CN102250207A - Novel human leukocyte antigen (HLA)-A2 limiting epitope polypeptide and use thereof - Google Patents

Abstract

The invention provides a novel HLA-A2 restricted epitope polypeptide and its application. Specifically, the present invention relates to the HLA-A2 restricted epitope polypeptide (i.e. 1F peptide) whose sequence is FLFCQGLEV, as well as the epitope and its related recombinant protein, coding nucleotide sequence, antigen presenting cell, composition in Use in tumor treatment and prevention expressing human phosphatidylethanolamine binding protein 4 (hPEBP4).

Description

A new HLA-A2 restricted epitope polypeptide 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, as well as the epitope and its related recombinant protein, coding nucleotide sequence, antigen-presenting cells, and compositions expressed in Use of human phosphatidylethanolamine binding protein (hPEBP4 protein) in tumor treatment and prevention.

Background technique

Human phosphatidylethanolamine (PE)-bindingprotein 4 (hPEBP4), its nucleotide sequence and amino acid can be found in Chinese patent CN02136556.3. It is a full-length bone marrow stromal cell (BMSC) isolated from normal adult bone marrow cells by primary culture in vitro, constructing a BMSC cDNA library, and using large-scale sequencing of the cDNA library. The gene, which belongs to the phosphatidylethanolamine-binding protein (PEBP) family, is registered in the GenBank/EMBL database (sequence number is AY037148). The hPEBP4 protein contains 227 amino acids, and its amino acid sequence at positions 75-195 is a typical phosphatidylethanolamine-binding conserved region (PBP).

Functional studies have found that hPEBP4 can inhibit TNFα-induced apoptosis by inhibiting the Ras/Raf/MEK/ERK pathway, JNK activation, and PE exversion, all of which are mediated by its PE-binding conserved domain (PBP). Down-regulation of hPEBP4 expression can promote TNF-α-induced apoptosis of breast cancer cells, prostate cancer cells and ovarian cancer cells, suggesting that hPEBP4 may be a new target for the treatment of tumors with high expression of hPEBP4.

The researchers confirmed by tissue microarray and immunohistochemistry that in clinical tumor specimens, hPEBP4 protein was highly expressed in more than 50% of breast cancer tumor tissues, but only in 4% of normal breast tissues. The dominant expression pattern of hPEBP4 protein was also found in human prostate cancer tissue and ovarian cancer tissue, suggesting that hPEBP4 is likely to be a tumor-specific/tumor-associated antigen relatively specifically expressed in tumor tissue.

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, the inventors have previously identified the tumor antigen epitope peptide p40-48 (TLFCQGLEV) (SEQ ID NO: 1) from hPEBP4, the amino acid sequence of which is shown in Chinese patent application CN200510030532.0. The epitope peptide has strong immunogenicity in HLA-A2.1/K ^b transgenic mice and peripheral blood of HLA-A2.1 positive breast cancer patients, and the effector cells induced by it can specifically kill hPEBP4+/ HLA-A2.1+ tumor cells.

Cellular immunity plays a key role in antitumor and antiviral immunity. The antigens recognized by T cells are peptides bound to MHC-class I or class II molecules on the cell surface, which are about 8-12 amino acids in length. As the main effector cell CTL (cytotoxic T lymphocytes, Cytotoxic TLymphocytes) to kill tumor cells, it recognizes endogenous peptides combined with MHC-I molecules. However, many natural tumor antigen peptides that can be recognized by CTLs are relatively weak in immunogenicity. The strength of immunogenicity is related to the level of affinity between peptides and MHC-I molecules. To a certain extent, the higher the affinity, the stronger its immunogenicity.

In order to enhance the immunogenicity of the peptide, amino acids at certain positions in the peptide sequence can be substituted to improve and optimize its affinity with MHC-I molecules. After amino acid substitution, the antigen specificity of the epitope peptide (ie, the ability to specifically recognize the wild-type peptide) should not be changed, but its immunogenicity (ie, the ability to induce specific CTL killing in vivo) should be enhanced. Antigenic peptides with amino acid substitutions have been used in cancer patients to improve the patient's anti-tumor immune response.

