CN104497123B

CN104497123B - EPS8 antitumor CTL epitope peptides and its application

Info

Publication number: CN104497123B
Application number: CN201410857469.7A
Authority: CN
Inventors: 李玉华; 周炜均
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2013-12-23
Filing date: 2013-12-23
Publication date: 2017-10-27
Anticipated expiration: 2033-12-23
Also published as: CN104497123A

Abstract

A kind of antitumor CTL epitope peptide in EPS8 sources, is nonapeptide, its sequence is：Phe‑Leu‑Phe‑Thr‑Pro‑Leu‑Asn‑Met‑Val.The invention also discloses application of the epitope peptide in medicine.The specific CTL that the present invention is induced can secrete certain IFN γ, and the T2A2 cells for the cells of MCF 7 and load EPS8 for expressing HLA A*0201 and EPS8 the positive show certain antigentic specificity lethal effect, the EPS8 being made expresses the therapeutical peptide vaccine of positive tumor, there is huge development and application potentiality in tumor-specific immunity therapy field.

Description

EPS8 antitumor CTL epitope peptides and its application

It is December 23, Application No. 201310717092.0, entitled EPS8 in 2013 applying date that the application, which is, The divisional application of antitumor CTL epitope peptide and its Chinese invention patent application of application.

Technical field

The invention belongs to the technical field of polypeptide in biochemical field, and in particular to it is antitumor that a kind of EPS8 originates CTL epitope peptides and its application in tumor is prepared.

Background technology

Malignant tumour is the first killer of the new century mankind.The traditional therapies such as operation, radiotherapy and chemotherapy are to some swollen The therapeutic effect of knurl especially late malignant tumour is still undesirable.In recent years, cytotoxic T lymphocyte (Cytotoxic T Lymphocyte, CTL) suppress tumour in effect greatly paid attention to, how effectively to excite CTL mediation specific cell Immune response, plays antitumor efficiency, has become the important topic that current tumor therapeutic polypeptide vaccine develops field.

The discovery of tumour antigen and its identification of CTL epitopes are tumor-specific immunity treatment and tumor therapeutic polypeptide The important prerequisite of vaccine development.EGF-R ELISA path substrate No.8 (Epidermal Growth Factor Receptor pathway substrate No.8, EPS8) it is the important kinase activity bottom of EGF-R ELISA (EGFR) One of thing.Research in recent years shows that EPS8 in a variety of solid tumors (such as lead by cervical carcinoma, colorectal cancer, hypophysoma, oral squamous cell carcinomas, pancreas Pipe cancer, breast cancer, thyroid cancer, the cancer of the esophagus and glioblastoma etc.) and hematological system tumor (such as Huppert's disease, acute Marrow series leukemia, mixed stocker leukaemia etc.) tissue and cell in abnormal expression raise, and normal tissue expression is very low or nothing Expression.Further studies have shown that EPS8 promotes the propagation of tumour cell by cell cycle regulation, by participating in cell pseudopodium Formation and with actin interaction promote tumour cell transfer, by influenceing tumour cell to the resistance to of chemotherapeutics By property it is related to the prognosis of patient (Li Y*,Xue T,He Y,Du J.A novel oncoprotein Eps8:new target for anti-cancer therapy.Future Oncology.2013(Accepted)).In addition, this seminar Found in the experimental study of early stage, recombined small-mouse rmHis-Eps8 protein vaccines can effectively induce lotus 4T1 breast cancer mouses to produce Raw specificity antineoplastic immunity response, suppresses the propagation of mouse interior tumor cell, extends its life span, and to mouse brain, The heart, liver, kidney, small intestine, epididymis tissue are without obvious toxic action.(He YJ,Zhou J,Li Y*,et al.Eps8vaccine exerts prophylactic antitumor effects in a murine model:a novel vaccine for breast carcinoma.Mol Med Rep.2013Aug；8(2):662-8).Therefore, EPS8 is diagnosing tumor and antitumor The novel targets for the treatment of, have great potential in immunotherapy of tumors.

