CN1288166C

CN1288166C - Modified tumor antigen peptide and use thereof

Info

Publication number: CN1288166C
Application number: CN 200410010990
Authority: CN
Inventors: 颜炜群; 王芳
Original assignee: JINLIN SHENGYUAN SCIENCE & TECHNOLOGY Co Ltd
Current assignee: JINLIN SHENGYUAN SCIENCE & TECHNOLOGY Co Ltd
Priority date: 2004-07-14
Filing date: 2004-07-14
Publication date: 2006-12-06
Anticipated expiration: 2024-07-14
Also published as: CN1721436A

Abstract

The present invention relates to a tumor antigenic peptide for stimulating an organic antitumor immunological reaction, particularly to a chiral tumor antigenic peptide based on tumor antigen HER2/neu modification and a preparation process thereof and an application thereof for producing a medicine or a vaccine for treating tumors, particularly mammary cancer.

Description

The tumor antigen peptide and the application thereof of modifying

Invention field

The present invention relates to be used to stimulate the tumor antigen peptide of antitumor immunity of organism reaction, particularly based on the chirality tumor antigen peptide of the modification of tumour antigen HER2/neu, its preparation method and be used for the treatment of the tumour particularly medicine of mammary cancer or the application in the vaccine in production.

Background of invention

Traditional treating malignant tumor method comprises surgical operation therapy, radiation cure and chemotherapy, the common drawback of these therapies is to be difficult to complete tumor resection tissue or thoroughly to kill tumour cell owing to malignant tumour is infiltrative growth, finally causes metastases and recurrence.And these treatment meanss lack specificitys, when killing tumour cell, and also injuring normal cell.Therefore need development new oncotherapy approach and means.

Cytotoxic T cell (CTL) is brought into play keying action in the process of infection of body defend against computer virus and removing intraindividual variation cell.Antigen and discerned by CTL and to realize in the activation of CTL is passed by MHC I quasi-molecule.Wherein be about 9～11 amino acid whose antigen peptide epi-positions mostly by the antigen of submission processed being processed in antigen presenting cell (APC).MHC I quasi-molecule causes the T cell activation after combining T cell surface receptor (TCR) with the mixture of these peptides, produces the CTL of peptide specific, and then can kill and wound specifically and express the antigenic tumour cell of these peptides.

Increasing tumour antigen and/or antigen peptide are identified, make the treatment application of tumor antigen protein matter become possibility.And the progress of genetic engineering technique and peptide synthesis technology has also promoted the development of immunotherapy of tumors medicine and tumour antigen peptide vaccine greatly.Compare with traditional vaccines such as dna vaccinations with tumour-cell vaccine, polypeptide vaccine has following advantage: the 1. designed polypeptide of chemosynthesis is not easily limited by material source; 2. be easy to preparation and purifying, a large amount of synthesis of high purity of energy, polypeptide with high performance reproducibility; 3. chemical property and thermodynamic property are more stable than protein, are convenient to transportation and preservation; 4. there is not the pollution of toxicity or infectious factor (as recombinant vectors DNA and encoded protein matter or peptide); 5. the downstream processing treatment is simple and cost is low.Because synthetic polypeptide vaccine has above these advantages, therefore become a kind of immunotherapy of tumors agent with broad prospect of application.

The neu gene is a kind of transforming gene (Shih C et al, Nature, 290:261-264,1981) of finding in the rat neuroblastoma that chemical substance is brought out at first.(epidermal growth factor receptor, gene erbB EGFR) have high homology (Coussens L et al, Science, 30 (4730): 1132-1139,1985) to the neu gene with the coding EGF-R ELISA.Be positioned on the 17th pair of karyomit(e) long-armed 21 (17q21) with the corresponding Human genome her2 of neu gene, it is a kind of proto-oncogene, the molecular weight that its expression product is made up of 1255 amino acid is the receptor tyrosine kinase of 185kD---HER2/neu albumen (is called for short HER2/neu, be called p185 again), its molecule by signal peptide, extracellular region, stride four parts of film district and intracellular region and form (Christianson TA et al, Cancer Res, 58 (22): 5123-5129,1999).The her2/neu gene mainly begins to express when the embryo is taken place, and after growing up, only can detect the HER2/neu albumen of minute quantity with the immunohistochemical methods method in the healthy tissues epithelial cell.In healthy tissues, her2/neu Chang Yidan copy form occurs.The amplification of her2/neu and/or proteic overexpression show on the tumour cell of mammary gland, ovary, lung, pancreas and gastroenteric tumor etc. usually, and often being accompanied by the patient shortens lifetime, recur in advance and prognosis mala (Slamon DJ, Science, 235 (4785): 177-182).Experiment in vivo and vitro is the result show, her2/neu crosses the vicious transformation that expression can cause non-tumor cell and the generation (Hoang M P et al, Am.J.Clin.Patho., 113 (6): 852-856,2000) of transgenic mouse milk cancer.Other member of expression product behind the her2/neu gene activation and Epidermal Growth Factor Receptor Family forms heterodimer, or spontaneous formation homodimer, these dimers are with after corresponding part combines, mediated cell signal transmission and cause in the born of the same parents tyrosine phosphorylation cascade reaction and proliferation signal is imported in the karyon finally causes the vicious transformation of cell.

Evidence suggests that some neoplastic disease human body internal memory is at HER2/neu specific antibody or ctl response, the expression product of HER2/neu can induce body to produce immune response (Disis ML et al, Cancer Res, 1; 54 (1): 16-20,1994), and proved that passive immunization therapy such as HER2/neu specific antibody Heceptin have antitumor action (Baselga J et al, Cancer Research, 58 (13): 2825.Therefore, HER2/neu can be used as the desirable target spot of malignant tumour active immunity treatment.

