CN101066990B - Antitumor CTL epitope peptide possessing population broad spectrum property - Google Patents
Antitumor CTL epitope peptide possessing population broad spectrum property Download PDFInfo
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- CN101066990B CN101066990B CN200710054307XA CN200710054307A CN101066990B CN 101066990 B CN101066990 B CN 101066990B CN 200710054307X A CN200710054307X A CN 200710054307XA CN 200710054307 A CN200710054307 A CN 200710054307A CN 101066990 B CN101066990 B CN 101066990B
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Abstract
The present invention discloses one kind of antitumor CTL epitope peptide comprising nine natural amino acid residues in the sequence of Ala-Leu-Tyr-Gly-Asp-Ile-Asp-Ala-Val (ALYGDIDAV). The antitumor CTL epitope peptide has COX-2 as the target antigen, and amino acid sequence quoted from GENEBANK. The present invention has the advantages of no limitation of MHC polymorphism and capacity of being submitted simultaneously by HLA-A2 and HLA-A3, effectiveness on epithelium tumors, and effectiveness on other tumor expressing COX-2.
Description
Technical field
The present invention relates to tumor vaccine, especially relate to a kind of antitumor CTL epitope peptide with population broad spectrum property.
Background technology
Progress along with tumour molecular immunology and molecular biology research, cytotoxic T cell (cytotoxic Tlymphocyte, CTL) Jie Dao specific cellular immunity becomes main antitumor mechanism, development tumour corresponding C TL epitope peptide vaccine, be a kind of approach of tumour-specific immunotherapy, seek the important prerequisite that effective advantage CTL epi-position becomes tumour-specific immunotherapy and therapeutical peptide vaccine research.Yet single HLA (human leucocyte antigen) restricted CTL epitope is subjected to the restriction of MHC (histocompatibility complex) polymorphism, and the tumour cell of not expressing this equipotential gene HLA molecule is lacked immunocompetence.Owing to the section H LA allelotrope down-regulated expression of malignant tumour, immune evasion takes place and loses immunocompetence in single HLA restricted epitope easily again.Therefore, explore HLA multiple alleles restricted CTL epitope, development not only can enlarge coverage rate but also can reduce the immune evasion risk of bringing because of the HLA down-regulated expression based on the wide spectrum vaccine of CTL epi-position.The overall average distribution frequency of two super types of HLA-A2 and HLA-A3 in Chinese just surpasses 85%, and the peptide sequence of same super type identification is closely similar.Therefore, the wide spectrum epi-position that adopts while and several super types to have avidity just might become the tumour epitope peptide that covers most of crowd, can overcome the obstacle of MHC polymorphism, lay the foundation for developing the tumour epiposition vaccine that covers most of crowd, but yet there are no all reports about this respect.
Summary of the invention
The object of the present invention is to provide a kind of antitumor CTL epitope peptide that tumour cell is had obvious killing activity with population broad spectrum property.
For achieving the above object, the present invention can take following technical proposals:
Antitumor CTL epitope peptide with population broad spectrum property of the present invention, this peptide is made up of nine natural amino acid residues, and its sequence is: Ala-Leu-Tyr-Gly-Asp-Ile-Asp-Ala-Val (ALYGDIDAV L-Ala-leucine-tyrosine-Gly-Asp-Isoleucine-aspartic acid-L-Ala-Xie Ansuan).
Select COX-2 (COX-2) as purpose antigen, aminoacid sequence draws from GENEBANK.
Described active test material and reagent are T2A2 cell, T2A3 cell, EC-1, EC-109 and EC-9706 tumor cell line, RPMI1640 substratum, foetal calf serum, macrophage colony stimulating factor of recombinant human granulocyte (GM-CSF), IL (interleukin)-2, IL-4, IL-7, nylon hair post, mouse-anti people β2Wei Qiudanbai, mouse-anti people HLA-A2, the sheep anti-mouse igg of marked by fluorescein isothiocyanate, Brefeldin A, HLA-A parting kit, ELISPOT test kit.
