CN106117336A - The CTL of tumor antigen NY ESO 1 identifies epitope peptide and application thereof - Google Patents

The CTL of tumor antigen NY ESO 1 identifies epitope peptide and application thereof Download PDF

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CN106117336A
CN106117336A CN201610483182.1A CN201610483182A CN106117336A CN 106117336 A CN106117336 A CN 106117336A CN 201610483182 A CN201610483182 A CN 201610483182A CN 106117336 A CN106117336 A CN 106117336A
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eso
cell
ctl
tumor antigen
epitope peptide
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唐小军
朱进
许国贞
刘振云
周荧
冯振卿
唐奇
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Sinobioway Cell Therapy Co Ltd
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K35/14—Blood; Artificial blood
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    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract

The invention discloses the antitumor CTL epitope peptide in a kind of NY ESO 1 source, for pentadecapeptide, its aminoacid sequence is Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly.Present invention additionally comprises the application in preparing tumor therapeutic DC CIK of the above-mentioned epitope peptide.Specific DC CIK cell prepared by the present invention demonstrates certain killing rate to melanoma cell A375, can be used for preparing the preparation of melanoma specific autoimmune cell.

Description

The CTL of tumor antigen NY-ESO-1 identifies epitope peptide and application thereof
Technical field
The present invention relates to epitope peptide technical field, the CTL particularly relating to a kind of specific tumor antigen NY-ESO-1 identifies Epitope peptide and application thereof, further relate to the preparation method and applications of a kind of tumor antigen NY-ESO-1 specificity DC cell, and A kind of preparation method and applications of tumor antigen NY-ESO-1 specificity DC-CIK cell.
Background technology
Tumor-testis antigen (Cancer-testis Antigen, CTA), mainly at normal testis and kinds of tumors tissue Middle expression, expresses in other normal structures hardly.NY-ESO-1 is the one in tumor-testis antigen family, is positioned X Chromosome, a kind of embrane-associated protein of coding, one of tumor antigen that the immunogenicity being known is the strongest, cell can be caused to exempt from simultaneously Epidemic disease and humoral immunization.NY-ESO-1 is such as multiple types such as the esophageal carcinoma, melanoma, breast carcinoma, carcinoma of prostate, pulmonary carcinoma, ovarian cancers Wide expression in the tumor of type, often can detect depositing of NY-ESO-1 antibody in the tumor patient body that NY-ESO-1 is positive , point out it can be as the potential target spot of oncotherapy.
Along with the fast development of biotechnology and going deep into tumor development Mechanism Study, immunotherapy of tumors is led Territory constantly obtains new achievement, and wherein, adoptive cellular immunotherapy is the focus in this field.Adoptive cellular immunotherapy is Refer to feed back again in patient body after autologous or allosome extract immunocyte and amplification cultivation reaches some in vitro, it is achieved anti-tumor The Therapeutic Method of effect.Adoptive cellular immunotherapy can avoid the number of mechanisms of in-vivo tumour dysimmunity, transfers body The immunologic function of self plays Synergistic action.
The cell being currently used for adoptive cellular immunotherapy mainly has NK, DC-CIK etc., wherein DC-CIK cellular immunization Therapy is exactly that DC cell and CIK cell co-culture a kind of new cell mass with specific antitumor activity of acquisition.DC- CIK cell can significantly resist the growth of tumor cell, propagation, helps the ability that body recovery is struggled with tumor cell, Immune function of human body is transferred on limits ground, and comprehensive preventing is recurred and transfer, hence it is evident that improves the quality of life of patient, effectively extends The life-span of patient.
The antigen being presently used for effectively inducing DC-CIK cell is extremely limited, find and prepare can efficiently stimulate swollen The CTL epitope polypeptide antigen of tumor specificity DC-CIK will improve the practicality of this technology.
Summary of the invention
The technical problem existed based on background technology, present invention aim at providing a kind of specific tumor antigen NY- The CTL of ESO-1 identifies epitope peptide.
The object of the invention also resides in the application providing the CTL of above-mentioned specific tumor antigen NY-ESO-1 to identify epitope peptide.
The object of the invention also resides in the preparation method providing a kind of tumor antigen NY-ESO-1 specificity DC cell.
The object of the invention also resides in the preparation method providing a kind of tumor antigen NY-ESO-1 specificity DC-CIK cell.
The object of the invention also reside in above-mentioned tumor antigen NY-ESO-1 specificity DC cell and DC-CIK cell be provided should With.
For achieving the above object, the present invention adopts the following technical scheme that:
The CTL of a kind of specific tumor antigen NY-ESO-1 that the present invention proposes identifies epitope peptide, and described CTL identifies epi-position Peptide is pentadecapeptide, and its aminoacid sequence is as follows: Gly-Ala-Ala-Arg-Ala-Ser-Gly-Pro-Gly-Gly-Gly-Ala- Pro-Arg-Gly, molecular weight is 1238.34.
