CN111620955B

CN111620955B - Multi-target-site composite antigen and application thereof

Info

Publication number: CN111620955B
Application number: CN202010627451.3A
Authority: CN
Inventors: 卢戌; 刘静维; 王跃; 刘雪松; 李君阳
Original assignee: Beijing Biohealthcare Cell Technology Co ltd
Current assignee: Beijing Biohealthcare Cell Technology Co ltd
Priority date: 2020-07-01
Filing date: 2020-07-01
Publication date: 2022-11-08
Anticipated expiration: 2040-07-01
Also published as: CN111620955A

Abstract

The invention relates to a multi-target composite antigen and application thereof, and aims to provide a multi-target composite antigen, namely a combined CTL epitope peptide. Another objective of the invention is to provide a multivalent vaccine against multiple target sites on the surface of tumor cells. It is still another object of the present invention to provide a multi-target complex antigen loaded CD8 ⁺ Cytotoxic T lymphocytes and a preparation method thereof. It is still another object of the present invention to provide an epitope-induced specific CTL effector cell.

Description

Multi-target-site composite antigen and application thereof

Technical Field

The invention relates to the fields of biology and medicine, in particular to a multi-target composite antigen and application thereof.

Background

In recent years, the important role of the immune system in tumor prevention and treatment has been widely agreed, and immunotherapy based on anti-tumor specific immune reconstitution is an important means which is internationally acknowledged at present and hopefully and completely eliminates tumor cells in vivo after surgery, radiotherapy and chemotherapy so as to radically treat tumors, and becomes a main direction of future tumor treatment. The immunotherapy mainly activates and mobilizes cells with cytotoxic activity and cytokines by supplementing, inducing and activating the inherent biological response regulation system of an organism in vitro, improves the functions of the autoimmune system of a patient, enhances the differentiation capacity of specific tumor killer cells, kills the tumor cells on the cellular level, effectively inhibits the metastasis, diffusion and recurrence of the tumor cells, has slight side reaction and good clinical treatment effect, and overcomes the defects of incomplete treatment, easy metastasis, great side effect and the like of the traditional tumor treatment mode. With the intensive research on tumor immunity at cellular and molecular level, tumor antigen-loaded Dendritic Cell (DCs) -sensitized tumor-specific Cytotoxic T Lymphocytes (CTLs) as an important part of immunotherapy can specifically recognize tumors, expand in cloning, form immunological memory in vivo, and mediate persistent anti-tumor specific immune effects. Activation of CTL requires the co-stimulation of a tumor antigen priming and an Antigen Presenting Cell (APC) provided co-stimulatory signals as well as a third class of signal molecules provided by cytokines. The existence of tumor-associated and specific antigens lays a theoretical and material foundation for specific immunotherapy. Only tumor antigens with strong immunogenicity and high specificity can effectively activate T lymphocytes with tumor specificity. In recent years, tumor polypeptide therapeutic vaccines developed aiming at tumor-associated antigens (TAA) or tumor-specific antigens (TSA) expressed by tumors are expected to overcome the defects of the therapy by virtue of the characteristics of strong specificity, safety, low price, easiness in storage and application and the like, so as to achieve the aim of effectively treating the tumors.

Tumor antigens must be degraded into short peptides and form peptide-MHC-TCR complexes in Antigen Presenting Cells (APC) to be recognized by T cells, triggering corresponding cytotoxic T lymphocyte responses. Since the polypeptide vaccine aims to deliver a high dose of tumor antigen polypeptides to MHC (major histocompatibility complex) molecules on the surface of APC, suitable and effective tumor antigen polypeptides are very important for the preparation of tumor vaccines. And the tumor antigen polypeptides are restricted by MHC, only patients with the same MHC class I molecule can use the same peptide, and some tumor antigen peptides may induce immune tolerance rather than activate immune response due to tumor heterogeneity. In addition, the weak immunogenicity is another weakness of polypeptide vaccines, and the immunogenicity can be effectively improved and stronger CTL activity can be induced by modifying epitope polypeptides (substitution of single amino acid, PMRI modification of polypeptide, lipopeptide and the like) or adopting multivalent vaccines.

