CN105753978B

CN105753978B - Epitope peptide derived from HLA-A11 restricted carcinoembryonic antigen and application thereof

Info

Publication number: CN105753978B
Application number: CN201610143157.9A
Authority: CN
Inventors: 刘书逊; 曹雪涛; 江金霞
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2016-03-14
Filing date: 2016-03-14
Publication date: 2020-02-07
Anticipated expiration: 2036-03-14
Also published as: CN105753978A

Abstract

The invention relates to the field of biomedicine, in particular to a carcinoembryonic antigen (CEA) -derived HLA-A11-restricted CTL epitope polypeptide, a gene for encoding the epitope polypeptide, a recombinant protein or a compound containing the epitope polypeptide, a sensitized antigen presenting cell and application of a specific immune effector cell aiming at the epitope polypeptide in preparing a medicine for treating CEA positive tumor. The polypeptide is a CEA-derived HLA-A11-restricted CTL epitope peptide, can safely and effectively induce immune response aiming at CEA, researches the pathogenesis of CEA-positive tumors, and has important significance for the development of known therapeutic preparations of the vaccine.

Description

Epitope peptide derived from HLA-A11 restricted carcinoembryonic antigen and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a carcinoembryonic antigen (CEA) -derived HLA-A11-restricted CTL epitope polypeptide, a gene for encoding the epitope polypeptide, a recombinant protein or a compound containing the epitope polypeptide, a sensitized antigen presenting cell and application of a specific immune effector cell aiming at the epitope polypeptide in CEA positive tumor treatment.

Background

Carcinoembryonic antigen (CEA: CEACAM-5 or CD66e) is a glycoprotein having a molecular weight of about 180 kDa. Structurally, CEA belongs to the immunoglobulin superfamily and is expressed on the cell surface via a Glycosylphosphatidylinositol (GPI) anchor. CEA has been found to be a tumor-associated antigen very early, and is expressed abundantly in large bowel cancer and present only in trace amounts in normal tissues. Many other tumors of tissue origin, such as gastric cancer, respiratory, genitourinary and breast tumors, are subsequently found to have elevated CEA. Serum CEA levels have been widely used as clinical markers for diagnosing and screening cancers, and for determining whether tumors have metastasized and recurred.

CEA is an ideal target antigen as a tumor-associated antigen. However, because of the sequence homology and structural similarity of CEA to other members of its family, and the presence of immunosuppressive factors in tumor patients, it is difficult to generate an immune response against CEA spontaneously in patients with CEA-positive tumors without intervention. Therefore, CEA-targeted tumor immunotherapy regimens need to consider two factors: one approach is to find precise CEA-specific epitopes, including T-cell epitopes and B-cell epitopes, for inducing antigen-specific Cytotoxic T Lymphocytes (CTLs) or antibodies; secondly, the technical scheme comprises a full-length or partial CEA antigen; in a second aspect, if there is a defined epitope, the treatment of CEA positive tumors can be achieved by inducing T, B cell clones specific for the epitope in vitro and then returning CTLs or antibodies, if there is no defined epitope, which would require the addition of adjuvants in vaccine design to overcome immunosuppression in vivo.

The central link in tumor immunity is the cellular immune response. In the cellular immune response, two major classes of immune effector cells with cytotoxic effects are: NK of the innate immune system, and CTL of the adaptive immune system. CD8⁺CTL recognition of target cells has MHC-I restriction and antigen specificity. This is due to the fact that antigen recognition receptors on the surface of CTL cells-TCR-need to bind to the complex formed by MHC-I molecules on the surface of target cells and antigenic peptides. TCRs need to recognize both MHC-I molecules and antigenic peptides bound in their grooves. The binding reaches a certain affinity, and can trigger the CD3 molecule connected with the TCR to transmit signals to the interior of the T cell, trigger the activation and proliferation of the T cell and achieve the amplification of clones.

MHC-I restricted antigenic peptides (also called epitope peptides) are composed of up to 9 amino acids and are bound in the groove of MHC-I molecules. Different MHC-I molecules bind different epitope peptide sequences. Empty groove MHC molecules that do not bind epitope peptides have a short half-life on the cell surface and are therefore expressed at low levels. One mechanism of immune escape for many tumors is that low levels of expression or no expression of MHC molecules, and therefore, do not activate tumor antigen-specific CTLs in vivo, thereby achieving the goal of evading CTL recognition and killing by the body. Therefore, under the condition of knowing the tumor antigen epitope peptide, on one hand, the epitope peptide is used for sensitizing antigen presenting cells, and then tumor antigen specific CTL is induced in vitro and is returned to the body; on the other hand, the epitope peptide is directly delivered to tumor tissues in vivo, so that the epitope peptide can be loaded on MHC-I molecules corresponding to the surface of tumor cells for recognition of the epitope peptide specificity CTL, thereby achieving the purpose of killing the tumor cells.