Immunogenicity is weak and immune tolerance significantly limit natural CTL epitopes answering in tumor therapeutic polypeptide vaccine With therefore, the immune tolerance for effectively improving the immunogenicity of natural CTL epitopes and breaking body turns into tumor therapeutic polypeptide epidemic disease The key of seedling development.In the enhancing strategy of numerous tumor therapeutic polypeptide vaccines, molecular modification quilt is carried out to natural CTL epitopes It is considered one of most effective method.Due to CTL epitopes and the knot of major histocompatibility complex (MHC)-class Ⅰmolecule Conjunction is the key factor for inducing CTL immune responses, and CTL epitopes are anchor residues with the important foundation that MHC- class Ⅰmolecules are combined, Therefore, it can reach that enhancing immunogenicity is special without influenceing by changing the amino acid residue of anchor residues or its adjacent position The purpose of property.Research is found, in nonapeptide CTL epitopes, second and the dominant anchor site that the 9th is HLA-A2.1 molecules, Two are Leu or Met, and nine are Leu, Val or Ile, and the ability that can be combined epitope with MHC improves 10~100 times, and one Position introduces Tyr then can be by the hydrophobic effect of its side-chain benzene ring and the hydrogen bond action enhancing epitope peptide of phenolic hydroxyl group and HLA molecules Interaction.

The method that the application is mainly combined using theoretical and experiment, modification and Preliminary Identification go out to be had with MHC- class Ⅰmolecules There are candidate's CTL epitope peptides in strong adhesion and the EPS8 of effectively inducing antitumor immunity response sources and the like.

The content of the invention

A technical problem to be solved by this invention is to provide a kind of tumour antigen for the above-mentioned state of the art The HLA-A*0201 restricted CTL epitopes in EPS8 sources, the epitope can be with high-affinity combination HLA-A*0201 molecules, institute's shape Into stable composite, can the specific CTL immune responses of induction peptide, show as stimulator polypeptide specific CTL secreting high levels IFN-γ and to tumour cell produce specific killing effect.

Another technical problem to be solved by this invention is to provide a kind of former in tumour for the above-mentioned state of the art Application of the HLA-A*0201 restricted CTL epitopes peptide in EPS8 sources in tumor is prepared.

The present invention solve the technical scheme that is used of above-mentioned technical problem for：

EPS8 antitumor CTL epitope peptides, are nonapeptide, and its sequence is：Trp-a-Gln-Asp-Met-Ile-Leu-Gln- Val, wherein, a is Thr or Leu；B-Leu-Asp-Asp-Ile-Glu-Phe-Phe-c, wherein, b is Ile or Tyr, c are Ile Or Val；Phe-Leu-Phe-Thr-Pro-Leu-Asn-Met-Val；D-Leu-Ala-Glu-Ser-Val-Ala-Asn-Val, Wherein, d is Gln or Tyr.

When a is Thr, sequence is that (i.e. WTQDMILQV, is designated as Trp-Thr-Gln-Asp-Met-Ile-Leu-Gln-Val EPS8-101)；

When a is Leu, sequence is that (WLQDMILQV is designated as Trp-Leu-Gln-Asp-Met-Ile-Leu-Gln-Val EPS8-101-2L)；

When b be Ile, c be Ile when, sequence be Ile-Leu-Asp-Asp-Ile-Glu-Phe-Phe-Ile (i.e. ILDDIEFFI, is designated as EPS8-276)；

When b be Ile, c be Val when, sequence be Ile-Leu-Asp-Asp-Ile-Glu-Phe-Phe-Val (i.e. ILDDIEFFV, is designated as EPS8-276-9V)；

When b be Tyr, c be Val when, sequence be Tyr-Leu-Asp-Asp-Ile-Glu-Phe-Phe-Val (i.e. YLDDIEFFV, is designated as EPS8-276-1Y9V)；

Phe-Leu-Phe-Thr-Pro-Leu-Asn-Met-Val (i.e. FLFTPLNMV, be designated as EPS8-360)；

When d is Gln, sequence is that (i.e. QLAESVANV, is designated as Gln-Leu-Ala-Glu-Ser-Val-Ala-Asn-Val EPS8-455)；

When d is Tyr, sequence is that (i.e. YLAESVANV, is designated as Tyr-Leu-Ala-Glu-Ser-Val-Ala-Asn-Val EPS8-455-1Y)。

In addition, the present invention covers peptide by modification, wherein substitute or with the addition of one, two or more amino acid and Obtained sequence, as long as the peptide by modification retains original CTL inducibilities.