With T2 cell, EC-1, EC-109 and EC-9706 cell strain and human PBMC's (peripheral blood lymphocytes), adopt the culture condition of conventional culturing cell to be 37 ℃, 5%CO
2Saturated humidity, routine goes down to posterity.
Utilization SYFPEITHI prediction is analyzed based on epi-position prediction algorithm (MHC-Pred) the structure affinity of structure again.The primary election nonapeptide carries out primary dcreening operation by proteasome cracking motif (Proteasomal cleavage motifs) restriction analysis, and analyzing through BLAST only has homology with COX-2.
Employing standard Fmoc scheme is synthesized, and behind HPLC (high performance liquid phase) purifying, its purity is greater than 90%, and mass spectroscopy confirms that its molecular weight meets theoretical value.
Analysis of experiments:
1, T2 cell combining power test: this peptide (50 μ g/mL) adding is contained β
2In the serum-free RPMI1640 substratum of microglobulin (3 μ g/mL) 37 ℃ hatch 18h-24h altogether after, cold PBA (isotonic phosphate buffer liquid) washing 3 times, add 100 μ L extent of dilution and be 1: 200 one anti-(the T2A2 cell adds monoclonal antibody BB7.2, and the T2A3 cell adds monoclonal antibody mouse-anti people β
2-M), place 30min for 4 ℃.After the cold PBA washing 3 times, add 50 μ L extent of dilution and be 1: 50 FITC-sheep anti-mouse igg solution, place 30min for 4 ℃, the washing back is detected in flow cytometer.In addition, certified Flu matrix
58-66(GILGFVFTL) and Flu NP
265-273(ILRGSVAHK) this experiment in respectively as the positive peptide of T2A2 and T2A3, PBS (phosphate buffered saline buffer) is as negative control.The result represents with fluorescence coefficient (FI):
Fluorescence coefficient (FI)=(epitope peptide average fluorescent strength-background average fluorescent strength)/background average fluorescent strength
2, peptide/HLA-I stable composite analysis: this peptide (50 μ g/mL) add in the serum-free RPMI1640 substratum contain β2Wei Qiudanbai (3 μ g/mL) 37 ℃ hatch 18h altogether after, unconjugated peptide is cleaned in cold PBA washing 4 times.The BrefeldinA that adds 10 μ g/mL is hatched 1h, washing.37 ℃, 5%CO
2Hatch 0,2,4,8h.Then, cold PBA washing 3 times, adding one, anti-(the T2A2 cell adds monoclonal antibody BB7.2, and the T2A3 cell adds monoclonal antibody mouse-anti people β
2-M), placing 30min for 4 ℃, cold PBA adds FITC-sheep anti-mouse igg solution after washing 3 times, places 30min for 4 ℃, and the washing back is detected in flow cytometer.The result is with DC
50(being the transformation period of peptide/HLA-I mixture) expression.
3, inducing of dendritic cell: the peripheral blood that extracts healthy donor, dilute after Ficoll-Paque density gradient centrifugation is separated with stroke-physiological saline solution, draw tunica albuginea layer PBMC, with PBMC adherent culture in 24 orifice plates, separate adherent monocyte, every hole adds 1640 substratum that 2mL contains 10%FBS (foetal calf serum), 100 μ g/L GM-CSF (granulocyte-macrophage colony stimutaing factor) and 50 μ g/L IL-4, in 37 ℃, 5%CO
2In the incubator, per three and half amounts are changed liquid, and be cultured to the 5th day and add LPS (lipopolysaccharides) and (100ng/mL) induce maturation, collecting cell behind the 48h, the morphocytology of observing in DC (dendritic cell) the inducing culture process with phase microscope changes.