Goal of the invention

An object of the present invention is to provide tumor antigen peptide based on the modification of HER2/neu sequence, be characterised in that they have aminoacid sequence shown in SEQ ID NO:1 in the sequence table and SEQ ID NO:2 respectively, and include a D-type amino acid separately at least.

According to a preferred embodiment of the invention, wherein said peptide obtains with chemical synthesis process.

Another object of the present invention provides HER2/neu specificity vaccine or pharmaceutical composition, and said vaccine or pharmaceutical composition are made up of the tumor antigen peptide that is defined as above and one or more pharmaceutically acceptable carriers.

According to a preferred embodiment of the invention, said vaccine or pharmaceutical composition also can contain and be selected from the complete freund adjuvant or the incomplete immunostimulant of freund adjuvant.

According to a preferred embodiment of the invention, said vaccine or pharmaceutical composition also can contain one or more cytokines that are selected from interleukin-22, IFN-and granulocyte-macrophage colony stimutaing factor.

A further object of the present invention relates to vaccine or the application of pharmaceutical composition in inducing the reaction of HER2/neu specificity antineoplastic immunity that is defined as above.

According to a preferred embodiment of the invention, the reaction of wherein said HER2/neu specificity antineoplastic immunity is a HER2/neu SC lymphocyte reaction.

According to a preferred embodiment of the invention, wherein said tumour is a mammary cancer.

Brief Description Of Drawings

Fig. 1 shows D-P _42-50The semipreparative column tomographic map.

Fig. 2 shows D-P _782-790The semipreparative column tomographic map.

Fig. 3 shows D-P _42-50Mass spectrum.

Fig. 4 shows D-P _782-790Mass spectrum.

Fig. 5 shows the influence of immunogenic peptide of the present invention to the mouse mammary tumor growth.

Fig. 6 shows acceptance tumour antigen vaccine (D-P of the present invention _42-50+ GM-CSF) treat after 35 days the histopathological examination result (200 *) of animal milk gland cancer transplanted tumor.

Fig. 7 shows acceptance tumour antigen vaccine (D-P of the present invention _782-790+ GM-CSF) treat after 35 days the histopathological examination result (200 *) of animal milk gland cancer transplanted tumor.

Fig. 8 shows acceptance tumour antigen vaccine (D-P of the present invention _42-50+ GM-CSF) treatment after 35 days, animal milk gland cancer transplanted tumor organize transmitted light spectroscopy result (10,000 *).

Fig. 9 shows acceptance tumour antigen vaccine (D-P of the present invention _782-790+ GM-CSF) treatment after 35 days, animal milk gland cancer transplanted tumor organize transmitted light spectroscopy result (10,000 *).

The particular content of invention

In people and mammiferous immune system, a series of cell immune responses such as the cell-mediated delayed allergy of T, graft rejection and cytotoxic activity. Some T cell and special cell cortex protein are that the MHC interaction of molecules is with submission antigen on cell surface. The corresponding antigens of the antigen presenting cell (APC) of specialization as expressing on macrophage and Dendritic Cells Induced cytotoxicity and helper cell propagation and the identification target cell surface, and then inducing T cell (CTL) or these target cells of other cell killings.

The T cell is CD8 particularly⁺And CD4⁺The T cell plays irreplaceable effect in the mediation anti-tumor immune response. The activation of T cell and amplification need the participation of a series of immunocytes and cell factor. BMDC (DC) to the T cell, is given the first signal of T cell activation with the antigen submission after picked-up, processing and the processing as the most powerful antigen presenting cell of function (APC). Simultaneously, behind the costimulatory molecules such as the CD80/CD86 on DC surface (B7-1/B7-2), CD40 and the corresponding acceptor of T cell surface or the ligand interaction, the secondary signal of mediation T cell activation. In addition, the activation of CTL also needs CD4⁺The interleukin-22 of T emiocytosis (IL-2), IFN-γ (IFN-γ) and granulocyte-macrophage colony stimutaing factor (GM-CSF) and etc. the participation of cell factor.

In order to make the specific tumour antigen load on the BMDC of maturation, be necessary the local optical activity by changing the epitope peptide molecule and give molecule with local chirality feature, with the resistance of enhancement antigen peptide to proteasome degradation, prolong the Half-life in vivo of molecule, improve its load capacity on antigen presenting cell, and then strengthen the ability of the former reaction of its inducing specific antineoplastic immune.

Therefore, an object of the present invention is to provide a kind of tumor antigen peptide, particularly a kind of antigenic peptides based on tumour antigen HER2/neu. The present invention further provides the preparation method of said antigenic peptides and said antigenic peptides and be used for the treatment of the tumour particularly medicine of breast cancer or the application in the vaccine in production.

In order to obtain tumor antigen peptide of the present invention, the inventor at first utilizes the HLA-A2 Restricted CTL peptide epitopes of the close HER2/neu antigen protein of SYFPEITHI motif method prediction and Breast Cancer, respectively chooses one section and be combined the highest peptide P of free energy with the HLA-A2 molecule from the extracellular region of HER2/neu albumen and intracellular region_42-50(Val-Val-Gln-Cys-Gly-Gln-Tyr-Leu-His) and P_782-790(Leu-Cys-Ile-Gly-Leu-Leu-Arg-Ser-Val). Do not affecting peptide under prerequisite that the MHC-I quasi-molecule is combined, we have carried out necessary modification to said peptide section. Said modification comprises P_42-50C-terminus residue L-Leu replace with D-Leu, with P_782-790C-terminus residue L-Ser replace with D-Ser, and obtain respectively the amino acid sequence shown in SEQ ID NO:1 in the sequence table and SEQ ID NO:2. The inventor wishes that these modifications can change the local optical activity of peptide molecule and give on the basis of molecule with local chirality feature, the enhancement antigen peptide is to the resistance of proteasome degradation, prolong the Half-life in vivo of molecule, increase its load capacity on antigen presenting cell, and then improve the ability of its inducing specific anti tumor immune response.

