CN101870725B - MAGE (Melanoma Antigen Gene)-4 anti-tumor CTL (Cytotoxic T Lymphocyte) epitope peptide and application thereof - Google Patents
MAGE (Melanoma Antigen Gene)-4 anti-tumor CTL (Cytotoxic T Lymphocyte) epitope peptide and application thereof Download PDFInfo
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Abstract
The invention relates to an MAGE (Melanoma Antigen Gene)-4 anti-tumor CTL (Cytotoxic T Lymphocyte) epitope peptide. The epitope peptide is a nonapeptide, and the sequence of the nonapeptide is a-b-Leu-Glu-His-Val-Val-Arg-c, wherein a is Lys or Tyr, b is Val or Leu, and c is Val or Leu; the sequence of an epitope peptide P286 is Lys-Val-Leu-Glu-His-Val-Val-Arg-Val; the sequence of an anti-tumor CTL epitope peptide P286-1Y2L is Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Val; and the sequence of an anti-tumor CTL epitope peptide P286-1Y2L9L is Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Leu. The specific CTL induced in the body of a transgenic mouse by the anti-tumor CTL epitope peptide has certain kill rate to tumor cells EC-9706; and the epitope peptide can be used for preparing therapeutic tumor polypeptide vaccines.
Description
Technical field
The invention belongs to the polypeptide technical field in the biochemical field, be specifically related to the antitumor CTL epitope peptide and the application in preparation tumor therapeutic polypeptide vaccine thereof in a kind of MAGE-4 source.
Background technology
MAGE-4 belongs to tumor-testis antigen, and (Cancer-Testis Antigen CTA), is one of member of MAGE gene family.It is reported that MAGE-4 expresses in multiple human malignancies, like ovarian cancer, tumor of head and neck, the esophageal carcinoma, colorectal carcinoma, lung cancer, bladder cancer etc.; At esophageal squamous cell carcinoma, lung cancer, tumor of head and neck; Expression degree in the bladder cancer is especially high, is respectively 74%, 59%, 53% and 45%.Zambon etc. discover in the adenocarcinoma of esophagus of 67% esophageal squamous cell carcinoma and 37.5% and express a kind of MAGE gene at least that wherein MAGE-4 more is more common in the squama cancerous tissue, thinks that thus MAGE-4 can be used as a specific index of esophageal squamous cell carcinoma.In immunotherapy of tumors, MAGE-4 is the target spot of an extraordinary diagnosing tumor and treatment.
Along with immunologic development of modern times, (Cytotoxic T lymphocyte, the vital role of CTL) in tumour, bringing into play more and more comes into one's own cytotoxic T lymphocyte.How effectively to excite the specificity cellular immunity response of CTL mediation, bring into play antitumor usefulness, become an important topic in current tumor therapeutic polypeptide vaccine development field.Previously discovering, cause the CTL immune response, is not complete tumour antigen molecule, but the specific CTL epi-position (Epitope) in antigen source.Behind the antigen presenting cell picked-up tumour antigen; Being processed into through proteolyzing is the polypeptide fragment of 8～10 amino acid lengths; Be the CTL epi-position, so with endoplasmic in mhc I (MHC-I) quasi-molecule combine to form polypeptide-MHC mixture (peptide-MHC complex, pMHC); And the pMHC submission supplies TXi Baoshouti (the T cell receptor of CD8+T cell surface to cell surface the most at last; TCR) identification, thereby activation CTL cell cause the CTL immunne response.
