CN1948333A - New hPEBP4 protein source HLA-A2 limiting epi-polypeptide and its application - Google Patents

Abstract

The present invention relates to an HLA-A2 restricted epitope polypeptide derived from hPEBP4 protein, as well as the epitope and its related recombinant protein, coding nucleotide sequence, antigen-presenting cell, composition and specificity for the epitope Use of immune effector cells in the treatment and prevention of tumors expressing hPEBP4.

Description

A new HLA-A2 restricted epitope polypeptide derived from hPEBP4 protein and its application

technical field

The present invention relates to the fields of biology and medicine, and more specifically relates to an HLA-A2 restricted epitope polypeptide derived from hPEBP4 protein, as well as the epitope and its related recombinant protein, coding nucleotide sequence, antigen presenting cell, The composition and the application of specific immune effector cells targeting the epitope in the treatment and prevention of hPEBP4-expressing tumors.

Background technique

hPEBP4 (SEQ ID NO: 12), hPEBP4 was originally called "hPEBP5", and its nucleotide sequence and amino acid are shown in Chinese patent application CN02136556.3. Bone marrow stromal cell (BMSC) is obtained from primary culture of normal adult bone marrow cells in vitro and a BMSC cDNA library is constructed. It is a new type of whole-body isolated from the BMSC cDNA library by means of large-scale sequencing of the cDNA library. The long new gene belongs to the phosphatidylethanolamine-binding protein (PEBP) family and is registered in the GenBank/EMBL database (sequence number is AY037148). The hPEBP4 protein encodes 227 amino acids (SEQ ID NO: 13). Its amino acid sequence at 75-195 is a typical phosphatidylethanolamine-binding conserved region (PBP).

Synthetic peptide vaccine is a new vaccine developed with the progress of molecular biology and immunology in recent years. It can induce the body to produce a specific immune response, and its side effects are mild and safe. It is the current vaccine research. A new direction, widely used with anti-tumor and anti-viral immunotherapy. At present, a variety of vaccines based on epitope peptides have entered clinical research or entered the market.

Cellular immunity plays a key role in antitumor and antiviral immunity. The antigen recognized by T cells is a peptide bound to MHC class I or class II molecules on the cell surface, and its length is about 8-12 amino acids. As the main effector cell CTL (cytotoxic T lymphocytes, Cytotoxic TLymphocytes) to kill tumor cells, it recognizes endogenous peptides combined with MHC-I molecules. Evidence has shown that synthetic peptides can directly bind to MHC-I molecules without processing and processing by APCs, and they have the same efficacy as natural endogenous peptides in activating the immune system.

Peptide vaccine has become a new strategy against malignant tumors, and it is also the most studied tumor therapeutic vaccine. More studies are: (1) gp100: peptides G9154 (sequence: KTWGQYWQV) and G9209 (sequence: ITDQVPPFSV) derived from gp100 can induce antigen-specific CTL. Using it to sensitize peripheral blood lymphocytes (PBL) from HLA-A2 ⁺ melanoma patients can significantly enhance its ability to induce specific CTL: the induced CTL can recognize unmodified gp100; (2) CEA: use Similar to the method described above, the research of Zaremba et al. showed that the peptide CAP1-6D of CEA can not only sensitize CEA-specific CTL in vitro, but its potency is 100-1000 times that of CAP1, and it can also induce CEA-specific CTL in vivo ( But CAP1 can't); and the sensitized CTL can also recognize CAP1. More importantly, these CTLs can dissolve homologous CEA-expressing human tumors; (3) MAGE-2: MAGE-2 is widely present in various tumors such as melanoma, laryngeal cancer, lung cancer, and sarcoma (except testis) , not present in normal tissue. In MAGE-2, three peptides have been found to form stable complexes with MHC-I molecules at 37°C, at least two of which can be processed and presented by HLA-A*0201, becoming new candidates for peptide vaccine research ; (4) P21 ^WAFI : the fusion of P21 ^WAFI peptide and endogenous peptide can inhibit the growth of tumor; (5) HER2/neu: the peptide derived from HER2/neu can sensitize specific CTL in vitro, and can induce specific CTL in small It can also induce specific CTL in mice, and can inhibit tumor growth. In addition, the use of HPV peptide vaccines has also entered clinical research.