Breast cancer antigen peptide carboxyl terminal of the present invention or amino terminal or both can be that carboxylic acid protective group or ammonia blocking group replace, and preferred blocking group should be the group that is conducive to the said peptide of cell delivery (for example the hydrophily by reducing peptide and improve lipophilicity). In addition, blocking group should be stable to the formation condition of peptide chain, can be easy to remove in the situation of not destroying the peptide chain that is extending simultaneously. Suitable blocking group comprises 9-fluorene methyl carbonyl (Fmoc), tert-butoxy carboxyl (Boc), benzyloxy carboxyl (Cbz), biphenyl isopropyl carbonyl, α; alpha-alpha-dimethyl-3; 5-dimethoxy benzyloxycarbonyl, and 2-cyano group-tertbutyloxycarbonyl etc. 9-fluorene methyl carbonyl (Fmoc) preferably wherein.

Can use the terminal blocking group of above-mentioned C or N; synthesize hybrid peptide of the present invention (for example referring to Steward according to solid-phase peptide synthetic technology known in the art; J.M.and Young J.D.; Solid Phase Peptide Synthesis; 2nd Ed.; Pierce Chemical Company, Rockford, I11. (1984)). In the solid-phase peptide synthetic method, at first the C end amino acid is connected on the suitable solid phase carrier or resin. For the synthesis of C terminal carboxyl group peptide preferred solid phase carrier be 4-hydroxymethyl phenoxy methyl-copolymerization (styrene-1% divinylbenzene). The preferred solid phase carrier that is used for the C terminal amide be 4-(2 ', 4 '-dimethoxy phenyl-Fmoc-aminomethyl)-phenoxy acetamide ethylamide resin (Apllied Biosystem Co.). Can be by N, N '-dicyclohexylcarbodiimide (DCC), N, N '-DIC (DIC) or 2-(1-hydrogen-1-BTA base)-1,1,3,3-tetramethylurea hexafluorophosphoric acid (HBTU) makes the C end amino acid be coupled to (50-100 ℃, reaction is 1 to 24 hour in carrene or DMF solvent) on the resin. When solid phase carrier be 4-(2 ', 4 '-dimethoxy phenyl-Fmoc-aminomethyl) during the phenoxy acetamide ethylamide resin, should with the coupling of above-mentioned C end amino acid before with secondary amine such as piperidines the cracking of Fmoc group is fallen. Can use adjacent 124 Triazole-1-base-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid (HBTU, 1 equivalent) in DFM with de-protected 4-(2 ', 4 '-dimethoxy phenyl-Fmoc-aminomethyl) coupling of phenoxy acetamide ethylamide resin.

When the solid phase end of synthesis, can use the polypeptide of lytic reagent (for example thioanisole, water, dithioglycol and trifluoroacetic acid) process resin combination, to remove synthetic peptide from resin and to make it protection. If the C end of peptide is alkylamide, can carries out the ammonia solution with alkylamine and go out resin with cracking. Perhaps, can carry out the peptide that transesterification processes to remove combination with ethanol, and then carry out the ammonia solution or directly turn amide groups processing.

Can use ion exchange chromatography, hydrophilic adsorption chromatography, silica gel adsorption chromatography, partition chromatography, a series of chromatography step purifying such as high performance liquid chromatography (HPLC), particularly reversed-phase HPLC de-protected synthetic peptide fully.

After synthetic peptide is purified, can use the Applied biosystems 477A type protein sequence instrument of being furnished with the 120A analyser, analyze the aminoacid sequence of the peptide of purifying with automatic Edman chemical method, and can use laser desorption mass spectrometry to measure the molecular mass of each peptide sequence.

Not homoallelic MHC-I quasi-molecule is in conjunction with having the different aminoacids polypeptide of sequence, and these amino acid whose variations mainly are confined to the N-terminal of polypeptide, that is to say, the aminoterminal amino-acid residue of polypeptide defines character (the Bjorkman PJ et al of specific MHC molecular recognition, Ann Rev Bioche, 59:253-288,1990).Therefore, at the N-terminal that does not change antigen peptide, promptly do not change with MHC-I quasi-molecule bonded prerequisite under, the C-terminal amino acid that we change synthetic polypeptide (comprises P _42-50Carboxyl terminal residue L-Leu replace with D-Leu, P _782-790Carboxyl terminal residue L-Ser replace with D-Ser), synthesized polypeptide D-P with local chirality feature _42-50And D-P _782-790Like this, just, can further improve its immunogenicity at the body internal stability that strengthens molecule and on the lip-deep bonded of antigen presenting cell basis.

