CN104497123A

CN104497123A - EPS8-derived antitumor CTL epitope peptide and application thereof

Info

Publication number: CN104497123A
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: 2015-04-08
Anticipated expiration: 2033-12-23
Also published as: CN104497123B

Abstract

The invention discloses an EPS8-derived antitumor CTL epitope peptide. The EPS8-derived antitumor CTL epitope peptide is a nonapeptide with the sequence of Phe-Leu-Phe-Thr-Pro-Leu-Asn-Met-Val. The invention also discloses an application of the epitope peptide to a drug. A specific CTL induced by the EPS8-derived antitumor CTL epitope peptide can secrete a certain IFN-gamma and has a certain antigen-specificity killing effect for HLA-A*0201, EPS8 positive-expression MCF-7 cells and EPS8-loaded T2A2 cells; and a prepared EPS8 positive-expression tumor treating polypeptide vaccine has huge development and application potentials in the field of specific immunotherapy of tumor.

Description

EPS8 antitumor CTL epitope peptide and application thereof

The divisional application that the application is the applying date is on December 23rd, 2013, application number is 201310717092.0, denomination of invention is the Chinese invention patent application of EPS8 antitumor CTL epitope peptide and application thereof.

Technical field

The invention belongs to the technical field of polypeptide in biochemical field, be specifically related to the antitumor CTL epitope peptide in a kind of EPS8 source and the application in preparation tumor thereof.

Background technology

Malignant tumour is first killer of the new millennium mankind.The traditional therapies such as operation, radiotherapy and chemotherapy are still undesirable to the result for the treatment of of some tumour especially late malignant tumour.In recent years, cytotoxic T lymphocyte (Cytotoxic TLymphocyte, CTL) effect in Tumor suppression is subject to very big attention, how effectively to excite the specificity cellular immunity response that CTL mediates, play antitumor usefulness, become an important topic in current tumor therapeutic polypeptide vaccine development field.

The discovery of tumour antigen and the qualification of CTL epi-position thereof are the important prerequisites of tumor-specific immunity treatment and the development of tumor therapeutic polypeptide vaccine.EGF-R ELISA path substrate No.8 (Epidermal Growth FactorReceptor pathway substrate No.8, EPS8) is one of important kinase activity substrate of EGF-R ELISA (EGFR).Research in recent years shows, EPS8 multiple solid tumor (as cervical cancer, colorectal cancer, pituitary tumor, oral squamous cell carcinomas, Pancreatic neoplasms, mammary cancer, thyroid carcinoma, the esophageal carcinoma and glioblastoma etc.) and hematological system tumor (as multiple myeloma, acute myeloid leukemia, mixed stocker leukemia etc.) tissue and cells is abnormal raises, and very low or without expression in normal tissue expression.Further research display, EPS8 promotes the propagation of tumour cell by cell cycle regulation, by participating in the formation of cell pseudopodium and promoting the transfer of tumour cell with the interaction of Actin muscle, by affect tumour cell to the tolerance of chemotherapeutics relevant with the prognosis of patient ( li Y*, Xue T, He Y, Du J.Anovel oncoprotein Eps8:new target for anti-cancer therapy.Future Oncology.2013 (Accepted)).In addition, this seminar finds in the experimental study in early stage, recombined small-mouse rmHis-Eps8 protein vaccine can effectively induce lotus 4T1 breast cancer mouse to produce specificity antineoplastic immunity response, suppress the propagation of mouse interior tumor cell, extend its survival time, and to mouse brain, the heart, liver, kidney, small intestine, epididymis tissue without obvious toxic action.(He YJ,Zhou J, Li Y*,et al.Eps8vaccine exerts prophylactic antitumor effects in a murinemodel:a novel vaccine for breast carcinoma.Mol Med Rep.2013Aug；8(2):662-8)。Therefore, EPS8 is the novel targets of diagnosing tumor and antineoplaston, in immunotherapy of tumors, have great potential.