Human MHC molecules (HLA) are currently recognized as the most highly polymorphic molecules. Because the epitope peptide recognized by the human CTL has HLA-I molecule restriction, the search of the CTL epitope peptide with certain HLA-I molecule restriction in the tumor antigen has guiding significance for the in vitro induction of antigen-specific CTL. The HLA-I restricted epitope peptide determined in the CEA at present is the HLA-A2 restricted CEA_605-613(CAP1)9 peptide, the other being HLA-A24 restricted CEA_652-660. However, no HLA-A11-restricted epitope for CEA origin has been reported so far. As is known, HLA-A2 is a common HLA-I molecule in Chinese population, and the data published by the http:// www.allelefrequencies.net website show that the positive frequency of the population in different areas of China is 19.3% (Yunnan) -31.3% (Shanghai); HLA-A2402 is lower than HLA-A2, and the positive frequency in people in different areas of China is 10.5% (Beijing Shijiazhuang Han nationality) -17.4% (Jiangsu Han nationality). In fact, HLA-A11 is a HLA-I molecule which is relatively common in Chinese people, and the distribution frequency in people in different areas of China is 15.9% (Qinghai) to 61.3% (Yunnan). Theoretically, the CTL therapeutic effect of a single epitope peptide is lower than that of the combination therapy of a plurality of epitope peptide-specific CTL clones. Therefore, the search for the CEA-derived HLA-A11-restricted epitope has guiding significance for inducing HLA-A11-restricted CEA-specific CTL in the future, and the combined HLA-A2-and HLA-A24-restricted CEA-specific CTL has undoubtedly a very broad prospect for treating CEA-positive tumors.

Disclosure of Invention

The invention aims to provide an HLA-A11 restrictive CEA-derived epitope polypeptide, and the purpose of treating CEA positive tumors is achieved.

The present inventors have conducted extensive and intensive studies and, as a result, have conducted on-line biological software analysis (SYFPEITHI, BIMAS and NetMHC) using HLA-A1101 for the primary sequence of CEA antigen, and have selectively synthesized 5 antigen peptides which are likely to bind to HLA-A11 and induce the body to produce CTLs. Through a binding experiment of the polypeptide and LoVo cells, one polypeptide is screened out to be the most probable HLA-A11 restrictive polypeptide, and the amino acid sequence is shown as SEQ ID NO. 1.

Further, dendritic cells derived from HLA-A11 are genetically modified by the polypeptide of SEQ ID NO. 1 itself or a recombinant expression vector carrying a gene sequence encoding the polypeptide of SEQ ID NO. 1; then repeatedly stimulating self T cells with antigen peptide sensitized or gene modified DC to generate CD8⁺The cell is subjected to a polypeptide activation experiment described by SEQ ID NO. 1, the activity of killing LoVo cells is detected in the presence of the polypeptide described by SEQ ID NO. 1, and whether the epitope polypeptide pentamer described by the artificially synthesized HLA-A11/SEQ ID NO. 1 is combined or not is detected. On the basis of obtaining a positive experiment result, the invention is formed.

In the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition of one or several amino acids at the C-or N-terminus does not generally alter the function of the protein. Therefore, the polypeptide of the present invention also includes a variant having 1 to 4 amino acids in the sequence, which has the same function as that of the present invention, and a deletion, insertion or substitution of one or more amino acids in number, and an addition of one or more amino acids to the C-or N-terminal (usually, 20 or less, preferably 10 or less, and more preferably 5 or less).

The polypeptide of the invention can be synthesized by conventional methods, can also be produced by recombinant methods, and can also be used for coupling with other proteins or immunological adjuvants so as to form a polypeptide complex.

In order to achieve the above objects, according to a first aspect of the present invention, there is provided an HLA-a 11-restricted CEA-derived epitope polypeptide capable of inducing CTLs having cytotoxic activity recognizing the polypeptide, the polypeptide being selected from the group consisting of:

(a) polypeptide having an amino acid sequence shown as sequence SEQ ID NO 1: or

(b) 1, and has HLA-A11-restricted epitope polypeptide which is derived from (a) and can induce CTL.