Among the present invention, we adopt Fmoc solid-phase polypeptide synthesis strategy, have synthesized altogether and have comprised L-P _42-50(the wild-type antigen nonapeptide that is equivalent to her2/neu albumen 42-50 amino acids), D-P _42-50(being equivalent to the antigen nonapeptide that her2/neu albumen 42-50 amino acids also comprises a D type aminoacid replacement at least), L-P _782-790(the wild-type antigen nonapeptide that is equivalent to her2/neu albumen 782-790 amino acids) and D-P _782-790Four kinds of antigen peptide of (being equivalent to the antigen nonapeptide that her2/neu albumen 782-790 amino acids also comprises a D type aminoacid replacement at least).After building-up process finishes, press anti-phase liquid chromatography that the thick peptide of synthetic is carried out purifying and analysis in the utilization.The result shows, peptide L-P _42-50And D-P _42-50Purity be respectively 99.2% and 98.9%, L-P _782-790And D-P _782-790Purity be respectively 99.1% and 99.0%.Utilize the desorption ionization flight time mass spectrum to measure the molecular weight of each chromatographic peak, obtain pure product peptide through rotary evaporation in vacuo and freeze-drying, the visible L-P of result _42-50And D-P _42-50Overall yield is respectively 63% and 45%, L-P _782-790And D-P _782-790Overall yield is respectively 58% and 46%.

Learn and the interior animal experiment confirmation through cell in vitro, the antigen peptide of modification of the present invention can promote the propagation of T lymphocyte and mammary cancer specific CTL effectively, excites the killing activity of immunity system to oncocyte, reaches the purpose that suppresses tumor growth.Particularly our comparative experiments shows, compares with wild-type sequence, and the antigen peptide that the present invention modifies can more effectively promote the propagation of T cell, improves the ability of T emiocytosis IFN-γ, thereby increases the killing activity of CTL to tumour cell.

Antigen peptide of the present invention can be made mineral acid or organic acid salt, these salt comprise but are not only limited to inorganic acid salts such as hydrochloride, vitriol, phosphoric acid salt, and organic acid salt such as acetate, alginates, Citrate trianion, benzoate, benzene sulfonate, succinate, maleate, fumarate, lactic acid salt, oxalate, tartrate, thereby can obtain oil-soluble or dispersible vaccine of water or pharmaceutical preparation.

Cytotoxic agents such as Pseudomonas exotoxin (PE40 or PE60), diphtheria intracellular toxin, Ricin or Toxins,exo-, cholera can be connected on T1249 of the present invention or its fragment, thereby a kind of means that can directed destroy or kill and wound with antigen peptide bonded cell of the present invention are provided.Can make the predetermined portion that reaches of the said peptide maximum possible that is connected with cytotoxic agent.For example can directly be delivered to target site, or enter in the blood vessel that leads to target site by the high affinity antigenic peptide fragment that intubate connects Ricin.Perhaps, also can be with synthetic immunogenic peptide of the present invention chemical coupling to macromolecular carrier protein for example on albumin or the relative hole key protein molecular, to induce the enhanced anti tumor immune response.

Can be according to known method in the medicine industry (as referring to Remington ' s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.1990), the pharmaceutically acceptable carrier of antigen peptide of the present invention and one or more, vehicle and/or immunostimulant are mixed mutually, make the vaccine or the pharmaceutical composition of the various different dosage forms that are suitable for the outer approach of various gi tract (for example in intravenously, intramuscular, the organ and intracutaneous, approach such as subcutaneous) administration.Pharmaceutically acceptable carrier comprises avirulent and activeconstituents is not had the liquid filling agent or the thinner of interference effect.Said immunostimulant can be complete Freund's adjuvant or incomplete Freund's adjuvant.

What should particularly point out here is, for the mammary cancer specific cell immunoreaction particularly of induced tumor more effectively, preferably also comprise the cytokine that one or more are selected from interleukin-22, IFN-and granulocyte-macrophage colony stimutaing factor in immunogenicity peptide vaccine of the present invention or the pharmaceutical composition.

For through said gi tract external administration, immunogenic peptide of the present invention can be dissolved in and make solution or suspension in water, salt solution, dextrose solution, ethanol or the oil.Employed carrier also can comprise other compositions such as sanitas, suspensoid, solubilizing agent, buffer reagent.

The dosage of medicine of the present invention or vaccine composition was generally 0.0001 to 200mg/kg/ day, and can divide administration several times every day with the sub-doses form.But definite dosage should be according to the character of disease to be treated, disease severity, patient's age and the general situation of health, and factor such as route of administration is determined by the clinician.

Antigen peptide of the present invention or its vaccine or pharmaceutical composition can be used for preventing or treat particularly breast tumor of former or the various solid tumors that shift.When being used for the prophylaxis of tumours transfer, can use separately or with tumor radiotherapy or chemotherapy combined.For example can behind the solid tumor radiotherapy, unite and use peptide compounds of the present invention and one or more conventional tumor chemotherapeutic drugs.Perhaps,, continue to use peptide compounds of the present invention through after operation, radiotherapy and the chemotherapy, removing the small metastases kitchen range that may exist in the body, or stable and suppress any remaining former hair-cream adenoncus knurl.