Enzyme-linked immunospot assay (ELISPOT) is a cellular immunological detection technique that can detect cytokine-secreting T cells from the single cell level. The principle of using ELISPOT to detect CTL secreting IFN-gamma is that the monoclonal antibody of IFN-gamma is coated on a 96-well plate attached with PVDF membrane, then T cells stimulated by immunogenic epitope polypeptide are added into micropores to be cultured, and the secreted IFN-gamma is combined with the coated antibody. After washing the cells and unbound components away, an enzyme-labeled IFN-gamma detection monoclonal antibody is added to bind with the captured IFN-gamma. Upon addition of the substrate, colored spots can be generated where there is IFN- γ, each spot representing one IFN- γ secreting cell. The number of the spots is counted by using a computer imaging analysis system, and the number of the IFN-gamma-secreting CTLs in the sample can be determined. The ELISPOT technology is highly sensitive, and due to the high concentration of cytokines in the vicinity of the cells, cells secreting 100 cytokines can also be found, which is sufficient to detect 1/105 IFN-. Gamma.secreting cells. And the ELISPOT technique is highly specific because two monoclonal antibodies recognizing two different epitopes of cytokines or antibodies are used.

The invention is based on the follow-up research of the Chinese invention patent with the application number of 201510270656. The documents cited in the present invention are as follows, which are incorporated by reference into the present patent application. Patent document 1: publication No.: CN101302537A; patent document 2: publication No.: CN 101854945A; patent document 3: publication No.: CN 102625832A.

Disclosure of Invention

It is an object of the present invention to provide a multi-target complex antigen, i.e., a combined CTL antigenAn epitope peptide. It is another object of the present invention to provide a multivalent vaccine against multiple targets on the surface of tumor cells. It is still another object of the present invention to provide a multi-target complex antigen loaded CD8 ⁺ Cytotoxic T lymphocytes and a preparation method thereof. It is still another object of the present invention to provide an epitope-induced specific CTL effector cell.

The invention provides a polypeptide, which consists of EBV, HPV, HSV and CMV, wherein:

the amino acid sequence of the EBV is SEQ ID NO: 1. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 or SEQ ID NO:13;

the amino acid sequence of the HPV is SEQ ID NO: 2. the amino acid sequence of SEQ ID NO: 14. SEQ ID NO: 15. the amino acid sequence of SEQ ID NO: 16. SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 18. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 20. the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22;

the HSV has an amino acid sequence shown as SEQ ID NO:3;

the amino acid sequence of CMV is SEQ ID NO:4 or SEQ ID NO:23.

wherein the amino acid sequence of the polypeptide is SEQ ID NO:5.

the invention also provides a nucleic acid molecule encoding the polypeptide of claim 2.

The nucleic acid is a DNA molecule, and the DNA molecule is a DNA molecule shown as a sequence 6 in a sequence table.

Recombinant expression vectors, expression cassettes, transgenic cell lines, recombinant bacteria or recombinant viral vectors containing said nucleic acid molecules should also be within the scope of the present invention.

The invention also provides a vaccine, wherein the active ingredients of the vaccine are any one of the following substances:

1) The polypeptide;

2) The nucleic acid molecule;

3) The recombinant expression vector;

4) The expression cassette;

5) The transgenic cell line;

6) The recombinant bacterium;

7) The recombinant viral vector.

The vaccine is a DC vaccine.

The invention also provides a multi-target composite antigen loaded CD8 ⁺ Cytotoxic T lymphocytes, the multi-target complex antigen loaded with CD8 ⁺ An epitope of a cytotoxic T lymphocyte comprises the polypeptide.

The invention also provides an epitope-induced specific CTL effector cell, which is characterized in that an antigenic epitope comprises the polypeptide.