Preferably, the epitope polypeptide is 8-20 amino acids in length, preferably 8-12 amino acids in length.

Most preferably, the epitope polypeptide has an amino acid sequence as shown in sequence SEQ ID NO. 1.

In a second aspect of the invention, there is provided a recombinant protein or complex comprising the HLA-A11-restricted epitope polypeptide of the invention.

The recombinant protein or the compound can be added with immune adjuvant molecules (such as heat shock protein molecules) and cytokines and other molecules on the basis of the epitope polypeptide to achieve the effect of enhancing the specific immune response against the epitope polypeptide.

In a third aspect of the present invention, there is provided a gene encoding an amino acid sequence shown by SEQ ID NO. 1, the gene having a nucleotide sequence shown by SEQ ID NO. 7(cac aca caa gtt ctc ttt atc gcc aaa).

In a fourth aspect of the present invention, there is provided a recombinant vector comprising a gene encoding the above HLA-A11-restricted epitope polypeptide of the present invention, wherein the recombinant vector comprises a nucleotide sequence represented by SEQ ID NO. 7(cac aca caa gtt ctc ttt atc gccaaa).

Preferably, the recombinant vector is selected from the group consisting of: a recombinant adenoviral vector, a recombinant adeno-associated viral vector, or a lentiviral vector.

In a fifth aspect of the present invention, there is provided an antigen presenting cell, wherein the antigen presenting cell is: the HLA-A11 restrictive epitope polypeptide, or the fusion protein containing the epitope polypeptide or the antigen presenting cell after composite sensitization; or, an antigen-presenting cell genetically modified with a gene or recombinant expression vector comprising a polypeptide encoding an HLA-A11-restricted epitope of the present invention.

Preferably, the antigen presenting cell is selected from the group consisting of: dendritic cells, monocytes, macrophages, monocytes, B cells, endothelial cells, fibroblasts, tumor cells.

In a sixth aspect of the present invention, there is provided a CTL specifically recognizing the epitope polypeptide of the present invention, which is CD8 obtained by repeatedly stimulating, inducing and activating the antigen-presenting cells of the present invention to thereby clonally proliferate the antigen-presenting cells⁺CTL。

In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) a safe and effective amount of CTL induced by the HLA-A11-restricted epitope polypeptide, fusion protein or complex of the present invention, the gene encoding the epitope polypeptide, recombinant vector, the sensitized antigen-presenting cell or the antigen-presenting cell sensitized with the HLA-A11-restricted epitope polypeptide; and

(b) a pharmaceutically acceptable carrier, excipient or diluent.

In an eighth aspect of the present invention, there is provided a use of the HLA-A11-restricted epitope polypeptide, fusion protein or complex of the present invention, or the gene or recombinant vector encoding the epitope polypeptide, or the sensitized antigen-presenting cell, or CTL induced by the HLA-A11-restricted epitope polypeptide-sensitized antigen-presenting cell of the present invention: is used for preparing the medicine for treating CEA positive tumor.

The CEA positive tumor can be gastric cancer, lung cancer, pancreatic cancer, genitourinary system tumor (such as ovarian cancer), breast cancer, medullary thyroid cancer, etc.

The invention has the advantages that: the epitope polypeptide is a CEA-derived HLA-A11-restricted CTL epitope peptide, can safely and effectively induce immune response aiming at CEA, and has important significance for researching pathogenesis of CEA-positive tumors and development of known treatment of vaccines thereof.

Drawings

FIG. 1 level of LoVo expressing HLA-A11 in the presence of different CEA nonapeptides: the epitope polypeptide specifically up-regulates the level of HLA-A11+ LoVo expressing HLA-A11.

FIG. 2 modification of epitope polypeptide of the present inventionLevels of IFN- γ secretion by DC-induced specific CTL. A: HLA-A11-derived AdCH-DC or AdLacZ-DC-induced autologous CD8⁺ELISPOT detection of T secretion of IFN γ in the presence of different peptides; b: HLA-A11 derived different nonapeptide-sensitized DC induced autologous CTLs were tested by ELISPOT for secretion of IFN γ in the presence of different peptides.

FIG. 3 HLA-A11-derived AdCH-DC or AdLacZ-DC, CAP1 or CEA_636-644Peptide-sensitized DC-induced binding of autologous CD8+ T cells to HLA-A11/CEA_636-644Detection of the pentamer: the specific CTL sensitized by the epitope polypeptide or DC modified by the encoded polypeptide gene recognizes HLA-A11/the epitope polypeptide pentamer.