The HLA-A*0201 restricted CTL epitopes in the tumour antigen EPS8 sources are preparing tumor therapeutic polypeptide vaccine In application.

Wherein, the EPS8 positive tumors include cervical carcinoma, colorectal cancer, hypophysoma, oral squamous cell carcinomas, Pancreatic neoplasms, Breast cancer, thyroid cancer, the cancer of the esophagus, glioblastoma, Huppert's disease, acute myeloid leukemia, mixed stocker leukaemia etc..

The beneficial effects of the present invention are：The invention discloses the natural CTL epitope peptides from tumour antigen EPS8 and Mimic epitope peptide, the CTL epitopes can peptide can the specific CTL immune responses of induction peptide, show as that peptide specific can be stimulated CTL secreting high levels IFN-γ simultaneously produces specific killing effect to EPS8 positive tumor cells；Because tumour antigen EPS8 is wide General to be expressed in different types of tumor tissues, therefore, CTL epitope peptides of the invention can attend class as active component, and pharmacy The carrier of receiving is grouped compound, or, attend class and connect with other extracts with pharmacological activity and/or synthetic drug and pharmacy The carrier received is grouped compound, and the conventional method according still further to pharmaceutical field prepares the tumor vaccine based on antigen EPS8, for controlling Treat and/or the positive tumour of prevention EPS8 expression, and/or prevent its recurrence after operation, have very in immunotherapy of tumors field Good application prospect.

Brief description of the drawings

Fig. 1 is the testing result of the specific CTL secretion of gamma-IFN ability of epitope inducing peptide of the present invention；

Fig. 2 is testing result of the specific CTL to tumor cell specific lethal effect of epitope inducing peptide of the present invention；

Fig. 3 is the specific CTL of epitope peptide EPS8-101 of the present invention inductions to tumor cell specific lethal effect The restricted testing results of HLA-A*0201；

Fig. 4 is the specific CTL of epitope peptide EPS8-276 of the present invention inductions to tumor cell specific lethal effect The restricted testing results of HLA-A*0201；

Fig. 5 is the specific CTL of epitope peptide EPS8-360 of the present invention inductions to tumor cell specific lethal effect The restricted testing results of HLA-A*0201；

Fig. 6 is the specific CTL of epitope peptide EPS8-455 of the present invention inductions to tumor cell specific lethal effect The restricted testing results of HLA-A*0201；

Fig. 7 is the specific CTL of epitope peptide EPS8-101-1Y of the present invention inductions to tumor cell specific lethal effect The restricted testing results of HLA-A*0201；

Fig. 8 epitope peptides of the present invention are the specific CTL of EPS8-276-9V inductions to tumor cell specific lethal effect The restricted testing results of HLA-A*0201；

Fig. 9 is the specific CTL of epitope peptide EPS8-276-1Y9V of the present invention inductions to tumor cell specific lethal effect The restricted testing results of HLA-A*0201；

Figure 10 is the specific CTL of epitope peptide EPS8-455-1Y of the present invention inductions to tumor cell specific lethal effect The restricted testing results of HLA-A*0201；

Embodiment

For below with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.

