CN104211771A - Compound of lung cancer-related peptides and heat shock proteins and use thereof - Google Patents

Abstract

The invention provides lung cancer antigens binding to heat shock protein. The lung cancer antigens comprise an IMP-3 antigen peptide and a MAGE-A3 antigen peptide. The invention also provides a compound of the lung cancer antigens and heat shock proteins gp96 and HSP78. The compound comprises a compound composed of the antigen peptides and the heat shock proteins gp96 and HSP78 binding to the antigen peptides by non-covalent bonds, and also comprises a fusion protein composed of the antigen peptides and the heat shock proteins gp96 and HSP78 binding to the antigen peptides by a covalent bond. The compound can be used for preparation of a therapeutic vaccine for treating lung cancer.

Description

The mixture that lung cancer related peptides and heat shock protein(HSP) are formed and application thereof

Technical field

The invention belongs to biological technical field, particularly the mixture of LuCA polypeptide and heat shock protein(HSP) gp96 and HSP78 and their purposes.

Background technology

Lung cancer is one of malignant tumour that M & M is the highest in the world.China's lung cancer morbidity rate in 2012 is 53.6/10 ten thousand, accounts for 18% of tumour total incidence; Mortality ratio is 45.6/10 ten thousand, accounts for 20% of tumour general mortality rate, has occupied first of kinds of tumor.Wherein nonsmall-cell lung cancer (non-small cell lung cancer, NSCLC) accounts for 80 ~ 85% of lung cancer, and 1/3 has been wherein Locally Advanced when making a definite diagnosis for the first time.Nonsmall-cell lung cancer root value criterion 5 years survival rates are about 25%.Advanced NSCLC is as without treatment, and median survival interval (median survival times, MST) is about 4 ~ 5 months, 1 year survival rate < 10%.

The first-line treatment scheme of advanced NSCLC patients's standard is platinum medicine associating third generation chemotherapeutics, but its objective efficient 30%, MST that is only about is only 8 ~ 9 months, and within 1 year, survival rate is only 30% ~ 40%.Clinical trial confirms, and adjuvant chemotherapy of patients significantly can reduce recurrence rate and lifetime is obviously extended.But because chemotherapy can not kill whole cancer cells in body, cancer whether recur finally depend on body self from steady function and immunologic function.And the toxic side effect that chemotherapy is brought to body, to kill more than chemotherapy the benefit that part cancer cells brings to body to the strike of body's immunity much bigger.

Target therapeutic agent emerging is in recent years grown by the signal path Tumor suppression that specific target is important to tumor growth, has the feature of high-efficiency low-toxicity.But the curative effect of targeted drug treatment lung cancer and therapeutic domain have certain limitation, also exist simultaneously and resistance, expensive problem easily occur.

The treatment of tumour in recent years makes great progress, and under the promotion of modern molecular biology develop rapidly, immunotherapy comes into one's own day by day, demonstrates good application prospect, becomes the new model of oncotherapy.Gp96 autoimmunity is as individuation, active immunity treatment, the clinical safety and efficacy having confirmed its treatment tumour abroad, our clinical trial also confirms that autologous gp96 activates the ability of patient's lung cancer specific CTL, and autologous gp96 immunity may be the new effective ways of of lung cancer therapy.

Gp96 is the important member in heat shock protein(HSP) HSP90 family.Heat shock protein(HSP) (Heat Shock Protein, HSP) is a class high conservative, is extensively present in the protein in protokaryon and eukaryote.Gp96 albumen all has wide expression in healthy tissues and tumour, is mainly positioned endoplasmic (Endoplasmic Reticulum).Gp96 is low expression level in cell under normal circumstances, but heat-shocked, glucose shortage, bacterium and virus infection etc. all can induce it to express increase.Gp96, as molecular chaperones, can stop protein aggregation, assists protein folding, stretching, extension, assembling and transhipment, suppresses the secretion of misfolded proteins matter.Gp96 molecule has the ability of Binding peptide, can in conjunction with 5-25 amino acid whose peptide sequence.

Heat shock protein(HSP) HSP78 is the member in tenuigenin in HSP70 family, and molecular weight is about 78kDa.HSP78 molecule can be combined by various small peptide as molecular chaperones in cell.

