CN104497125B

CN104497125B - EPS8 antitumor CTL epitope peptides and its application

Info

Publication number: CN104497125B
Application number: CN201410857508.3A
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: 2018-04-27
Anticipated expiration: 2033-12-23
Also published as: CN104497125A

Abstract

A kind of antitumor CTL epitope peptide in EPS8 sources, is nonapeptide, its sequence is：D Leu Ala Glu Ser Val Ala Asn Val, wherein, d is Gln or Tyr.The invention also discloses application of the epitope peptide in medicine.The specific CTL that the present invention induces can secrete certain IFN γ, and certain antigentic specificity lethal effect is shown to the T2A2 cells of positive 7 cells of MCF of HLA A*0201 and EPS8 expression and load EPS8, the therapeutical peptide vaccine of manufactured EPS8 expression 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 a kind of EPS8 sources it is antitumor 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) effect in tumour is suppressed greatly paid attention to, the specific cell for how effectively exciting CTL to mediate 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 influencing 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 period, 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 becomes 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 to induce the key factor of CTL immune responses, and CTL epitopes are anchor residues with the important foundation that MHC- class Ⅰmolecules are combined, Therefore, can be special without influencing to reach enhancing immunogenicity by varying 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, the ability that epitope is combined with MHC can be improved 10~100 times, and one Position, which introduces Tyr, can then rely on 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 Have strong with reference to power and candidate's CTL epitope peptides in EPS8 sources of effective inducing antitumor immunity response 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 shapes 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 provided 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.

Technical solution is used by the present invention solves above-mentioned technical problem：

EPS8 antitumor CTL epitope peptides, are nonapeptide, 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 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, for Trp-Thr-Gln-Asp-Met-Ile-Leu-Gln-Val, (i.e. WTQDMILQV, is denoted as sequence EPS8-101)；

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

When b is Ile, and c is Ile, sequence for Ile-Leu-Asp-Asp-Ile-Glu-Phe-Phe-Ile (i.e. ILDDIEFFI, is denoted as EPS8-276)；

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

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

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

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

When d is Tyr, for Tyr-Leu-Ala-Glu-Ser-Val-Ala-Asn-Val, (i.e. YLAESVANV, is denoted as sequence 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；Since tumour antigen EPS8 is wide General to be expressed in different types of tumor tissues, therefore, CTL epitope peptides of the invention can be used as active ingredient, and pharmacy to attend class The carrier of receiving is grouped compound, alternatively, attending class and connecing with other extracts with pharmacological activity and/or synthetic drug and pharmacy The carrier received is grouped compound, the tumor vaccine based on antigen EPS8 is prepared according still further to the conventional method of pharmaceutical field, for controlling Treat and/or prevention EPS8 expresses positive tumour, 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 attached drawing, the preferred embodiment of the present invention is 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, is used 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 denoted as EPS8-101), ILDDIEFFI (are denoted as EPS8- 276), FLFTPLNMV (being denoted as EPS8-360) and QLAESVANV (being denoted as EPS8-455) is used 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 are replaced, make The unnecessary ammonia for being unfavorable for combining of amino acid residue that CTL epitopes are unfavorable for the amino acid residue combined or are conducive to combine Base acid residue, thus designs 4 simulation CTL epitopes：WLQDMILQV (being denoted as EPS8-101-2L), ILDDIEFFV (are denoted as EPS8-276-9V), YLDDIEFFV (being denoted as EPS8-276-1Y9V) and QLAESVANV (being denoted 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.It is the results show that each 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 are 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) extremely final concentration of 50 μ g/ml (including 3 μ g/ml of β2-microglobulin), 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 measures average fluorescent strength at wavelength 488nm, and calculates fluorescence Coefficient FI：FI=(sample average fluorescence intensity-background average fluorescent strength)/background average fluorescent strength detects.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 combination power 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 3 times with ice PBS, and 1640 culture mediums of serum-free RPMI (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) 1640 culture medium of serum-free 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) after being incubated, 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 measures 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 peptides/HLA-A2.1 compounds is all higher than 6h.