Immunogenicity is weak significantly limit the application of natural CTL epi-position in tumor therapeutic polypeptide vaccine with immunological tolerance, therefore, the immunogenicity of natural CTL epi-position is effectively improved and the immunological tolerance breaking body becomes the key of tumor therapeutic polypeptide vaccine development.In the enhancing strategy of numerous tumor therapeutic polypeptide vaccine, molecular modification is carried out to natural CTL epi-position and is considered to one of the most effective method.Because the combination of CTL epi-position and major histocompatibility complex (MHC)-class Ⅰmolecule is the key factor that elicit CTL immune is replied, and the important foundation that CTL epi-position is combined with MHC-class Ⅰmolecule is anchor residues, therefore, the amino-acid residue by changing anchor residues or its consecutive position does not affect specific object to reach enhancing immunogenicity.Research finds, in nonapeptide CTL epi-position, second and the 9th are the dominant anchor site of HLA-A2.1 molecule, two is Leu or Met, nine is Leu, Val or Ile, the ability that epi-position can be combined with MHC improves 10 ~ 100 times, introduces Tyr the hydrophobic interaction of its side-chain benzene ring and the hydrogen bond action of phenolic hydroxyl group then can be relied on to strengthen the interaction of epitope peptide and HLA molecule at one.

The application mainly adopts theoretical and tests the method combined, and modifies and preliminary evaluation goes out to have strong bonding force with MHC-class Ⅰmolecule and the EPS8 that effectively inducing antitumor immunity the is replied candidate CTL epitope peptide of originating and analogue thereof.

Summary of the invention

A technical problem to be solved by this invention provides for the above-mentioned state of the art HLA-A*0201 restricted CTL epitope that a kind of tumour antigen EPS8 originates, this epi-position can with high-affinity in conjunction with HLA-A*0201 molecule, the stable composite formed, the CTL immunne response of peptide specific can be induced, show as the IFN-γ of stimulator polypeptide specific CTL secreting high levels and specificity kill and wounding effect is produced to tumour cell.

Another technical problem to be solved by this invention provides a kind of application of HLA-A*0201 restricted CTL epitope peptide in preparation tumor in the former EPS8 source of tumour.

The present invention solves the problems of the technologies described above adopted technical scheme:

EPS8 antitumor CTL epitope peptide, be nonapeptide, its sequence is: Trp-a-Gln-Asp-Met-Ile-Leu-Gln-Val, and wherein, a is Thr or Leu; B-Leu-Asp-Asp-Ile-Glu-Phe-Phe-c, wherein, b is Ile or Tyr, c is 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 Trp-Thr-Gln-Asp-Met-Ile-Leu-Gln-Val (i.e. WTQDMILQV, is designated as EPS8-101);

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

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

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

When b be Tyr, c is Val, sequence is 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, is designated as EPS8-360);

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

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

In addition, the peptide through modifying is contained in the present invention, wherein substitutes or with the addition of one, two or more amino acid and the sequence that obtains, as long as retain original CTL inducibility through the peptide modified.

The HLA-A*0201 restricted CTL epitope that described tumour antigen EPS8 originates is preparing the application in tumor therapeutic polypeptide vaccine.

Wherein, described EPS8 positive tumor comprises cervical cancer, colorectal cancer, pituitary tumor, oral squamous cell carcinomas, Pancreatic neoplasms, mammary cancer, thyroid carcinoma, the esophageal carcinoma, glioblastoma, multiple myeloma, acute myeloid leukemia, mixed stocker leukemia etc.

Beneficial effect of the present invention is: the invention discloses the natural CTL epitope peptide and analogue epi-peptide that derive from tumour antigen EPS8, this CTL epi-position can induce the CTL immunne response of peptide specific by peptide, and showing as can stimulator polypeptide specific CTL secreting high levels IFN-γ produce specificity kill and wounding effect to EPS8 positive tumor cell; Because tumour antigen EPS8 wide expression is in dissimilar tumor tissues, therefore, CTL epitope peptide of the present invention can be used as activeconstituents, with pharmacy attend class accept vehicle group become composition, or, with other, there is the extract of pharmacologically active and/or synthetic drugs and the pharmacy vehicle group accepted of attending class and become composition, the tumor vaccine based on antigen EPS8 is prepared again according to the ordinary method of pharmaceutical field, be used for the treatment of and/or prevent EPS8 to express positive tumour, and/or prevent its recurrence after operation, in immunotherapy of tumors field, there is good application prospect.