HLA-A2 is a kind of MHC-I molecule with a relatively high distribution in the Chinese population, the positive rate is between 40-60%, accounting for the first place in each subgroup of MHC-I molecules, because HLA-A* 0201 is the site with the highest occurrence frequency in the population, and the motif composition is clear, so it is the preferred related molecule in vaccine design. With the in-depth understanding of the interaction between MHC-I molecules and peptide epitope molecules, scientists have established a relatively mature epitope identification technology route. The main steps are as follows: (1) According to the epitope and MHC-I molecule According to the characteristics of the interaction, predict the restricted CTL epitopes of MHC-I molecules, and use computer molecular simulation technology to further screen the predicted epitopes; (2) determine the binding force between the epitopes and MHC-I molecules; (3) use In vitro cytotoxicity analysis or immunization of tumor-bearing transgenic mice to identify whether the peptide epitope can induce CTL, and seek the best dominant epitope. Based on this technical route, a variety of HLA-A*0201-restricted CTL epitopes have been identified, some of which have shown good clinical efficacy.

T2 cells are one of the tool cells used to determine the binding ability of epitopes to HLA-A*0201 molecules. It is a HLA-A*0201 cell line with antigen-presenting transporter defects. The HLA-A*0201 molecule of the endogenous antigen molecule can be used to determine the affinity between the HLA-A0201 molecule and the target epitope by using its binding degree to the target peptide.

At present, there is still a lack of therapeutic vaccines for effective prevention and treatment of anti-tumor, especially synthetic peptide vaccines with high safety. Peptide vaccine.

Contents of the invention

The purpose of the present invention is to provide a synthetic peptide with high safety and high immunogenicity and its application.

In the first aspect of the present invention, there is provided a hPEBP4-derived HLA-A2 restricted epitope polypeptide, characterized in that the polypeptide has the activity of inducing cytotoxic T cell killing, and is selected from the following group:

(a) a polypeptide comprising an amino acid sequence in general formula I as follows:

X _aa1 -TLFCQGLEV-X _aa2 (I)

In the formula, each of X _aa1 and X _aa2 is 0, 1, 2 or 3 optional amino acids;

(b) The HLA-A2 restricted epitope derived from (a) formed by substituting, deleting or adding 1-4 amino acid residues to the amino acid sequence of the general formula, and having the function of inducing cytotoxic T cell killing activity peptide.

More preferably, the amino acid is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr , Val.

More preferably, said X _aa1 is absent, D, ED, or DED; and X _aa2 is absent, F, FY or FYP.

Preferably, the amino acid sequence is substituted, deleted or added by one or more amino acids.

In one embodiment of the invention, the polypeptide is TLFCQGLEV (SEQ ID NO: 1).

The second aspect of the present invention relates to a recombinant protein containing the HLA-A2 restricted epitope polypeptide derived from hPEBP4 as described above.

The third aspect of the present invention relates to an isolated nucleic acid encoding the HLA-A2 restricted epitope polypeptide derived from hPEBP4 as described above.

The fourth aspect of the present invention relates to an antigen-presenting cell sensitized by the HLA-A2 restricted epitope polypeptide derived from hPEBP4 as described above.

Preferably, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, B cells, fibroblasts, and endothelial cells.

The fifth aspect of the present invention relates to a composition, which contains: (a) 0.001-99.99wt% of the HLA-A2 restricted epitope polypeptide derived from hPEBP4 as described above or the antigen-presenting polypeptide as described above cells; and (b) an acceptable carrier, diluent or excipient.

Preferably, the composition is a pharmaceutical composition, and the carrier, diluent or excipient is a pharmaceutically acceptable carrier, diluent or excipient.

The sixth aspect of the present invention relates to the use of the aforementioned HLA-A2 restricted epitope polypeptide derived from hPEBP4 or antigen-presenting cells in the preparation of drugs for preventing and treating tumors.