4, ripe DC phenotype flow cytometry analysis: collecting the 8th day DC and adjusting cell concn is 1 * 10
6/ ml, add the fluidic cell detector tube, 100 μ L/ pipe, add traget antibody (anti-CD83-FITC, anti-CD1a-PE, anti-CD80-FITC, anti-HLA-DR-PE) final concentration is 5 μ g/ml, mark 40min is left standstill in 4 ℃ of dark places, and the PBS damping fluid is washed 2 times, does negative homotype contrast with the mouse IgG of FITC (fluorescein isothiocyanate), PE mark.Each detects sample institute analysis of cells number greater than 10000.
5, nylon Mao Zhufa separates T lymphocyte and purity evaluation thereof: the peripheral blood that extracts healthy donor, dilute after Ficoll-Paque density gradient centrifugation is separated PBMC with stroke-physiological saline solution, make PBMC adherent in 24 orifice plates, collect not attached cell, adjusting cell count with the RPMI1640 nutrient solution is 5 * 10
7/ mL is added to nylon Mao Zhuzhong with cell suspension, 37 ℃, 5%CO
2Hatch 1h under the saturated humidity condition, wash post 2 times, be rich in the T lymphocyte in the elutriant, identify T lymphocyte purity through α-Yi Suannaizhi enzyme (ANAE) staining with the RPMI1640 nutrient solution of 37 ℃ of pre-temperature.
6, tumour cell HLA-I class somatotype: parting kit is used in the experiment of HLA-I class somatotype.The HLA gene type adopts the method for sequence specific primers-polymerase chain reaction (PCR-SSP) to carry out somatotype.Basic step is: extract tumour cell EC-1, behind the DNA of EC-109 and EC-9706, carry out conventional pcr amplification with sequence specific primers, the agarose electrophoresis observations is carried out HLA-A site phenotype analytical.Result: EC-1 is HLA-A3
+, EC-109 is HLA-A3
+, EC-9706 is HLA-A68
+(belonging to the super type of HLA-A2).
7, RT-PCR detects the expression of COX-2 in tumor cell line: the sequence of primer is as follows: β-actin:sense:5 ' ACC AAC TGG GAC GAC ATG GAG AAA ATC 3 ', antisense:5 ' GTA GCC GCG CTC GGT GAG GAT CTT CAT 3 '; COX-2:sense:5 ' ACCAAC TGGGAC GAC ATG GAG AAA ATC 3 ', antisense:5 ' GTA GCC GCG CTC GGT GAG GAT CTTCAT 3 '.β-actin and the segmental length of COX-2 purpose are respectively 409bp and 242bp.The PCR condition is as follows: behind 95 ℃ of sex change 10min, and the circulation beginning, 94 ℃ of sex change 60s → 55 ℃ annealing 60s → 72 ℃ extension 60s, totally 30 circulations, last 72 ℃ are extended 10min.RT-PCR result: EC-1 and EC-9706 cell are all expressed COX-2 albumen, and the EC-109 cell does not detect expression COX-2 albumen.
8, cytotoxic T cell is external evoked: DC is induced to the 5th day, adds peptide to be measured, and concentration reaches 50 μ g/mL, and (LPS 100ng/mL), gathered in the crops DC on the 7th day, and adjusting cell count is 1 * 10 to add lipopolysaccharides simultaneously
5ML
-1Nylon hair post separates fresh T lymphocyte, and adjusting cell count is 1 * 10
6ML
-1, in 24 orifice plates, add 1 * 10
5/ hole T lymphocyte is as reacting cells, and 1 * 10
3/ hole DC added IL-2 to 100U/mL on the 2nd day as irritation cell, and every pore volume 1mL puts 37 ℃, 5%CO
2Cultivate under the saturated humidity condition, per three and half amounts are changed liquid.Press preceding method repetitive stimulation 1 time again with the DC of peptide load, add among the CTL once more.Results CTL was used for cellulotoxic experiment in the 12nd day.