Research in recent years finds, the small peptide that HSP combines determines the antigenicity of HSP mixture.The combining small peptide of HSP, in passing MHC I quasi-molecule, activates specificity and memory t cell, causes Cellular Immunity reaction.Gp96 can also promote the expression of MHC I class and MHC II quasi-molecule and costimulating factor by activating dendritic cells (DC), thus improves immune response.Therefore gp96 can the effective polypeptide that combines of submission, the specific ctl response of efficient activating peptide.The mixture of this gp96-polypeptide and HSP78-polypeptide complex can be used for control autologous tumor and some communicable disease.

Confirm that gp96 and HSP78 molecular energy is in conjunction with plurality of antigens polypeptide at present, comprised vesicular stomatitis virus antigen district peptide, the ovalbumin epitope peptide of mouse H-2Kb restriction, the leukemia epitope peptide, antigen of hepatitis B virus peptide etc. of H-2La restriction.But have no the antigenic peptide that isolation identification is combined with HSP from lung cancer tumor tissue so far.

Lung cancer is malignant tumour the most serious at present.Although chemicotherapy, targeted therapy obtain very large progress, within 1 year, survival rate brings up to 50%, and due to the personalization of tumour, resistance, the median survival interval of lung cancer was at about 1 year.Lung cancer still can not be cured on the whole.Existing autologous gp96 mixture immunotherapy clinical trial also shows the disposable limiting factor being the method and promoting prepared by the requirement of tumor tissues size and vaccine.

Summary of the invention

Therefore, the technical problem to be solved in the present invention is the specific peptide section of screening lung cancer.Overcome the reduced immunogenicity that tumour cell immunoediting causes, can at healthy individuals and patients with lung cancer inducing producing specificity cytotoxic T lymphocyte (also known as killer T cell, CTL) lung carcinoma cell is killed and wounded, to normal cell and tissue without destruction, produce very strong anti-lung cancer effect.

An object of the present invention is to provide a kind of mixture of the LuCA be combined with gp96 and HSP78.Described LuCA can be the aminoacid sequence of 62-70 position on lung cancer tumor antigen I MP-3 albumen respectively, and its sequence can be ELHGKPIEV, or its series of variation; The aminoacid sequence of 357-365 position on lung cancer tumor antigen I MP-3 albumen, its sequence can be SMNLQAHLI, or its series of variation; The aminoacid sequence of 210-218 position on lung cancer tumor antigen I MP-3 albumen, its sequence can be IIGKEGATI, or its series of variation; The aminoacid sequence of 138-146 position on lung cancer tumor antigen I MP-3 albumen, its sequence can be KLNGFQLEN, or its series of variation; The aminoacid sequence of 15-23 position on lung cancer tumor antigen MAGE-A3 albumen, its sequence can be GLEARGEAL, or its series of variation; The aminoacid sequence of 141-149 position on lung cancer tumor antigen MAGE-A3 albumen, its sequence can be GNWQYFFPV, or its series of variation.

Mixture of the present invention had both comprised heat shock protein(HSP) and had been combined with polypeptide with non covalent bond, comprised again heat shock protein(HSP) and was combined with polypeptide with covalent linkage.The invention provides the method for the mixture of preparation antigenic peptide described above of the present invention and heat shock protein(HSP) gp96 and HSP78.

brief Description Of Drawings

Fig. 1 is with 9 peptides and gp96-9 peptide complex (0,1,3 week) immune mouse BALB/c (HLA-A2 ⁺) cause afterwards specific CTL reaction.Effector cell CTL to the cracking percentage of specificity target cell with 4 hours standards ⁵¹cr discharges mensuration, effector cell and target cell ratio be respectively 10,25,50 and 100, figure in cleavage rate be 10 mouse mean values.

Fig. 2 PC-9 cell tumor bearing nude mice tumor growth curve.Nude mice by subcutaneous inoculation human lung carcinoma cell PC-9, lotus knurl transfers the mouse spleen lymphocyte of different sources after 7 days.The acceptance of peptide section 1 group (n=20) is through the BALB/c (HLA-A2 of gp96-peptide section 1 mixture immunity ⁺) spleen lymphocyte of mouse; Irrelevant peptide group (n=20) accepts to have nothing to do peptide (HBcAg through gp96- _82-90) BALB/c (HLA-A2 of mixture immunity ⁺) spleen lymphocyte of mouse.