In the seventh aspect of the present invention, the use of the HLA-A2 restricted epitope polypeptide derived from hPEBP4 of the present invention is provided, which is used to prepare a recombinant protein (such as a fusion protein) comprising the restricted epitope polypeptide, Or for the preparation of sensitized antigen-presenting cells.

Description of drawings

Figure 1: P40-48 peptide-sensitized dendritic cells (DCs) in vivo induced the killing activity of cytotoxic T lymphocytes in HLA-A2.1/K ^b transgenic mice. The effector cells in A are from cytotoxic T lymphocytes induced in vivo by homologous DC alone; the effector cells in B are from cytotoxic T lymphocytes induced in vivo by P40-48 peptide-sensitized DC.

Figure 2: IFN-γ secretion of cytotoxic T lymphocytes induced by P40-48 peptide-sensitized dendritic cells (DC) in vivo in HLA-A2.1/K ^b transgenic mice. The effector cells in A are from cytotoxic T lymphocytes induced in vivo by homologous DC alone; the effector cells in B are from cytotoxic T lymphocytes induced in vivo by P40-48 peptide-sensitized DC.

Detailed ways

After extensive and in-depth research, the inventors found that hPEBP4 is highly expressed in certain tumor cells and can inhibit TNFα-induced apoptosis by inhibiting Ras/Raf/MEK/ERK pathway, JNK activation and PE eversion. Therefore, the use of hPEBP4-specific antigenic peptides can induce the body to produce specific CTLs to kill tumor cells that highly express hPEBP4. Accordingly, on the basis of computer simulation analysis of hPEBP4, the inventors selectively synthesized a number of hPEBP4-specific antigenic peptides that may bind to HLA-A*0201 and induce the body to produce CTL. , screened the epitope peptide with strong affinity to HLA-A*0201, and evaluated its immunogenicity, and found ^that it could not only induce It produces specific, HLA-A*0201-restricted cytotoxic T lymphocytes, and is an immunogenic polypeptide that is naturally processed and presented by cells. The present invention has been accomplished on this basis.

Specifically, the inventor's research shows that the amino acid sequence of hPEBP4 at position 75-195 is a typical phosphatidylethanolamine-binding conserved region (PBP). hPEBP4 can inhibit TNFα-induced apoptosis by inhibiting Ras/Raf/MEK/ERK pathway, JNK activation and PE eversion, all of which are mediated by its PE-binding conserved domain (PBP).

RT-PCR showed that hPEBP4, as an anti-apoptotic molecule, was highly expressed in prostate cancer PC-3, ovarian cancer CaoV-3, breast cancer MCF-7 and other tumor cells, while A549 cells derived from lung cancer and LoVo cells derived from colon cancer were as large as Some solid tumor cells have no expression. In clinical tumor samples, hPEBP4 protein is highly expressed in more than 50% of breast cancer tumor tissues, but only in 4% of normal breast tissues. In addition, the experimental results also showed that the down-regulation of hPEBP4 expression enhanced the sensitivity of breast cancer, ovarian cancer and other tumor cells to TNFα-induced apoptosis, indicating that hPEBP4 is a potential target for the treatment of breast cancer and other tumors.

Therefore, if hPEBP4 is used as the target to immunize the body, the body will generate a specific immune response against hPEBP4 protein, so that the body can effectively inhibit and eliminate tumor cells, and provide measures for the prevention and treatment of related tumors.

Accordingly, on the basis of computer simulation analysis of hPEBP4, the inventors selectively synthesized a number of hPEBP4-specific antigenic peptides that might bind to HLA-A*0201 and induce the body to produce CTLs. , screened the epitope peptide with strong affinity to HLA-A*0201, and evaluated its immunogenicity, and found ^that it could not only induce It produces specific, HLA-A*0201-restricted cytotoxic T lymphocytes, and is an immunogenic polypeptide that is naturally processed and presented by cells.

The identification of the HLA-A2-restricted cytotoxic T lymphocyte epitope peptide derived from the tumor-associated antigen hPEBP4 is of great significance for the development of tumor vaccines and therapeutic preparations.

