complex, thus inducing specific effector cell, for example T cell (CD particularly
8 +t cell), then described antigen presenting cell and/or immune effector cell are returned in patient body for prevention or treatment cancer.In one aspect of the invention, described patient is HLA I type, is preferably HLA A2 or HLA A3 type, and more preferably HLA A2 type, most preferably is A*0201 or A*0202 type.
Immune effector cell of the present invention, for example T cell (CD particularly
8 +t cell), can be used as being used for the treatment of or the vaccine of prophylaxis of tumours or the activeconstituents of pharmaceutical composition.
The polypeptide of the invention described above or nucleic acid, and the antigen presenting cell of the invention described above or immune effector cell, can be used for prevention or treatment cancer.Described cancer comprises leukemia, solid tumor etc., more particularly, comprise lung cancer, malignant lymphoma (reticulum cell sarcoma for example, lymphosarcoma, Hodgkin's disease (Hodgkin's disease) etc.), digestion organs cancer (cancer of the stomach for example, carcinoma of gallbladder, cholangiocarcinoma, carcinoma of the pancreas, liver cancer, colorectal carcinoma, the rectum cancer etc.), mammary cancer, ovarian cancer, flesh and bone sarcoma (musculoskeletal sarcoma) (such as osteosarcoma (osteosarcoma) etc.), bladder cancer, leukemia (acute leukemia for example, comprise chronic granulocytic leukemia acute attack), kidney, prostate cancer etc., be preferably digestion organs cancer, cancer of the stomach for example, carcinoma of gallbladder, cholangiocarcinoma, carcinoma of the pancreas, liver cancer, colorectal carcinoma, the rectum cancer etc.Preferably, be liver cancer.
The present invention also provides foregoing polypeptide of the present invention or its variant, or foregoing nucleic acid of the present invention, or foregoing antigen presenting cell of the present invention, foregoing immune effector cell of the present invention for the preparation for the treatment of or the vaccine of preventing cancer or the purposes in pharmaceutical composition.Described cancer comprises leukemia, solid tumor etc., more particularly, comprise lung cancer, malignant lymphoma (reticulum cell sarcoma for example, lymphosarcoma, Hodgkin's disease (Hodgkin's disease) etc.), digestion organs cancer (cancer of the stomach for example, carcinoma of gallbladder, cholangiocarcinoma, carcinoma of the pancreas, liver cancer, colorectal carcinoma, the rectum cancer etc.), mammary cancer, ovarian cancer, flesh and bone sarcoma (musculoskeletal sarcoma) (such as osteosarcoma (osteosarcoma) etc.), bladder cancer, leukemia (acute leukemia for example, comprise chronic granulocytic leukemia acute attack), kidney, prostate cancer etc., be preferably digestion organs cancer, cancer of the stomach for example, carcinoma of gallbladder, cholangiocarcinoma, carcinoma of the pancreas, liver cancer, colorectal carcinoma, the rectum cancer etc.Preferably, be liver cancer.
Embodiment
By following examples, further illustrate the present invention, but these embodiment are not in office, where face limits the present invention.
Synthesizing of embodiment 1 antigen peptide
Artificial synthetic polypeptide MTPGTQSPFFLLLLLTVLTVVTGS(SEQ ID NO:1), be designated hereinafter simply as antigen peptide.Synthetic peptide purification to 97%.The antigen peptide of freeze-drying is dissolved in to methyl-sulphoxide (25mM), is stored in-80 ℃.
With the synthetic polypeptide FLLLLLTVLT(SEQ ID NO:2 of same method), LLLLLTVLTV(SEQ ID NO:3), LLLLTVLTVV(SEQ ID NO:4), FFLLLLLTVL(SEQ ID NO:5), GTQSPFFLLL(SEQ ID NO:6), TQSPFFLLLL(SEQ ID NO:7), respectively referred to as antigen peptide 1, antigen peptide 2, antigen peptide 3, antigen peptide 4, antigen peptide 5 and antigen peptide 6.
The preparation of embodiment 2 dendritic cell (Dendritic Cells, DCs)
Mononuclearcell in peripheral blood (PBMC) comprises lymphocyte and monocyte, and its volume, shape proportion are 1.075 left and right.The Ficoll-Hypaque parting liquid that is 1.077 with proportion carries out density gradient centrifugation, makes the cell of certain weight proportion by corresponding density Gradient distribution, can various hemocytes are in addition separated.