The CTL of above-mentioned specific tumor antigen NY-ESO-1 identifies that epitope peptide uses solid-phase synthesis to synthesize.Substantially Flow process is as follows: be first connected on insoluble solid phase carrier Wang resin by the aminoacid of Fmoc radical protection by an amino, Then taking off the protection group of amino, first aminoacid is i.e. connected on solid phase carrier；Secondly by second amino by Fmoc base The amino acid whose carboxyl condensing agent activation of group's protection, second aminoacid carboxyl after activation again be connected on the of solid phase carrier One amino acid whose amino reaction forms peptide bond, has now been generated as a dipeptides with protection group on solid phase carrier.Weight Multiple above-mentioned peptide bond forms reaction, makes peptide chain grow to N end from C end, until it reaches required peptide chain length, finally cuts To purpose peptide, then through HPLC purification, its purity is more than 90%.
The CTL of the above-mentioned specific tumor antigen NY-ESO-1 that the present invention also proposes identifies that epitope peptide has NY-in preparation The application of the tumor therapeutic polypeptide vaccine that ESO-1 expresses.
Preferably, the CTL of above-mentioned specific tumor antigen NY-ESO-1 identifies that epitope peptide is in preparation melanoma therapeutic Application in polypeptide vaccine, especially has lethality to melanoma cell A375.
The preparation method of a kind of tumor antigen NY-ESO-1 specificity DC cell that the present invention also proposes, uses above-mentioned special Property tumor antigen NY-ESO-1 CTL identify that epitope peptide adds DC cell and carries out cultivation and obtain.
The specificity that the preparation method of the above-mentioned tumor antigen NY-ESO-1 specificity DC cell that the present invention also proposes obtains The application in preparation treatment melanoma medicine of the DC cell.
The preparation method of a kind of tumor antigen NY-ESO-1 specificity DC-CIK cell that the present invention also proposes, uses above-mentioned The CTL of specific tumor antigen NY-ESO-1 identifies that epitope peptide carries out induction and obtains.
The spy that the preparation method of the above-mentioned tumor antigen NY-ESO-1 specificity DC-CIK cell that the present invention also proposes obtains The application in preparation treatment melanoma medicine of the opposite sex DC-CIK cell.
The present invention is ingenious utilizes NY-ESO-1 at multiple kinds of tumor such as neuroblastoma, synovial sarcoma, maligna All there is survivin (NY-ESO-1) unconventionality expression in element tumor, epithelial ovarian cancer, carcinoma of prostate, bladder cancer, breast carcinoma etc., but just Often adult's tissue is not expressed or in low expression in addition to testis.The method screening that theoretical and experiment combines obtains tumor associated antigen Epitope peptide, gained epitope peptide has no that document is reported, for developing tumor vaccine based on antigen NY-ESO-1 or tumour-specific The preparation of CTL cell is provided fundamental basis, and the antigenic peptides vaccine construction for follow-up multivalence lays the foundation.
Accompanying drawing explanation
Fig. 1 is the mass spectral analysis of the CTL identification epitope peptide of a kind of specific tumor antigen NY-ESO-1 that the present invention proposes Figure.
Fig. 2 is the embodiment of the present invention 1 gained P1, P2, P3 ... the Specific CTL Cells secretion that each self-induction of P20 obtains The ability comparison diagram of INF-γ.
Fig. 3 is that the Specific CTL Cells that the embodiment of the present invention 1 each self-induction of gained P3, P5, P7 obtains is thin to melanoma Born of the same parents A375 kills ability comparison diagram.
The CTL of a kind of specific tumor antigen NY-ESO-1 that Fig. 4 is proposed by the present invention identifies and obtains prepared by epitope peptide The statistical analysis figure of flow cytomery data of Specific CTL Cells immunocyte group.
Detailed description of the invention
Below, by specific embodiment, technical scheme is described in detail.
Embodiment 1: synthesis epitope peptide
Use the method integrated theory with practice, according to the primary structure of antigen, integrated use Immunoinformatics means, SYFPEITHI, BIMAS, NetCTL, WAPP and EpiJen comprehensively antigens c TL epi-position to NY-ESO-1 is used to be predicted point Analysis, the peptide sequence choosing scoring front 20 carries out experiment sieving, the most named P1, P2, P3 ... P20.
Basic procedure is as follows: first by the aminoacid of Fmoc radical protection, one amino is connected to insoluble solid phase carrier On Wang resin, then taking off the protection group of amino, first aminoacid is i.e. connected on solid phase carrier；Secondly by second ammonia Base is activated by the amino acid whose carboxyl condensing agent of Fmoc radical protection, and second aminoacid carboxyl after activation is consolidated with being connected on again First amino acid whose amino reaction of phase carrier forms peptide bond, has now been generated as one on solid phase carrier with protection group Dipeptides.Repeat above-mentioned peptide bond and form reaction, make peptide chain grow to N end from C end, until it reaches required peptide chain length, Finally cutting obtains purpose epitope peptide crude product.Through HPLC purification, purpose epitope peptide crude product is obtained purpose epitope peptide fine peptide, and it is pure Degree is more than 90%, and mass spectral analysis confirms its molecular weight coincidence theory value.
The CTL of a kind of specific tumor antigen NY-ESO-1 that the present invention proposes identifies that epitope peptide uses Fmoc solid phase synthesis Method synthesizes, and described CTL identifies that epitope peptide is pentadecapeptide, and numbered P7, its aminoacid sequence is as follows: Gly-Ala-Ala- Arg-Ala-Ser-Gly-Pro-Gly-Gly-Gly-Ala-Pro-Arg-Gly.P7 is carried out mass spectral analysis, and its mass spectral analysis is tied Fruit is as shown in Figure 1, it can be verified that its molecular weight is 1238.34g/mol, coincidence theory value.