Accompanying drawing explanation

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

Fig. 2 is that the specific CTL of epitope peptide of the present invention induction is to the detected result of tumor cell specific lethal effect;

Fig. 3 is the restricted detected result of the HLA-A*0201 of specific CTL to tumor cell specific lethal effect that epitope peptide EPS8-101 of the present invention induces;

Fig. 4 is the restricted detected result of the HLA-A*0201 of specific CTL to tumor cell specific lethal effect that epitope peptide EPS8-276 of the present invention induces;

Fig. 5 is the restricted detected result of the HLA-A*0201 of specific CTL to tumor cell specific lethal effect that epitope peptide EPS8-360 of the present invention induces;

Fig. 6 is the restricted detected result of the HLA-A*0201 of specific CTL to tumor cell specific lethal effect that epitope peptide EPS8-455 of the present invention induces;

Fig. 7 is the restricted detected result of the HLA-A*0201 of specific CTL to tumor cell specific lethal effect that epitope peptide EPS8-101-1Y of the present invention induces;

Fig. 8 epitope peptide of the present invention is the restricted detected result of the HLA-A*0201 of specific CTL to tumor cell specific lethal effect of EPS8-276-9V induction;

Fig. 9 is the restricted detected result of the HLA-A*0201 of specific CTL to tumor cell specific lethal effect that epitope peptide EPS8-276-1Y9V of the present invention induces;

Figure 10 is the restricted detected result of the HLA-A*0201 of specific CTL to tumor cell specific lethal effect that epitope peptide EPS8-455-1Y of the present invention induces;

Embodiment

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

One, the prediction of EPS8HLA-A*0201 restricted CTL epitope

1, biological software prediction CTL epi-position

The antitumor CTL epitope peptide analogue in EPS8 source of the present invention, it is the primary structure according to antigen, adopt Immunoinformatics means, move online biological software SYFPEITHI, the HLA-A*0201 of BIMAS and NetMHC 3.2 pairs of EPS8 antigenic structures is restricted carries out forecast analysis, screening WTQDMILQV (being designated as EPS8-101), ILDDIEFFI (being designated as EPS8-276), FLFTPLNMV (being designated as EPS8-360) and QLAESVANV (being designated as EPS8-455) alternatively CTL epitope peptide, and according to the impact of different aminoacids residue on HLA-A*0201 restricted epitope motif and other position on epi-position and HLA-A*0201 molecule avidity, the above-mentioned natural dominant anchor residues of CTL epi-position or the amino-acid residue of its consecutive position are replaced, CTL epi-position is not comprised and is unfavorable for the amino-acid residue combined or the unnecessary amino-acid residue being unfavorable for combining of amino-acid residue being conducive to combining, design 4 simulation CTL epitope: WLQDMILQV (being designated as EPS8-101-2L) thus, ILDDIEFFV (being designated as EPS8-276-9V), YLDDIEFFV (being designated as EPS8-276-1Y9V) and QLAESVANV (being designated as EPS8-455).

Two, the synthesis of candidate's simulation CTL epitope

Candidate's simulation CTL epitope entrusts Zhongtai Bio-Chem. Co., Ltd., Hangzhou to adopt standard Fmoc scheme to synthesize, and adopts reversed-phased high performace liquid chromatographic to carry out purifying and purity check, and mass spectroscopy carries out identifying and molecular weight determination.Result shows, and the purity of each candidate's simulation CTL epitope is all higher than 95%, and molecular weight conforms to theoretical value.

Gained candidate CTL epitope peptide dimethyl sulfoxide (DMSO) (DMSO) dissolves and makes storing solution, puts temperature-70 DEG C preservation, interim front phosphate buffered saline buffer (PBS) dilution.Solid phase synthesis step can with reference to existing technology.

Three, the CTL epitope peptide of above-mentioned preparation, can be used for preparing tumor therapeutic polypeptide vaccine, its application experiment is as follows:

3.1, candidate CTL epitope peptide and HLA-A*0201 molecule avidity are tested

(1) 800rpm collected by centrifugation HLA-A2.1 expresses the positive and the T2 cell (ATCC company) of deleting endogenous antigen processing treatment ability, and PBS washs 3 times, and serum-free RPMI-1640 substratum re-suspended cell is 1 × 10 to cell density ⁶/ ml, is inoculated in 24 orifice plates;

(2) every hole adds CTL epitope peptide, positive control peptide (influenza matrix protein IMP _58-66: GILGFVFTL) or negative control peptide (Idiotype _98-106: AHTKDGFNF) to final concentration be 50 μ g/ml (including β2-microglobulin 3 μ g/ml), 37 DEG C, 5%CO ₂, hatch 18h under saturated humidity condition;