From Hong Kong red cross blood center, obtain healthy volunteer's white corpuscle concentrated solution, with PBS, diluted suitable multiple, then join on Ficoll-Hypaque parting liquid surface 18-20 ℃, 800g density gradient centrifugation 30min, centrifugal complete, centrifuge tube content is divided into three layers, and upper strata is blood plasma (including thrombocyte), and middle layer is parting liquid, bottom is red corpuscle and granulocyte, at PBMC layer upper, that liquid separation surface place, middle level can see oyster white muddiness.Take out PBMC layer, be resuspended in PBS, the centrifugal 10min of 400g, repeat step, cell precipitation is resuspended in the RPMI1640 that contains 10% foetal calf serum, cultivate 2 hours for 37 ℃, take out non-adherent cell, for separating of T cell, in adherent cell, add the RPMI1640 substratum containing 1000U/ml hGM-CSF and 5OOU/ml hIL-4, cultivate 1 week for 37 ℃, induction monocyte is divided into DCs, wherein within every two days, change a subculture and cytokine, gather in the crops non-absorption and the more loose cell of absorption, be DCs.
The preparation of embodiment 3T cell (the cotton method of nylon)
T cell surface fine hair is short and lack, and B cell fine hair is many and grow, and due to cell surface smooth degree difference, B cell is easily killed and invested on nylon cotton fibre in the time of 37 ℃, and T cell does not have this ability.Utilize this characteristic, separable T cell and B cell.
The preparation of nylon cotton column: after 1g nylon cotton/post sterilising treatment, add RPMI1640 substratum, place 2 hours for 37 ℃, allow RPMI1640 flow out.Above-mentioned non-adherent cell is resuspended in the RPMI1640 of proper volume, making cell concn is 0.5-1.0x10
8/ ml, then joins in prepared nylon cotton column, 2ml/ post, and 37 ℃ are incubated 1 hour, in nylon cotton column, add RPMI1640,10ml/ post, wash-out is not adsorbed onto the cell on nylon cotton, collects elutriant, is T cell suspension.The centrifugal 5min of 400g, is resuspended in cell precipitation in cells frozen storing liquid, frozen standby.
Embodiment 4T cell proliferating determining (3H-TdR mixes method)
The prerequisite of cell proliferation is tenuigenin and nuclear copying, and a general cell cycle is roughly divided into 4 periods, i.e. G1 phase, S phase, G2 phase, M phase.Wherein, the S phase is DNA synthesis phase, and major function is to carry out DNA to synthesize.3H-TdR i.e. (methyl-3H) thymidylic acid is the synthetic precursor of DNA, after being added in cell culture fluid, can, by cellular uptake, can be used as the synthetic raw material of DNA.The synthetic DNA of cell is more, and the 3H-TdR mixing is also more, detects the 3H-TdR mixing and just can reflect the degree of cell proliferation.
In the nutrient solution of DCs, add the antigen of proper concn as each antigen peptide of this experiment, 37 ℃ of incubations 4 hours, results DCs, and wash once with PBS, according to certain cell proportion, DCs from same volunteer and T cell are joined in the 96 flat culture plates in hole, cultivate altogether after 5 days for 37 ℃, add 3H-TdR, 1uCi/ hole, 37 ℃ are incubated 18 hours, utilize bull cell harvestor, by cell harvesting on glass fiber filter paper, and use successively PBS, 5% trichoroacetic acid(TCA) and absolute ethanol washing each three times, dry filter paper, filter paper is put in scintillation solution, on β liquid scintillation counter, measure cpm value.
Embodiment 5 cytotoxic T lymphocytes (CTLs) are measured (LDH method)
Serum lactic dehydrogenase (LDH) is that the endochylema of viable cell includes one of enzyme, under normal circumstances can not permeate through cell membranes, but be subject to that effector cell attacks and when impaired when target cell, and the permeability changes of cytolemma, LDH will be discharged in medium.And LDH generates in the process of pyruvic acid at catalysis lactic acid, can make to be oxidized nadide (NAD) and become dihydrocoenzyme I (NADH), the latter is again by hydrogen carrier PMS (PMS) reduction iodine nitro chlorination four oxygen azoles (INT), INT accepts H2+ and is reduced into mauve first the part between the ribs and the hips compound, this compound has a high absorption peak at 490nm place, the A490nm that utilization is read is as index, the degree that known target cell is killed and wounded by effector cell.
In the nutrient solution of DCs, add the antigen of proper concn as each antigen peptide of this experiment, 37 ℃ of incubations 4 hours, results DCs, and wash once with PBS, according to certain ratio, the DCs from same volunteer and T cell are joined in 24 well culture plates, cultivate altogether 7-10 days for 37 ℃, wherein in nutrient solution, add 20U/ml hIL-2, utilize Ficoll-Hypaque density gradient centrifugation to collect T cell with action effect cell.5mM EDTA digestion tumor cell line (for example SMMC-7721 or K562) cell is usingd as target cell.