First, the prediction of EPS8HLA-A*0201 restricted CTL epitopes

1st, biological software prediction CTL epitopes

The antitumor CTL epitope peptide analog in EPS8 sources of the present invention, is the primary structure according to antigen, uses Immunoinformatics means, move online biological software SYFPEITHI, BIMAS and NetMHC 3.2 to EPS8 antigenic structures HLA-A*0201 is restricted to be predicted analysis, and screening WTQDMILQV (being designated as EPS8-101), ILDDIEFFI (are designated as EPS8- 276), FLFTPLNMV (being designated as EPS8-360) and QLAESVANV (being designated as EPS8-455) be as candidate's CTL epitope peptides, and according to Different aminoacids residue is affine to epitope and HLA-A*0201 molecules on HLA-A*0201 restricted epitopes motif and other positions The influence of power, the amino acid residue of dominant anchor residues or its adjacent position to above-mentioned natural CTL epitopes is replaced, and is made CTL epitopes are unfavorable for the amino acid residue combined or are conducive to the unnecessary ammonia for being unfavorable for combining of the amino acid residue combined Base acid residue, thus designs 4 simulation CTL epitopes：WLQDMILQV (being designated as EPS8-101-2L), ILDDIEFFV (are designated as EPS8-276-9V), YLDDIEFFV (being designated as EPS8-276-1Y9V) and QLAESVANV (being designated as EPS8-455).

2nd, the synthesis of candidate's simulation CTL epitope

Candidate's simulation CTL epitope entrusts Zhongtai Bio-Chem. Co., Ltd., Hangzhou to be synthesized using standard Fmoc schemes, and adopts Purified with reversed-phased high performace liquid chromatographic and purity analysis, mass spectrography is identified and molecular weight determination.As a result show, respectively The purity of candidate's simulation CTL epitope is above 95%, and molecular weight is consistent with theoretical value.

Storing solution is made in gained candidate's CTL epitope peptides dimethyl sulfoxide (DMSO) (DMSO) dissolving, puts -70 DEG C of preservations of temperature, faces Shi Qianyong phosphate buffers (PBS) dilute.Synthesis in solid state step refers to existing technology.

3rd, the CTL epitope peptides of above-mentioned preparation, available for tumor therapeutic polypeptide vaccine is prepared, its application experiment is as follows：

3.1st, candidate CTL epitope peptides and HLA-A*0201 molecules affinity are tested

(1) the T2 cells of the HLA-A2.1 expression positive and deleting endogenous antigen working process ability are collected by centrifugation in 800rpm (ATCC companies), PBS is washed 3 times, and it is 1 × 10 that cell to cell density, which is resuspended, in serum-free RPMI-1640 culture mediums⁶/ ml, inoculation In 24 orifice plates；

(2) CTL epitope peptides, positive control peptide (influenza matrix protein IMP are added per hole_58-66：GILGFVFTL it is) or negative right According to peptide (Idiotype_98-106：AHTKDGFNF) to final concentration of 50 μ g/ml (including the μ g/ml of β2-microglobulin 3), 37 DEG C, 5% CO₂, be incubated 18h under the conditions of saturated humidity；

(3) after ice PBS is washed 3 times, the HLA-A2.1 antibody of FITC marks is added, 4 DEG C of lucifuges are incubated 30min；

(4) after ice PBS is washed 3 times, flow cytometer determines average fluorescent strength at wavelength 488nm, and calculates fluorescence Coefficient FI：FI=(sample average fluorescence intensity-background average fluorescent strength)/background average fluorescent strength is detected.FI ＞ 1.5 are High-affinity, 1.0 ＜ FI ＜ 1.5 are medium affinity, and FI ＜ 1.0 are low-affinity.

As a result：As shown in table 1, EPS8-276, EPS8-360 and EPS8-455 epitope peptide and HLA-A2.1 molecules have height Affinity, EPS8-101 epitope peptides have medium affinity；And the adhesion of transformation peptide and HLA-A2.1 molecules is above parent peptide.

3.2nd, candidate CTL epitope peptides/HLA-A2.1 molecular complex stability analyses

(1) T2A2 cells are collected by centrifugation in 800rpm, are washed with ice PBS 3 times, and the culture mediums of serum-free RPMI 1640 (contain 100ng/ml human β-2microglobulins) be resuspended to cell density be 1.0 × 10⁶/ ml, is inoculated in 24 well culture plates；

(2) CTL epitope peptides are added per hole to final concentration of 100 μm of ol/ml, 37 DEG C, 5%CO₂And under the conditions of saturated humidity It is incubated 18h；

(3) ice PBS washs cell 4 times, removes uncombined free peptide；

(4) Brefeldin A (BFA, Sigma company) are added per hole to final concentration of 10 μ g/ml, 37 DEG C, 5%CO₂And 1h is incubated under the conditions of saturated humidity；