(3), after ice PBS washs 3 times, add the HLA-A2.1 antibody of FITC mark, 4 DEG C of lucifuges hatch 30min;

(4) after ice PBS washs 3 times, flow cytometer measures average fluorescent strength in wavelength 488nm place, and calculates the detection of fluorescence coefficient FI:FI=(sample average fluorescence intensity-background average fluorescent strength)/background average fluorescent strength.FI > 1.5 is high-affinity, and 1.0 < FI < 1.5 are medium avidity, and FI < 1.0 is low-affinity.

Result: as shown in table 1, EPS8-276, EPS8-360 and EPS8-455 epitope peptide and HLA-A2.1 molecule have high-affinity, the medium avidity of EPS8-101 epitope peptide tool; And the bonding force of transformation peptide and HLA-A2.1 molecule is all higher than parent peptide.

3.2, candidate CTL epitope peptide/HLA-A2.1 molecular complex stability analysis

(1) 800rpm collected by centrifugation T2A2 cell, washs 3 times with ice PBS, serum-free RPMI 1640 substratum (containing 100ng/ml human β-2microglobulin) resuspended to cell density be 1.0 × 10 ⁶/ ml, is inoculated in 24 well culture plates;

(2) every hole adds CTL epitope peptide to final concentration is 100 μm of ol/ml, 37 DEG C, 5%CO ₂and hatch 18h under saturated humidity condition;

(3) ice PBS washed cell 4 times, removes unconjugated free peptide;

(4) every hole adds Brefeldin A (BFA, Sigma company) to final concentration is 10 μ g/ml, 37 DEG C, 5%CO ₂and hatch 1h under saturated humidity condition;

(5) ice PBS washed cell 1 time;

(6) serum-free 1640 substratum containing 0.5 μ g/ml BFA is added, in 37 DEG C, 5%CO ₂and hatch 0h, 2h, 4h, 6h, 8h respectively under saturated humidity condition;

(7) after hatching end, ice PBS washs 3 times, adds the HLA-A2.1 antibody of FITC mark, 4 DEG C of lucifuge reaction 30min;

(8), after ice PBS washs 3 times, flow cytometer measures average fluorescent strength in wavelength 488nm place.The transformation period DC50 of result epitope peptide/HLA-A2.1 molecular complex represents.

Result: as shown in table 1, the transformation period of 8 epitope peptide/HLA-A2.1 mixtures is all greater than 6h.

Table 1 epitope peptide and HLA-A2.1 molecular binding affinities and stability test result

Natural epitopes peptide	FI	DC ₅₀/h	Analogue epi-peptide	FI	DC ₅₀/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

3.3, the specific CTL secretion of gamma-IFN ability of CTL epitope peptide induction detects

1, the preparation of effector cell

(1) conventional Ficoll-Hypaque density gradient method is separated the anti-freezing fresh whole blood of HLA-A*0201 positive healthy volunteer, obtaining peripheral blood mononuclear cell (PBMC), is 1.0 × 10 with the RPMI-1640 perfect medium adjustment cell concn containing 10% foetal calf serum ⁶/ ml, is inoculated in 24 orifice plates, every hole 1ml;

(2) often organize that to add CTL epitope peptide to final concentration be respectively 10 μ g/ml next day, set up PBS group (substituting epitope peptide using PBS) as negative control group simultaneously;

It is 50U/ml that (3) the 3rd days every holes add IL-2 to final concentration, and every 2 ~ 3 days half amounts are changed liquid and supplemented IL-2 is 50U/ml to final concentration;

(4) respectively at the 7th day, fortnight carries out second taking turns, third round lotus peptide stimulates, and namely often organizes that to add CTL epitope peptide/PBS respectively to final concentration be 10 μ g/ml, adding IL-2 next day to final concentration is 50U/ml;

(5) third round stimulates latter 3 days, obtains effector cell CTL.