The change of 9 peptide-specific T-cell after Fig. 3 patients with lung cancer immunity autologous tumor gp96 mixture.9 peptide-specific T-cell in peripheral blood in patients are detected with the ELISPOT method of IFN-γ.Irrelevant peptide HBcAg _82-90peptide is negative control.

embodiment:

Further illustrate the present invention by embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.

Embodiment 1. is purifying gp96 albumen from lung tissue

By centrifugal after five parts of refreshing cancerous lung tissues and the homogenate of a lung healthy tissues, with 30%/70% saturation ratio (NH ₄) ₂sO ₃precipitation, ConA Sepharose(GE company is adopted after resolution of precipitate) carry out phytohemagglutinin affinity purification, with the albumen of the α-methylglucoside elution of bound of 10%, glucosides elutriant carries out polishing purification with Hitrap-Q anion-exchange column (GE Healthcare Life Science), obtains the gp96 albumen of >95% purity through this three steps purifying.Gp96 albumen gp96/grp94 monoclonal antibody (Santa Cruz company) carries out Western qualification.Its purity SDS-PAGE, silver dye and reversed-phase HPLC qualification.

Embodiment 2. is from the polypeptide of gp96 protein delivery Non-covalent binding

The gp96 albumen of purifying being added trifluoroacetic acid (TFA) makes its final concentration reach 0.2%(pH about 2.0), then use ultrafiltration process (molecular weight cut-off is 30kDa, Millipore company) isolated polypeptide, polypeptide mixture is carried out mALDI-TOFmass spectrum (ABI 4700) is analyzed, and polypeptide molecular weight is mostly between 600-1100 Da.

Embodiment 3. analysed by reverse phase HPLC polypeptide

Be dissolved in solution A (0.065% TFA, 2% acetonitrile) after polypeptide mixture freeze-drying, be splined on C18 reversed-phase column (Sephasil peptide C18; 5 um granularities; 4.6 X 250 mm, GE company), carry out gradient elution from 0 to 65% solution B (0.05% TFA, 100% acetonitrile), flow velocity is 1 mL/min, uses 214nm wavelength detecting.Comparison of tumor tissue and healthy tissues HPLC collection of illustrative plates, find the peptide peak that five parts of lung cancer tumor tissues are total and do not have in healthy tissues.

Embodiment 4. polypeptide microsequencing and sequential analysis

Collect this special peptide peak, identify its purity with MALDI-TOF mass spectrum (PE company Voyager-DE system), only find that molecular weight is the simple spike of 1021 Da.To this peptide microsequencing (ABI, Procise 491 Protein Sequencer), its aminoacid sequence is " ELHGKPIEV ", and the sequence that 5 parts of tumor tissues obtain is consistent.By this sequence in online albumen database (NCBI) inquiry, find that it is positioned at the 62-70 position of IMP-3 albumen.

The external rapid-assembling of polypeptide of embodiment 5. gp96 albumen and synthetic

Synthetic 9 peptide " ELHGKPIEV " (entrusting the synthesis of gill biochemical company limited), carries out assembled in vitro by gp96 albumen and polypeptide, external combination anchor:

2.7 mmol/L KCl, 1.47 mmol/L KH ₂pO ₄, 8.1 mmol/L Na ₂hPO ₄, 138 mmo1/L NaCl, 10% (V/V) glycerine, 3.0 mol/L 9 peptides, 0.421 mmol/L gp96 albumen, 60 DEG C are reacted 10 minutes, and ultrafiltration process (molecular retention 30 KDa, Millipore company) removes unconjugated polypeptide.

The fusion rotein of secreting, expressing lung cancer tumor antigen peptide 1 and heat shock protein(HSP) gp96 in embodiment 6 Bichi yeast system

This example provides antigen peptide 1(ELHGKPIEV) method of fusion rotein that formed with covalent linkage with heat shock protein(HSP) gp96.We have done optimization for Pichia anomala expression to corresponding to the nucleotide sequence of this peptide, and this sequence of synthetic (

GAA TTG CAT GGT AAA CCA ATT GAA GTT), introduce restriction enzyme site Bgl II the nucleotide sequence of this 9 peptide is connected with the people gp96 gene 5 ' end T4 ligase enzyme optimized for pichia spp preferred codons, connect 5 ' and 3 ' end introducing 2 restriction enzyme site EcoR I and Xho I of product, to be connected in expression vector pPICZaA secreting, expressing in pichia spp, expression product is the fusion rotein of gp96 and this 9 peptide.