Embodiment

EXAMPLE l: tumour specific antigen peptide of the present invention synthetic

Basically in accordance with known methods, use synthetic respectively the nonapeptide of Advenced ChemTech 90 type peptide synthesizers, i.e. D-P with aminoacid sequence shown in SEG ID NO:1 and the SEG ID NO:2 _42-50And D-P _782-790, do not comprise the amino acid whose corresponding wild-type nonapeptide of D type in the composition sequence respectively simultaneously, i.e. L-P _42-50And L-P _782-790

For this reason, at first take by weighing solid-phase resin (Wang Resin-Val-Fmoc resin and each 0.3mmol of Wang Resin-Leu-Fmoc resin), and with the abundant swelling of methylene dichloride (DMF).Use DIC and HOBt to make coupling agent then, in the DMF solvent, repeat following synthesis cycle step, add the amino acid shown in the above-mentioned peptide fragment successively:

1, at first with the Fmoc-amino acid in DMF washing (5 minutes) reactor that contains 20% piperidines;

2, use 20% piperidines that is dissolved among the DMF to handle once more 25 minutes, to remove the Fmoc blocking group from aminoacid functional group;

3, wash 2 times each 2 minutes with DMF;

4, use methyl alcohol (MeOH) to wash totally 2 minutes 1 time;

5, wash 2 times each 2 minutes once more with DMF;

6, in the presence of DIC and HOBt coupling agent, add the protected amino acid that is equivalent to the amino 3 times of amounts of resin, reaction is 1 hour under the room temperature;

7, wash 2 times each 2 minutes with DMF;

8, use methyl alcohol (MeOH) to wash totally 2 minutes 1 time;

9, wash 2 times each 2 minutes once more with DMF.

After synthetic the finishing, wash resin (5 minutes) to remove DMF with tetrahydrofuran (THF) (THF), air-drying resin under argon gas and nitrogen environment then, thus obtain the peptide of resin-bonded.

(trifluoroacetic acid: water: phenol: with disulfide: mixture thioanisole=82.5: 5: 5: 2.5: 5) continued stir about 120 minutes again in about 0 ℃ of following stir about 15 minutes under room temperature with the peptide of resin-bonded and the lytic reagent of pre-preparation (5 to-10 ℃ of preservations).Leach resin then also with purified trifluoroacetic acid drip washing.The resin that leaches and washed is added to by every part of 0.5ml in the centrifuge tube that contains the 8ml diethyl ether of having an appointment, behind the centrifugal resulting suspension, removes supernatant.Can repeatedly repeat this process comes out up to all peptides are all precipitated.Wash sedimentary filtrate with ether, air-dry then and lyophilize it.

After cracking is finished, the thick peptide (using the 5-100% acetonitrile gradient) (result is referring to Fig. 1 and 2), the peptide of lyophilize purifying like this then that use Sephasil peptide C18 post as above to obtain through 60 minutes wash-outs with HPLC method purifying.With resulting peptide with after the 0.1% trichoroacetic acid(TCA) dissolving, with desorption ionization time-of-flight mass spectrometer determining molecular weight (result is referring to Fig. 3 and 4).

In order finally to make vaccine composition of the present invention, can in advance or use the preceding interim immunological adjuvant (for example complete or incomplete Freund's adjuvant) that in said antigen peptide vaccine, adds proper concn, and/or cytokine (for example interleukin-22, IFN-and/or granulocyte-macrophage colony stimutaing factor).

Embodiment 2: vaccine composition of the present invention is to the influence of mouse lymphocyte multiplication capacity and specific CTL killing activity

Present embodiment use inoculated breast cancer cell D2F2 (transfection the clone of people HER2/neu overall length cDNA, be so kind as to give by Wei-Zen doctor Wei) mouse inbred lines as experimental subjects, observe the influence of immunogenic peptide of the present invention or its vaccine composition respectively to mouse lymphocyte multiplication capacity and specific CTL killing activity.

Select 100 inoculations of female BALB/c mouse D2F2 cell, after 7 days animal is divided into 10 groups: according to group, GM-CSF group, L-P _42-50, L-P _42-50+ GM-CSF group, D-P _42-50, D-P _42-50+ GM-CSF group, L-P _782-790, L-P _782-790+ GM-CSF group, D-P _782-790And D-P _782-790+ GM-CSF group.Every group of 5 animals.Every mouse back immune peptide 30 μ g, weekly, totally twice, and subcutaneous injection GM-CSF 300ng every other day.

(1) mensuration of mouse spleen weight: immunity was for the second time put to death animal after 7 days, aseptic get spleen and wipe out spleen around reticular tissue the weighing spleen is heavy then.The results are summarized in down in the tabulation 1.

Table 1 tumor antigen peptide of the present invention to the influence of immune mouse spleen weight (X ± SD, n=6)

Group	Spleen heavy (g)
Group	Spleen heavy (g)	Blank	0.202±0.02
GM-CSF	0.219±0.04	Blank	0.202±0.02
GM-CSF	0.219±0.04	L-P _42-50	0.221±0.05
L-P _42-50+GM-CSF	0.273±0.09 ^a	L-P _42-50	0.221±0.05
L-P _42-50+GM-CSF	0.273±0.09 ^a	D-P _42-50	0.223±0.06
D-P _42-50+GM-CSF	0.344±0.13 ^ce	D-P _42-50	0.223±0.06
D-P _42-50+GM-CSF	0.344±0.13 ^ce	L-P _782-790	0.218±0.04
L-P _782-790+GM-CSF	0.282±0.12 ^b	L-P _782-790	0.218±0.04
L-P _782-790+GM-CSF	0.282±0.12 ^b	D-P _782-790	0.224±0.05
D-P _782-790+GM-CSF	0.352±0.15 ^df	D-P _782-790	0.224±0.05

A, b: compare P＜0.05:c with control group, d: compare P＜0.01 with control group:

E: compare P＜0.05 with a group; F: compare P＜0.05 with b.