FIG. 4 shows that specific CTL induced by DC sensitized with the epitope polypeptide of the present invention or modified with the gene encoding the polypeptide kills HLA-A11-restricted CEA⁺A tumor. A: killing of LoVo by HLA-A11-derived AdCH-DC or AdLacZ-DC-induced self CTL in the presence of different peptides; b: HLA-A11 derived killing of LoVo by autologous CTLs induced by different nonapeptide-sensitized DCs in the presence of different peptides.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example 1 screening of HLA-A11 high affinity Polypeptides

First, on-line biological software analysis (SYFPEITHI, BIMAS and NetMHC) was performed on the primary sequence of CEA antigen using HLA-A1101, and 5 antigen peptides which could bind to HLA-A11 and induce the body to produce CTL were selectively synthesized. The sequences are shown in Table 1. We have screened these polypeptides using LoVo cells because:

the LoVo cell is a colon tumor cell derived from HLA-A11 and highly expressing CEA. However, the levels of MHC-I and HLA-A11 expression by LoVo were very low as measured by flow cytometry. It is well known in the field of immunology that all nucleated cells in the body express MHC-I molecules and can be counted as antigen presenting cells in a broad sense. Tumor cells express low MHC-I, which on the one hand does not efficiently induce the body to produce CTLs, and on the other hand, evades recognition by CTLs and is thus cleared. One mechanism for the low expression of MHC-I molecules by tumor cells is the loss of TAP1/2 function. TAP1/2 is responsible for the transport of antigenic peptides to the MHC-I antigen binding groove, and when its function is lost, the MHC-I molecule has a very short half-life on the cell surface because it cannot load antigenic peptides. Therefore, we speculate that LoVo may underexpress MHC-I due to the above mechanism.

Therefore, we combined the candidate polypeptide (50. mu.g/ml) with β 2m (2.5. mu.g/ml) and LoVo cells (3X 10)⁵Per ml) at 37 ℃ 5% CO₂Culturing in an incubator overnight; then, each group of cells was labeled with HLA-A11, and the CEA described in SEQ ID NO:1 was found by flow cytometry_636-644The polypeptide can obviously up-regulate the level of HLA-A11 expressed by LoVo, but other polypeptides do not have the effect, which indicates that CEA_636-644Possibly an HLA-A11 restricted epitope (see FIG. 1).

TABLE 1 candidate HLA-A11 restricted CEA derived epitope polypeptide (9 peptide)

The initial position of the CEA amino acid sequence	Amino acid sequence	SEQ ID NO
			CEA_118-126	GFYTLHVIK	2
CEA_241-249	TISPLNTSY	3
			CEA_420-428	ISPSYTYYR	4
CEA_617-625	SASNPSPQY	5
			CEA_636-644	HTQVLFIAK	1

And (4) conclusion: the polypeptide described in SEQ ID NO. 1 may be HLA-A11 restricted epitope polypeptide located in the 636-644 segment of CEA antigen. Is named as: CEA_636-644。

Examples 2-4 Induction of HLA-A11-restricted CEA_636-644Generation of specific CTL

1. Preparation of human monocytic dendritic cells (MoDC)

Separation of HLA-A11 positive healthy human-derived peripheral blood CD14 by immunomagnetic bead method⁺The monocytes are induced by human recombinant GM-CSF (50ng/ml) and IL-4(10ng/ml) for 5 days to obtain immature MoDC.

Sensitization of DCs

The method comprises two methods:

2.1 will carry CEA-containing vector_636-644CEA of peptide fragment_576-669Recombinant adenovirus (AdCEA) of fusion gene (SEQ ID NO:6) of segment and heat shock protein 70-like molecule 1(HSP70L1)_576-669HSP70L1, called AdCH for short) vector infects human immature MoDC, and after 48h, the human immature MoDC infected by AdCH (AdCH-DC) is collected, namely the DC sensitized in cells. Controls used recombinant adenovirus-infected MoDC carrying the LacZ gene (AdLacZ-DC).

2.2 direct application of CEA_636-644The peptide fragment (50 mu g/ml) is cultured with human immature MoDC overnight, thus obtaining extracellular CEA_636-644Peptide-sensitized DC (CEA)_636-644-DC). EBV restricted with known HLA-A11_416-424Peptides and HLA-A2-restricted CAP1 sensitized MoDC were controls.

2.3 the two methods sensitized DC are divided into 3 parts, and another part of the non-sensitized DC is reserved for standby. 1 part was co-cultured with autologous T cells, and 2 parts and non-sensitized DCs were frozen in a frozen stock solution containing 5% DMSO and 95% FBS at-80 ℃.

