CN102250208A - New HLA-A2 restrictive epitope polypeptide and application thereof - Google Patents
New HLA-A2 restrictive epitope polypeptide and application thereof Download PDFInfo
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Abstract
The invention provides a new HLA-A2 restrictive epitope polypeptide and its application thereof. In particular, the invention relates to the HLA-A2 restrictive epitope polypeptide (i.e. 1Y3W6L peptide) with a sequence of YLWCQLLEV, its epitope and its relevant recombinant protein, coded nucleotide sequence, antigen presenting cell, as well as the application of its composition in preventing and treating tumors with the expression of human phosphatidylethanolamine (PE)-bindingprotein 4 (hPEBP4).
Description
Technical field
The present invention relates to biology and medical field, relate more specifically to a kind of HLA-A2 restriction epi polypeptide, and this epi-position and relevant recombinant protein, coding nucleotide sequence, antigen presenting cell, the purposes of composition in (hPEBP4 albumen) oncotherapy of expressing human phosphotidylethanolabinding binding protein and prevention thereof.
Background technology
(human phosphatidylethanolamine (PE)-bindingprotein 4, hPEBP4), its nucleotide sequence and amino acid can be referring to Chinese patent CN02136556.3 for human phosphotidylethanolabinding binding protein 4.It is by supporting acquisition marrow stromal cell (Bone marrowstromal cell from external former being commissioned to train of normal adult medullary cell, BMSC) and make up BMSC cDNA library, utilize the means of cDNA library large scale sequencing, a kind of full-length gene that from BMSC cDNA library, is separated to, belong to phosphotidylethanolabinding binding protein (PEBP) family, login (sequence number is AY037148) in the GenBank/EMBL database.HPEBP4 albumen comprises 227 amino acid, and its aminoacid sequence is that typical phosphatidylethanolamine is in conjunction with conservative region (PBP) in the 75-195 position.
Functional study finds that hPEBP4 can turn up by inhibition Ras/Raf/MEK/ERK approach, JNK activation and PE and suppress TNF α inductive apoptosis, and these functions mediate in conjunction with conservative region (PBP) by its PE.Can promote TNF-α inductive breast cancer cell, prostate cancer cell and ovarian cellular apoptosis behind the hPEBP4 down-regulated expression, prompting hPEBP4 is likely a new action target spot of treatment hPEBP4 high expression level tumour.
The investigator confirms by organization chip and immunohistochemical methods, and in the clinical tumor sample, hPEBP4 albumen high expression level and only is expressed in 4% the normal galactophore tissue in the breast cancer tumour tissue more than 50%.Also find the proteic predominant expression pattern of hPEBP4 in human prostate cancerous tissue and ovarian cancer tissue, prompting hPEBP4 is likely that a relative specificity is expressed in the tumour-specific/tumor associated antigen of tumor tissues.
Therefore,, body is carried out immunity, body is produced at the proteic specific immune response of hPEBP4, thereby make body can suppress, remove tumour cell effectively, for the prevention and the treatment of related neoplasms provides measure if be target with hPEBP4.
In view of the above, the inventor has identified the tumour antigen epitope peptide p40-48 (TLFCQGLEV) (SEQ ID NO:1) from hPEBP4 early stage, and its aminoacid sequence is seen Chinese patent application CN200510030532.0.This epitope peptide is at HLA-A2.1/K
bIn the transgenic mouse body and in the positive breast cancer disease human peripheral of HLA-A2.1 stronger immunogenicity is arranged, its effector cell who induces can express hPEBP4 by specific killing
+/ HLA-A2.1
+Tumour cell.
Cellular immunization plays a crucial role in antitumor and antiviral immunity.The antigen of T cell recognition is and the MHC-I class or the II quasi-molecule bonded peptide of cell surface that its length is about 8-12 amino acid.And then discern and MHC-I quasi-molecule bonded endogenous peptide as the main effects cell CTL (cytotoxic T cell, Cytotoxic TLymphocytes) of killing tumor cell.But, the immunogenicity of the many natural tumor antigen peptides that can be discerned by CTL all relatively a little less than.Immunogenic power is relevant with the height of peptide and MHC-I quasi-molecule avidity.To a certain extent, avidity is high more, and its immunogenicity is strong more.