The application of any one of the following substances in preparing the medicine for treating cancer also falls within the protection scope of the present invention:

1) The polypeptide;

2) The nucleic acid molecule;

3) The recombinant expression vector;

4) The expression cassette;

5) The transgenic cell line;

6) The recombinant bacterium;

7) The recombinant viral vector;

8) The vaccine as described;

9) The multi-target composite antigen loaded with CD8 ⁺ Cytotoxic T lymphocytes.

The multi-Target complex antigen (MTCA) provided by the invention is a mode of combining a plurality of epitopes, the combination mode is that at least four polypeptide combinations matched with human leukocyte antigens are selected from a series of candidate polypeptides according to genetic gene structure and function difference to prepare tumor vaccines, so that specific immune response of a patient body to tumors is stimulated, the survival time of the tumor vaccines is prolonged, and the MTCA has the unique advantages of bypassing immune diversity and tumor heterogeneity compared with other tumor vaccines, and can effectively induce stronger CTL killing activity.

In an embodiment of the present invention, CTLs for a plurality of tumor antigens and methods of preparing the same are provided. The present invention can be used for any tumor, including but not limited to: lung cancer, breast cancer, kidney cancer, stomach cancer, colorectal cancer, pancreatic cancer, liver cancer, cervical cancer, bladder cancer, prostate cancer, melanoma, head and neck tumor, etc., and can also be used for cancer related to blood and bone marrow.

In a preferred embodiment of the invention, the CTL product of the invention is expressed as an antigen that specifically corresponds to that exhibited by the tumor. Thus, co-infection of DC cells with a combination of multiple epitope polypeptides in combination with lentivirus Lenti-hGM-CSF provides a means to generate a single CTL product with specific tumor independent antigenic characteristics.

The invention has important significance in that: provides a brand-new cell immunotherapy concept, namely an autoimmune cell therapy technology on the basis of breaking tumor immune tolerance, and establishes a new technology and a new method for implementing efficient specific tumor killing on the basis of breaking tumor immune tolerance. The technology solves the bottleneck problems of short survival time of the effector cells in vivo, low tumor killing efficiency, poor clinical curative effect and the like in the prior tumor immunotherapy, greatly improves the clinical curative effect of the cell immunotherapy, and has very wide application prospect.

In an embodiment of the invention, a lentiviral vector is constructed by the following method: providing PCR primers aiming at CDS region sequence of human peripheral blood hGM-CSF gene (GeneID: NM-000758.3), adding enzyme cutting sites at 5 'end and 3' end of the gene through PCR amplification, and amplifying hGM-CSF gene fragment by PCR method with plasmid pORFhGM-CSF as template; the hGM-CSF gene is directionally cloned into a lentivirus linked vector system through a PacI restriction endonuclease site to construct a recombinant vector. And mixing the recombinant vector, the envelope plasmid and the packaging structure plasmid to form a lentivirus vector system. The lentivirus of the invention is preferably Lenti-hGM-CSF.

In an embodiment of the invention, the DC vaccine is prepared by the following method: separating out monocyte, preferably separating out monocyte from peripheral blood, inducing rhGM-CSF, rhIL-4 and TNF-alpha to obtain DC cell, and infecting DC cell with the combined antigen epitope polypeptide (preferably SEQ ID NO: 5) and the lentivirus of the present invention to obtain DC vaccine.

In an embodiment of the present invention, theThe DC vaccine obtained after the DC cell is infected by the combined antigen epitope polypeptide (preferably SEQ ID NO: 5) and the lentivirus of the invention at the same time is incubated with the T lymphocyte to obtain the sensitized cytotoxic T lymphocyte. Preferably, the MTCA-DC/APC-CTL immune cell therapy technique is to express CD3 ⁺ CD8 ⁺ Predominantly cytotoxic T cells are the primary effector cells.