Embodiment 2: polypeptide Function detection
Above-mentioned gained P7 and remaining 19 epitope peptide can be used for preparing the tumor therapeutic polypeptide with NY-ESO-1 expression Vaccine, its application experiment is as follows:
1, above-mentioned CTL identifies that epitope peptide inducing specific CTL cell, IFN-γ secretion and the killing to tumor target cell are real Test detection: the peripheral blood of extraction patient separates through density gradient centrifugation, it is thus achieved that PBMCs, add cytokine cultivate DC cell and CTL cell, uses the NY-ESO-1 epitope peptide of the DC cell loading present invention further, and co-culturing with CTL stimulates special CTL to expand Increase, use CTL secretion of INF-γ under specific antigen stimulates that ELISAPOT and LDH experiment detection is special the most in vitro And the lethal effect to melanoma cell A375.
Concrete grammar is as follows:
(I), the separation of PBMC and induction:
1) peripheral blood 50mL anticoagulant processed, 2000rpm is centrifuged 10min；
2) collect upper plasma frozen, dilute remaining hemocyte with PBS (pH=7.4)；
3) hemocyte of dilution is joined on isopyknic lymph separation liquid liquid level；
4) 20 DEG C of centrifugal 20min, close centrifuge brake；
5) after centrifugal, it is divided into four layers, draws tunica albuginea layer (i.e. the second layer) with suction nozzle glass dropper；
6) the tunica albuginea layer PBS taken out washs twice；
7) by cell with 2～5 × 106/ mL is inoculated in 6 orifice plates, after 2h, reclaims the most adherent cell, with pre-coated The culture plate of anti-CD3IgG and anti-CD28IgG carries out activating to be cultivated；
8) adherent cell adds GM-CSF with IL-4 stimulates cultivation to induce into DC cell in 5 days, within the 3rd day half, changes liquid；
9) the 5th day, collect DC cell, 10 μ g embodiment 1 gained purpose epitope peptide fine peptides are added in DC cell, after 1h, The T cell of DC cell with activation is co-cultured, is simultaneously introduced IL2 and IL-15；
10), after continuing to cultivate 5 days, the CTL cell obtaining antigen-specific carries out cytokine secretion and to tumor cell Killing experiments.
(II), IFN-γ cytokine secretion detection: Human IFN-gamma Platinum ELISA (IFN-γ ELISA detection kit, eBioscience company) IFN-γ of detection CTL emiocytosis, step is as follows:
1), after CTL cell being removed cytokine cultivation 24h, it is inoculated in 96 orifice plates；
2) after in cell, addition stimulates corresponding polypeptide again to stimulate 24h, centrifugal segregation cell, collect cell conditioned medium；
3) using the expression of IFN-γ in ELISA detection supernatant, the CTL epitope peptide selecting IFN-γ secretion more kills Wound experiment.
Result, as in figure 2 it is shown, the DC cell of P7 and P3, P5 load all can preferably induce Specific CTL Cells, is secreted The IFN-γ of higher amount.
(III), tumor cytotoxicity experiment: use lactic acid dehydrogenase (LDH) release detection method, use CytoTox 96Non-Radioactive Cytotoxicity Assay (citotoxicity detection kit, Promega company) carries out LDH examination Test detection cellkilling capacity.Step is as follows:
1) detection culture plate (100 μ L/ hole) is set up
A. experimental group is set up: with melanoma cell A375 of NY-ESO-1 positive expression as target cell, by effector lymphocyte Above-mentioned characteristic CTL cell is added than for 5:1,10:1,20:1 with target cell
B. effector lymphocyte's spontaneous release group is set up
C. target cell spontaneous release group is set up
D. target cell maximum release group is set up
E. ground control group is set up
2) cell cracking and results supernatant
A.37 DEG C 5%CO2Co-culture 5h
B. adding lysate, after 45min, all of hole in target cell maximum release group, 250g/min is centrifugal collects supernatant
3) LDH detection
A. transferase 45 0 μ L of supernatant is to another 96 orifice plate
B. every hole adds the substrate mixture of 50 μ L dilutions, and room temperature lucifuge hatches 30min
C. 50 μ L stop buffers are added in every hole
D. under 490nm, detect light absorption value OD
Cell killing rate computing formula is as follows:
Killing rate (%)=[(ODExperimental group-ODEffector lymphocyte's spontaneous release group-ODTarget cell spontaneous release group)/(ODTarget cell maximum release group-ODTarget cell spontaneous release group)] × 100%
Result is as it is shown on figure 3, Fig. 3 is that the specific CTL that the embodiment of the present invention 1 each self-induction of gained P3, P5, P7 obtains is thin Born of the same parents kill ability comparison diagram to melanoma cell A375；From the figure 3, it may be seen that the CTL cell of P3 and P7 induction is thin to melanoma Born of the same parents A375 is respectively provided with preferable fragmentation effect.
2, using the percentage ratio shared by various immunocytes in the special CTL of flow cytometer showed acquisition, result is as shown in Figure 4. As shown in Figure 4, containing substantial amounts of CTL cell in the immunocyte group that the present invention is prepared by P7, also contain a definite proportion simultaneously The NKT cell of example, i.e. this immunocyte group have preferable immunocompetence, have the energy of powerful killing specific tumors cell Power.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto, Any those familiar with the art in the technical scope that the invention discloses, according to technical scheme and Inventive concept equivalent or change in addition, all should contain within protection scope of the present invention.