In order to strengthen the immunogenicity of peptide, can replace to improve, to optimize the avidity of itself and MHC-I quasi-molecule the amino acid in some site in the peptide sequence.Through behind the amino-acid substitution, should keep the antigen-specific specific recognition capability of wild type peptide (promptly to) of epitope peptide not change, but strengthen its immunogenicity (being the kill capability of inductor internal specific CTL).Antigen peptide behind the amino-acid substitution has been used to cancer patient, to have improved patient's anti-tumor immune response.
Yet, because the subtle change of polypeptide amino acid inside will cause tremendous influence to its 26S Proteasome Structure and Function, make polypeptide lose original activity (for example binding affinity) even, and to amino acid whose modification in the peptide sequence with shift gears and make up numerous and complicated mixed and disorderlyly, from numerous modifications, screen and obtain modified polypeptide not a duck soup with enhanced activity (for example immunocompetence).
Therefore, still press in this area on the basis of the sequence of known epitope polypeptide, to develop and have the immunogenic modification epitope polypeptide of enhanced.
Summary of the invention
Purpose of the present invention just provides a kind of synthetic peptide and application thereof with high immunogenicity.
In a first aspect of the present invention, a kind of HLA-A2 restriction epi polypeptide is provided, described polypeptide has the inducing cytotoxic T cell killing activity, and described polypeptide has the sequence of YLWCQLLEV (SEQ IDNO:2).
In an embodiment of the invention, the sequence of described polypeptide is YLWCQLLEV (SEQ IDNO:2).
In a second aspect of the present invention, a kind of recombinant protein is provided, described recombinant protein contains foregoing HLA-A2 restriction epi polypeptide.
In a preference, the sequence of described recombinant protein is shown in SEQ ID NO:2.
In a third aspect of the present invention, relate to a kind of antigen presenting cell of sensitization, described antigen presenting cell is by foregoing HLA-A2 restriction epi polypeptide sensitization.
In a preference, described antigen presenting cell is selected from down group: dendritic cell, scavenger cell, B cell, inoblast or endotheliocyte.
In a fourth aspect of the present invention, a kind of method for preparing the antigen presenting cell of sensitization is provided, described method comprises: with HLA-A2 restriction epi polypeptide sensitization antigen presenting cell of the present invention.
In a preference, described antigen presenting cell is selected from down group: dendritic cell, scavenger cell, B cell, inoblast or endotheliocyte.
In a fifth aspect of the present invention, a kind of composition is provided, described composition contains:
(a) antigen presenting cell of foregoing HLA-A2 restriction epi polypeptide of 0.001-99.99wt% or foregoing sensitization; With
(b) immunology or pharmaceutically acceptable carrier, thinner or vehicle.
In an embodiment of the invention, described composition is a pharmaceutical composition, and described carrier, thinner or vehicle are pharmaceutically acceptable carrier, thinner or vehicle.
In a preference, the content of described component (a) is 0.001-99.99wt%, preferred 0.1-99.0wt%, more preferably 1-90wt%.
In another preference, described composition also comprises the other medicines of one or more treatment tumours, preferred described medicine is selected from: alkylating agent, antimetabolite, antitumor antibiotics, plant kind anti-cancer drugs, hormone or immunological reagent, more preferably: antitumor antibiotics, as Zorubicin.
In a sixth aspect of the present invention, the purposes of the antigen presenting cell of a kind of usefulness HLA-A2 restriction epi polypeptide of the present invention or sensitization is provided, it is used to prepare the medicine that prevents and/or treats tumour.
In an embodiment of the invention, described tumour is selected from down group: prostate cancer, mammary cancer, liver cancer, glioma, colorectal carcinoma, cervical cancer, nonsmall-cell lung cancer, lung cancer, carcinoma of the pancreas, cancer of the stomach or bladder cancer or ovarian cancer.
In a seventh aspect of the present invention, the purposes of HLA-A2 restriction epi polypeptide of the present invention is provided, it is used to prepare the recombinant protein (as fusion rotein) that comprises this restriction epi polypeptide, or is used to prepare the antigen presenting cell of sensitization.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Below in conjunction with accompanying drawing the present invention is further set forth.
Fig. 1: induce HLA-A2.1/K in dendritic cell (DC) body of 1Y3W6L peptide sensitization
bThe killing activity of transgenic mice cytotoxic T cell.