3. Sensitized DC and self CD3⁺Mixed culture of T lymphocytes

The above-mentioned DC sensitized by different methods and the control DC were each collected in one portion and then mixed with its own CD3⁺The T lymphocytes are mixed and cultured according to the proportion of 1:20, after 7 days of culture, one frozen sensitized DC and a control DC are recovered, the sensitized DC and the control DC are added into a mixed culture system according to the same proportion, after 3 days of culture, human IL-2(500U/ml) + IL-7(50ng/ml) is added every two days, and the culture is continued. The 3 rd cycle of stimulation was performed as described above. The culture was carried out for 21 days in total.

4. After 21 days, CD8 in each culture system was separated by immunomagnetic bead method⁺T cells on HLA-A11-restricted CEA_636-644Detection of specific CTLs:

4.1 IFN-. gamma./ELISPOT assay

Autologous DCs without any antigen sensitization were revived, following a 5: 1 ratio to CD8 in the above system⁺T cells were cultured in each well of a 96-well plate coated with anti-human IFN-. gamma.s. Adding human CEA into the system_636-644Polypeptide or known HLA-A11-restricted EBV_416-424Peptides (10. mu.g/ml), incubated for 24 hours and then terminated, and IFN-. gamma.secretion spots were detected according to the ELISPOT protocol.

As shown in FIG. 2A, the AdCH-DC group induced CD8 compared to AdLacZ-DC⁺T cells in CEA_636-644In the presence of the IFN-gamma spot, the number and the intensity of the IFN-gamma spot are obviously increased; however, CD8 induced by AdCH-DC group⁺T cells in EBV_416-424In the presence of the peptide, no significant spots were produced.

As shown in FIG. 2B, CEA_636-644Peptides or EBV_416-424Peptide-sensitized DC-induced CD8⁺T cells, in the presence of the corresponding peptide only, produce significant IFN-. gamma.spots, whereas in the presence of the other peptides, they do not.

And (4) conclusion: CEA_636-644Peptide-sensitized DC or DC containing gene modification encoding the peptide capable of inducing CEA_636-644Specific CD8⁺T cell production.

4.2 flow cytometry detection of HLA/CEA peptide pentamers

Synthetic PE-coupled HLA-A11/CEA_636-644A pentamer. The pentamer and anti-human CD8 antibodies were labeled with CD8 in the above culture system⁺T cells, and then flow cytometry detection.

The results are shown in FIG. 3, which shows that HLA-A11-derived AdCH-DC induces CD8 compared to HLA-A11-derived AdLacZ-DC group⁺T cells obviously contain recognition HLA-A11/CEA_636-644The population of (1); CEA in comparison to CAP 1-sensitized HLA-A11-derived DCs_636-644Peptide-sensitized HLA-A11-derived DC-induced CD8⁺T cells apparently containing recognition HLA-A11/CEA_636-644The population of (1).

And (4) conclusion: CEA_636-644The DC sensitized by the peptide or the DC modified by the gene coding the peptide can induce and recognize HLA-A11/CEA_636-644CD8 of Complex⁺T cell production.

4.3 cytotoxic Effect on LoVo cells

LoVo cells were pre-CFSE labeled and separately combined with CEA_636-644Peptide, CAP1, EBV_416-424Peptides were incubated at 37 ℃ and 5% CO₂Incubating in an incubator overnight; then the cells were incubated with CD8 in the above culture system⁺Cells were cultured according to 1:20 for 12 hours; and (4) performing flow cytometry detection, adding PI before loading, and detecting the death of the CFSE positive LoVo cell.

The results are shown in FIG. 4A, which shows that AdCH-DC induced CD8 compared to the AdLacZ group⁺CTL cell pair only passing through CEA_636-644The peptide-pretreated LoVo cells have obvious killing effect, and the peptide-pretreated LoVo cells, CAP1 or EBV cells have obvious killing effect_416-424The treated LoVo cells did not significantly kill. Possible mechanisms for the above results are: AdCH-DC cells express the fusion protein CH, which contains CEA_636-644Fragment, therefore it is possible to use HLA-A11/CEA_636-644The complex form is expressed on the DC surface, and then the self T cell is stimulated to generate CEA_636-644Specific CTL; in the absence of peptide load on LoVo cells, empty MHC-I cells express very low levels of HLA-A11 due to instability of MHC-I molecules and are therefore not killed by CTL recognition; the CAP1 antigen peptide is HLA-A2 restricted and cannot be HLA-A11⁺The presentation of DC can not induce CTL specific to CAP1, and in addition, CAP1 is not combined with MHC-I on the surface of LoVo cells, so that the LoVo cells are not recognized and killed; EBV_416-424Although it could be loaded into HLA-A11 on the surface of LoVo cells, the CTL induced by AdCH-DC did not recognize the CTL specific to the antigen peptide and thus was not killed; therefore, only load CEA_636-644LoVo cells of (1), surface-expressed high levels of HLA-A11/CEA_636-644Complexes, can be recognized and killed.