However, small changes in the amino acids of the polypeptide will have a huge impact on its structure and function, and even cause the polypeptide to lose its original activity (such as binding affinity), and the modification and change of amino acids in the polypeptide sequence and combinations are numerous It is complicated, and it is not easy to screen a modified polypeptide with enhanced activity (such as immune activity) from numerous modifications.

Therefore, there is still an urgent need in the art to develop modified epitope polypeptides with enhanced immunogenicity based on the sequences of known epitope polypeptides.

Contents of the invention

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

In the first aspect of the present invention, an HLA-A2 restricted epitope polypeptide is provided, the polypeptide has the activity of inducing cytotoxic T cell killing, and the polypeptide has the sequence of FLFCQGLEV (SEQ ID NO: 2).

In one embodiment of the present invention, the sequence of the polypeptide is FLFCQGLEV (SEQ ID NO: 2).

In the second aspect of the present invention, a recombinant protein is provided, which contains the aforementioned HLA-A2 restricted epitope polypeptide.

In a preferred example, the sequence of the recombinant protein is shown in SEQ ID NO: 2.

The third aspect of the present invention relates to a sensitized antigen-presenting cell, wherein the antigen-presenting cell is sensitized by the aforementioned HLA-A2 restricted epitope polypeptide.

In a preferred example, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, B cells, fibroblasts or endothelial cells.

In the fourth aspect of the present invention, a method for preparing sensitized antigen-presenting cells is provided, the method comprising: sensitizing the antigen-presenting cells with the HLA-A2 restricted epitope polypeptide of the present invention.

In a preferred example, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, B cells, fibroblasts or endothelial cells.

In a fifth aspect of the present invention, a composition is provided, which contains:

(a) 0.001-99.99 wt% of the aforementioned HLA-A2 restricted epitope polypeptide or the aforementioned sensitized antigen-presenting cells; and

(b) Immunologically or pharmaceutically acceptable carriers, diluents or excipients.

In one embodiment of the present invention, the composition is a pharmaceutical composition, and the carrier, diluent or excipient is a pharmaceutically acceptable carrier, diluent or excipient.

In a preferred example, the content of the component (a) is 0.001-99.99wt%, preferably 0.1-99.0wt%, more preferably 1-90wt%.

In another preferred example, the composition also includes one or more other drugs for treating tumors, preferably the drugs are selected from: alkylating agents, anti-metabolites, anti-tumor antibiotics, plant-based anticancer drugs, hormones , or immune preparations, more preferably: antitumor antibiotics, such as doxorubicin.

In the sixth aspect of the present invention, a use of the HLA-A2 restricted epitope polypeptide or sensitized antigen-presenting cells of the present invention is provided for preparing a drug for preventing and/or treating tumors.

In one embodiment of the present invention, the tumor is selected from the group consisting of prostate cancer, breast cancer, liver cancer, glioma, colon cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, or bladder cancer cancer, or ovarian cancer.

In the seventh aspect of the present invention, the use of the HLA-A2 restricted epitope polypeptide 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 Prepare sensitized antigen-presenting cells.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

The present invention will be further elaborated below in conjunction with the accompanying drawings.

Figure 1: Dendritic cells (DCs) sensitized by 1F peptides induce the killing activity of cytotoxic T lymphocytes in HLA-A2.1/ ^Kb transgenic mice in vivo.

Figure 2: IFN-γ-secreting cells of cytotoxic T lymphocytes induced by 1F peptide-sensitized dendritic cells (DC) in vivo in HLA-A2.1/K ^b transgenic mice.

Detailed ways

Based on the analysis (such as computer simulation analysis, etc.) of the epitope peptide P40-48 derived from hPEBP4, the present inventors selectively synthesized many candidate antigenic peptides by replacing the amino acids of the epitope peptide P40-48. It is expected to screen out antigenic peptides that bind to HLA-A*0201 with high affinity and can induce stronger specific CTL responses in the body.