Respectively effector cell and target cell are resuspended in the analysis substratum (containing the RPMI1640 of 1%BSA) of certain volume, in different ratios, effector cell (100ul) and target cell (100ul) are joined at the bottom of 96 hole circles in culture plate to 37 ℃ of insulations 5 hours, take out supernatant 100ul/ hole, and join in 96 hole enzyme plates, add 100ul/ hole LDH reaction solution, reflection 30min in dark place under room temperature, add 1MHCL stop buffer, 50ul/ hole, measures A490nm, and A630nm is as with reference to wavelength.Calculate cytotoxicity: cytotoxicity (%) ﹦ [(the spontaneous release aperture of the spontaneous release aperture-effector cell of fragmentation test hole-target cell)/(the spontaneous release aperture of maximum release aperture-target cell)] * 100%.
Note: killing experiments hole is effector cell+target cell; The spontaneous release aperture of target cell is target cell+analysis substratum; The spontaneous release aperture of effector cell is effector cell+analysis substratum; Maximum release aperture is target cell+2%TritionX100.
Embodiment 6ELISA-spot(ELISPOT) method is measured IFN γ
Cytokine ELISPOT method is for measuring the quantity of single cell suspension cytokine secreting cell, and its detection speed is fast, has very high sensitivity, and easy handling.Its principle is first the antibody of the antibacterial agent of high-affinity to be coated on ELISPOT plate, after cell to be measured is joined in ELISPOT plate, its secreted cytokine will be arrived by coated antibody capture, like this after removing cell suspension to be measured, the antibody that adds the another kind of antibacterial agent of mark, then add after corresponding colouring reagents, just can on ELISPOT plate, produce the spot that represents cytokine secretion quantity and position.
In the nutrient solution of DCs, add the antigen of proper concn as each antigen peptide of this experiment, 37 ℃ of incubations 4 hours, results DCs, and wash once with PBS, according to certain ratio, to add in 24 well culture plates from same volunteer DCs and T cell, cultivate altogether 7-10 days for 37 ℃, wherein in nutrient solution, add 20U/ml hIL-2, utilize Ficoll-Hypaque density gradient centrifugation and collect T cell with action effect cell, utilize 5mM EDTA digestion SMMC-7721, and process and using as target cell with gamma Rays, respectively effector cell and target cell are resuspended in the substratum of certain volume, according to a certain percentage, effector cell (100ul) and target cell (100ul) are joined in the 96 hole ELISPOT Sptting plates that have been coated with in advance IFN gamma antibodies, 37 ℃ of incubations 18 hours, remove cell suspension, PBST washes 10 times, add biotin labeled IFN gamma antibodies, 37 ℃ of incubations 2 hours, PBST washes 10 times, the antibody that adds the antibiotin of enzyme labelling, 37'C incubation 2 hours, PBST washes 10 times, add substrate solution, reaction 15-30min in dark place under room temperature, utilize ELISPOT automatic reading instrument to count.
Embodiment 7 Flow cytometries (FACS)
Prepare single cell suspension: gather in the crops cell to be measured, and with cold PBS washed cell, cell is resuspended in 50ul/ pipe PBS, adds corresponding antibody, place on ice 1 hour, with cold PBS washing 2 times, add two of FITC mark to resist, place 30min on ice, with cold PBS washing 2 times, cell is resuspended in 2% paraformaldehyde, for Flow cytometry.
Embodiment 8Western method is identified the tumor cell line with MUC1 albumen and HLA-A2 albumen
Adopt general SDS-PAGE and the Western immunoblotting in this area, be that protein is transferred on nitrocellulose after separation after unidirectional electrophoresis, then with the specific antibody of radioactivity or enzyme labelling, detect the method for the existence of corresponding antigens, identify the albumen that tumour cell contains.
Tumor cell line SMMC-7721 and SMMC-7721-0201 have been used in experiment.SMMC-7721 is the commercially available tumor cells of hepatocellular carcinoma system (referring to Lu J., Xu R.B.and Doung R.C. (1985) The glucocorticoid receptors and the induction of tyrosine aminotransferase by glucocorticoid in the human liver cancer cell line (SMMC-7721) in vitro.Shi Yan Sheng Wu Xue Bao18:231-238) that Shanghai Institute of Cell Biology provides.SMMC-7721-0201 is by the clone at SMMC-7721 transfection and stably express human HLA-0201 gene.
First use 0.25% tryptic digestion tumour cell SMMC-7721 and SMMC-7721-0201, then use 1640 perfect mediums (containing 10%FBS and 1% green grass or young crops-Streptomycin sulphate) to dispel cell, be collected in 15ml centrifuge tube.1000rpm, centrifugal 5min, abandoning supernatant.With 80 μ l PBS re-suspended cells (cell concentration is many can suitably increase PBS volume), then add 5 * SDS-PAGE loading Buffer, mix rear boiling water bath and boil 5min.