9, cellulotoxic experiment: inductive CTL action effect cell, EC-1, EC-109 and EC-9706 cancer cells added in 96 orifice plates every hole 1 * 10 of target cell as target cell by imitating target than 20: 1
3Individual, establish independent target cell hole and independent CTL hole, every group 3 multiple hole, every hole 200 μ L put 37 ℃ of 5%CO
2, cultivate 4h under the saturated humidity condition, MTT (tetrazolium bromide) method detects each hole A
570nmValue.
Calculate kill rate as follows: kill rate=[1-(test holes A value-effector cell hole A value)/target cell hole A value] * 100%
Cellulotoxic experiment result: for COX-2
+Esophageal cancer cell strain EC-1 (HLA-A3
+) and EC-9706 (HLA-A68
+), this peptide has tangible killing activity (P＜0.01), for COX-2
-Esophageal cancer cell strain EC-109 (HLA-A3
+), this peptide also has certain killing activity (P＜0.05).
10, the detection of the T cell count of secretion of gamma-IFN (Interferon, rabbit): adopt the ELISPOT test.Target cell is respectively EC-1, EC-109 and EC-9706.The density of each target cell is 1 * 10
3/ mL, the ratio of imitating target cell is 20: 1, and target cell and effector cell are respectively got 100 μ L, joins to wrap in advance by in the 96 hole flat undersides of anti-IFN-γ mAb, in 37 ℃, 5%CO
2Cultivate 24h in the incubator, wash plate with reference to the specification sheets in the ELISPOT test kit after, add successively that biotin labeled anti-IFN-γ is how anti-, the streptavidin of alkali phosphatase enzyme mark and developer BCIP/NBT etc.Count the spot number that every hole forms in 96 orifice plates with the ELISPOT Quantimet, calculate spot number/1 * 10
5The percentage of the T cell of CTL and secretion of gamma-IFN.
ELISPOT detects the cell count of secretion of gamma-IFN among the antigen-specific CTL: the result of ELISPOT test shows that the CTL of this inducing peptide is to COX-2
+EC-1 (HLA-A3
+) and EC-9706 (HLA-A68
+) the cell antigen of offering can reply and activate, and the percentage 0.293% and 0.415% of T cell that can secretion of gamma-IFN is apparently higher than control group 0.169% and 0.220% (P＜0.05).
Table 1 a peptide-mediated CTL cytotoxicity test result:
*P＜0.05 **P＜0.01
EC-1：COX-2
+HLA-A3
+；TE-1：COX-2
+HLA-A2
+；EC109：COX-2
-HLA-A3
+
The IFN-γ ELISPOT detected result of table 2 a peptide-mediated CTL
*P＜0.05 **P＜0.01
The invention has the advantages that: 1, break through the restriction of MHC polymorphism, can be offered by HLA-A2 and HLA-A3 simultaneously; 2, the epithelium tumour had general adaptability; 3, other tumour of expressing COX-2 is suitable for too.
Description of drawings
Fig. 1 is mass spectrometry results figure of the present invention.
Fig. 2 is peptide of the present invention/HLA-I bonding force and stability result figure.
Embodiment
Antitumor CTL epitope peptide with population broad spectrum property of the present invention selects COX-2 (604aa) as purpose antigen, and aminoacid sequence draws from GENEBANK.
T2A2 cell, T2A3 cell, EC-1, EC-109 and EC-9706 tumor cell line are the conventional preservation in this laboratory.The RPMI1640 substratum is available from GIBCO company, and foetal calf serum is purchased the company in Hyclone, and recombinant human GM-CSF, IL-2, IL-4, IL-7 purchase in R﹠amp; D company, nylon hair post is purchased the company in Wako, and mouse-anti people β2Wei Qiudanbai is purchased the company in Biolegend, and the sheep anti-mouse igg and the Brefeldin A of mouse-anti people HLA-A2 (BB7.2), fluorescein isothiocyanate (FITC) mark purchase the company in Sigma.The HLA-A parting kit is purchased the company in BAG, and the ELISPOT test kit is purchased the company in Millipore.