(2) T lymphproliferation response experiment (MTT colorimetry): after immune 7 days for the second time, six mouse of every group of picked at random, the aseptic spleen of getting with 400 purpose steel mesh mechanical separation splenocytes, is regulated cell concn to 2.0 * 10 with containing the 10%RPMI RPMI-1640 ⁶/ ml adds 96 well culture plates, every hole 0.1ml (2.0 * 10 ⁵/ hole), adds synthetic peptide (final concentration 5 μ g/ml, 10 μ g/ml, 20 μ g/ml) in the experimental port, organize positive contrast with ConA (5 μ g/ml), the negative contrast of blank nutrient solution group.Establish three multiple holes for every group, at 37 ℃, 5%CO ₂Cultivated 72 hours under the condition.20 μ l MTT (5 μ g/ml) are added in every afterwards hole, at 37 ℃, and 5%CO ₂Cultivate 4h under the condition, centrifugal 5 minutes of 1000rpm inhales and to abandon supernatant, and methyl-sulphoxide 150 μ l are added in every hole, and light shaking 10 minutes is with the dissolve purple crystalline deposit.Measure light absorption value and calculate the average OD value and the stimulation index SI in each hole at wavelength 490nm then.Shown in the following tabulation 2 of result.

Table 2 tumor antigen peptide of the present invention to the influence of immune mouse lymphocyte proliferation activity (X ± SD, n=6)

Group	HER2(5μg/ml)	HER2(10μg/ml)	HER2(20μg/ml)
Group	HER2(5μg/ml)	HER2(10μg/ml)	HER2(20μg/ml)	Negative control	1.032±0.17	1.046±0.19	1.026±0.15
GM-CSF	1.112±0.22	1.195±0.25	1.145±0.23	Negative control	1.032±0.17	1.046±0.19	1.026±0.15
GM-CSF	1.112±0.22	1.195±0.25	1.145±0.23	L-P _42-50	1.102±0.21	1.168±0.24	1.107±0.22
L-P _42-50±GM-CSF	1.285±0.31 ^a	1.365±0.40 ^a	1.305±0.36 ^a	L-P _42-50	1.102±0.21	1.168±0.24	1.107±0.22
L-P _42-50±GM-CSF	1.285±0.31 ^a	1.365±0.40 ^a	1.305±0.36 ^a	D-P _42-50	1.157±0.22	1.202±0.27	1.136±0.23
D-P _42-50+GM-CSF	1.494±0.42 ^ce	1.685±0.56 ^ce	1.505±0.49 ^ce	D-P _42-50	1.157±0.22	1.202±0.27	1.136±0.23
D-P _42-50+GM-CSF	1.494±0.42 ^ce	1.685±0.56 ^ce	1.505±0.49 ^ce	L-P _782-790	1.128±0.22	1.142±0.23	1.116±0.20
L-P _782-790+GM-CSF	1.262±0.30 ^b	1.373±0.38 ^b	1.284±0.32 ^b	L-P _782-790	1.128±0.22	1.142±0.23	1.116±0.20
L-P _782-790+GM-CSF	1.262±0.30 ^b	1.373±0.38 ^b	1.284±0.32 ^b	D-P _782-790	1.168±0.24	1.175±0.26	1.187±0.27
D-P _782-790+GM-CSF	1.396±0.33 ^df	1.726±0.69 ^df	1.502±0.54 ^df	D-P _782-790	1.168±0.24	1.175±0.26	1.187±0.27

A, b: compare P＜0.05 with control group; C, d: compare P＜0.01 with control group;

E: compare P＜0.05 with a group; F: compare P＜0.05 with b.

(3) mensuration of T emiocytosis IFN-γ (enzyme linked immunological spotting method, ELISPOT method): by 96 orifice plates (50 μ l/ hole), 4 ℃ are spent the night then with the PBS bag that contains 10 μ g/ml rat anti-mouse IFN-gamma antibodies.After washing culture plate 3 times with PBS, add the RPMI RPMI-1640 that 100 μ l contain 10% foetal calf serum, 37 ℃ of insulations were sealed in 30 minutes.After discarding confining liquid and washing culture plate 3 times, with 50 μ l (2 * 10 with PBS ⁷/ ml) splenocyte adds in 96 well culture plates (establishing three multiple holes for every group) to, and every then hole adds 50 μ l synthetic peptide of the present invention (10 μ g/ml), in 37 ℃, 5%CO ₂Cultivated 72 hours under the condition.ConA (final concentration 5 μ g/ml) is added in the positive control hole, and negative control hole adds 50 μ l, 10% foetal calf serum.The reaction back is washed culture plate 3 times with PBS and is added the anti-IFN-γ second antibody of 50 μ l (5 μ g/ml) enzyme labelling, and 4 ℃ are spent the night.Wash plate 3 times with PBS, the PBS solution that 100 μ l contain 0.1% alkaline phosphatase is added in every hole, and 37 ℃ are incubated 1 hour.Wash plate 3 times with PBS, add the BCIP gelating soln that 50 μ l contain 140 μ g/ml, room temperature reaction 30 minutes produces blue spot.Conventional then phase microscope calculates the spot number.Shown in the following tabulation 3 of result.

Table 3 tumor antigen peptide of the present invention to the influence of immune mouse T emiocytosis IFN-γ (X ± SD, n=6)

Group	The blue spot number
Group	The blue spot number	Negative control	2.42±0.9
GM-CSF	12.09±2.4	Negative control	2.42±0.9
GM-CSF	12.09±2.4	L-P _42-50	4.15±1.3
L-P _42-50+GM-CSF	37.62±5.6 ^a	L-P _42-50	4.15±1.3
L-P _42-50+GM-CSF	37.62±5.6 ^a	D-P _42-50	6.21±1.8
D-P _42-50+GM-CSF	69.75±7.8 ^ce	D-P _42-50	6.21±1.8
D-P _42-50+GM-CSF	69.75±7.8 ^ce	L-P _782-790	5.26±1.5
L-P _782-790+GM-CSF	35.84±5.2 ^b	L-P _782-790	5.26±1.5
L-P _782-790+GM-CSF	35.84±5.2 ^b	D-P _782-790	7.36±2.3
D-P _782-790+GM-CSF	57.66±5.3 ^df	D-P _782-790	7.36±2.3

A, b: compare P＜0.05 with control group; C, d: compare P＜0.01 with control group:

E: compare P＜0.05 with a group; F: compare P＜0.05 with b.