In an embodiment of the present invention, antigen-specific CTLs are prepared as follows: the DC cell loaded with the tumor-associated antigen obtained by the method is mixed with T lymphocyte, GM-CSF, IL-4 and IL-2 are added into a culture medium for continuous culture, and the cell is collected to obtain the epitope-induced specific CTL effector cell.

The specific CTL of the invention has obviously improved IFN-gamma secretion capability. In the test of the killing effect of MTCA-multi-target complex antigen induced antigen specific CTL on specific tumor cells, the antigen specific CTL induced by the MTCA-multi-target complex antigen can form specific lysis on target cells in the co-culture process of effective target cells, namely the CTL induced by the MTCA-multi-target complex antigen can kill target cells loaded with control peptides at the same time, and the application range of experimental polypeptide is expanded.

Drawings

FIG. 1 is a diagram of the construction of recombinant vector FattbC31UGW-hGM-CSF by targeted cloning of hGM-CSF gene into a lentiviral ligation vector system via pacl restriction endonuclease sites.

FIG. 2 is the envelope plasmid VSVG.

FIG. 3 shows the packaging construct plasmid CMV Δ 8.9-D64N/D116N.

FIG. 4 is a bar graph comparing the ability of the multi-target complex antigen of the present invention to induce antigen-specific CTL to produce IFN-. Gamma..

FIG. 5 is a bar graph comparing the killing effect of the multi-target complex antigen-induced antigen-specific CTLs on specific tumor cells according to the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Synthesis of polypeptides

The polypeptides in the following examples were synthesized using standard Fmoc protocol by Shanghai intense Biotechnology Ltd, and subjected to purification and purity analysis by high performance liquid chromatography, identification by mass spectrometry, and molecular weight determination. The results show that the purity of the synthesized polypeptide is higher than 95%, and the molecular weight is consistent with the theoretical value.

This example provides a polypeptide designated MTCA consisting of EBV, HPV, HSV and CMV wherein:

the amino acid sequence of the HPV is SEQ ID NO: 2. SEQ ID NO: 14. the amino acid sequence of SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO:21 or SEQ ID NO:22;

the HSV has an amino acid sequence shown as SEQ ID NO:3;

the amino acid sequence of CMV is SEQ ID NO:4 or SEQ ID NO:23.

wherein when the amino acid sequence of EBV is SEQ ID NO: 1. the amino acid sequence of the HPV is SEQ ID NO: 2. the HSV has an amino acid sequence shown as SEQ ID NO:3 and the amino acid sequence of CMV is SEQ ID NO:4, and connecting the N terminal to the C terminal in sequence to obtain the polypeptide MTCA. The amino acid sequence of MTCA is SEQ ID NO:5 and the sequence of the corresponding coding gene is the DNA molecule shown in the sequence 6.

Example 2 construction of LentihGM-CSF, a lentivirus

1. PCR primer F1 (F1' -G) was designed based on the CDS region sequence of human peripheral blood hGM-CSF gene (GeneID: NM-000758.3)TTAATTAACATGTGGCTGCTGCAGAGCCTGCTGCTCT-3 ') and R1 (R1: 5' -G)TTAATTAACTCACTCCTGGACTGGCTCCCAGCAG-3 '), primers F1 and R1 can be amplified by PCR to add enzyme recognition sites at both the 5' end and the 3' end of the above genes. In the above primers, the underlined part is an enzyme recognition site.

2. Using plasmid pORFhGM-CSF (purchased from Invivogen, cat # porf-hgmcsf) as a template, and amplifying by PCR method through primers F1 and R1 to obtain hGM-CSF gene fragment; then, the hGM-CSF gene is directionally cloned into a lentivirus ligation vector FattbC31UGW (Purpurin biotechnology (Beijing) Co., ltd., product number Biovector912008, the structure of which is shown in figure 1) through a PacI restriction endonuclease site to obtain a recombinant vector FattbC31UGW-hGM-CSF. The recombinant vector FattbC31UGW-hGM-CSF is obtained by inserting hGM-CSF gene into the PacI restriction endonuclease site of the lentivirus linking vector FattbC31UGW and keeping other sequences of the lentivirus linking vector FattbC31UGW unchanged.