Specifically, the inventors initially obtained possible candidate sequences through in silico analysis. However, the number of candidate polypeptides obtained through computer simulation prediction ranges from hundreds to thousands. Moreover, the inventors have proved through experiments that the immunogenicity of candidate epitope peptides may not be effectively improved compared with wild-type by computer simulation analysis of candidate epitope peptides with substitutions at different positions in the known sequence, even including some of the same positions. Even on the contrary, it is significantly lower, which suggests the higher uncertainty of computer simulation predictions in complex biological systems.

Therefore, it is not easy to obtain a polypeptide sequence with high affinity, high immunogenicity, and the ability to induce a stronger hPEBP4-specific cellular immune response from such a large number of candidate polypeptides with high uncertainty in biological effects.

The inventor further screened the epitope peptide with strong affinity to HLA-A*0201 from many sequences through the T2 peptide binding experiment, and evaluated its immunogenicity, and finally found that the optimized epitope peptide 1F was The HLA-A0201-restricted hPEBP4 antigen-specific T cell epitope can induce cytotoxic T effector cells that are significantly stronger than wild peptides (P40-48) in vivo and in vitro.

As an amino acid-modified peptide with improved immunogenicity but unchanged antigen specificity, epitope peptide 1F has potential application value in the immunotherapy of breast cancer or other tumors with high expression of hPEBP4. At the same time, it also provides a new experimental basis for preparing a more effective tumor vaccine, which is of great significance for the development of its tumor vaccine and therapeutic preparations.

On this basis, the present inventors have completed the present invention.

HLA-A2 restricted epitope polypeptide

As used herein, "polypeptide of the present invention", "1F peptide", "replaced specific polypeptide" and the like can be used interchangeably, referring to the amino acid sequence P40-48 (SEQ ID NO : The first amino acid residue in 1) is substituted into the specific polypeptide obtained by "F", and the polypeptide sequence is shown in SEQID NO: 2, namely FLFCQGLEV. In addition, the term also includes a sequence formed by adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at its C-terminal and/or N-terminal. For example, in the art, the addition of one or several amino acids at the C-terminus and/or N-terminus usually does not alter the function of the protein.

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 the 1F peptide, and/or at Its downstream addition corresponds to 1 or 2 or 3 or 4 amino acids (such as F, FY, FYP or FYPE) at positions 49-52 in hPEBP4.

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.

combination

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.

Advantages of the invention

The main advantage of the present invention is that: the epitope peptide of the present invention is obtained by amino acid substitution on the basis of the known HLA-A2-restricted cytotoxic T lymphocyte epitope peptide, which is similar to the known epitope peptide This is not only of great significance for further in-depth study of tumor pathogenesis, but also for the development of more effective tumor therapeutic vaccines and therapeutic agents.

Example

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.

Percentages and parts are by weight unless otherwise indicated. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

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 medium (purchased from Invitrogen), and adjust the cell concentration to 2×10 ⁵ cells/ ml, spread in 24-well plate, 0.5ml/well. Then, 50 μM candidate polypeptide and 2.5 μg/ml β2 microglobulin (purchased from R&D Company) were added and co-incubated for 18 hours at 37° C. in a 5% CO ₂ incubator. After incubation, the cells were washed three times with ice-cold PBS (pH 7.2, PBS was used as a buffer, self-prepared), added FITC-labeled HLA-A2 specific mAb BB7.2 (Sterotec Ltd, Oxford, UK), and kept on ice for 45 After washing with PBS, the average fluorescence intensity was detected by a flow cytometer (FACSCalibur, Beckton Dickinson Company).

The positive known peptide CEA HLA-A2 restricted epitope polypeptide CAP-1 (SEQ ID NO: 11, the sequence is YLSGANLNL) was used as a positive control, and the simple T2 cells stimulated without peptide were used as a background control.