Fig. 2: induce HLA-A2.1/K in dendritic cell (DC) body of 1Y3W6L peptide sensitization
bTransgenic mice cytotoxic T cell IFN-γ secretory cell situation.
Embodiment
The inventor analyzes at the epitope peptide P40-48 to hPEBP4 source on the basis of (for example computer simulation analysis etc.), by the amino acid of epitope peptide P40-48 is replaced, optionally synthesized numerous candidate antigens peptides, combined and can induce body to produce the antigen peptide that stronger specific CTL is replied in the hope of therefrom filtering out with the HLA-A*0201 high-affinity.
Particularly, the contriver has obtained possible candidate sequence by computer simulation analysis at first.Yet, hundreds of at least of the quantity of the candidate's polypeptide that obtains by the computer simulation forecasting institute, thousands of at most.And, the contriver is by evidence: at the different positions of known array even comprise the part same position and carry out metathetical computer simulation analysis candidate epitope peptide, its immunogenicity is compared with wild-type and may effectively do not improved, even obviously reduce on the contrary, this has just pointed out the higher uncertainty of computer simulation and forecast in the living things system of complexity.
Therefore, be from quantity so numerous and biological effect have the peptide sequence not a duck soup that screening obtains high-affinity, high immunogenicity higher probabilistic candidate's polypeptide, can induce stronger hPEBP4 specificity cellular immunity response.
The contriver further passes through the T2 peptide in conjunction with experiment, filter out the epitope peptide that has strong affinity with HLA-A*0201 in the multisequencing of comforming, and its immunogenicity estimated, finally find that the epitope peptide 1Y3W6L after displacement is optimized is the t cell epitope of the restrictive hPEBP4 antigen-specific of HLA-A0201, it all can induce the cytotoxic T effector cell who obviously is better than wildtype peptide (P40-48) in vivo, outward.
Improve and the constant amino acid modified peptide of antigen-specific as immunogenicity, epitope peptide 1Y3W6L has the potential using value in the immunotherapy of the tumour of mammary cancer or other high expression levels hPEBP4.Simultaneously also the tumor vaccine of efficient provides new experimental basis in order to prepare more, and the development of its tumor vaccine and treatment preparation is had important meaning.
On this basis, the inventor has finished the present invention.
The HLA-A2 restriction epi polypeptide
As used herein, " polypeptide of the present invention ", " 1Y3W6L peptide ", " metathetical specific polypeptide " etc. are used interchangeably, the 1st amino acids residue of the aminoacid sequence P40-48 of the HLA-A2 restriction epi polypeptide that finger will have been differentiated (SEQ ID NO:1) is replaced into " Y ", the 3rd amino acids residue and is replaced into the specific polypeptide that " W " and the 6th amino acids residue are replaced into " L " gained, this peptide sequence is gone into shown in the SEQ ID NO:2, i.e. YLWCQLLEV.
In addition, this term also is included in its C-terminal and/or N-terminal interpolation one or several (being generally in 20, preferably is in 10, more preferably is in 5) formed sequences of amino acid.For example, in the art, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.
A kind of polypeptide of preferably deriving is 1 or 2 or 3 or 4 amino acid (as D, ED, DED or LDED) of 36-39 position in the upstream of 1Y3W6L peptide is added corresponding to hPEBP4, and/or in its downstream is added corresponding to hPEBP4 1 or 2 or 3 or 4 amino acid (as F, FY, FYP or FYPE) of 49-52 position.
Polypeptide of the present invention can be used the ordinary method synthetic, also available recombination method production.
Polypeptide of the present invention also can be used for the albumen coupling with the BSA equimolecular quantity, thereby forms the polypeptide conjugate.Usually, described conjugate is made of polypeptide, linking agent and BSA, the preferred glutaraldehyde of wherein said linking agent, EDAC.
Composition
Polypeptide of the present invention and conjugate also can be used for preparing curative pharmaceutical composition or preventative and curative vaccine composition.
Therefore, on the other hand, the present invention also provides a kind of composition, and it contains polypeptide of the present invention, conjugate or its composition of (a) safe and effective amount; And (b) pharmaceutically acceptable carrier or vehicle.The quantity of polypeptide of the present invention is generally 10 micrograms-100 milligram/agent, preferably is 100-1000 microgram/agent.