After sample preparation, carry out according to a conventional method SDS-polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) and Western immunoblot experiment.Primary antibodie in Western immunoblot experiment is respectively anti-Muc1 monoclonal antibody and anti-HLA-A2 monoclonal antibody (BB7.2 cell conditioned medium), and two resist for sheep anti-mouse igg-HRP.
Fig. 1 a and Fig. 1 b are SDS-PAGE and Western immunoblot experiment result.
Wherein the 1st swimming lane of Fig. 1 a is SMMC-7721-0201 cell strain sample, and the second swimming lane is SMMC-7721 cell strain sample.
Wherein the first swimming lane of Fig. 1 b is SMMC-7721-0201 cell strain sample, and the second swimming lane is SMMC-7721 cell strain sample.
As shown in Figure 1a, tumor cell line SMMC-7721 and SMMC-7721-0201 have expressed MUC1 albumen.
As shown in Figure 1 b, tumor cell line SMMC-7721 is that HLA-A2 is negative; SMMC-7721-0201 is that HLA-A2 is positive.
The expression of MHC I in SMMC-7721 and SMMC-7721-0201 has also been tested in experiment in addition, proves that cell strain SMMC-7721 and SMMC-7721-0201 are that MHC I is positive.
Embodiment 9 loads the DCs of each antigen peptide stimulate the propagation of T cell
Utilize the antigen peptide of preparation in embodiment 1, be MTPGTQSPFFLLLLLTVLTVVTGS(SEQ ID NO:1) stimulate DCs, then utilize the load obtain the DCs of antigen peptide stimulate self T cell, measure load the DCs of antigen peptide can stimulate the propagation of self T cell.
By 10ug/ml antigen peptide, carry out load DCs, loading condition is 37 ℃ of incubations 4 hours, then DCs carries out common cultivation from self T cell with different ratio (DCs:T=1:10 and DCs:T=1:3), after 5 days, add 3H-TdR, after 18 hours, measure the absorption of 3H-TdR, to measure the propagation situation of T cell.In contrast, T cell and the not DCs of load antigen peptide cultivate altogether, or T cell single culture.As shown in Figure 2 a, each propagation level of organizing T cell is visibly different, when DCs:T=1:10, load the propagation level of the T cell that stimulates of the DCs of antigen peptide be 10 times of T cell proliferation level that load DCs does not stimulate, and 15 times of the T cell of single culture; When DCs:T is brought up to 1:3, the propagation level of T cell also obviously raises.These experimental results show: load the DCs of antigen peptide can effectively stimulate the propagation of self T cell, the DCs of load antigen peptide can not stimulate the propagation of self T cell.
In addition, as shown in Fig. 2 b and 2c, in form, through load the T cell that stimulates of the DCs of antigen peptide be obviously different from the T cell of control group, present obvious state of activation, as cell volume increases, number increases etc.
Antigen peptide 1, antigen peptide 2, antigen peptide 3, antigen peptide 4, antigen peptide 5 and antigen peptide 6 are carried out to same test, load the DCs of antigen peptide can effectively stimulate the propagation of self T cell.
Embodiment 10 loads the DCs of each antigen peptide can activate the CTLs of tumor cell specific
In the antineoplastic immunne response of body, CTLs is bringing into play extremely important effect.Utilize in embodiment 1 antigen peptide of preparation to stimulate DCs, then utilize the load that makes the DCs of antigen peptide stimulate self T cell, measure load the DCs of antigen peptide can activate the CTLs of tumor cell specific.
By the antigen peptide of different concns, carry out load DCs, 37 ℃ of incubations 4 hours, then DCs and T cell carry out common cultivation with the ratio of DCs:T=1:5, after 7-10 days, utilize LDH method to measure T cell to expressing the lethality of the liver cancer cell SMMC-7721-0201 of MUC1 albumen.In experiment, the DCs of T cell and load antigen peptide cultivates altogether, and in contrast, T cell and the not DCs of load antigen peptide cultivate or T cell single culture altogether.As shown in Figure 3, through load the T cell that stimulates of the DCs of antigen peptide can effectively kill and wound the tumour cell of expressing MUC1 albumen, with the T cell of T cell that DCs of load antigen peptide does not cultivate altogether and single culture can not killing tumor cell.As shown in Figure 4, when increasing the concentration of antigen peptide of load DCs, the T cell stimulating through this DCs also strengthens the kill capability of tumour cell thereupon.