As shown in FIG. 4B, CEA_636-644Peptide-sensitized DC-induced CD8⁺T cells, only to CEA-passed cells_636-644The peptide-pretreated LoVo cells have obvious killing effect, and the peptide-pretreated LoVo cells, CAP1 or EBV cells have obvious killing effect_416-424No significant killing of the treated LoVo cells was evident, suggesting CEA_636-644Peptide-sensitized DCs were likely to present HLA-A11/CEA_636-644Complex, induced CEA_636-644Generation of specific CTL by recognizing HLA-A11/CEA on LoVo cell surface_636-644The complex kills LoVo cells; the CAP1 antigen peptide is restricted by HLA-A2, so that the DC cannot be used for sensitizing HLA-A11 sources, and the generation of CAP1 specific CTL cannot be induced, so that the LoVo cells treated by different peptides shown in the figure are not killed; EBV_416-424Is HLA-A11 restricted, EBV_416-424Peptide-sensitized DC-induced CD8⁺T cells, EBV only_416-424Peptide-treated LoVo cells had significant killing, but not other peptide-pretreated LoVo cells.

And (4) conclusion: CEA_636-644Peptide-sensitized DC or DC-induced CD8 containing gene modification encoding the peptide⁺CTL cell pair only to CEA_642-660Peptide-loaded HLA-A11⁺LoVo cells have an obvious killing effect.

5. To sum up, the conclusion is that: CEA_636-644Is a CTL epitope restricted by HLA-A11.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Claims

1. A compound containing HLA-A11 restrictive epitope polypeptide shown as sequence SEQ ID NO. 1 is HLA-A11/CEA_636-644And (c) a complex.

2. An antigen presenting cell, wherein said antigen presenting cell is: antigen presenting cells sensitized by HLA-A11 restricted epitope polypeptide as shown in SEQ ID NO. 1 or the complex of claim 1; or antigen presenting cells modified by genes encoding HLA-A11 restrictive epitope polypeptides shown as sequence SEQ ID NO. 1 or recombinant vectors containing the genes; the cell is a dendritic cell, a monocyte macrophage, a B cell, an endothelial cell, a fibroblast or a tumor cell expressing human HLA-A11; the tumor cell is colon cancer Lovo cell.

3. A cytotoxic T lymphocyte specifically recognizing HLA-A11-restricted epitope polypeptide as shown in SEQ ID NO. 1, wherein the cytotoxic T lymphocyte is activated by repeated stimulation of the antigen-presenting cell of claim 2 to obtain clone-proliferated CD8⁺CTL。

4. A pharmaceutical composition, comprising:

(a) the complex of claim 1, or the antigen-presenting cell of claim 2, or the cytotoxic T lymphocyte of claim 3; and

(b) a pharmaceutically acceptable carrier, excipient or diluent.

5. Use of the complex of claim 1 for the preparation of a medicament for the treatment of a CEA positive tumor, said use comprising:

the complex is used as a tumor vaccine;

the application of the compound as a tool for detecting CTL (cytotoxic T lymphocyte) for identifying the compound in preparation of medicines for treating CEA (CEA-positive tumor) positive tumors.

6. Use of the antigen presenting cell of claim 2 in the preparation of a medicament for the treatment of a CEA positive tumor, the use comprising:

the antigen presenting cell is used as a tumor vaccine;

inducing CTLs that recognize the complex of claim 1 by the antigen-presenting cell;

the antigen-presenting cells were used as target cells for detecting the killing activity of CTLs recognizing HLA-A11-restricted CEA specificity.

7. Use of the cytotoxic T lymphocyte of claim 3 in the preparation of a medicament for the treatment of a CEA positive tumor, said use comprising: the cytotoxic T lymphocyte acts as an effector cell; the cytotoxic T lymphocytes are used as a test tool for screening tumor vaccines which target and recognize the specificity of the HLA-A11 restricted CEA antigen.