Term used herein " significant quantity " refers to the therapeutical agent treatment, alleviates or prevent the amount of target disease or situation, or shows the amount of detectable treatment or preventive effect.Depend on the nature and extent of the build of this object and healthy state, illness and the therapeutical agent selecting to give and/or the combination of therapeutical agent for the accurate significant quantity of a certain object.Therefore, specifying accurately in advance, significant quantity is useless.Yet, for certain given situation, can determine this significant quantity with normal experiment, the clinicist can judge.
For the purposes of the present invention, effective dosage is for giving individual about 0.01 mg/kg to 50 mg/kg, the preferably polypeptide of the present invention of 0.05 mg/kg to 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
Pharmaceutical composition also can contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration.This term refers to like this some medicament carriers: they itself do not induce generation to accepting the individual deleterious antibody of said composition, and do not have undue toxicity after the administration.These carriers are well known to those of ordinary skill in the art.
(Mack Pub.Co. can find discussing fully about pharmaceutically acceptable vehicle in N.J.1991) at Remington ' s Pharmaceutical Sciences.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol, adjuvant and combination thereof.
Acceptable carrier can contain liquid on the therapeutic composition Chinese materia medica, as water, salt solution, glycerine and ethanol.In addition, also may there be complementary material in these carriers, as wetting agent or emulsifying agent, pH buffer substance etc.In addition, can also contain immunological adjuvant in the immune composition.
Usually, therapeutic composition can be made injectable agent, for example liquor or suspension; Also can be made into before injection, be fit to allocate in solution or the suspension, the solid form of liquid vehicle.
In case be made into composition of the present invention, can directly give object with it.The object of waiting to prevent or treating can be an animal; Especially people.
Treatment or the prophylactic medicament (comprising vaccine) that contains polypeptide of the present invention of the present invention can oral administration, mode such as subcutaneous, intracutaneous, intravenous injection uses.The therapeutic dose scheme can be single agent scheme or multi-agent scheme.
Advantage of the present invention
Major advantage of the present invention is: epitope peptide of the present invention is to get through amino-acid substitution on the basis of the restrictive cytotoxic T cell epitope peptide of known HLA-A2, it is compared with known epitope peptide, can more effectively cause immunne response at tumour cell, this is not only significant to further further investigation tumor invasion mechanism, and the development of stronger effective tumor therapeutic vaccine and treatment preparation is all had important meaning.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Unless otherwise indicated, otherwise per-cent and umber calculate by weight.Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The screening of embodiment 1.HLA-A*0201 high-affinity peptide
Present embodiment is selected the epitope peptide that has high-affinity with HLA-A*0201 by peptide in conjunction with testing sieve.
Testing sequence
At first, collect T2 cell (a kind of cell that lacks the antigen working ability is bought from ATCC:CRL-1991), after giving a baby a bath on the third day after its birth time with serum-free 1640 substratum (available from Invitrogen company), accent cell concn to 2 * 10
5Individual/ml, be laid in 24 orifice plates 0.5ml/ hole.Then, add candidate's polypeptide of 50 μ M, the β2Wei Qiudanbai of 2.5 μ g/ml is (available from R﹠amp; D company) in 37 ℃, 5%CO
2Hatch 18h in the incubator altogether.Cell after hatching is with icing PBS (pH7.2, PBS is a damping fluid, autogamy) gives a baby a bath on the third day after its birth time, specific mAb BB7.2 (the Sterotec Ltd of HLA-A2 that adds the FITC mark, Oxford, UK), ice bath 45 minutes, PBS are washed the back and are detected average fluorescent strength with flow cytometer (the Beckton Dickinson FACSCalibur of company).
As positive control, the simple T2 cell that not adding peptide stimulates contrasts as a setting with positive known peptide CEA HLA-A2 restriction epi polypeptide CAP-1 (SEQ ID NO:9, sequence is YLSGANLNL).
Detection method
The situation that combines of immunofluorescence technique detection of peptides and HLA-A*0201 molecule, the expression amount that this method can make its surperficial MHC-I quasi-molecule based on exogenous polypeptid and combining of T2 cell surface MHC-I quasi-molecule increases, both are in conjunction with firm more, expression amount that then can detected MHC-I quasi-molecule is many more, with the average fluorescent strength is to detect index.The result with fluorescence coefficient (FI) as measurement index.