As used herein, "polypeptide of the present invention", "specific polypeptide", and "HLA-A2 restricted epitope polypeptide" are used interchangeably, referring to the polypeptide shown in the amino acid sequence P40-48 (SEQ ID NO: 1). In addition, variant forms of the sequence of SEQ ID NO: 1 having the same function as P40-48 (SEQ ID NO: 1) are also included. These variations include (but are not limited to): one or more (usually 1-20, preferably 1-10, more preferably 1-5, and most preferably 1-3) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active derivatives of P40-48.

A preferred derivative polypeptide is the addition of 1 or 2 or 3 or 4 amino acids (such as D, ED, DED or LDED) corresponding to positions 36-39 in hPEBP4 upstream of SEQ ID NO: 1, and / Or add 1 or 2 or 3 or 4 amino acids (such as F, FY, FYP or FYPE) corresponding to the 49-52 position in hPEBP4 downstream of SEQ ID NO: 1.

The polypeptide of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.

The polypeptide of the present invention can also be used to couple with proteins with molecular weights such as BSA to form polypeptide conjugates. Usually, the conjugate is composed of a polypeptide, a cross-linking agent, and BSA, wherein the cross-linking agent is preferably glutaraldehyde and EDAC.

In addition, the polypeptides and conjugates of the present invention can also be used to prepare therapeutic pharmaceutical compositions or preventive and therapeutic vaccine compositions.

Therefore, in another aspect, the present invention also provides a composition, which contains (a) a safe and effective amount of the polypeptide of the present invention, a conjugate or a composition thereof; and (b) a pharmaceutically acceptable carrier or excipient agent. The amount of the polypeptide of the present invention is usually 10 μg-100 mg/dose, preferably 100-1000 μg/dose.

As used herein, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or exhibits a detectable therapeutic or preventive effect. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agents and/or combination of therapeutic agents chosen for administration. Therefore, it is not useful to prespecify an exact effective amount. However, the effective amount can be determined by routine experimentation, within the judgment of the clinician, for a given situation.

For the purposes of the present invention, an effective dosage is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the present invention administered to an individual. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. The term refers to pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration. These vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids, such as water, saline, glycerol and ethanol. In addition, there may also be auxiliary substances in these carriers, such as wetting agents or emulsifying agents, pH buffering substances and the like. In addition, immune adjuvants may also be included in the immune composition.

Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension, in liquid carriers prior to injection can also be prepared.

Once formulated, the compositions of the invention can be administered directly to a subject. The subject to be prevented or treated can be an animal; especially a human.

The therapeutic or preventive pharmaceutical composition (including vaccines) containing the polypeptide of the present invention can be applied orally, subcutaneously, intradermally, or intravenously. The therapeutic dosage regimen can be a single dose regimen or a multiple dose regimen.

The main advantage of the present invention is that: the epitope peptide of the present invention is derived from hPEBP4 protein and HLA-A2 restricted cytotoxic T lymphocyte epitope peptide, which can safely and effectively induce immune response against tumor cells, and not only to tumor cells It is of great significance to the study of pathogenesis, and to the development of tumor therapeutic vaccines and therapeutic preparations.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacture conditions recommended by the manufacturer.

Example

Example 1 Screening of HLA-A*0201 High Affinity Peptides

In this example, epitope peptides with high affinity to HLA-A*0201 were screened out through peptide binding experiments.

experiment procedure

First collect T2 cells (a cell lacking antigen processing ability, purchased from ATCC: CRL-1991), wash three times with serum-free 1640, adjust the cell concentration to 2×10 ⁵ /ml, spread in 24-well plates, 0.5 ml/well. Then co-incubate with 50 μM candidate polypeptide and 2.5 μg/ml β2 microglobulin at 37° C. in a 5% CO ₂ incubator for 18 hours. The incubated cells were washed three times with ice-cold PBS, FITC-labeled HLA-A2-specific mAb BB7.2 (Sterotec Ltd, Oxford, UK) was added, kept on ice for 45 minutes, and the average fluorescence was detected by flow cytometry after washing with PBS strength. A positive known peptide CEA HLA-A2 restricted epitope polypeptide CAP-1 (SEQ ID NO: 10, sequence YLSGANLNL) was used as a positive control, and simple T2 cells stimulated without peptide were used as a background control.