(5) ice PBS washs cell 1 time；

(6) culture medium of serum-free 1640 containing 0.5 μ g/ml BFA is added, in 37 DEG C, 5%CO₂And under the conditions of saturated humidity 0h, 2h, 4h, 6h, 8h are incubated respectively；

(7) it is incubated after terminating, ice PBS is washed 3 times, adds the HLA-A2.1 antibody of FITC marks, 4 DEG C of lucifuge reactions 30min；

(8) after ice PBS is washed 3 times, flow cytometer determines average fluorescent strength at wavelength 488nm.As a result epitope is used The half-life period DC50 of peptide/HLA-A2.1 molecular complexes is represented.

As a result：As shown in table 1, the half-life period of 8 epitope peptide/HLA-A2.1 compounds is all higher than 6h.

The epitope peptide of table 1 and HLA-A2.1 molecular binding affinities and stability test result

Natural epitopes peptide	FI	DC₅₀/h	Mimic epitope peptide	FI	DC₅₀/h
						EPS8-101	1.03	6~8	EPS8-101-2L	2.51	＞ 8
EPS8-276	2.10	＞ 8	EPS8-276-9V	4.95	＞ 8
						EPS8-360	1.99	＞ 8	EPS8-276-1Y9V	4.54	＞ 8
EPS8-455	2.07	＞ 8	EPS8-455-1Y	2.75	＞ 8

3.3rd, the specific CTL secretion of gamma-IFN ability detection of CTL epitopes inducing peptide

1st, the preparation of effector cell

(1) anti-freezing of conventional Ficoll-Hypaque density gradient method separation HLA-A*0201 positive healthy volunteers is new Fresh whole blood, obtains PMNC (PBMC), adjusts thin with the RPMI-1640 complete mediums containing 10% hyclone Born of the same parents' concentration is 1.0 × 10⁶/ ml, is inoculated in 24 orifice plates, per hole 1ml；

(2) every group of next day is separately added into CTL epitope peptides to final concentration of 10 μ g/ml, (is substituted while setting up PBS groups with PBS Epitope peptide) it is used as negative control group；

IL-2 to final concentration of 50U/ml is added per hole within (3) the 3rd days, every 2~3 days half amounts change liquid and supplement IL-2 to end Concentration is 50U/ml；

(4) carrying out the second wheel, third round lotus peptide respectively at the 7th day, fortnight stimulates, i.e., every group is separately added into CTL tables Position peptide/PBS to final concentration of 10 μ g/ml, next day adds IL-2 to final concentration of 50U/ml；

(5) 3 days after third round is stimulated, effector cell CTL is obtained.

2nd, ELISPOT is tested：Use Human IFN-gamma ELISPOT kit (ELISPOT kits, U-Cytech Company) detection of IFN-γ secretion is carried out, key step is following (referring to kit specification)：

(1) it is coated with：

1. add 70% ethanol to PVDF plates to prewet, 15 μ l/ holes；

2. add deionized water to wash 2 times, 100 μ l/ holes；

3. the coated antibody that PBS has diluted is added, 50 μ l/ holes, 4 DEG C of coatings are stayed overnight；

(2) close：

1. coating buffer is toppled over；

2. PBS is washed 3 times, and 100 μ l/ holes are patted dry on the blotting paper of sterilizing for the last time；

3. the confining liquid diluted, 200 μ l/ holes, 37 DEG C of closing 1h are added；

(3) cell loading：

1. topple over confining liquid, PBS washed once, patted dry on the blotting paper of sterilizing；

2. cell upper plate：The CTL of above-mentioned induction is used as effector cell (1 × 10⁵/ hole), the T2A2 cells of lotus peptide are used as thorn Swash cell (1 × 10⁵/ hole) bed board, 100 μ l/ holes.Positive control group is set：Cell concentration is 1 × 105/ hole, adds PHA dense to end Spend for 1~4 μ l/ml, the secretion of the concentration energy effective stimulus IFN-γ；Background negative control group is set：100 μ l are added containing 10% tire The RPMI-1640 culture mediums of cow's serum；