Detection method

The combination of peptides and HLA-A*0201 molecules is detected by immunofluorescence. This method 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 of the two, the more the expression of MHC-I molecules can be detected, and the average fluorescence intensity is used as the detection index. The results were measured by fluorescence coefficient (FI).

Among them, a polypeptide with FI>1 is considered as 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 peptides shown in the table were simulated and analyzed by computer software (via the National Institutes of Health Bioinformatics and Molecular Analysis Division (BIMAS) HLA peptide binding prediction website http://bimas.dcrt.nih.gov/molbio/hla_bind/index. html) part of the candidate sequences were artificially synthesized by Shanghai Gil Biochemical Co., Ltd.

The inventors screened out HLA-A2 high-affinity epitope 1F from several optimized peptides based on the substitution of epitope p40-48, and its sequence is FLFCQGLEV (SEQ ID NO: 2). The affinity between the epitope peptide and the HLA-A*0201 molecule is significantly higher than that of the wild-type epitope peptide p40-48 (SEQ ID NO: 1).

Table 1 T2-HLA-A*0201 Binding Affinity of hPEBP4 Protein-derived Polypeptides

Example 2. Induction of hPEBP4-derived HLA-A2-restricted polypeptide-specific cytotoxic T lymphocytes in HLA-A2.1/K ^b transgenic mice

Preparation of effector cells

Dendritic cells derived from bone marrow of HLA-A2.1/K ^b transgenic mice were prepared according to conventional methods (referring to Inaba laboratory method, [Inaba, K. et al., J Exp Med. 1992; 176: 1693-1702]) ( DC). Collect the DCs cultured (referring to the Inaba laboratory method, as above) to the seventh day, adjust the cell concentration to 1×10 ⁶ cells/ml, add epitope peptides (each epitope peptide in Table 1 in Example 1, the final concentration 10 μM/ml) and β2 microglobulin (final concentration 3 μg/ml), incubated at 37° C., 5% CO ₂ incubator for 3 h.

Collect the peptide-sensitized DCs, wash them three times with PBS (pH7.2, PBS is the buffer solution, self-prepared), adjust the cell concentration to 1×10 ⁶ cells/0.2ml (0.2ml for immunization) and immunize the transgenic mice (HLA-A2.1/K ^b transgenic mice, purchased from JaxMice, USA, 4-6 weeks old, female, 5 mice in each group). Each mouse was intraperitoneally injected with 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 cells/ml) and peptide-sensitized autologous (namely the same mouse) dendritic cells irradiated with ⁶⁰ Co γ-rays (total dose of 30Gy) were mixed at a ratio of 10:1 (quantity than) cultured in RPMI 1640 complete medium. After 6 days in culture, effector cells were harvested.

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 P40-48WT epitope peptide, T2 cells loaded with irrelevant peptide SSp-1 (irrelevant control, SEQ ID NO: 12) and T2 cells without any polypeptide load were used as target cells (i.e. as an example target cells of the effector cells prepared in 2), ⁵¹ Cr (Na ₂ ⁵¹ CrO ₄ , 100 μCi/10 ⁶ cells) was added, placed in a 37° C. water bath for labeling for 90 minutes, and mixed gently every 15 minutes. Wash 3 times with PBS to thoroughly remove residual Na ₂ ⁵¹ CrO ₄ , adjust the cell concentration of the labeled target cells to 1×10 ⁵ cells/ml with complete 1640 medium, add 100 μl per well to a 96-well round bottom plate. Add effector cells according to three different effect-target ratios of 50:1, 25:1 and 12.5:1 (quantity ratio), incubate at 37°C for 4 hours, collect 100 μl of supernatant from each well, and detect the cpm value with a gamma counter (Wallac Company 1470). (counts per minute counts per minute).

Calculate the specific lysis rate or killing rate according to the following formula:

Among them, each well of the maximum release group is a single target cell plus 100 μl 1% SDS; the spontaneous release well is a single target cell plus 100 μl complete medium.