Detection method

Immunofluorescence detection of the combination of peptides and HLA-A*0201 molecules is based on the fact that the combination of exogenous peptides and MHC-I molecules on the surface of T2 cells can increase the expression of MHC-I molecules on the surface. The more stable the combination, the more the expression of MHC-I class molecules can be detected, and the average fluorescence intensity is used as the detection index. The results were measured by fluorescence coefficient (FI). A polypeptide with FI > 1 is considered a high affinity epitope.

test results

Table 1 shows the binding affinity results of hPEBP4 protein-derived polypeptides to T2-HLA-A*0201 measured by immunofluorescence. The present inventor screened out the high-affinity epitope P40-48 of HLA-A2 from several polypeptides derived from hPEBP4 protein, and its sequence is TLFCQGLEV (SEQ ID NO: 1).

T2-HLA-A*0201 binding affinity of table 1 hPEBP4 protein source polypeptide name start site sequence FI value SEQ ID NO P40-48R1R2R4R5R7R8R9R10 401112163452113127153 TLFCQGLEVALLLGLMMVLLLGLMMVVSLLPKENKTTMRLVTAALELGNIGCKVWLVTDIKGAKIQGQELSAFVYLQEGKV 1.630.421.040.030.350.370.290.390.27 123456789

High affinity: FI>1

Example 2 Preparation of Adenovirus (pAdPEBP4) Expressing hPEBP4

The operation was performed according to the instructions of the Adeasy Adenovirus Vector System Kit of Stratagene Company. The brief steps are:

Using the plasmid containing human hPEBP4 cDNA obtained in Example 1 of Chinese patent application CN02136556.3 as a template, digested with SalI-NotI, and then recombined with plasmid pShuttle-CMV (Stratagene Company) according to a conventional method and transformed into a competent state Escherichia coli BL21, the positive clones were picked and identified, purified and sequenced (ABI company, BigDye Terminator kit). The human hPEBP4-pShuttle-CMV vector with the correct sequence was linearized with Pme I and transformed into Escherichia coli BJ5183 (Stratagene) together with the pAdeasy adenovirus backbone plasmid (Stratagene). Positive clones were identified by PacI digestion, and the products were analyzed by 0.8% agarose gel electrophoresis. It was demonstrated that an adenoviral vector containing the human hPEBP4 coding sequence has been produced recombinantly.

After the adenovirus vector was linearized by PacI, the AD293 cells (Stratagene) were transfected with Lipofectamine transfection reagent (Invitrogen). After 7-10 days, the cells were completely rounded, and the cells were collected, and the virus supernatant was obtained by freezing and thawing repeatedly. Gradient centrifugation was used to purify a large amount of virus, so as to obtain recombinant adenovirus (pAdhPEBP4) expressing hPEBP4, and the virus titer was 10×10 ¹³ .

Example 3 Induction of hPEBP4-derived HLA-A2-restricted polypeptide-specific cytotoxic T lymphocytes in HLA-A2.1/ ^Kb transgenic mice

Preparation of effector cells

Dendritic cells (DC) derived from bone marrow of HLA-A2.1/K ^b transgenic mice were prepared according to conventional methods. Collect DCs cultured to day 7, adjust the cell concentration to 1×10 ⁶ cells/ml, add P40-48 (final concentration 10 μM/ml) and β2 microglobulin (final concentration 3 μg/ml), 37°C, 5 Incubate for 3 hours in a %CO ₂ incubator. The peptide-sensitized DCs were collected, washed three times with PBS, and the cell concentration was adjusted to 1×10 ⁶ /0.2ml to immunize mice. Each mouse was intraperitoneally injected with 1×10 ⁶ cells/0.2ml, and immunized three times with an interval of one week. Seven days after the last immunization, the mouse spleen was removed aseptically, and the red blood cells were dissolved to make a single cell suspension. Splenocyte suspension (5×10 ⁶ /ml) and peptide-sensitized irradiated autologous dendritic cells were cultured in RPMI 1640 complete medium at a ratio of 10:1. After 6 days of culture, the effector cells were collected,

Detection method

(1) Detection of specific killing activity

In this example, a standard 4-hour ⁵¹ Cr release test was used to detect specific killing activity.