In order to measure effector cell, to the lethality of tumour cell, be owing to having activated the special CTLs of tumour cell rather than due to the existence of non-specific killer cell NK cell, in cytotoxic assay, utilize commercially available NK cellular sensitivity cell line k562 (ATCC.Cat.no.:CCL-243) as target cell, as shown in Figure 5, through load the T cell that stimulates of the DCs of antigen peptide can effectively kill and wound the liver cancer cell SMMC-7721 that expresses MUC1 albumen, and the SMMC-7721 derived cell of antigen peptide just.But through load the T cell that stimulates of the DCs of antigen peptide can not kill and wound K562 cell.What show killing hepatoma cell is specific CTLs rather than non-specific NK cell.
In order further to measure the specificity to liver cancer cell killing activity, in cytotoxic assay, utilized people's hepatoma cell line SMMC-7721-0201 and SMMC-7721 as target cell, in addition, in order to measure killing activity, whether be that MHC I is restrictive, antibody sealing by the MHC I on target cell surface with anti-MHC I, as described in the result of embodiment 8, wherein SMMC-7721-0201 and SMMC-7721 are that MHC I and antigen peptide are positive.As Fig. 6 experimental result shows, CTLs to the killing activity of SMMC-7721-0201 apparently higher than the killing activity to SMMC-7721, after the MHC on SMMC-7721-0201 surface I is closed, CTLs has also disappeared to its lethality, contrary, after the MHC on SMMC-7721 surface I is closed, CTLs does not but have considerable change to its lethality.This shows that CTLs is that MHC I is restricted to the lethality of SMMC-7721, thereby has illustrated the killing activity of SMMC-7721 is mediated by CTLs.
Because the cell SMMC-7721 as antigen source and the T cell of action effect cell are not from same individuality, so T cell may be allotype reaction (allogeneic response) to the killing activity of SMMC-7721.In order to get rid of the possibility of this allotype reaction, in cytotoxic assay, the utilized load DCs of antigen peptide and unstressed DCs is as target cell.As shown in Figure 7, because DCs and T cell are to come from same individuality, therefore T cell does not have killing activity to unstressed DCs, contrary, T cell to load the DCs of antigen peptide show obvious killing activity, this be due to load the DCs of antigen peptide the oncopeptide of antigen peptide institute combination carried out to processing presented, at cell surface expression tumour antigen, this shows that CTLs is to be specific to the anti-tumor immune response of tumour antigen rather than allotype reaction to the killing activity of SMMC-7721.
Above experimental result shows: antigen peptide is successfully processed by DCs and is in conjunction with tumour antigen and passs T cell, thereby has effectively activated the special CTLs of tumour cell.
Antigen peptide 1, antigen peptide 2, antigen peptide 3, antigen peptide 4, antigen peptide 5 and antigen peptide 6 are carried out to same test, and it can effectively activate the special CTLs of tumour cell.
Embodiment 11 loads the DCs of antigen peptide can stimulate T emiocytosis cytokine IFN γ
In antineoplastic immunne response, cell-mediated antitumor replying plays keying action, and wherein, the secretion of CTLs reaction and IFN γ is the principal character of cell response.
Measured load the DCs of antigen peptide can stimulate self T emiocytosis cytokine IFN γ.Press the method that embodiment 6 describes, with the antigen peptide load DCs of 40ug/ml, 37 ℃ of incubations 4 hours, then DCs and T cell carry out common cultivation with the ratio of DCs:T=1:5, after 7-10 days, the situation of mensuration T emiocytosis IFN γ.In contrast, T cell and the not DCs of load antigen peptide cultivate altogether, or T cell single culture.Self the T cell stimulating is gathered in the crops by gradient centrifugation, and IFN γ secretion is measured with ELISPOT.Self T cell and not load antigen peptide DCs cultivate altogether and self T cell self in contrast.As shown in Figure 8, through load the T cell that stimulates of the DCs of antigen peptide when running into target cell SMMC-7721, IFN γ that can secreting high levels, and the T cell of the T cell stimulating through the DCs of load antigen peptide not and single culture is when running into target cell SMMC-7721, there is no obvious IFN γ secretion.This experimental result is consistent with above-mentioned CTLs experimental result, show load the DCs of antigen peptide can activate the special T cell of tumour cell, the cytokine IFN γ that this T cell can secreting high levels.
Embodiment 12 antigen peptide stimulate DCs ripe
As mentioned above, load the DCs of antigen peptide can activate the CTLs of tumor cell specific, this shows that DCs has processed the tumour antigen of having presented the combination of antigen peptide institute, and has caused specific anti-tumor immune response.Due to the Phenotype Transition of DCs, by Immature DC s, be transformed into mature DCs, be the prerequisite that DCs plays a role, thus can release load the DCs of antigen peptide must there is Phenotype Transition.In order to detect this inference, measured the maturation that can antigen peptide stimulate DCs.