Embodiment 7. immune mouse

Select the genetically modified BALB/c mouse (HLA-A2 of HLA-A2 in growth 8-10 week ⁺) for this experiment.

Immunization ways adopts nape subcutaneous injection in PBS sample being dissolved in 100 uL.Subcutaneous injection operation is relatively simple, requires that immunizing dose is moderate, therefore adopts this kind of immunization ways.Simultaneously at the endoxan of each immunity first 1 day abdominal injection 0.4 mg.Low-dose cyclophosphamide can suppress in gp96 immunity, play inhibiting Treg cell thus can strengthen immune effect.

The suitableeest immunizing dose of gp96 albumen-9 peptide complex is 0.1 nmol.

Immunization time is 0,1,3 week reinforced immunological carrying out three times, is better than immunity once or the effect of secondary.

Therefore subcutaneous injection immunity is adopted, 9 peptides are dissolved in damping fluid (90% PBS 10% DMSO 0.1% TFA), gp96-peptide 1 mixture is dissolved in PBS, thermal agitation 1 minute before injection, every mouse immune dosage peptide section 1 is respectively 1 nmol and 10 nmol, by immune mouse after peptide and freund's adjuvant emulsification.Gp96-peptide section 1 mixture immunizing dose is 0.05 nmol respectively, 0.10 nmol and 0.50 nmol, adopts nape subcutaneous injection, and carry out second time immunity after immune 1 week of first time, booster immunization after 2 weeks, carried out cytotoxicity analysis after 3 days.Each process use 10 mouse.

Embodiment 8. cytotoxicity analysis

Mouse booster immunization is after 3 days, from every mouse gather in the crops about 3 × 10 ⁷splenocyte is suspended from containing 10 mM HEPES damping fluids, in microbiotic and 10% (V/V) FCS nutrient solution, in culturing bottle with penetrate through width the T2 cell (3:1) of (4500 Rad) and 1 ug/mL peptide in perfect medium 37 DEG C cultivate.Within 6 days, collect splenocyte afterwards and carry out 4 hours standards ⁵¹cr release experiment (concrete grammar is shown in Kuhrober, A, et al. 1997. International Immunology, 9 (8): 1203-1212) measures cellular cytoxicity activity.In brief, target cell adds the effector cell of different quantities after 30 minutes in 37 DEG C of sensitization with 10ug/mL peptide, reaction system is the perfect medium of 100 uL.37 DEG C of Dual culture 4 h before harvest supernatants measure specific lysis rate.

From ⁵¹cr release experiment can find out that gp96-peptide 1 mixture and independent peptide section 1 all can stimulate mouse to produce specific cytotoxic t lymphocytes, but the immune effect of gp96 mixture makes more than 100 times of independent 9 peptides.Gp96-peptide 1 mixture of every mouse immune 0.1 nmol (about 10ug) can bring out body and produce strong cell immune response, the cleavage rate of cytotoxic assay target cell is more than 60%, and the pre-treatment of endoxan can strengthen this cellulotoxic effect (Fig. 1) further.Experimental result shows that gp96-peptide 1 mixture can be developed into the medicine into a kind of novel lung cancer.

Embodiment 9. cell transfusions tumor inhibition

With HLA-A2 positive human lung carcinoma cell PC-9 subcutaneous injection nude mice.Transfer the spleen lymphocyte (n=20), the gp96-that obtain from gp96-LuCA 9 peptide 1 mixture immunity HLA-A2 transgenic mice after 7 days to have nothing to do peptide complex immune mouse lymphocyte (n=20), transfer weekly once, totally 3 weeks.Detect the growth (Fig. 2) that lymphocyte that tumor size can find only to transfer gp96-LuCA 9 peptide 1 mixture immune mouse obviously can suppress lung carcinoma cell.

The autologous gp96 complex therapies tumour of embodiment 10. patients with lung cancer immunity

Patients with lung cancer is postoperative accepts gp96 treatment and doctor's conventional treatment (chemicotherapy).