2, ELISPOT experiment: use Human IFN-gamma ELISPOT kit (ELISPOT test kit, U-Cytech company) to carry out the detection of IFN-γ secretion, key step following (referring to test kit specification sheets):

(1) bag quilt:

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

2. deionized water wash is added 2 times, 100 μ l/ holes;

3. add the coated antibody that PBS has diluted, 50 μ l/ holes, 4 DEG C of bags are spent the night;

(2) close:

1. coating buffer is toppled over;

2. PBS washs 3 times, and 100 μ l/ holes, pat dry for the last time on the thieving paper of sterilizing;

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

(3) cell loading:

1. topple over confining liquid, PBS washing once, the thieving paper of sterilizing pats dry;

2. cell upper plate: the CTL action effect cell (1 × 10 of above-mentioned induction ⁵/ hole), the T2A2 cell of lotus peptide is as irritation cell (1 × 10 ⁵/ hole) bed board, 100 μ l/ holes.Arrange positive control group: cell concn is 1 × 105/ hole, adding PHA to final concentration is 1 ~ 4 μ l/ml, the secretion of this concentration energy effective stimulus IFN-γ; Background is set and bears control group: add the RPMI-1640 substratum of 100 μ l containing 10% foetal calf serum;

3. after adding all samples, cover plate lid, 37 DEG C, 5%CO2, under saturated humidity condition, hatch 18 ~ 24h;

(4) operation after cultivating:

1. Low Osmotic Method lysing cell: pouring aperture inner cell and substratum, adds ice-cold deionized water, 200 μ l/ holes, 4 DEG C of ice bath 10min;

2. wash: liquid in pouring aperture, add the washing buffer diluted and wash 5 ~ 7 times, 200 μ l/ holes, stop 30 ~ 60s at every turn, for the last time, thieving paper pat dry;

3. antibody incubation is detected: add the biotin labeling diluted and detect antibody, 100 μ l/ holes, 4 DEG C of overnight incubation;

4. wash: liquid in next day pouring aperture, add the washing buffer washing of dilute 5 ~ 7 times, 200 μ l/ holes, stop 30 ~ 60s at every turn, for the last time, thieving paper pat dry;

5. enzyme connection avidin is hatched: add the enzyme connection avidin working fluid diluted, 100 μ l/ holes, hatch 1h for 37 DEG C;

6. wash: liquid in pouring aperture, adds the washing buffer diluted and wash 5 times, and 200 μ l/ holes, stop 30 ~ 60s at every turn, for the last time, thieving paper pats dry;

7. develop the color: add the AEC nitrite ion configured, 100 μ l/ holes, room temperature lucifuge leaves standstill 15 ~ 30min;

8. color development stopping: liquid in pouring aperture, deionized water wash 3 ~ 5 times, color development stopping process;

9. ELSPOT image analyzer counts the spot number that every hole is formed.

Result: as shown in Figure 1, the ability of the specific CTL secretion of gamma-IFN of natural CTL epitope peptide EPS8-101, EPS8-276, EPS8-360, EPS8-455 and analogue epi-peptide EPS8-101-2L, EPS8-276-9V, EPS8-276-1Y9V, EPS8-455-1Y induction thereof is apparently higher than negative control group (P < 0.001).

3.4, the specific CTL of CTL epitope peptide induction is to the detection of tumor cell specific lethal effect

(1) experiment grouping:

1. effector cell: be divided into large group of 9 samples: EPS8-101, EPS8-276, EPS8-360, EPS8-455 and EPS8-101-2L, EPS8-276-9V, EPS8-276-1Y9V, EPS8-455-1Y, the specific CTL of inducing with each epitope peptide is respectively for effector cell;

2. target cell: human breast cancer cell line Bcap-37 (the HLA-A*0201 expression positive, EPS8 express the positive), lotus peptide T2A2 cell (the HLA-A*0201 expression positive, EPS8 express the positive), T2 cell (HLA-A*0201 expresses the positive, and EPS8 expresses feminine gender)

(2) effector cell's preparation: the specific CTL preparing the induction of CTL epitope peptide according to aforementioned same method, is adjusted to proper concn, action effect cell with the RPMI-1640 substratum containing 5% foetal calf serum.