Wherein, the FI of polypeptide＞1 is considered to the epi-position of high-affinity.
Test-results
The T2-HLA-A*0201 bonded avidity result of the hPEBP4 dietary protein origin polypeptide that immunofluorescence technique records is as shown in table 1.Polypeptide shown in the table is to analyze the part candidate sequence that (by NIH's bioinformation and analysis of molecules portion (BIMAS) hla peptide in conjunction with prediction website http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html) obtains by computer software simulation, by the biochemical company limited of Shanghai gill synthetic.
The inventor filters out the epi-position 1Y3W6L of HLA-A2 high-affinity from several optimization peptides that displacement obtains based on epi-position p40-48, its sequence is YLWCQLLEV (SEQ ID NO:2).This epitope peptide significantly improves than wild-type epitope peptide p40-48 (SEQ ID NO:1) with the avidity of HLA-A*0201 molecule.
The T2-HLA-A*0201 bonded avidity of table 1hPEBP4 dietary protein origin polypeptide
Embodiment 2.HLA-A2.1/K
bInducing of the restricted polypeptid specificity cytotoxic T cell of HLA-A2 of originating at hPEBP4 in the transgenic mice body
Effector cell's preparation
According to a conventional method (with reference to the Inaba laboratory method, [Inaba, K. etc., J Exp Med.1992; 176:1693-1702]) preparation HLA-A2.1/K
bThe dendritic cell of transgenic mice derived from bone marrow (DC).Collect to cultivate (, the same) to the 7th day DC, transfer cell concn to 1 * 10 with reference to the Inaba laboratory method
6Individual cell/ml adds epitope peptide (each epitope peptide in embodiment 1 table 1, final concentration 10 μ M/ml) and β2Wei Qiudanbai (final concentration 3 μ g/ml), and 37 ℃, 5%CO
2Hatch 3h in the incubator.
Collect the DC of peptide sensitization, give a baby a bath on the third day after its birth time, transfer cell concn to 1 * 10 with PBS (pH7.2, PBS are damping fluid, autogamy)
6Individual/0.2ml (immune consumption is 0.2ml) immune transgenic mouse (HLA-A2.1/K
bTransgenic mice, female age in 4-6 week available from U.S. JaxMice, 5 every group).Every mouse peritoneal injection 0.2ml, immunity is three times altogether, at interval a week.
Back 7 days of last immunity, mouse spleen is won in aseptic technique, and lysed erythrocyte is made single cell suspension.With splenocyte suspension (5 * 10
6Individual/ml) with the warp of peptide sensitization
60Co gammairradiation (total dose is 30Gy) place RPMI 1640 perfect mediums to cultivate from body (being same mouse) dendritic cell with 10: 1 ratios (quantity than).Cultivate after 6 days, collect the effector cell.
Detection method
(1) the active detection of specific killing
Present embodiment has adopted 4 hours of standard
51Cr release test detection specificity killing activity.
Use respectively load P 40-48WT epitope peptide T2 cell, load irrelevant peptide SSp-1 (irrelevant contrast, SEQ ID NO:10) T2 cell and the T2 cell that do not add any polypeptide load add as target cell (promptly as the effector cell of embodiment 2 preparations target cell)
51Cr (Na
2 51CrO
4, 100 μ Ci/10
6Individual cell), put in 37 ℃ of water-baths mark 90 minutes, the 15 minutes light mixings in every interval once.PBS washes 3 times, thoroughly the remaining Na of flush away
2 51CrO
4, it is 1 * 10 that the target cell that mark is good is adjusted cell concn with complete 1640 substratum
5Individual cell/ml adds 96 hole circle base plates, every hole 100 μ l.
Added effector cells by 50: 1,25: 1 with 12.5: 1 (quantity ratio) three different effect targets ratios, hatched 4 hours for 37 ℃, collect each 100 μ l of each hole supernatant, detect cpm value (countsper minute count per minute) with γ calculating instrument (Wallac company 1470).
Be calculated as follows specificity cleavage rate or kill rate:
Wherein, to organize each hole be that independent target cell adds 100 μ l 1%SDS in maximum release; Spontaneous release aperture is that independent target cell adds 100 μ l perfect mediums.