Operating process

Choose II, III A phase patients with lung cancer of plan row operation;

Obtain " Informed Consent Form " and " Operation Agreement Letters " that patient signs voluntarily;

The preoperative comprehensive inspection of row is with clear and definite radical surgery feasibility;

Operation excised tumor tissue;

A) by organize in sterile tube;

B) sterile tube having a tumor tissues was delivered to GMP and is prepared workshop under 0 DEG C of condition in 2 hours.

Qualification tumor tissues quality, confirms to extract enough heat shock protein(HSP) gp96-polypeptide complexes;

Heat shock protein(HSP) gp96-polypeptide complex isolation and determination;

Gp96 treatment and conventional treatment;

Heat shock protein(HSP) gp96-polypeptide complex subcutaneous injection flow process:

A) the 1st injection first 3 days domestic demands check routine blood test, blood biochemistry and electrocardiogram(ECG.

B) pre-treatment is injected: each gp96 injects first 1-3 days to patient's intravenous injection endoxan.

C) carry out gp96 protein injection by research nurse, formulate dosage according to the past foreign study data, add physiological saline and dilute, upper arm and the subcutaneous injection of thigh left and right after mixing, 1 time weekly.

D), after the injection of the 1st, 2 immune protein, need remain in a hospital under observation 24 hours, after need observation 1 hour after injection several times, can leave after having no adverse reaction.

E) the 2nd time and the 8th injection latter 3 days domestic demand inspections routine blood test, blood biochemistry and electrocardiogram(ECGs.

F), before immunotherapy and after immunity terminates, gather 10ml blood samples of patients, carry out the mensuration of amynologic index.

If palindromia during l patient treatment, by investigator's well-documented history patient disease recurrence, and give corresponding treatment according to clinic diagnosis specification;

Follow up a case by regular visits to;

In postoperative 2 years, per March makes a house call 1 time, and within 2 years, make a house call 1 time per June afterwards, until patient disease recurrence or dead, content of specifically making a house call is as follows:

1. within 2 years every 3 months, check 1 time:

A) the 3rd, 9,15,21 month check content:

Tumor markers (CA19-9, CEA, CA125);

Chest CT, Abdominal B type ultrasonography.

B) the 6th, 12,18,24 month check content:

Tumor markers (CA19-9, CEA, CA125);

Chest CT, Abdominal B type ultrasonography;

Head CT, bone scanning (ECT).

2. within after 2 years every 6 months, check 1 time, check content:

Tumor markers (CA19-9, CEA, CA125);

Chest CT, Abdominal B type ultrasonography;

Head CT, bone scanning (ECT).

If palindromia during follow-up of patients, by investigator's well-documented history patient disease recurrence, and give corresponding treatment according to clinic diagnosis specification (as NCCN guide etc.).

Embodiment 11. ELISPOT method detection specificity T cell

Epitope specificity CTL in ELISPOT detection of lung cancer patient PBMC.Operate according to the specification sheets of ELISPOT test kit.First close the pre-coated plate of ELISPOT one hour with PRMI 1640 substratum containing 10% foetal calf serum, outwell confining liquid before use, add 100uL containing 1 × 10 ⁵the PBMC(of people directly cultivate 7 days with fresh PBMC or by PBMC amplification in vitro).Add 9 peptides 1 of 10ug/mL in experimental group, PHA that positive control adds 4ug/mL stimulates, negative control adds irrelevant peptide (HBcAg _82-90).Cultivate in cell culture incubator after 18 hours, the formation of detection specificity spot, and carry out statistical study.We choose the patients with lung cancer of the HLA-A2 positive as study group, detect epitope specificity CTL in patient's fresh blood by ELISPOT method, and the frequency of specific CTL is determined by counting amount of speckle.In the HLA-A2 positive lung cancer patient of autologous gp96 mixture immunity, high-frequency ELHGKPIEV specific CTL all detected in 4 examples, and increase significantly than before immunity, irrelevant peptide HBcAg do not detected simultaneously _82-90(negative control) specific T-cells, illustrates that ELISPOT result is epi-position special (Fig. 3).

The immunocompetence of other gp96-polypeptide complex of embodiment 12. measures

Except above-mentioned antigen 9 peptide 1, we choose again other 5 LuCA polypeptide and gp96 assembled in vitro and measure its immunocompetence, and these 5 antigenic peptides are 1 respectively) SMNLQAHLI, 2) IIGKEGATI, 3) KLNGFQLEN, 4) GLEARGEAL, 5) GNWQYFFPV.