DCs after 4 hours, continues to cultivate 48 hours in 37 ℃ of incubations in the antigen peptide with 40ug/ml, then utilizes Flow Cytometry to measure the expression situation of HLA-DR, CD80, CD86, CD83 and the CD40 on DCs surface.As shown in Figure 9, after antigen peptide stimulates, the expression level of the HLA-DR on DCs surface, costimulatory molecules CD80 and CD86 and cell maturation marker molecule CD83 and CD40 obviously improves.This shows, antigen peptide can effectively stimulate DCs ripe, is the effective activation molecule of DCs.
Each experimental result shows above, and from tumour cell, each antigen peptide of separation and purification combines tumour antigen, can effectively stimulate the maturation of DCs; And load the DCs of antigen peptide can effectively process and present tumour antigen, and activate the antitumor cell immunne response of tumor cell specific, comprising stimulate T cell propagation, activate the special CTLs of tumour cell and stimulate T emiocytosis cytokine IFN γ etc.
Embodiment 13 loads the DCs of antigen peptide can activate the CTLs of tumor cell specific and effectively attack tumour cell
This experiment test the HLA phenotype specificity of each antigen peptide of the present invention.
Experiment has adopted the lethal mensuration of carrying out cytotoxic T lymphocyte purchased from the test kit of PerkinElmer (AD0116):
With 0.25% tryptic digestion target cell (tumour cell SMMC-7721 and SMMC-7721-020, wherein SMMC-7721 and SMMC-7721-0201 are MUC1 protein positive; SMMC-7721 is that HLA-A2 is negative; SMMC-7721-0201 is that HLA-A2 is positive) it is rear that with 1640 perfect mediums (containing 10%FBS and 1% green grass or young crops-Streptomycin sulphate), washing is once; Use again 1640 perfect medium re-suspended cells, adjust cell and count to 1 * 10
6cells/ml.Get 1ml cell and add 1 μ l BATDA, 37 ℃, 5%CO
2in incubator, place 15min.Then 200g, centrifugal 5min, supernatant discarded, with the PBS washing containing 2%FBS 5 times, each centrifugal 5min of 200g.Finally use 1640 perfect medium re-suspended cells, and to adjust cell count be 5 * 10
4cells/ml.
By the method described as embodiment 9, to the antigen that adds respectively proper concn in the nutrient solution of DCs, comprise that antigen peptide and antigen peptide 2 stimulate DCs, then utilize the load that obtains the DCs of described antigen peptide stimulate self T cell.
Collect the CD8+T cell having been stimulated, 1000rpm is centrifugal, abandons supernatant, with the resuspended CD8+T cell of 1640 perfect medium, adjusts cell and counts to 1 * 10
6cells/ml.
Get 100 μ l CD8+T cells and 100 μ l tumour cells (BATDA-loaded) join in 96 orifice plates.Set up blank group (substratum contrast) simultaneously, the maximum release group (10 μ l substratum+100, μ l lysate+90 μ l tumour cell) of the spontaneous release group of tumour cell (100 μ l tumour cell+100 μ l substratum) and tumour cell, T cell is set up not stimulating group.Experimental group need to be set up 3 repeating holes.
96 orifice plates are put in to 37 ℃, in 5%CO2 incubator, cultivate after 2h, the centrifugal 5min of 200g, draws 20 l supernatants (test kit carries) in Elisa plate, and adds 200 l Europium Solution, and room temperature water yawing moves 15min.Use the detecting instrument (Victor2V multilabel counter) of PerkinElmer company to detect, exciter filter (emission filter) is selected 615nm.
The calculating of experimental result:
Result is as shown in Figure 10 a and Figure 10 b.What wherein in Figure 10 a, show is to use the result of attacking tumour cell after antigen peptide load DCs, and Figure 10 b is for being used the result of attacking tumour cell after antigen peptide 2 load DCs.
Experimental result demonstration, the T cell that the DCs of Antigen (comprising antigen peptide and antigen peptide 2) stimulates can kill and wound SMMC-7721 and the SMMC-7721-0201 of MUC1 protein positive.The ability of wherein killing and wounding SMMC-7721-0201 cell (HLA-A2+, MUC1+) will be higher than SMMC-7721 cell (HLA-A2-, MUC1+).Prove that antigen peptide of the present invention more effectively attacks HLA-A2 positive cell.
Embodiment 14 experimentation on animalies: load the DCs of antigen peptide in Mice Body, suppress tumour
1. build the B16(of stably express MUC1 albumen for experimentation on animals) clone
B16 is commercially available mouse melanin tumor cell strain (ATCC CAT.NO.CRL-6322).Contain the plasmid of MUC1 gene purchased from Origene company (Cat.No.RC221390).