Yet the weak existence with immunological tolerance of immunogenicity is the two big obstacles that the CTL epi-position is used as the tumor therapeutic polypeptide vaccine.Therefore, how to improve the immunogenicity of CTL epi-position and break body the immunological tolerance of CTL epi-position is become tumor therapeutic polypeptide vaccine development key.In the enhancing strategy of numerous tumor therapeutic polypeptide vaccines, the CTL epi-position is carried out molecular modification be considered to one of the most promising method.To the transformation of CTL epi-position, can improve the avidity of epi-position and MHC and the stability of pMHC, thereby reach the purpose of enhances immunogenicity through transforming epi-position MHC binding site; Also can improve pMHC and TCR bonded stability, and then improve the CTL activity and overcome the CTL tolerance through transforming epi-position TCR binding site.Recent research shows, though can improve the immunogenicity of vaccine to a certain extent based on the polypeptide vaccine of epi-position MHC binding site transformation, in immunotherapy of tumors, do not reach the ideal tumor killing effect; And be expected to through strengthening the stimulation ability of low-affinity T cell or recruiting the new T cell with cross reactivity of a group and break the tumour immunity tolerance and reach the ideal tumor killing effect based on the polypeptide vaccine of epi-position TCR binding site transformation.
The HLA-A2.1 molecule mainly combines with nonapeptide; Its two and nine is the main anchored site of MHC; Jorg Ruppert (Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules.Cell.1993; 74 (5): 929-37.) in the advantage amino acid of the main anchored site of research, find: two are L or M, and nine is L, V or I, can epi-position and MHC bonded ability be improved 10～100 times.(A general strategy to enhanceimmunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification ofcryptic tumor epitopes.Eur J Immunol.2000,30 (12): the introducing of 3411-21.) pointing out a Tyr mainly relies on the hydrogen bond action of hydrophobic interaction and the phenolic hydroxyl group of its side-chain benzene ring to strengthen the interaction of epitope peptide and HLA molecule to Tourdot.
The application mainly be adopt the theoretical method that combines with experiment modify and preliminary evaluation go out can with MHC molecule binding ability strong the candidate CTL epitope peptide and the analogue thereof in MAGE-4 source.We have found the cancer testis antigen MAGE-4 of high expression level in esophageal squamous cell carcinoma through to the screening of CT datebase, and its positive expression rate in esophageal squamous cell carcinoma has reached 74%, are a unusual esophageal carcinoma immunotherapy of ideal target antigens in the CT antigen.According to antigenic primary structure, adopt the immunologic information section of learning to do, utilization SYFPEITHI, BIMAS and NetCTL 1.2 DBs have carried out forecast analysis to the HLA-A2.1 restricted CTL epitope of antigen MAGE-4.
Summary of the invention
The object of the invention is with artificial synthetic method a kind of antitumor CTL epitope peptide with MAGE-4 source of antitumor action to be provided, and the preparation method of epitope peptide is provided.
Another purpose of the present invention is to provide the application of this MAGE-4 antitumor CTL epitope peptide in preparation tumor therapeutic polypeptide vaccine.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
The MAGE-4 antitumor CTL epitope peptide is nonapeptide, and its sequence is: a-b-Leu-Glu-His-Val-Val-Arg-c; Wherein, a is Lys or Tyr, and b is Val or Leu; C is Val or Leu.
When a is Lys, b is Val, and when c was Val, sequence was Lys-Val-Leu-Glu-His-Val-Val-Arg-Val (is KVLEHVVRV, is designated as P286), and molecular weight is 1078.3.
When a is Tyr, b is Leu, and when c was Val, sequence was Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Val (is YLLEHVVRV, is designated as P286-1Y2L), and molecular weight is 1127.4.
When a is Tyr, b is Leu, and when c was Leu, sequence was Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Leu (is YLLEHVVRL, is designated as P286-1Y2L9L), and molecular weight is 1141.4.