(2) Detection of peptide-specific CTL IFN-γ release

ELISPOT detection was performed with a commercial kit (purchased from R&D Company). Add 1×10 ⁵ effector cells to a 96-well flat-bottomed nitrocellulose plate coated with specific anti-mouse IFN-γ monoclonal antibody, and at the same time add P40-48 wild-type epitope peptide-stimulated T2 cells (1× 10 ⁴ ), T2 cells stimulated by irrelevant peptide SSp-1 or unstimulated T2 cells were cultured in a 5% carbon dioxide incubator at 37°C for 24 hours. Remove the cells, wash the plate 4 times and dry it, add biotinylated anti-mouse IFN-γ specific antibody, incubate overnight at 4°C; wash the plate 4 times and dry it, then add alkaline phosphatase-labeled streptavidin , act at room temperature for 2 hours; wash the plate 4 times and shake dry, add the substrate 5-bromo-4-chloro-3-indole phosphoric acid/nitroblue tetrazolium (BCIP/NBT), act in the dark at room temperature for 1 hour, discard the color development solution, washed with double distilled water, and terminated the reaction. The T cell colonies secreting IFN-γ were detected according to the method in the kit instruction manual.

Three replicate wells were set for each of the above tests, and the average value of the three replicate wells was used for counting. The value shows the number of detected IFN-γ-specific CTLs.

test results

The results showed that after immunizing mice with dendritic cells sensitized with optimized epitope peptide 1F, the CTL produced could effectively kill T2 cells loaded with wild-type epitope peptide, and the effect of killing target cells with optimized epitope peptide 1F was significantly higher than the wild-type epitope peptide (see Figure 1, *, P<0.01).

The number of effector cells (CTL) with significant IFN-γ release detected after stimulation with the optimized epitope peptide 1F was also significantly higher than that of the wild-type epitope peptide (see Figure 2, *, P<0.01).

The above results show that the ability of the optimized epitope peptide 1F to induce CTLs that specifically recognize the wild-type epitope peptide in vivo (ie, immunogenicity) is significantly higher than that of the wild-type epitope peptide.

In this test, effector cells are used to kill target cells. The target cells are T2 cells loaded with wild-type epitope peptides. The effector cells are the dendritic cells derived from transgenic mice sensitized by the polypeptide prepared in Example 1 and stimulated in vivo. Sexual T cells (present in spleen cells).

Dendritic cells sensitized with the optimized epitope peptide 1F stimulated T cells can specifically kill T2 cells loaded with the wild-type epitope peptide, which means that the optimized epitope peptide 1F can induce more effectively than the wild-type epitope peptide hPEBP4 wild-type epitope peptide-specific killer T cells, thus proving that the optimized epitope peptide 1F has higher immunogenicity while maintaining antigen specificity, and can induce stronger hPEBP4-specific cells immune response.

In addition, the above results also show: the combination of different positions and amino acid substitutions (such as SEQ ID NOs: 3-10), even including part of the same position substitution (such as the polypeptide of SEQ ID NO: 7) or the same amino acid substitution (such as SEQ ID NOs: 7) : 3 or 4), the immunogenicity of other optimized epitope peptides has not been effectively improved compared with the wild type, and even has been significantly reduced, which suggests that in complex biological systems, computer simulation predictions have a higher Uncertainty.

Moreover, the number of candidate polypeptides that can be obtained through computer simulation prediction ranges from hundreds to thousands. It is necessary to screen such a large number of candidate polypeptides with high uncertainty in biological effects to obtain high-affinity, high-immunity The identification of original, polypeptide sequences capable of inducing a stronger hPEBP4-specific cellular immune response was not straightforward.