T2 cells loaded with P40-48, SSp-1 (irrelevant control, sequence: RLNEVAKNL (SEQ ID NO: 11)), T2 cells infected with recombinant adenovirus (pAdPEBP4) expressing hPEBP4 (positive control) and unloaded Add ⁵¹ Cr (100 μCi/10 ⁶ cells) to the T2 cells as target cells, put them in a 37°C water bath for 90 minutes, mix gently every 15 minutes, wash 3 times, and thoroughly wash away residual Na ₂ ⁵¹ CrO ₄ , the marked target cells were adjusted to a cell concentration of 1×10 ⁵ cells/ml with complete medium, and added to a 96-well round bottom plate, 100 μl per well. Add effector cells at three different effector-target ratios of 50:1, 25:1, and 12.5:1, incubate at 37°C for 4 hours, collect 100 μl of supernatant from each well, and detect the cpm value with a gamma counter. For each well of the maximum release group, individual target cells were added with 100 μl of 1% SDS; for spontaneous release wells, individual target cells were added with 100 μl of complete medium.

(2) Detection of intracellular IFN-γ in peptide-specific CTL

20 μM of the corresponding peptide was added to the induced effector cells, and after 48 hours, routine intracellular staining was performed.

test results

The results showed that hPEBP4-derived HLA-A2-restricted polypeptide P40-48 sensitized dendritic cells could significantly induce HLA-A2.1/K ^b transgenic mice to produce restricted polypeptide-specific cytotoxic T lymphocytes and killing activity (see Figure 1), and can stimulate IFN-γ production (see Figure 2).

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

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Claims (10)

1. the HLA-A2 restriction epi polypeptide in hPEBP4 source is characterized in that described polypeptide has the inducing cytotoxic T cell killing activity, and is selected from down group:

(a) comprise the polypeptide of the aminoacid sequence in the following general formula I:

X _aa1-TLFCQGLEV-X _aa2 (I)

In the formula, X _Aa1And X _Aa2Respectively be 0,1,2 or 3 optional amino acid;

(b) the general formula aminoacid sequence is formed through replacement, disappearance or the interpolation of 1-4 amino-acid residue, and have inducing cytotoxic T cell killing activity function by (a) deutero-HLA-A2 restriction epi polypeptide.

2. polypeptide as claimed in claim 1 is characterized in that, described aminoacid sequence is through one or more amino acid whose replacements, disappearance or interpolation.

3. polypeptide as claimed in claim 1 is characterized in that, described polypeptide is TLFCQGLEV.

4. a recombinant protein is characterized in that, it contains the HLA-A2 restriction epi polypeptide in the described hPEBP4 of claim 1 source.

5. an isolating nucleic acid is characterized in that, the HLA-A2 restriction epi polypeptide in the described hPEBP4 of its coding claim 1 source.

6. an antigen presenting cell is characterized in that, described antigen presenting cell is by the HLA-A2 restriction epi polypeptide sensitization in the described hPEBP4 of claim 1 source.

7. antigen presenting cell as claimed in claim 6 is characterized in that, described antigen presenting cell is selected from down group: dendritic cell, scavenger cell, B cell, inoblast, endotheliocyte.

8. composition is characterized in that it contains:

(a) the HLA-A2 restriction epi polypeptide or the described antigen presenting cell of claim 6 in each described hPEBP4 source of 0.001-99.99wt% claim 1-3; With

(b) acceptable carrier, thinner or vehicle.

9. composition as claimed in claim 8 is characterized in that described composition is a pharmaceutical composition, and described carrier, thinner or vehicle are pharmaceutically acceptable carrier, thinner or vehicle.

10. the HLA-A2 restriction epi polypeptide in each described hPEBP4 source of claim 1-3 or the purposes of the described antigen presenting cell of claim 6 is characterized in that, are used to prepare the medicine of prevention and treatment tumour.

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