(2) detection of peptide specific CTL IFN-γ release
Carrying out ELISPOT with business-like test kit detects (available from R﹠amp; D company).With 1 * 10
5Individual effector cell's adding is coated with in the flat nitrocellulose plate in 96 holes of the anti-mouse IFN-of specificity γ monoclonal antibody, adds the T2 cell (1 * 10 that P40-48 wild-type epitope peptide stimulates simultaneously
4Individual), the T2 cell that stimulates of irrelevant peptide SSp-1 or the T2 cell that does not stimulate, cultivated 24 hours in 37 ℃ of 5% carbonic acid gas incubator.Remove cell, wash plate and dry for 4 times, add biotinylated anti-mouse IFN-γ specific antibody, 4 ℃ of overnight incubation; Wash plate and dry for 4 times, add the streptavidin of alkali phosphatase enzyme mark again, room temperature effect 2 hours; Wash plate and dry for 4 times, add substrate 5-bromo-4-chloro-3-indoles phosphoric acid/nitroblue tetrazolium(NBT) (BCIP/NBT), room temperature lucifuge effect 1h discards colour developing liquid, and distilled water cleans, termination reaction.The method of reference reagent box specification sheets detects the T cell colony of secretion of gamma-IFN.
More than each detects and all to establish 3 multiple holes, get the average in 3 multiple holes during counting.This numerical value shows the detected specific CTL quantity that discharges IFN-γ.
Test-results
The result shows, after optimizing the dendritic cell vaccination mouse of epitope peptide 1Y3W6L sensitization, the CTL of generation can effectively kill and wound the T2 cell of load wild-type epitope peptide, and the effect of killing and wounding target cell of optimization epitope peptide 1Y3W6L (is seen Fig. 1 apparently higher than the wild-type epitope peptide, *, P＜0.01).
Optimize and detect effector cell (CTL) quantity of tangible IFN-γ release after epitope peptide 1Y3W6L stimulates also apparently higher than wild-type epitope peptide (seeing Fig. 2, *, P＜0.01).
Above result shows: optimizing the ability (being immunogenicity) that epitope peptide 1Y3W6L induces specific recognition wild-type epitope peptide CTL in vivo will be apparently higher than the wild-type epitope peptide.
This test is to kill and wound target cell with the effector cell, target cell is the T2 cell of load wild-type epitope peptide, and the effector cell is the killer T cell (being present in the splenocyte) that the transgenic mice source dendritic cell body internal stimulus of the polypeptide sensitization of preparation among the embodiment 1 obtains.
The dendritic cell of optimizing epitope peptide 1Y3W6L sensitization stimulates and obtains the T2 cell that the T cell can the specific killing load has the wild-type epitope peptide, epitope peptide 1Y3W6L is optimized in this expression can more effectively induce the specific killer T cell of hPEBP4 wild-type epitope peptide than wild-type epitope peptide, thereby proved and optimized epitope peptide 1Y3W6L, can induce stronger hPEBP4 specificity cellular immunity response having kept having higher immunogenicity on the antigen-specific basis of invariable.
In addition, The above results also shows: different positions (as SEQ ID NOs:3-8) in addition comprise part same position displacement (as the polypeptide of SEQ ID NO:3) other optimize epitope peptide, its immunogenicity is not compared with wild-type and is effectively improved, even obviously reduce on the contrary, this has just pointed out, and the computer simulation and forecast has higher uncertainty in the living things system of complexity.
And, can obtain hundreds of at least of the quantity of candidate's polypeptide by computer simulation prediction, thousands of at most, be from quantity so numerous and biological effect have the peptide sequence not a duck soup that screening obtains high-affinity, high immunogenicity higher probabilistic candidate's polypeptide, can induce stronger hPEBP4 specificity cellular immunity response.
The originate cultivation of DC of the human peripheral blood mononuclear cell that embodiment 3. optimizes epitope peptide 1Y3W6L sensitization
(Ficoll-Histopaque 1.077 by lymphocyte separation medium for HLA-A2.1+/hPEBP4+ mammary cancer patient anti-freezing periphery whole blood, available from Sigma company) density gradient centrifugation (room temperature, 400 * g, 30 minutes), get the interface cell, insert in the 50ml centrifuge tube, with no calcium magnesium PBS (pH7.2)-EDTA (2mM) suspension cell, centrifugal afterwards (300 * g, 10 minutes) wash cell once, abandon supernatant, no calcium magnesium PBS re-suspended cell, recentrifuge (200 * g, 5 minutes) is washed cell 2 times, obtains human peripheral blood single nucleus cell (PBMC).