Synthetic method:
Synthesizing of this peptide: adopt standard Fmoc scheme to synthesize: ALYGDIDAV
The first step: C holds first amino acid V to receive on the Wang resin,
1. claim Wang resin 0.3 gram to place synthesizer, use DMF swelling ten minutes earlier.
2. weighing: Fmoc-Val:0.4254g (2.5eq)
Hobt：0.1694g(2.5eq)
DIC：0.1528mL
Add synthesizer, stirred 10 minutes
Add DMAP:0.1531g (0.5eq)
Stir: 4 hours
3. washing: DMF 2 minutes 2 times, MeOH 2 minutes 3 times, DCM 2 minutes 3 times, DMF 2 minutes 2 times.
4. end socket: acetic anhydride: pyridine=1: 1, stirred 30 minutes
5. washing: with step 3
6. taking-up resin, dried overnight is weighed: 0.8357g
7. calculate substitution value (loading)
Loading＝(W2-W1)/(MWa-MWb)/W1(mmol/g)
Wherein: the W1=weight resin
The weight of W2=Fmoc-amino acid-resin
MWa=Fmoc-amino acid molecular amount
MWb＝18
Second step: add second amino acid: A
1. deprotection: 20% piperidines/DMF solution 2 * 15min,
2. washing: the same
3. free amino group detects: reagent A: 5% ethanol solution of ninhydrin (g/L)
Reagent B:0.1%KCN 2mL adds 98mL pyridine liquid
Reagent C: 80% phenol ethanolic soln
Be sequentially added into three kinds of reagent 1: 2: 1, boiling water bath.Show the blue expression free amino acid positive.
Detected result: mazarine
4.Fmoc-Ala-OH：0.2578g(2.5eq)
Hobt：0.1119g(2.5eq)
DIC：0.1045mL(2.5eq)
5. stir: 4 hours
6. washing: the same
7. free amino group detects: yellow
The 3rd step: add the 3rd amino acid: D (Otbu protection)
1, deprotection: 20% piperidines/DMF solution 2 * 15min,
2, washing: the same
3, free amino group detects: result: blueness
4、Fmoc-Asp(Otbu)-OH：0.3407g(2.5eq)
Hobt：0.1119g(2.5eq)
DIC：0.1045mL(2.5eq)
5, stir: 4 hours
6, washing: the same
7, free amino group detects: yellow
The 4th step: add the 4th amino acid: I
1, deprotection: 20% piperidines/DMF solution 2 * 15min,
2, washing: the same
3, free amino group detects: result: blueness
4、Fmoc-Ile-OH：0.2926g(2.5eq)
Hobt：0.1119g(2.5eq)
DIC：0.1045mL(2.5eq)
5, stir: 4 hours
6, washing: the same
7, free amino group detects: yellow
The 5th step: add five amino acid: D (Otbu protection)
1. deprotection: 20% piperidines/DMF solution 2 * 15min,
2. washing: the same
3. free amino group detects: result: blueness
4、Fmoc-Asp(Otbu)-OH：0.3407g(2.5eq)
Hobt：0.1119g(2.5eq)
DIC：0.1045mL(2.5eq)
5, stir: 4 hours
6, washing: the same
7, free amino group detects: yellow
The 6th step: add the 6th amino acid: G
1. deprotection: 20% piperidines/DMF solution 2 * 15min,
2. washing: the same
3. free amino group detects: result: blueness
4.Fmoc-Gly-OH：0.2462g(2.5eq)
Hobt：0.1119g(2.5eq)
DIC：0.1045mL(2.5eq)
5, stir: 4 hours
6, washing: the same
7, free amino group detects: yellow
The 7th step: add seven amino acid: Y
1. deprotection: 20% piperidines/DMF solution 2 * 15min,
2. washing: the same
3. free amino group detects: result: blueness
4.Fmoc-Tyr-OH：0.3858g(2.5eq)
Hobt：0.1119g(2.5eq)
DIC：0.1045mL(2.5eq)
5. stir: 4 hours
6. washing: the same
7. free amino group detects: yellow
The 8th step: add the 8th amino acid: L
1. deprotection: 20% piperidines/DMF solution 2 * 15min,