(4) specific CTL detects (cytotoxic T cell granzyme method for releasing): back 7 days of immunity for the second time, and put to death the animal separating spleen and prepare splenocyte suspension (20 * 10 ⁷/ ml).Getting the 1ml suspension is added in and sneaks into antigen peptide of the present invention (5 μ g/ml) and IL-2 (final concentration 10U/ml), 37 ℃ and 5%CO in the culture plate ₂Cultivate under the condition and obtained the effector cell in 8 days.In each hole of 96 porocyte culture plates, add the RPMI RPMI-1640 that 50 μ l contain 10% foetal calf serum and obtain effect CTL cell suspension (2 * 10 ⁶/ ml).In detecting hole (E) by different effector cells: target cell ratio (E: T) (40: 1,20: 1,10: 1) interpolation 50 μ l D2F2 cells (50 μ l).Always discharge at enzyme simultaneously and add 40 μ l complete culture solutions and 10 μ l 1%TritonX-100 in the control wells (R), in blank hole (B), add complete culture solution (50 μ l).Establish three multiple holes for every group, culture plate is put 37 ℃ and 5%CO ₂Cultivate under the condition.After 4 hours, centrifugal 5 minutes of 1100rpm.Draw 50 μ l culture supernatant then and change in the test tube, add the BLT substrate of the interim preparation of 950 μ l and use liquid.37 ℃ of water-baths placed ice bath with test tube after 20 minutes, added PMSF (for a kind of irreversible serine ester enzyme inhibitors) 10 μ l (final concentration 0.05mol/L) immediately.Measure light absorption value down in the 412nm wavelength then, calculate the activity of tumor antigen peptide inductive specific CTL of the present invention.Shown in the following tabulation 4 of result.

Table 4 tumor antigen peptide of the present invention to the influence (%) of immune mouse CTL kill rate (X ± SD, n=6)

Group	E∶T
	E∶T			40∶1	20∶1	10∶1
	Blank	8.9±1.1	7.4±0.9	40∶1	20∶1	10∶1	5.2±0.5
GM-CSF	Blank	8.9±1.1	7.4±0.9	16.2±3.8	10.6±2.7	8.9±2.2	5.2±0.5
GM-CSF	L-P _42-50	10.5±2.8	8.8±1.9	16.2±3.8	10.6±2.7	8.9±2.2	5.7±1.8.
L-P _42-50+GM-CSF	L-P _42-50	10.5±2.8	8.8±1.9	36.7±5.4 ^a	29.3±4.6 ^a	24.4±3.9 ^a	5.7±1.8.
L-P _42-50+GM-CSF	D-P _42-50	14.7±3.3	9.8±2.6	36.7±5.4 ^a	29.3±4.6 ^a	24.4±3.9 ^a	8.8±1.9
D-P _42-50+GM-CSF	D-P _42-50	14.7±3.3	9.8±2.6	58.4±7.9 ^ce	47.2±6.2 ^ce	36.9±4.9 ^ce	8.8±1.9
D-P _42-50+GM-CSF	L-P _782-790	14.3±3.4	12.4±2.8	58.4±7.9 ^ce	47.2±6.2 ^ce	36.9±4.9 ^ce	10.7±2.4
L-P _782-790+GM-CSF	L-P _782-790	14.3±3.4	12.4±2.8	35.2±5.1 ^b	28.4±4.2 ^b	22.7±.3.2 ^b	10.7±2.4
L-P _782-790+GM-CSF	D-P _782-790	13.6±3.0	11.2±2.7	35.2±5.1 ^b	28.4±4.2 ^b	22.7±.3.2 ^b	9.7±1.8
D-P _782-790+GM-CSF	D-P _782-790	13.6±3.0	11.2±2.7	56.5±7.4 ^df	44.3±5.7 ^df	35.4±4.8 ^df	9.7±1.8

E: compare P＜0.05 with a group; F: compare P＜0.05 with b.

No matter by the result shown in the above table 1-4 as can be seen, compare with negative control group, be natural wild-type HER2/neu epitope peptide (L-P _42-50And L-P _782-790) or the new HER2/neu epitope peptide (D-P of the present invention's modification _42-50And D-P _782-790) during respectively with the GM-CSF combined utilization, all can promote lymphopoiesis, increase IFN-γ secretion property T lymphocyte number, and kill and wound the D2F2 breast cancer cell specifically.And in the presence of GM-CSF, the HER2/neu epitope peptide that the present invention modifies is D-P _42-50And D-P _782-790Show respectively than natural wild-type HER2/neu epitope peptide and have the ability of more significant inducing antitumor cell immune response, for example have more significant short T cell-proliferation activity, short T emiocytosis activity and specific CTL and activate activity etc.This is because the tumor antigen peptide D-P that the present invention modifies _42-50And D- _P782-790The molecule part be endowed the chirality feature, had the constitutional features that is different from its natural parent HER2/neu peptide, improved immunogenicity to a certain extent, thereby made its generation and the release of inducing tumor-specific CTL more effectively.