The preparation method of said MAGE-4 antitumor CTL epitope peptide can adopt solid-phase synthesis to synthesize the CTL epitope peptide.Basic procedure is following: at first an amino is connected on the insoluble solid phase carrier Wang resin by the amino acid of Fmoc radical protection, and the protection base of desamidizate then, first amino acid promptly is connected on the solid phase carrier; Secondly amino is used the condensing agent activation by second amino acid whose carboxyl of Fmoc radical protection; Amino acid after the activation forms peptide bond with first amino group of amino acids reaction that is connected on solid phase carrier again, on solid phase carrier, has just generated a dipeptides that has the protection base this moment.Repeat above-mentioned peptide bond and form reaction, peptide chain is grown to the N end from the C end, until reaching needed peptide chain length, cutting at last obtains the purpose peptide.Behind the HPLC purifying, its purity is greater than 90%, and mass spectroscopy also confirms that its molecular weight meets theoretical value.(referring to: 1. yellow only moral, Chen Changqing work, polypeptide is synthetic, Science Press, 1985.2. .N. Xiu Ede, H.D. Jia Kubuke show, and Liu Keliang etc. translate, peptide: chemistry and biology, Science Press, 2005.)
The application of said MAGE-4 antitumor CTL epitope peptide in preparation tumor therapeutic polypeptide vaccine.
Advantage of the present invention: be utilized in high expression level in the esophageal carcinoma and the antigen MAGE-4 that in human normal tissue, do not express; Filter out epitope peptide with anti-tumor activity; The nonapeptide of identifying is not all seen bibliographical information; For development is provided fundamental basis based on the tumor therapeutic polypeptide vaccine of antigen MAGE-4, and lay the foundation for follow-up polyvalent antigen peptide vaccine construction.
Description of drawings
Fig. 1 is the mass spectroscopy collection of illustrative plates of epitope peptide P286 of the present invention;
Fig. 2 is the mass spectroscopy collection of illustrative plates of epitope peptide P286-1Y2L of the present invention;
Fig. 3 is the mass spectroscopy collection of illustrative plates of epitope peptide P286-1Y2L9L of the present invention;
Fig. 4 be epitope peptide of the present invention in the transgenic mice body inductive specific CTL to the detected result of tumour cell EC-9706 lethal effect.
Laboratory apparatus title and model:
Electron spray(ES)-ion trap multi-stage ms appearance BRU KER Esquire-3000 Germany Brooker dalton instrument company
Flow cytometer Calibur U.S. company BD
Embodiment
Below through embodiment the present invention is done further explanation, but protection scope of the present invention is not limited thereto.
Embodiment 1: the preparation of antitumor CTL epitope peptide P286 (Lys-Val-Leu-Glu-His-Val-Val-Arg-Val)
Adopt the Fmoc solid-phase synthesis, prolong one by one from C end → N end.Protect amino acid whose alpha-amino group with fluorenes methoxy carbonyl acyl group (Fmoc); Various Fmoc protect that amino acid whose Side chain protective group is respectively Arg (Pbf), His (trt), Glu (OtBu), (Pbf represents 2 to Lys (Boc); 2; 4,6, on behalf of trityl, OtBu, 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl, trt represent uncle's fourth fat, Boc to represent tertbutyloxycarbonyl).With first amino acid of C end of alpha-amino group protection, promptly Fmoc-Val-OH makes condensing agent with N, N '-DIC (DIC), and adds 1-hydroxy benzo triazole (HOBt), and Fmoc-Val-OH is connected on the wang resin earlier; Successively with DMF, anhydrous methanol, methylene dichloride, DMF washing; Use piperidines-DMF mixed solution (V then
Piperidines: V
DMF=1: 4) remove Fmoc protection base, successively with DMF, anhydrous methanol, methylene dichloride, DMF washing.Be that Fmoc-Arg (Pbf)-OH makes condensing agent with DIC with second amino acid of C end again, and add HOBt, Fmoc-Arg (Pbf)-OH is connected on the amino of Val, with piperidines-DMF mixed solution (V
Piperidines: V
DMF=1: 4) slough Fmoc protection base, successively with DMF, anhydrous methanol, methylene dichloride, DMF washing.Get the Arg-Val-Wang resin.Be connected with Fmoc-Val-OH again, repeat above-mentioned steps, connect successively and go up Val, His, Glu, Leu, Val, Lys, peptide chain is progressively prolonged, by sequence from C end → N end with piperidines-DMF mixed solution (V
Piperidines: V
DMF=1: 4) slough Fmoc protection base, with cutting reagent (V
Trifluoroacetic acid: V
Thioanisole: V
Water: V
Phenol: V
1=82.5: 5: 5: 5: 2.5) P286 is downcut from the Wang resin, and slough Pbf, trt, OtBu, Boc and protect base, obtain antitumor CTL epitope peptide P286 bullion.