(3) LDH detects: use CytoTox non-Radioactive Cytotoxicity Assay (citotoxicity detection kit, Promega company) carries out LDH testing inspection cytotoxic activity, key step following (referring to test kit specification sheets):

<1> sets up check-out console (100 μ l/ hole)

1. experimental group is set up: (optimum target cell number is for 5 × 10 as target cell using MCF-7, lotus peptide T2A2, T2A2 cell ³/ hole), add above-mentioned effector cell CTL by different effect targets than 10:1,20:1,40:1, various concentrations of cells is inoculated in 96 well culture plates with 50 μ l/ holes, final volume 100 μ l;

2. set up the spontaneous release group of effector cell: each group effector cell adds 96 orifice plates with 50 μ l/ holes, supplement the RPMI-1640 substratum of 50 μ l containing 5% foetal calf serum to final volume 100 μ l;

3. set up the spontaneous release group of target cell: each group target cell adds 96 orifice plates with 50 μ l/ holes, supplement the RPMI-1640 substratum of 50 μ l containing 5% foetal calf serum to final concentration 100 μ l;

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

5. ground control group is set up: add the RPMI-1640 substratum 100 μ l containing 5% foetal calf serum;

6. volume correction control group is set up: add the RPMI-1640 substratum 100 μ l containing 5% foetal calf serum;

<2> lysis and results supernatant

1. after plating cells, the centrifugal 4min of 250g, 37 DEG C, 5%CO ₂, under saturated humidity condition, hatch 4h;

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

3. the centrifugal 4min of 250g, results supernatant;

<3>LDH detects

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

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

3. the reaction of stop buffer color development stopping is added, 50 μ l/ holes;

4. bubble in extraction hole, detects 490nm light absorption value OD in microplate reader in 1h.

(4) cell killing rate is calculated

Kill rate (%)=[(experimental group OD value-effector cell spontaneous release group OD value-target cell spontaneous release group OD value)/(target cell maximum release group OD value-target cell spontaneous release group OD value)] × 100%

(note: the OD value of all experimental group, effector cell's spontaneous release group, the spontaneous release group of target cell all should subtracting background mean absorbance; Target cell maximum release group absorption value should deduct the mean absorbance of volume correction control group)

Result: as shown in Figure 2, natural CTL epitope peptide EPS8-101, EPS8-276, EPS8-360, EPS8-455 and analogue epi-peptide EPS8-101-2L, EPS8-276-9V, EPS8-276-1Y9V, EPS8-455-1Y all have specific killing action to the T2A2 cell that HLA-A*0201 and EPS8 expresses positive MCF-7 cell and load EPS8, and have no specific killing action to the T2A2 cell of not expressing EPS8 and only express HLA-A*0201.As shown in Figures 3 to 10, after expressing with the HLA-A2.1 molecule of anti-HLA-A2.1 antibody honey MCF-7 cell surface or the EPS8 of reticent MCF-7, the specific CTL of each EPS8 antitumor CTL epitope peptide induction is showed no specific killing at each comparison of effect target MCF-7/A2Ab, MCF-7/EPS8-cell, show its specific CTL of inducing to MCF-7 be antigen-specific, MHC is restricted kills and wounds.

Based on above-mentioned experimental result, can draw the following conclusions: natural CTL epitope peptide EPS8-101, EPS8-276, EPS8-360, EPS8-455 and analogue epi-peptide EPS8-101-2L, EPS8-276-9V, EPS8-276-1Y9V, EPS8-455-1Y and HLA-A*0201 molecule have good avidity and combination stability, simultaneously, the specific CTL of its induction all can secrete certain IFN-γ, and all shows certain antigen-specific lethal effect to the T2A2 cell of MCF-7 cell and load EPS8 that HLA-A*0201 and EPS8 expresses the positive; Because tumour antigen EPS8 wide expression is in the tumor tissues that different times is dissimilar, therefore, EPS8 antitumor CTL epitope of the present invention can as activeconstituents, composition is become with pharmaceutically acceptable vehicle group, or, with other, there is the extract of pharmacologically active and/or synthetic drugs becomes composition with pharmaceutically acceptable vehicle group, make according to the ordinary method of pharmaceutical field the therapeutical peptide vaccine that EPS8 expresses positive tumor again, have huge Application and Development potentiality in tumor-specific immunity treatment field.

Claims

1. an EPS8 antitumor CTL epitope peptide, be nonapeptide, its sequence is: Phe-Leu-Phe-Thr-Pro-Leu-Asn-Met-Val.

2. EPS8 antitumor CTL epitope peptide according to claim 1, is characterized in that: adopt solid-phase synthesis to prepare CTL epitope peptide.

3. the application of EPS8 antitumor CTL epitope peptide according to claim 1 in preparation tumor.