Claims (7)

1. the CTL of a specific tumor antigen NY-ESO-1 identifies epitope peptide, it is characterised in that described CTL identifies epitope peptide For pentadecapeptide, its aminoacid sequence is:
Gly-Ala-Ala-Arg-Ala-Ser-Gly-Pro-Gly-Gly-Gly-Ala-Pro-Arg-Gly。
2. the CTL of specific tumor antigen NY-ESO-1 described in claim 1 identifies that epitope peptide has NY-ESO-1 table in preparation The application of the tumor therapeutic polypeptide vaccine reached.
3. the CTL of specific tumor antigen NY-ESO-1 described in claim 1 identifies that epitope peptide is in preparation melanoma therapeutic Application in polypeptide vaccine.
4. the preparation method of a tumor antigen NY-ESO-1 specificity DC cell, it is characterised in that use such as claim 1 institute The CTL stating specific tumor antigen NY-ESO-1 identifies that epitope peptide addition DC cell carries out cultivation and obtains.
The specificity DC that the most according to claim 4, the preparation method of tumor antigen NY-ESO-1 specificity DC cell obtains is thin Born of the same parents' application in preparation treatment melanoma medicine.
6. the preparation method of a tumor antigen NY-ESO-1 specificity DC-CIK cell, it is characterised in that use right such as to want The CTL seeking specific tumor antigen NY-ESO-1 described in 1 identifies that epitope peptide carries out induction and obtains.
The specificity that the most according to claim 6, the preparation method of tumor antigen NY-ESO-1 specificity DC-CIK cell obtains The application in preparation treatment melanoma medicine of the DC-CIK cell.
CN201610483182.1A 2016-06-24 2016-06-24 The CTL of tumor antigen NY ESO 1 identifies epitope peptide and application thereof Pending CN106117336A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101381402A (en) * 2008-10-20 2009-03-11 中国人民解放军第三军医大学 NY-ESO-1 tumour antigen mimic epitope and use thereof
WO2013172926A1 (en) * 2012-05-14 2013-11-21 Yale University Immune biomarkers and assays predictive of clinical response to immunotherapy for cancer
CN104341530A (en) * 2014-10-28 2015-02-11 重庆沁涟生物医药科技股份有限公司 Vnsak polypeptide and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101381402A (en) * 2008-10-20 2009-03-11 中国人民解放军第三军医大学 NY-ESO-1 tumour antigen mimic epitope and use thereof
WO2013172926A1 (en) * 2012-05-14 2013-11-21 Yale University Immune biomarkers and assays predictive of clinical response to immunotherapy for cancer
CN104341530A (en) * 2014-10-28 2015-02-11 重庆沁涟生物医药科技股份有限公司 Vnsak polypeptide and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
刘志青等: "NY-ESO-1癌症疫苗研究进展", 《国际肿瘤学杂志》 *
罗彬等: "癌-睾丸抗原NY-ESO-1的表位鉴定及其抗原肽疫苗研究", 《中国免疫学杂志》 *

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