HPLC separation and purification: use C
18The separation and purification of preparation property HPLC post (Partisil 10ODS-39.4 * 250mm, Whatman company).Get above-mentioned P286 bullion 30mg and be dissolved among the aqueous solution a (the TFA volume content is 0.1%, and the CAN volume content is 30%) that 3ml contains trifluoroacetic acid (TFA), acetonitrile (CAN), carry out the constant gradient wash-out with a, flow velocity 5ml/min collects main peak, and lyophilize gets smart peptide.Product is carried out mass spectroscopy see Fig. 1, the result confirms that molecular weight is 1078.3, is consistent with theoretical value.
Embodiment 2: the preparation of antitumor CTL epitope peptide P286-1Y2L (Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Val)
Adopt the compound method same with embodiment 1, difference only is to substitute second amino acid Val of N end with Leu, and Tyr substitutes Lys.
Product is carried out mass spectroscopy see Fig. 2, the result confirms that molecular weight is 1127.4, is consistent with theoretical value.
Embodiment 3: the preparation of antitumor CTL epitope peptide P286-1Y2L9L (Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Leu)
Adopt the compound method same with embodiment 1, difference only is to substitute second amino acid Val of N end with Leu, substitutes N with Leu and holds the 9th amino acid Val, and Tyr substitutes Lys.
Product is carried out mass spectroscopy see Fig. 3, the result confirms that molecular weight is 1141.4, is consistent with theoretical value.
The CTL epitope peptide of above-mentioned preparation can be used for preparing the tumor therapeutic polypeptide vaccine, and its application experiment is following:
1, epitope peptide and HLA-A2.1 molecule combining power test
(1) the centrifugal collection of 800rpm HLA-A2.1 expresses the T2 cell (ATCC company) of the positive and endogenous antigen processing treatment anergy; The phosphate buffered saline buffer of PH7.2 (PBS) washing 3 times, serum-free RPMI 1640 substratum (Gibico company) resuspended to cell density be 1 * 10
6/ mL is inoculated in 24 orifice plates.
(2) every hole adds 50 μ g epitope peptides and 0.5 μ g people β
2Microglobulin (Sigma company) is hatched 18h for 37 ℃ altogether;
(3) with 4 ℃ PBA (with the 2g bovine serum albumin; 0.2g sodiumazide is dissolved among the PBS of 100ml PH7.4 and promptly gets PBA) washing 3 times; Add the monoclonal antibody BB7.2 (Sigma company) of the mouse-anti people HLA-A2.1 molecule of 100 μ l after with 100 times of dilutions of PBS of PH 7.4,4 ℃ of lucifuges are hatched 40min;
(4) with 4 ℃ PBA washing 3 times, add the sheep anti-mouse igg (Beijing Bo Aosen Bioisystech Co., Ltd) of fluorescein isothiocyanate (FITC) mark of 50 μ l after with 50 times of dilutions of PBS of PH 7.4,4 ℃ of lucifuges are hatched 30min;
(5) PBA washing back is detected in flow cytometer.The result representes with fluorescence coefficient FI, sees table 1 for details.
The result shows that three epitope peptides all demonstrate medium bonding force, and transforms peptide P286-1Y2L and P286-1Y2L9L all is higher than parent peptide P286.