3. add after all samples, cover plate lid, 37 DEG C, 5%CO2 is incubated 18~24h under the conditions of saturated humidity；

(4) operated after cultivating：

1. Low Osmotic Method cell lysis：Pouring aperture inner cell and culture medium, add ice-cold deionized water, 200 μ l/ holes, 4 DEG C Ice bath 10min；

2. wash：Liquid in pouring aperture, adds the washing buffer diluted and washs 5~7 times, 200 μ l/ holes, often 30~60s of secondary stop, last time, is patted dry on blotting paper；

3. antibody incubation is detected：Add the biotin labeling detection antibody diluted, 100 μ l/ holes, 4 DEG C of overnight incubations；

4. wash：Liquid in next day pouring aperture, adds the washing buffer diluted and washs 5~7 times, 200 μ l/ Hole, stops 30~60s every time, and last time is patted dry on blotting paper；

5. enzyme-linked Avidin is incubated：Add the enzyme-linked Avidin working solution diluted, 100 μ l/ holes, 37 DEG C of incubation 1h；

6. wash：Liquid in pouring aperture, adds the washing buffer diluted and washs 5 times, 200 μ l/ holes are stopped every time 30~60s is stayed, last time is patted dry on blotting paper；

7. develop the color：The AEC nitrite ions configured are added, 100 μ l/ holes, room temperature lucifuge stands 15~30min；

8. color development stopping：Liquid in pouring aperture, deionized water is washed 3~5 times, color development stopping process；

9. ELSPOT image analyzers count the spot number formed per hole.

As a result：As shown in figure 1, natural CTL epitope peptides EPS8-101, EPS8-276, EPS8-360, EPS8-455 and its mould Intend the specific CTL secretion of epitope peptide EPS8-101-2L, EPS8-276-9V, EPS8-276-1Y9V, EPS8-455-1Y induction The ability of IFN-γ is apparently higher than negative control group (P ＜ 0.001).

3.4th, detection of the specific CTL of CTL epitopes inducing peptide to tumor cell specific lethal effect

(1) experiment packet：

1. effector cell：It is divided into 9 big group of samples：EPS8-101, EPS8-276, EPS8-360, EPS8-455 and EPS8- 101-2L, EPS8-276-9V, EPS8-276-1Y9V, EPS8-455-1Y, respectively using the specific CTL of each epitope inducing peptide as Effector cell；

2. target cell：Human breast cancer cell line Bcap-37 (HLA-A*0201 expression is positive, EPS8 expression is positive), lotus peptide T2A2 Cell (HLA-A*0201 expression is positive, EPS8 expression is positive), T2 cells (HLA-A*0201 expression is positive, and EPS8 expression is negative)

(2) prepared by effector cell：The specific CTL of CTL epitope inducing peptides is prepared according to foregoing same method, with containing 5% The RPMI-1640 culture mediums of hyclone are adjusted to debita spissitudo, are used as effector cell.

(3) LDH is detected：Use CytoToxNon-Radioactive Cytotoxicity Assay (cell toxicants Property detection kit, Promega companies) carry out LDH testing inspection cytotoxic activities, key step (refers to kit to say as follows Bright book)：

<1>Set up detection plate (100 μ l/ holes)

1. experimental group is set up：Using MCF-7, lotus peptide T2A2, T2A2 cell as target cell (optimal target cell number as 5 × 10³/ hole), compare 10 by different effect targets:1、20:1、40:1 adds above-mentioned effector cell CTL, and various concentrations of cells are with 50 μ l/ holes It is inoculated in 96 well culture plates, the μ l of final volume 100；

2. the spontaneous release group of effector cell is set up：Each group effector cell adds 96 orifice plates with 50 μ l/ holes, and 50 μ l of supplement contain The RPMI-1640 culture mediums of 5% hyclone are to the μ l of final volume 100；

3. the spontaneous release group of target cell is set up：Each group target cell adds 96 orifice plates with 50 μ l/ holes, and 50 μ l of supplement contain 5% tire The RPMI-1640 culture mediums of cow's serum are to the μ l of final concentration 100；