3. Mixing a recombinant vector FattbC31UGW-hGM-CSF, an envelope plasmid VSVG (prohibitin biotechnology (Beijing) Co., ltd., product No. Biovector912007, the structure of which is shown in FIG. 2) and a packaging structure plasmid pCMVAR8.91-D64N/D116N (prohibitin biotechnology (Beijing) Co., ltd., product No. Biovector912006, the structure of which is shown in FIG. 3) to form a lentivirus vector system, transfecting 293T cells by using liposome LipofectamineTM, observing under a fluorescence microscope after 24-48h, collecting virus supernatant after 72h appears in a large amount of fluorescence, obtaining lentivirus particles Lenti-hGM-CSF encoding hGM-CSF, concentrating the collected virus supernatant, and subpackaging for later use or immediate use.

4. When the activity of the recombinant lentivirus is determined, the concentrated virus stock solution is diluted in different proportions, cells are infected for 72 hours, and then fluorescence counting is carried out under a fluorescence microscope to determine the titer. The lentivirus ligation vector FattbC31UGW was used as a control. The results showed that the site-specific integrating lentivirus titer was 7.1X 10 ^-7 TU/ml。

The method for detecting the virus titer comprises the following steps: taking logarithmic growth HT1080 cells, spreading in a 96-well plate in 6 × 103 cells/well, adding DMEM medium containing 10 FBS into each well, diluting the fluorescent expression virus with serum-free DMEM medium in a round bottom 96-well plate in 10-fold gradient in the next day, repeating three wells in each gradient, discarding 90ul of the medium in each well of the 96-well plate with HT1080 cells, adding 90ul of the diluted virus solution in the round bottom 96-well plate correspondingly, changing into 100ul of the DMEM medium containing 10 FBS after 24h, after 72h infection, counting the number of fluorescence in the wells with moderate fluorescence expression, wherein the volume ratio of the fluorescence to the virus solution in the wells is the titer of the fluorescent expression virus, the titer of the fluorescent expression virus is a standard curve, and the cq value of the fluorescent expression virus and the target virus obtained by combining the QPCR method is combined, so as to calculate the titer (TU/mL) of the target virus.

Example 3 preparation of DC vaccine

Monocytes were isolated from human peripheral blood, DC cells were obtained by induction with rhGM-CSF, rhIL-4 and TNF- α, and DC vaccines were obtained by infecting DC cells with the multi-target complex antigen peptide MTCA (SEQ ID NO: 5) synthesized in example 1 and the lentivirus Lenti-hGM-CSF prepared in example 2 simultaneously after identification by phase contrast microscopy and flow cytometry. The method specifically comprises the following steps:

1) Collecting peripheral blood of volunteer, separating autologous plasma (inactivated at 56 deg.C for 30min and preserved at 4 deg.C) and PBMC (peripheral blood mononuclear cell) by density gradient separation method using lymphocyte separation liquid (Ficoll-Hypaque, polysucrose-diatrizoate), and treating PBMC with serum-free RPMI-1640 (5-7) x 10 ⁷ After 1h, suspension cells are collected and used for separating T lymphocytes, adherent cells are replaced by 1640 culture medium containing rhGM-CSF (1000U/ml), rhIL-4 (1000U/ml) and TNF-alpha (500U/ml) to stimulate and induce the monocyte to differentiate to the DC.

2) Immature DCs collected in 24-well plates in suspension were infected on day 5 with the polypeptide MTCA in example 1 and Lenti-hGM-CSF concentrate of lentiviral particles in example 2, and the volume required for lentivirus was calculated according to the pre-experimental optimal MOI values. After 24 hours of transfection, the medium was replaced with fresh DC induction medium and rhGM-CSF (1000U/ml), rhIL-4 (1000U/ml) and TNF- α (500U/ml) were added to promote differentiation and maturation of DC, to obtain DC vaccine.