2, epitope peptide/HLA-A 2.1 molecular complex stability analyses
(1) the centrifugal collection of 800rpm T2A2 cell, the PBS of PH7.2 washing 3 times, serum-free RPMI 1640 substratum resuspended to cell density be 1 * 10
6/ mL is inoculated in 24 orifice plates;
(2) every hole adds 50 μ g epitope peptides and 0.5 μ g people β
2Microglobulin was hatched 18 hours for 37 ℃ altogether;
The PBS of (3) 4 ℃ of PH7.2 washs 4 times to remove unconjugated peptide;
(4) every hole adds 10 μ g BrefeldinA (BFA, Sigma company) and hatches 1h, the PBS washing;
(5) 37 ℃, 5%CO
2Hatch 0h, 2h, 4h, 6h respectively;
(6) hatch end after, with 4 ℃ PBA washing 3 times, add the monoclonal antibody BB7.2 (is anti-) of the mouse-anti people HLA-A2.1 molecule of 100 μ l after with 100 times of dilutions of PBS of PH 7.4,4 ℃ are reacted 30min;
(7) 4 ℃ PBA washing 3 times, the sheep anti-mouse igg that adds the FITC mark of 50 μ l after with 50 times of dilutions of PBS of PH 7.4 reacts 30min for 4 ℃;
(8) 4 ℃ PBA washing back is detected in flow cytometer.The result is with the transformation period DC of epitope peptide/HLA-A2.1 molecular complex
50Expression sees table 1 for details.
Can know that by table 1 transformation period of three epitope peptides is all greater than 2h.
Table 1 epitope peptide and HLA-A2.1 bonding force and stability test result
| Epitope peptide | FI | DC 50/h |
| P286 | 0.39 | 2～4 |
| P286-1Y2L | 0.98 | 2～4 |
| P286-1Y2L9L | 0.69 | 2～4 |
Annotate: FI=(epitope peptide average fluorescent strength-background average fluorescent strength)/background average fluorescent strength
FI＞1.5: epitope peptide and HLA-A2.1 have strong bonding force;
0.5＜FI＜1.5: medium bonding force; FI＜0.5: weak bonding force;
DC
50Be that 50% epitope peptide/HLA-A2.1 molecular complex dissociates the required time.
3. transgenic mice in vivo tests
3.1CTL induce in the body:
Immunization protocol: picked at random 8～12 week HLA-A2.1/Kb transgenic mices in age (second professor Cao Xuetao of medical university of army is so kind as to give), male and female at random, every group of 4 mouse.With 100 μ g epitope peptides, 140 μ g Th epitope peptide (I-A
bThe Th cell epitope TPPAYRPPNAPIL in HBcAg source; Can adopt conventional Fmoc solid-phase synthesis preparation or buy the commercially available prod), 50 μ l Freund's incomplete adjuvant (IFA; Sigma company) and the immunological reagent that obtains of the PBS mixing and emulsifying of 50 μ l PH7.2 in the subcutaneous injection of mouse tail root, dosage be 100 μ l/ only, injected respectively at the 1st day, the 6th day, the 11st day; Inject altogether 3 times, got the mouse spleen cell on the 12nd day.PBS group (promptly not adding epitope peptide and Th epitope peptide) and Th+PBS group (promptly not adding epitope peptide) is set does negative control.
3.2 effect CTL preparation:
(1) mouse of above-mentioned injection three times was taken off neck in the 12nd day and cause death, after alcohol-pickled 2～3min sterilization, aseptic taking-up mouse spleen in super clean bench;
(2) 200 order steel meshes grind, and PBS (PH7.2) flushing gets splenocyte suspension;
(3) 800rpm is centrifugal, abandons supernatant;
(4) add 5ml erythrocyte cracked liquid (Beijing ancient cooking vessel state company), re-suspended cell is hatched 5min for 4 ℃;
(5) 800rpm is centrifugal, collecting cell, PBS (PH7.2) washing 3 times;
(6) cell be resuspended in RPMI 1640 substratum (Gibico company) that 10ml contains 10% foetal calf serum (Hangzhou SIJIQING company) cell suspension, then it is cultivated every hole 5ml in 6 orifice plates;
(7) next day, every hole added 250U Recombinant mouse IL-2 (PROSPEC company) and 50 μ g epitope peptides;
(8) vitro culture is gathered in the crops effector cell CTL after 6 days, carries out LDH and detects.