4. the maximum release group of target cell is set up：Cell loading is with the spontaneous release group of target cell；

5. ground control group is set up：Add the μ l of RPMI-1640 culture mediums 100 containing 5% hyclone；

6. volume correction control group is set up：Add the μ l of RPMI-1640 culture mediums 100 containing 5% hyclone；

<2>Cell is cracked and harvest supernatant

1. after plating cells, 250g centrifugation 4min, 37 DEG C, 5%CO₂, 4h is incubated under the conditions of saturated humidity；

2. 45min before harvest supernatant, lysate (10 is added respectively at the maximum release group of target cell and volume correction control group ×), 10 μ l/ holes；

3. 250g centrifuges 4min, harvests supernatant；

<3>LDH is detected

1. supernatant is shifted to another 96 orifice plate, 50 μ l/ holes；

2. the Substrate cocktail that rapid addition dilutes, 50 μ l/ holes, room temperature lucifuge reaction 30min；

3. the reaction of addition terminate liquid color development stopping, 50 μ l/ holes；

4. take out in bubble in hole, 1h in ELIASA detection 490nm light absorption values OD.

(4) cell killing rate is calculated

Killing rate (%)=[(the spontaneous spontaneous release group OD of release group OD values-target cell of experimental group OD values-effector cell Value)/(the target cell maximum release group OD values-spontaneous release group OD values of target cell)] × 100%

(note：All experimental groups, the spontaneous release group of effector cell, the OD values of the spontaneous release group of target cell all should subtract background Mean absorbance；Target cell maximum release group absorption value should subtract the mean absorbance of volume correction control group)

As a result：As shown in Fig. 2 natural CTL epitope peptides EPS8-101, EPS8-276, EPS8-360, EPS8-455 and its mould Intend epitope peptide EPS8-101-2L, EPS8-276-9V, EPS8-276-1Y9V, EPS8-455-1Y to HLA-A*0201 and EPS8 tables T2A2 cells up to positive MCF-7 cells and load EPS8 are respectively provided with specific killing action, and to not expressing EPS8 table T2A2 cells up to HLA-A*0201 have no specific killing action.As shown in Figures 3 to 10, with anti-HLA-A2.1 antibody honey After the EPS8 expression of the HLA-A2.1 molecules or silence MCF-7 of MCF-7 cell surfaces, each EPS8 antitumor CTL epitope peptides induction Specific CTL it is each effect target compare MCF-7/A2Ab, MCF-7/EPS8- cell be showed no specific killing, show its induce Specific CTL to MCF-7 be antigentic specificity, the restricted killings of MHC.

Based on above-mentioned experimental result, it can draw the following conclusions：Natural CTL epitope peptides EPS8-101, EPS8-276, EPS8- 360th, EPS8-455 and its mimic epitope peptide EPS8-101-2L, EPS8-276-9V, EPS8-276-1Y9V, EPS8-455-1Y with HLA-A*0201 molecules have good affinity and combination stability, meanwhile, its specific CTL induced can secrete certain IFN-γ, and express HLA-A*0201 and EPS8 positive MCF-7 cells and load EPS8 T2A2 cells and show Go out certain antigentic specificity lethal effect；Because tumour antigen EPS8 wide expressions are in the different types of tumor group of different times In knitting, therefore, EPS8 antitumor CTL epitopes of the invention can be constituted as active component, and pharmaceutically acceptable carrier Composition, or, it is grouped with other extracts with pharmacological activity and/or synthetic drug and pharmaceutically acceptable carrier The therapeutical peptide vaccine that EPS8 expresses positive tumor is made in compound, the conventional method according still further to pharmaceutical field, in tomour specific Property immunotherapy field has huge development and application potentiality.

Claims

1. a kind of EPS8 antitumor CTL epitope peptides, are nonapeptide, its sequence is：Phe-Leu-Phe-Thr-Pro-Leu-Asn- Met-Val。

2. EPS8 antitumor CTL epitope peptides according to claim 1, it is characterised in that：CTL is prepared using solid-phase synthesis Epitope peptide.

3. application of the EPS8 antitumor CTL epitope peptides in tumor is prepared described in claim 1.