With perfect medium (RPMI1640 that contains 10% foetal calf serum) suspension PBMC, by 1 * 10
7Cells/well is laid on 6 orifice plates, 37 ℃, 5%CO
2After hatching 2 hours, the sucking-off suspension cell is carefully washed 6 orifice plates three times (this part cell is frozen standby together with the suspension cell of sucking-off) with the substratum of pre-temperature gently, and remaining cell is adherent monocyte.Attached cell is recombinated rhIL-4 (10ng/ml) (available from R﹠amp containing recombinate rhGM-CSF (500U/ml) and people of people; D company) 37 ℃, 5%CO in the perfect medium
2Cultivate.The 3rd day additional perfect medium continues to cultivate.
Collect the above-mentioned DC that is cultured to the 6th day hPEBP4+/HLA-A*0201+ patient with breast cancer peripheral blood lymphocytes source, it is 2 * 10 that personnel selection DC perfect medium (RPMI 1640 perfect mediums, 500U/mlrhGM-CSF, 10ng/ml rhIL-4) is adjusted cell concn
5Individual cell/ml concentration, 1ml/ hole branch is gone into 24 orifice plates, adds 20 μ M and optimizes epitope peptide 1Y3W6L, collecting cell after 4 hours is abandoned the substratum supernatant, with twice in RPMI1640 substratum centrifuge washing cell to remove the stimulator that exists in the former substratum, at last with 2 * 10
5Individual cell suspension is the human peripheral blood mononuclear cell who the optimizes epitope peptide 1Y3W6L sensitization DC that originates in 0.5ml people DC perfect medium.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Second Military Medical University, PLA
＜120〉a kind of new HLA-A2 restriction epi polypeptide and application thereof
<130>102223
<160>10
<170>PatentIn?version?3.3
<210>1
<211>9
<212>PRT
＜213〉artificial sequence
<400>1
Thr?Leu?Phe?Cys?Gln?Gly?Leu?Glu?Val
1 5
<210>2
<211>9
<212>PRT
＜213〉artificial sequence
<400>2
Tyr?Leu?Trp?Cys?Gln?Leu?Leu?Glu?Val
1 5
<210>3
<211>9
<212>PRT
＜213〉artificial sequence
<400>3
Tyr?Leu?Trp?Cys?6ln?Ile?Leu?Glu?Val
1 5
<210>4
<211>9
<212>PRT
＜213〉artificial sequence
<400>4
Tyr?Leu?Trp?Cys?Gln?Gly?Phe?Glu?Val
1 5
<210>5
<211>9
<212>PRT
＜213〉artificial sequence
<400>5
Tyr?Leu?Phe?Cys?Gln?Leu?Phe?Glu?Val
1 5
<210>6
<211>9
<212>PRT
＜213〉artificial sequence
<400>6
Tyr?Leu?Phe?Cys?Gln?Gly?Leu?Glu?Val
1 5
<210>7
<211>9
<212>PRT
＜213〉artificial sequence
<400>7
Tyr?Leu?Phe?Cys?Gln?Gly?Tyr?Glu?Val
1 5
<210>8
<211>9
<212>PRT
＜213〉artificial sequence
<400>8
Tyr?Leu?Phe?Cys?Gln?Gly?Phe?Glu?Val
1 5
<210>9
<211>9
<212>PRT
＜213〉artificial sequence
<400>9
Tyr?Leu?Ser?Gly?Ala?Asn?Leu?Asn?Leu
1 5
<210>10
<211>9
<212>PRT
＜213〉artificial sequence
<400>10
Arg?Leu?Asn?Glu?Val?Ala?Lys?Asn?Leu
1 5
Claims (10)
1. a HLA-A2 restriction epi polypeptide is characterized in that, described polypeptide has the inducing cytotoxic T cell killing activity, and the sequence of described polypeptide is YLWCQLLEV shown in SEQ ID NO:2.