LDH detects: use
Non-Radioactive Cytotoxicity Assay (cytotoxicity detection kit, Promega company) to carry out the LDH test and detect cytotoxic activity.Step (sees the test kit specification sheets for details) as follows:
1) sets up check-out console (100 μ l/ hole)
(1) set up experimental group: with tumour cell EC-9706 (ATCC company) as target cell (optimum target cell number: 5000) by the different targets of imitating than 20: 1,40: 1,80: the 1 above-mentioned effector cell CTL of adding
(2) set up the spontaneous release group of effector cell
(3) set up the spontaneous release group of target cell
(4) set up the maximum release group of target cell
(5) set up the volume correction control group
(6) set up the background control group
2) lysis and results supernatant
(1) 37 ℃, 5%CO
2Hatch check-out console (5h)
(2) add lysate in maximum release group of target cell and the volume correction control group, add 10 μ l lysates (10 *) in per 100 μ l substratum, 45min adds lysate before the results supernatant
(3) the centrifugal 4min of 250g, the results supernatant
3) LDH detects
(1) transferase 45 0 μ l supernatant is to another orifice plate
The substrate mixed solution of dilution is added in (2) 50 μ l/ holes rapidly, and the room temperature lucifuge is hatched 30min
(3) add 50 μ l stop baths
(4), detect 490nm absorption value OD in one hour with the bubble removal that contains in the hole.
Cell killing rate calculation formula is following:
Kill rate (%)=[(OD
Experimental group-OD
The spontaneous release group of effector cell-OD
The spontaneous release group of target cell)/(OD
The maximum release group of target cell-OD
The spontaneous release group of target cell)] * 100% (annotate: the mean absorbance that the absorption value of all experimental group, the spontaneous release group of effector cell, the spontaneous release group of target cell all should the subtracting background control group; The maximum release group of target cell absorption value should deduct the mean absorbance of volume correction control group)
The result sees Fig. 4, can be known by Fig. 4, than 80: 1 o'clock three epitope peptide inductive specific CTLs tumour cell EC-9706 is all demonstrated certain kill rate at the effect target.Thus, can know that the antitumor CTL epitope peptide that MAGE-4 originates can be used in preparation tumor therapeutic polypeptide vaccine.
Claims (3)
1.MAGE-4 antitumor CTL epitope peptide is characterized in that, said antitumor CTL epitope peptide is a nonapeptide, and its sequence is Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Val or Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Leu.
2. the preparation method of the said MAGE-4 antitumor CTL epitope peptide of claim 1 is characterized in that, adopts solid-phase synthesis to prepare the CTL epitope peptide.
3. the application of the said MAGE-4 antitumor CTL epitope peptide of claim 1 in preparation tumor therapeutic polypeptide vaccine.
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Non-Patent Citations (3)
| Title |
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| Sabrina Ottaviani et al.A new MAGE-4 antigenic peptide recognized by cytolytic T lymphocytes on HLA–A24 carcinoma cells.《Cancer Immunol Immunother》.2006,第55卷867–872. * |
| T. Kobayashi et al.New MAGE-4 antigenic peptide recognized by cytolytic T lymphocytes on HLA-A1 tumor cells.《Tissue Antigens》.2003,第62卷426–432. * |
| 张秀敏 等.肿瘤抗原MAGE-A 亚家族HLA-A2 限制性CTL 表位的预测及分析.《第四军医大学学报》.2004,第25卷(第24期),2222-2225. * |
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