2. washing: the same
3. free amino group detects: result: blueness
4.Fmoc-Leu-OH：0.2926g(2.5eq)
Hobt：0.1119g(2.5eq)
DIC：0.1045mL(2.5eq)
5. stir: 4 hours
6. washing: the same
7. free amino group detects: yellow
The 9th step: add the 9th amino acid: A
1. deprotection: 20% piperidines/DMF solution 2 * 15min,
2. washing: the same
3. free amino group detects: result: blueness
4.Fmoc-Ala-OH：0.2578g(2.5eq)
Hobt：0.1119g(2.5eq)
DIC：0.1045mL(2.5eq)
5. stir: 4 hours
6. washing: the same
7. free amino group detects: yellow
The tenth step: cut from resin
1. deprotection: 20% piperidines/DMF solution 2 * 15min,
2. washing: the same
3. free amino group detects: result: blueness
4, join cutting reagent: 10mL (TFA8.25mL+ water 0.5mL+ phenol 0.5mL+ thioanisole 0.5mL+ dithioglycol 0.25mL) under the ice bath
5. filter a small amount of TFA washing
6. collect and filter after product, slowly add pre-cold diethyl ether, thermal agitation seals, and-20 ℃ are spent the night
7. B filters, and the ice ether washs to weight
8. dry, weigh
9. productive rate: 47.7%.
Claims (1)
1. antitumor CTL epitope peptide with population broad spectrum property, it is characterized in that: this peptide is made up of nine natural amino acid residues, and its sequence is: Ala-Leu-Tyr-Gly-Asp-Ile-Asp-Ala-Val.
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|---|---|---|---|---|
| CN102153624A (en) * | 2011-03-10 | 2011-08-17 | 郑州大学 | Antitumor cytotoxic T lymphocyte (CTL) epitope peptide analog derived from COX-2 |
| CN105949302A (en) * | 2016-05-27 | 2016-09-21 | 郑州大学 | FAP(fibroblast activation protein)-sourced anti-tumor CTL (cytotoxic T lymphocyte) epitope peptide P639 and application thereof |
| CN111088228B (en) * | 2019-12-27 | 2021-02-23 | 华农（肇庆）生物产业技术研究院有限公司 | Duck dendritic cell separation and culture method |
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| WO2005040212A2 (en) * | 2003-10-24 | 2005-05-06 | Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa | Orthogonal gene switches |
| CN1688605A (en) * | 2002-08-12 | 2005-10-26 | 昆士兰医学研究所理事会 | Novel immunogenic lipopeptides comprising T-helper and cytotoxic T lymphocyte (CTL) epitopes |
| CN1711279A (en) * | 2002-11-07 | 2005-12-21 | 昆士兰医学研究所理事会 | Epstein Barr virus peptide epitopes, polyepitopes and delivery system therefor |
| CN1711025A (en) * | 2002-10-03 | 2005-12-21 | 艾普免疫公司 | Optimized multi-epitope constructs and uses thereof |
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| CN1688605A (en) * | 2002-08-12 | 2005-10-26 | 昆士兰医学研究所理事会 | Novel immunogenic lipopeptides comprising T-helper and cytotoxic T lymphocyte (CTL) epitopes |
| CN1711025A (en) * | 2002-10-03 | 2005-12-21 | 艾普免疫公司 | Optimized multi-epitope constructs and uses thereof |
| CN1711279A (en) * | 2002-11-07 | 2005-12-21 | 昆士兰医学研究所理事会 | Epstein Barr virus peptide epitopes, polyepitopes and delivery system therefor |
| WO2005040212A2 (en) * | 2003-10-24 | 2005-05-06 | Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa | Orthogonal gene switches |
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