EXAMPLE 4 preparation of antigen-specific CTL

Collecting peripheral blood of volunteers, subjecting to density gradient centrifugation with lymphocyte separation liquid (Ficoll-Hypaque), collecting interface cells rich in mononuclear cells, suspending in RPMI-1640 culture medium, placing at 37 deg.C, and 5% CO ₂ Culturing in incubator for 2 hr, collecting non-adherent suspension cells, re-suspending with RPMI-1640 medium containing 3% serum, and regulating cell density to 1 × 10 ⁶ And each ml, the suspension is rich in T lymphocytes. The DC vaccine prepared in example 3 was mixed with T lymphocytes in a ratio of 1 ⁶ Per ml; and collecting the cells after culturing for 1 week to obtain epitope-induced specific CTL effector cells named CTL-MTCA.

EXAMPLE 5 preparation of control samples

The polypeptide MTCA in example 1 was replaced with the polypeptide EBV (amino acid sequence SEQ ID NO: 1), the polypeptide HPV (amino acid sequence SEQ ID NO: 2), the polypeptide HSV (amino acid sequence SEQ ID NO: 3) and the polypeptide CMV (amino acid sequence SEQ ID NO: 4), respectively, according to the methods of examples 1 to 4; specific CTL effector cells induced by the epitopes are prepared and named as CTL-EBV, CTL-HPV, CTL-HSV and CTL-CMV respectively.

EXAMPLE 6 measurement and comparative experiment of the ability of MTCA-Multi-target Complex antigen of the present invention to induce antigen-specific CTL to produce IFN-. Gamma.

The experimental groups are: 1. EBV antigen control group (CTL-EBV cell to be detected); 2. HPV antigen control group (CTL-HPV is the cell to be detected); 3. HSV antigen control group (tested cell is CTL-HSV); 4. a CMV antigen control group (the cell to be detected is CTL-CMV); 5. MTCA-multi-target complex antigen group (the cell to be detected is CTL-CMV); 6. blank control group (100 ul of RPMI-1640 medium only). The specific experimental method is as follows:

MTCA-multi-target complex antigen group: the capability of CTL-MTCA to secrete IFN-gamma is detected by an enzyme-linked immunospot method (ELISPOT): an ELISPOT assay kit was used, and a 96-well plate was added with PBS diluted at 1100ul of IFN-. Gamma.capture antibody was incubated at 4 ℃ overnight, washed with PBS, and then 100ul of a test cell (CTL-MTCA prepared in example 4) solution (the content of the test cell was adjusted by RPMI-1640 medium) was added thereto, and the amount of the antibody was 1X 10 ⁶ Cell/well, at 37 ℃,5% CO ₂ After 24h incubation under conditions, washed with PBS containing 0.1% Tween 20, the biotin-labeled anti-IFN-. Gamma.antibody diluted by 1 ₂ Incubate for 2h under conditions, incubate for 1h with streptavidin-labeled alkaline phosphatase 100ul diluted with 1% BSA in PBS at 1. The experiment was performed in triplicate.

1. EBV antigen control group: the same procedure was followed except that the test cells in the MTCA-multi-target complex antigen group were replaced with CTL-EBV prepared in example 5.

2. HPV antigen control group (the cell to be tested is CTL-HPV);

3. HSV antigen control group (tested cell is CTL-HSV);

4. a CMV antigen control group (the cell to be detected is CTL-CMV);

5. MTCA-multi-target complex antigen group (the cell to be detected is CTL-CMV);

6. blank control group: the procedure was the same except that 100ul of the cell sap to be tested was replaced with 100ul of RPMI-1640 medium.

The results are shown in FIG. 4 and show that: the capacity of the specific CTL (cytotoxic T lymphocyte) for secreting IFN-gamma in the MTCA-multi-target compound antigen group is obviously higher than that in the other 5 groups, the capacity of inducing and secreting IFN-gamma is strongest, and the capacity of secreting IFN-gamma is improved by more than 3 times compared with that of a control group in which a single antigen peptide loads the specific CTL.