2. a recombinant protein is characterized in that, it comprises the described HLA-A2 restriction epi polypeptide of claim 1.
3. the antigen presenting cell of a sensitization is characterized in that, described antigen presenting cell is by the described HLA-A2 restriction epi polypeptide of claim 1 sensitization.
4. antigen presenting cell as claimed in claim 3 is characterized in that, described antigen presenting cell is selected from down group: dendritic cell, scavenger cell, B cell, inoblast or endotheliocyte.
5. method for preparing the antigen presenting cell of sensitization, described method comprises: with the described HLA-A2 restriction epi polypeptide of claim 1 sensitization antigen presenting cell.
6. method as claimed in claim 5 is characterized in that, described antigen presenting cell is selected from down group: dendritic cell, scavenger cell, B cell, inoblast or endotheliocyte.
7. composition is characterized in that it contains:
(a) antigen presenting cell of described HLA-A2 restriction epi polypeptide of 0.001-99.99wt% claim 1 or the described sensitization of claim 3; With
(b) acceptable carrier, thinner or vehicle.
8. composition as claimed in claim 7 is characterized in that described composition is a pharmaceutical composition, and described carrier, thinner or vehicle are pharmaceutically acceptable carrier, thinner or vehicle.
9. the purposes of the antigen presenting cell of described HLA-A2 restriction epi polypeptide of claim 1 or the described sensitization of claim 3 is characterized in that, is used to prepare the medicine that prevents and/or treats tumour.
10. purposes as claimed in claim 9 is characterized in that, described tumour is selected from down group: prostate cancer, mammary cancer, liver cancer, glioma, colorectal carcinoma, cervical cancer, nonsmall-cell lung cancer, lung cancer, carcinoma of the pancreas, cancer of the stomach or bladder cancer.
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| CN2010101770250A CN102250208A (en) | 2010-05-18 | 2010-05-18 | New HLA-A2 restrictive epitope polypeptide and application thereof |
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| CN2010101770250A CN102250208A (en) | 2010-05-18 | 2010-05-18 | New HLA-A2 restrictive epitope polypeptide and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104761636A (en) * | 2015-04-14 | 2015-07-08 | 孙伟红 | HLA-A33 restricted eEF2 epitope polypeptide and application thereof |
| CN106645677A (en) * | 2016-11-15 | 2017-05-10 | 恒瑞源正（深圳）生物科技有限公司 | Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine |
| CN111978375A (en) * | 2020-08-28 | 2020-11-24 | 深圳市乐土生物医药有限公司 | Protein or polypeptide having cytotoxic T cell-inducing ability |
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| CN1948333A (en) * | 2005-10-14 | 2007-04-18 | 中国人民解放军第二军医大学 | New hPEBP4 protein source HLA-A2 limiting epi-polypeptide and its application |
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| CN1948333A (en) * | 2005-10-14 | 2007-04-18 | 中国人民解放军第二军医大学 | New hPEBP4 protein source HLA-A2 limiting epi-polypeptide and its application |
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| 《中国博士学位论文全文数据库（医药卫生科技辑）》 20060913 孙伟红 人磷脂酰乙醇胺结合蛋白4（hPEBP4）的HLA-A0201限制性CD8CTL表位的鉴定及其功能研究 摘要 1-10 , 第9期 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104761636A (en) * | 2015-04-14 | 2015-07-08 | 孙伟红 | HLA-A33 restricted eEF2 epitope polypeptide and application thereof |
| CN104761636B (en) * | 2015-04-14 | 2018-04-27 | 青岛市中心医院 | A kind of restricted eEF2 epitope polypeptides of HLA-A33 and its application |
| CN106645677A (en) * | 2016-11-15 | 2017-05-10 | 恒瑞源正（深圳）生物科技有限公司 | Method and kit for in vitro detection of tumor neoantigen specificity T cells and tumor vaccine |
| CN106645677B (en) * | 2016-11-15 | 2019-08-09 | 恒瑞源正（上海）生物科技有限公司 | Method, kit and the tumor vaccine of vitro detection tumor neogenetic T cells with antigenic specificity |
| CN111978375A (en) * | 2020-08-28 | 2020-11-24 | 深圳市乐土生物医药有限公司 | Protein or polypeptide having cytotoxic T cell-inducing ability |
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