Embodiment 3: antitumor activity in the body of tumour antigen peptide vaccine of the present invention

Present embodiment uses the mouse inbred lines that has inoculated D2F2 breast cancer cell (as transfection the clone of people HER2/neu overall length cDNA) as experimental subjects, observe vaccine composition of the present invention to the tumor-bearing mice tumor growth in vivo influence.

Basically the method according to embodiment 2 prepares animal model for tumour and grouping, put to death animal in 21 days and measure tumor weight, tumour inhibiting rate and gross tumor volume behind the inoculated tumour cell.The result is shown in table 5 and accompanying drawing 5.In addition, go back the tumor resection tissue in the experiment and carried out histopathologic examination's (result is not shown).

The restraining effect that table 5 tumor antigen peptide of the present invention is grown to mouse D2F2 breast tumor (X ± SD, n=6)

Group	Tumor weight (g)	Tumour inhibiting rate (%)
Group	Tumor weight (g)	Tumour inhibiting rate (%)	Negative control	1.358±0.44
GM-CSF	1.255±0.29	7.5	Negative control	1.358±0.44
GM-CSF	1.255±0.29	7.5	L-P _42-50	1.294±0.49	4.7
L-P _42-50+GM-CSF	0.945±0.28 ^a	30.4 ^a	L-P _42-50	1.294±0.49	4.7
L-P _42-50+GM-CSF	0.945±0.28 ^a	30.4 ^a	D-P _42-50	1.324±0.45	2.5
D-P _42-50+GM-CSF	0.785±0.33 ^ce	42.1 ^ce	D-P _42-50	1.324±0.45	2.5
D-P _42-50+GM-CSF	0.785±0.33 ^ce	42.1 ^ce	L-P _782-790	1.322±0.36	2.6
L-P _782-790+GM-CSF	0.974±0.38 ^b	28.4 ^b	L-P _782-790	1.322±0.36	2.6
L-P _782-790+GM-CSF	0.974±0.38 ^b	28.4 ^b	D-P _782-790	1.318±0.24	2.9
D-P _782-790+GM-CSF	0.824±0.39 ^df	39.3 ^df	D-P _782-790	1.318±0.24	2.9

E: compare P＜0.05 with a group; F: compare P＜0.05 with b.

By the result shown in above table 5 and the accompanying drawing 5 as can be seen, no matter be natural wild-type HER2/neu epitope peptide (L-P _42-50And L-P _782-790) or the new HER2/neu epitope peptide (D-P of the present invention's modification _42-50And D-P _782-790), when during respectively with the GM-CSF combined utilization, all making the weight and volume of tumour be starkly lower than control group them.And in the presence of GM-CSF, the HER2/neu epitope peptide that the present invention modifies is D-P _42-50And D-P _782-790Show respectively than natural wild-type HER2/neu epitope peptide have the activity of more significant anti-tumor in vivo immunity and suppress tumor growth ability.

Pathological observation is found, the new HER2/neu epitope peptide (D-P that the present invention modifies _42-50And D-P _782-790) during respectively with the GM-CSF combined utilization, visible tumor tissues central authorities boundary clear, large-area necrosis region being arranged, cell cytosol is red to be dyed, and cellularstructure disappears.But the profile of weave construction is still preserved, the nuclear chromatin disintegration is small shreds and see have nuclear membrane to break.(referring to accompanying drawing 6 and 7).

Further tumor tissue cell's transmission electron microscope Ultrastructural observation as seen, the new HER2/neu epitope peptide (D-P that the present invention modifies _42-50And D-P _782-790) during respectively with the GM-CSF combined utilization, neoplastic cell nuclei is irregular, nuclear membrane depression, heterochromatin slightly increase, the pyknosis of part tumour cell, mitochondrial swelling, neoplastic cell nuclei are irregular, the kytoplasm engrain is the early stage constructional feature of apoptosis.(referring to accompanying drawing 8 and 9).

Above result shows, compare with wild-type (L-HER2/neu) antigen peptide, the HER2/neu antigen peptide that the present invention modifies can more effectively promote the propagation of T cell, increase the IFN-γ secretion of T cell, further improve the killing activity of CTL, thereby reach the purpose that suppresses tumor growth tumour cell.

Sequence table

＜110〉the holy first Science and Technology Ltd. in Jilin

＜120〉tumor antigen peptide of Xiu Shiing and application thereof

<140>

<141>

<160>2

<210>1

<211>9

＜212〉amino acid

＜213〉artificial sequence

<220>

＜223〉HLA-A2 restricted CTL epitope (nonapeptide) sequence of synthetic HER2/neu tumour antigen.

<400>1

ValValGlnCysGlyGlnTyrD-LeuHis

1 5

<210>2

<211>9

＜212〉amino acid

＜213〉artificial sequence

<220>

<400>2

LeuCysIleGlyLeuLeuArgD-SerVal

1 5

Claims

1,, is characterised in that they have aminoacid sequence shown in SEQ ID NO:1 in the sequence table and SEQ ID NO:2 respectively, and includes a D-type amino acid separately at least based on the tumor antigen peptide of the modification of HER2/neu sequence.

2, HER2/neu specific tumour vaccine or pharmaceutical composition, said vaccine or pharmaceutical composition are made up of the tumor antigen peptide of claim 1 and one or more pharmaceutically acceptable carriers.

3, according to the tumor vaccine or the pharmaceutical composition of claim 3, wherein also can contain the immunostimulant that is selected from complete Freund's adjuvant or incomplete Freund's adjuvant.