With 0.25% tryptic digestion B16 cell, and dispel cell with 1640 perfect mediums (containing 10%FBS and 1% green grass or young crops-Streptomycin sulphate).Then get appropriate cell and be layered in 24 orifice plates and (inoculate altogether three holes), after 36h, utilize the transfection of liposome transfection method to enter (plasmid is 0.5 μ g/ hole) in B16 cell containing the plasmid of MUC1 gene.Cell after 0.25% tryptic digestion transfection for second day, is switched in 24 all holes, continues to cultivate.Within the 3rd day, change liquid, add 1640 perfect mediums containing 1.2mg/ml G418 to continue to cultivate.
Every day observation of cell death condition, change every other day liquid; After four days, survivaling cell is stable gradually, and cell is carried out to limiting dilution, is inoculated in 96 orifice plates, and cell concentration is 0.5 cells/well (connecing 10-20 piece 96 orifice plates).Observe the upgrowth situation of cell in 96 orifice plates, find monoclonal cell, and do lower mark.To covering with the monoclonal cell of cell hole, carry out enlarged culturing, after collecting cell, adopt the cell of the foregoing Western method identification of M UC1 positive.To identifying that positive cell carries out enlarged culturing frozen.
In Figure 11, the B16 cell strain that the 1st swimming lane is untransfected, the 2nd swimming lane is MUC1-4 monoclonal cell, the 3rd swimming lane is MUC1-6 monoclonal cell.
From Figure 11 result, MUC1-4 and MUC1-6 bis-strain monoclonal cells have obvious band, test positive.So determine that MUC1-4 and MUC1-6 bis-strain monoclonal cells are the B16 clone of stably express MUC1 albumen.
2. experimentation on animals
Experiment mice (C57BL/6 mouse, 6-8 week age) is divided into two groups: treatment group and immunoprotection group.Wherein treatment group, for first giving mouse tumor cell, then gives polypeptide of the present invention (being respectively antigen peptide and antigen peptide 2).Wherein immunoprotection group, for first giving mouse polypeptide of the present invention (being respectively antigen peptide and antigen peptide 2), then gives tumour cell.
Treatment group: totally 32 of experiment mices, be divided into 4 groups, 8 every group, and be labeled as A, B, C, D.Wherein 8 injected in mice B16 cells of A group, remain 3 groups and all inject B16-MUC1-4 cell.Inoculation position is that the right armpit of forelimb is subcutaneous, every injected in mice 1 * 10
4cell, dosage is 200 μ l.Start injection polypeptide after one week, two groups of A and B be totally 16 injected in mice PBS, and two groups of C and D inject respectively two peptide species: antigen peptide and antigen peptide 2, every injected in mice 40 μ g/100 μ l.Later two weeks each groups are duplicate injection same polypeptide once more weekly, and dosage is identical, observes mouse tumor growing state every day, and record data.
Figure 12 a is that antigen peptide is at Mice Body internal therapy tumor effect.
Wherein,
B16-g:A group, injection B16 cell;
B16-mg:B group, injection B16-MUC1-4 cell;
B16-mg53:C group, injection B16-MUC1-4 cell and antigen peptide 2;
B16-mg64:D group, injection B16-MUC1-4 cell and antigen peptide;
Because two groups of B16-g and B16-mg come to the same thing, so lines are overlapping.
Figure 12 b is antigen peptide 2 immunoprophylaxis tumor effect in Mice Body.
Wherein
B16-g:E group, injection B16 cell;
B16-mg:F group, injection B16-MUC1-4 cell;
B16-mg53:G group, injection B16-MUC1-4 cell and antigen peptide 2;
B16-mg64:H group, injection B16-MUC1-4 cell and antigen peptide.
From experimental result, draw, and control group comparison, the mouse of injection polypeptide group can prolongs life over two weeks.Give the comparison between antigen peptide and antigen peptide 2, high to give the survival rate of antigen peptide group mouse.From experiment grouping relatively, in injection polypeptide group, the survival rate of immune group mouse is higher than treatment group.
Each experimental result shows above, polypeptide provided by the invention, antigen peptide and its fragment of comprising the aminoacid sequence shown in SEQ ID NO:1, comprise its continuous 8-10 amino acid, continuous 10 amino acid whose fragments (polypeptide FLLLLLTVLT for example particularly, LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL), can effectively stimulate the maturation of the DCs of tumour-specific, and load the DCs of antigen peptide can effectively process and present tumour antigen, and activate the antitumor cell immunne response of tumor cell specific, comprising the propagation that stimulates T cell, activate the special CTLs of tumour cell and stimulate T emiocytosis cytokine IFN γ etc.