Example 3. Optimizing the cultivation of epitope peptide 1F-sensitized human peripheral blood mononuclear cell-derived DC

Anticoagulated whole blood from HLA-A2.1+/hPEBP4+ breast cancer patients was passed through lymphocyte separation medium (Ficoll-Histopaque 1.077, purchased from Sigma) density gradient centrifugation (room temperature, 400 × g, 30 minutes), and the interfacial cells were collected. Place in a 50ml centrifuge tube, suspend the cells with calcium-magnesium-free PBS (pH7.2)-EDTA (2mM), then centrifuge (300×g, 10 minutes) to wash the cells once, discard the supernatant, and resuspend the cells in calcium-magnesium-free PBS , and centrifuged again (200×g, 5 minutes) to wash the cells twice to obtain human peripheral blood mononuclear cells (PBMC).

Suspend PBMCs with complete medium (RPMI1640 containing 10% fetal bovine serum), spread 1×10 ⁷ cells/well on a 6-well plate, incubate at 37°C and 5% CO ₂ for 2 hours, gently suck out the suspended cells, and use Carefully wash the 6-well plate three times with the pre-warmed medium (this part of the cells, together with the aspirated suspension cells, are frozen for future use), and the remaining cells are the adherent monocytes. Adherent cells were cultured at 37° C., 5% CO ₂ in complete medium containing human recombinant rhGM-CSF (500 U/ml) and human recombinant rhIL-4 (10 ng/ml) (purchased from R&D Company). On the third day, the complete medium was supplemented, and the culture was continued.

Collect DCs derived from peripheral blood mononuclear cells from hPEBP4+/HLA-A*0201+ breast cancer patients cultured to day 6 above, and use human DC complete medium (RPMI 1640 complete medium, 500 U/ml rhGM-CSF, 10 ng/ml rhIL -4) Adjust the cell concentration to 2×10 ⁵ cells/ml, divide 1ml/well into a 24-well plate, add 20 μM optimized epitope peptide 1F, collect the cells after 4 hours, discard the medium supernatant, and use RPMI1640 medium Centrifuge and wash the cells twice to remove the stimulants present in the original medium, and finally suspend 2× ¹⁰⁵ cells in 0.5ml of human DC complete medium, which is human peripheral blood mononuclear cells sensitized with the optimized epitope peptide 1F Cell-derived DC.

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. An HLA-A2 restricted epitope polypeptide, characterized in that the polypeptide has the activity of inducing cytotoxic T cell killing, and the sequence of the polypeptide is FLFCQGLEV as shown in SEQ ID NO: 2. 2. A recombinant protein, characterized in that it comprises the HLA-A2 restricted epitope polypeptide according to claim 1. 3. A sensitized antigen-presenting cell, characterized in that the antigen-presenting cell is sensitized by the HLA-A2 restricted epitope polypeptide according to claim 1. 4. The antigen-presenting cell according to claim 3, wherein the antigen-presenting cell is selected from the group consisting of dendritic cells, macrophages, B cells, fibroblasts or endothelial cells. 5. A method for preparing sensitized antigen-presenting cells, the method comprising: sensitizing antigen-presenting cells with the HLA-A2 restricted epitope polypeptide according to claim 1. 6. The method of claim 5, wherein the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages, B cells, fibroblasts or endothelial cells. 7. A composition characterized in that it contains: (a) 0.001-99.99 wt% of the HLA-A2 restricted epitope polypeptide of claim 1 or the sensitized antigen-presenting cell of claim 3; and (b) acceptable carrier, diluent or excipient. 8. The composition of claim 7, wherein the composition is a pharmaceutical composition, and the carrier, diluent or excipient is a pharmaceutically acceptable carrier, diluent or excipient Forming agent. 9. The use of the HLA-A2 restricted epitope polypeptide according to claim 1 or the sensitized antigen-presenting cell according to claim 3, characterized in that it is used for the preparation of medicaments for preventing and/or treating tumors . 10. The use according to claim 9, wherein the tumor is selected from the group consisting of prostate cancer, breast cancer, liver cancer, glioma, colon cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer cancer, stomach cancer, or bladder cancer.

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