1 epitope peptide of table 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 peripheral blood mononuclear cells (PBMC), is adjusted with the RPMI-1640 complete mediums containing 10% hyclone thin 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, while sets up PBS groups and (substituted 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) respectively at the 7th day, fortnight carry out the second wheel, third round lotus peptide stimulates, i.e., every group is separately added into CTL tables Position peptide/PBS to final concentration of 10 μ g/ml, next day add IL-2 to final concentration of 50U/ml；

(5) 3 days after third round stimulates, 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. adding the coated antibody that PBS has diluted, 50 μ l/ holes, 4 DEG C of coatings are overnight；

(2) close：

1. topple over coating buffer；

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

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

(3) cell loading：

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

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

3. after adding all samples, covering plate lid, 37 DEG C, 5%CO2,18~24h is incubated 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, pats dry on blotting paper；

3. detect antibody incubation：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, last time, pats dry on blotting paper every time；

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, stop every time 30~60s is stayed, last time, pats dry on blotting paper；

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

8. color development stopping：Liquid in pouring aperture, deionized water are 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, as effector cell.

(3) LDH is detected：Use CytoToxNon-Radioactive Cytotoxicity Assay (cytotoxicities Detection kit, Promega companies) LDH testing inspection cytotoxic activities are carried out, key step (refers to kit explanation as follows Book)：

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

1. set up experimental group：Using MCF-7, lotus peptide T2A2, T2A2 cell as target cell (optimal target cell number as 5 × 10³/ hole), by different effect targets than 10:1、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, 100 μ l of final volume；

2. set up the spontaneous release group of effector cell：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 100 μ l of final volume；

3. set up the spontaneous release group of target cell：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 100 μ l of final concentration；

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

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

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

<2>Cell cracking 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. harvesting 45min before supernatant, lysate (10 is added respectively at target cell maximum release group and volume correction control group ×), 10 μ l/ holes；

3. 250g centrifuges 4min, supernatant is harvested；

<3>LDH is detected

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

2. diluted Substrate cocktail is added rapidly, 50 μ l/ holes, room temperature lucifuge reaction 30min；

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

4. bubble in hole is taken out, in microplate reader detection 490nm light absorption values OD in 1h.

(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 should all 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 induction Specific CTL to MCF-7 for antigentic specificity, the restricted killings of MHC.

Based on above-mentioned experimental result, 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 be secreted certain IFN-γ, and to the positive MCF-7 cells of HLA-A*0201 and EPS8 expression and load the T2A2 cells of EPS8 and show Go out certain antigentic specificity lethal effect；Since 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 used as active ingredient, and pharmaceutically acceptable carrier composition Composition, alternatively, being grouped with other extracts with pharmacological activity and/or synthetic drug and pharmaceutically acceptable carrier Compound, is made the therapeutical peptide vaccine of EPS8 expression positive tumors, in tomour specific according still further to the conventional method of pharmaceutical field Property immunotherapy field has huge development and application potentiality.

Claims

1. a kind of EPS8 antitumor CTL epitope peptides, are nonapeptide, its sequence is：d-Leu-Ala-Glu-Ser-Val-Ala-Asn- Val, wherein, d is Gln or Tyr.

2. EPS8 antitumor CTL epitope peptides according to claim 1, it is characterised in that：When d is Gln, its sequence is： Gln-Leu-Ala-Glu-Ser-Val-Ala-Asn-Val。

3. EPS8 antitumor CTL epitope peptides according to claim 1, it is characterised in that：When d is Tyr, its sequence is： Tyr-Leu-Ala-Glu-Ser-Val-Ala-Asn-Val。

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

5. the EPS8 antitumor CTL epitope peptides described in claims 1 to 3 any one claim are preparing treatment breast cancer drug Application in thing.