Be not bound by any theory, contriver has inquired into treatment and the prophylactic effect of antigenic peptide of the present invention.Calculating householder method is becoming a kind of maturation in recent years, and the method for effective and efficient analyzing proteins or polypeptide structure and function, in being widely used in comprising the analysis of protein of analyzing epitope and having predicted.Those skilled in the art can sum up and according to the structure function research of albumen, identify for the residue in activity or the important similar polypeptide of structure, thus the effect of amino-acid residue in predicted protein matter, and then the activity of analysing protein aminoacid sequence and function.Calculate householder method and in recent years becoming a kind of comparative maturity, effective and efficient defined antigen epi-position method.Calculate auxiliary defined antigen epi-position by the arrangement of available data, analysis, according to the rule obtaining, set up epi-position activity model, by computer approach analysis and prediction, there is the combination activity of the sequence of antigenic epi-position and itself and antigen or MHC.
In general antigenicity identified region possess hydrophilic, be positioned at the feature such as dimensional instability in protein surface and structure.Because in most natural (nature) environment, hydrophilic region tends to concentrate on protein surface, and hydrophobic region is usually wrapped in active site of protein, as a same reason, antibody can only interact with the identified region of finding at protein surface, and when these identified regions have enough structure dimensional instabilities and transfer to the position that antibody can contact, will and antibody between have very high affinity.Continuous and discontinuous identified region are the identified regions consisting of continuous or discrete aminoacid sequence (residue).A lot of antibody is the epi-position for continuous identified region, and antibody capable combines with very high avidity with this class epi-position.Discontinuous identified region is that representative has certain one section of folding peptide sequence, or the identified region of the antibody that two sections of polypeptide of separating are connected together.In some cases, for the antibody of discontinuous like this identified region, also can produce, but these antigenic peptides possess the secondary structure similar to this discontinuous identified region, and the length of sequence need to meet relevant requirement.The length of epi-position is conventionally between 8-20 amino-acid residue.
Applying biological Informatics Method is analyzed, and uses Antigen Epitope Prediction website as http://www.cbs.dtu.dk/services/BepiPred/; Http:// www.epitope-informatics.com/Links.htm; Http:// bio.dfci.harvard.edu/Tools/index.html etc., and SYFPEITHI, BIMAS, IMMUNE EPITOPE, the various software such as MHC2PRED are analyzed antigen peptide of the present invention.
By above-mentioned means analysis different HLA-A,-B, the difference of the bonding force of-C and antigen peptide of the present invention or its fragment and epitope sequences varient thereof, particularly HLA A2 molecule and HLA A3 molecule, find to exist and have efficient activation CD in described antigen peptide of the present invention (SEQ ID NO:1)
8 +the epi-position of T cell, comprises its continuous 8-10 amino acid, particularly continuous 10 amino acid whose fragments (polypeptide FLLLLLTVLT for example, LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL etc.), and exist and there is efficient activation CD in described antigen peptide
4 +the epi-position of T cell, comprising: MTPGTQSPFF LLLLLTVLTV VTGS, MTPGTQSPFF LLLLL and SPFF LLLLLTVLTV VTGS etc.These epi-positions are higher with the bonding force of HLA molecule in HLA A2 (comprising A*0201, A*0202, A*0204, A*0205, A*0206, A*0207 and A*0208) and HLA A3 (comprising A*0301, A*1101, A*3101 and A*6801).
The present invention has found antigen peptide and its fragment of the aminoacid sequence shown in SEQ ID NO:1 unexpectedly, comprise its continuous 8-10 amino acid, continuous 10 amino acid whose fragments (polypeptide FLLLLLTVLT for example particularly, LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL), can effectively stimulate the maturation of the DCs of tumour-specific, and load the DCs of antigen peptide can effectively process and present tumour antigen, and activate the antitumor cell immunne response of tumor cell specific, can effectively treat or prophylaxis of tumours in animal body.Therefore, the invention provides TPA as the application of cancer vaccine.Simultaneously, the invention provides the described polypeptide DC tumor vaccine that sensitization dendritic cell obtains effectively, or the T cell tumour vaccine made from the dendritic cell ciita T cell of above-mentioned TPA sensitization, and directly apply the Preparation method and use that these polypeptide or composition are tumor vaccine.
Unless otherwise noted, practice of the present invention, by using the routine techniques of biotechnology, organic chemistry, inorganic chemistry etc., obviously, except describing especially in above-mentioned explanation and embodiment, can also other mode realize the present invention.Other aspect within the scope of the present invention will be apparent to those skilled in the art in the invention with improvement.According to instruction of the present invention, many changes and variation are feasible, so it within the scope of the present invention.All patents mentioned in this article, patent application and technical paper are all attached to herein accordingly by reference.