This 5 peptide species of synthetic respectively, the reaction system described in embodiment 7 is adopted to be combined with gp96 in vitro, this 5 peptide species and gp96 all have higher avidity, and by measuring association reaction equilibrium constant K, in 5 kinds of peptides and gp96 association reaction, K value is all more than 4.5.The mixture that the method described in embodiment 5 of employing is formed with above-mentioned 5 kinds of LuCA polypeptide and gp96, with the fusion rotein immune mouse respectively of above-mentioned gp96 and 5 kind of LuCA polypeptide, and adopt the method described in embodiment 7 to carry out CTL analysis to these 5 kinds of mixtures respectively, from ⁵¹cr release experiment can find out that the mixture that these 5 kinds of LuCA polypeptide and gp96 are formed all can stimulate HLA-A2 transgenic mice to produce specific cytotoxic t lymphocytes, and the immunocompetence of the mixture that 5 peptide species are formed with gp96 than independent polypeptide height 120-200 doubly.Every mouse immune dosage can bring out body when being 0.1 nmol (about 10ug) and produce strong cell immune response, finds that the cleavage rate of target cell is between 50%-65% by the cytotoxic assay of the mixture formed this 5 peptide species and gp96.

Above experimental result shows that mixture that gp96 and LuCA polypeptide assembled in vitro are synthesized can be developed into the medicine into a kind of novel lung cancer.Because experiment condition limits patent of the present invention can not accomplish to carry out determination of activity to each lung cancer related polypeptide, but we make research object by choosing 6 kinds of representational lung cancer antigen polypeptides, be combined with gp96 in vitro and carry out immunocompetence mensuration, great many of experiments shows that the mixture that gp96 and these antigenic peptides are formed all can stimulate mouse to produce strong immune response, gp96, as the novel good adjuvant of antigenic peptide, can be developed into as a kind of novel therapeutic vaccine.Therefore, except above-mentioned 6 kinds of antigenic peptides, other any LuCA polypeptide and gp96 are combined as new generation vaccine also should within protection scope of the present invention.

Embodiment 13. HSP78-immunogenicity of polypeptides measures

By the mixture that HSP78 and 6 peptide species assembled in vitro are formed, the same gp96 of reaction system (see embodiment 5), by fusion rotein (embodiment 6) immune mouse of the mixture of external synthesis and HSP78 and 6 peptide species.The same gp96 of mouse immune mode, immunizing dose and immune programme for children (see embodiment 7) cytotoxicity (CTL) analytical procedure is shown in embodiment 8 with gp96().From ⁵¹cr release experiment can find out that HSP78-peptide complex can stimulate mouse to produce specific cytotoxic t lymphocytes, the immunogenicity of HSP78-peptide complex is more than 100 times of antigen alone peptide, every mouse immune 0.1 nmol (about 10ug) can be brought out body and be produced strong cell immune response, and the cleavage rate of cytotoxic assay target cell is more than 50%.Experiment shows that HSP78-9 peptide complex can be developed into the medicine into a kind of novel lung cancer.

Kang Ernuo Bioisystech Co., Ltd of <110> Shenzhen

The mixture that <120> lung cancer related peptides and heat shock protein(HSP) are formed and application thereof

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<170> PatentIn version 3.3

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Glu Leu His Gly Lys Pro Ile Glu Val

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Claims (9)

1. the epitope peptide be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.1 in sequence table.

2. the epitope peptide be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.2 in sequence table.

3. the epitope peptide be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.3 in sequence table.

4. the epitope peptide be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.4 in sequence table.

5. the epitope peptide be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.5 in sequence table.

6. the epitope peptide be combined with heat shock protein(HSP), is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.6 in sequence table.

7. the mixture be covalently or non-covalently connected of the epitope peptide as described in any one of claim 1-6 and gp96 or HSP78.

8. the epitope peptide as described in any one of claim 1-6 forms mixture as claimed in claim 7 purposes separately or in co-immunization human body inductive formation killer T cell separately or with heat shock protein(HSP) in vitro.

9. as right wants the purposes of immune induction killer T cell in prevention or treatment lung cancer as described in 8.