Example 7 MTCA-Multi-target Complex antigen of the present invention induces killing Effect of antigen-specific CTL against specific tumor cells

The experimental groups are: MTCA-multi-target complex antigen group:

MTCA-multi-target complex antigen group: time resolved fluorescence cytotoxicity assays were used for the analysis. CTL-MTCA prepared in examples 4 and 5 were used as effector cells (E) and T2+ EBV, \8230; target cells \8230Specific killing activity of CTL was examined for target cells (T). At 37 ℃,5% CO ₂ Under the condition, effector cells and target cells are mixed according to the quantitative ratio of 50:1 co-incubation for 4h, setting 3 multiple wells for each sample, taking an average value as a detection result, and calculating the cell killing activity of CTL-MTCA according to the following formula, wherein the specific lysis rate = (fluorescence intensity of the well to be detected-fluorescence intensity of the natural release well)/(fluorescence intensity of the maximum release well-fluorescence intensity of the natural release well) × 100%.

The experimental groups are: 1. EBV antigen control group (CTL-EBV); 2. HPV antigen control group (CTL-HPV); 3. HSV antigen control group (CTL-HSV); 4. a CMV antigen control group (CTL-CMV); 5. MTCA-Multi-target Complex antigen group (CTL-MTCA).

The results are shown in fig. 5, antigen-specific CTLs (CTL-MTCA) induced by MTCA-multi-target complex antigen can form specific lysis to target cells during the co-culture of effector target cells, while effector cells induced by each control polypeptide can only kill target cells loaded with the corresponding peptide, i.e., CTLs induced by MTCA-multi-target complex antigen can simultaneously kill target cells loaded with the above four control peptides, thus expanding the application range of experimental polypeptides. The MTCA-multi-target compound antigen group has higher specific cracking rate (more than 70%) for four groups of target cells T2+ EBV, T2+ HSV, T2+ CMV and T2+ HPV, among other four groups, the EBV antigen control group has the highest specific cracking rate for the target cells T2+ EBV, the HSV antigen control group has the highest specific cracking rate for the target cells T2+ HSV, the CMV antigen control group has the highest specific cracking rate for the target cells T2+ CMV, the HPV antigen control group has the highest specific cracking rate for the target cells T2+ HPV, but the highest value is smaller than the specific cracking rate corresponding to the MTCA-multi-target compound antigen group.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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Claims

1. A polypeptide consisting of EBV, HPV, HSV and CMV the amino acid sequence is SEQ ID NO:5, the CTL induced by the polypeptide composite antigen has a broad spectrum type for killing target cells loaded with at least one control peptide of EBV, HPV, HSV and CMV at the same time.

2. A nucleic acid molecule for encoding the polypeptide of claim 1, wherein the nucleic acid is a DNA molecule, and the DNA molecule is shown as a sequence 6 in a sequence table.

3. A recombinant expression vector, expression cassette, transgenic cell line, recombinant bacterium or recombinant viral vector comprising the nucleic acid molecule of claim 2.

4. The vaccine is characterized in that the active ingredients of the vaccine are any one of the following substances:

1) A polypeptide according to claim 1;

2) The nucleic acid molecule of claim 2;

3) The recombinant expression vector of claim 3;

4) The expression cassette of claim 3;

5) The transgenic cell line of claim 3;

6) The recombinant bacterium of claim 3;

7) The recombinant viral vector of claim 3.

5. The vaccine of claim 4, wherein the vaccine is a DC vaccine.

6. An epitope-induced specific CTL effector cell, wherein the epitope comprises the polypeptide of claim 1, and said CTL effector cell is capable of recognizing EBV, HPV, HSV and CMV simultaneously with the polypeptide of claim 1.

7. Use of any one of the following in the manufacture of a medicament for the treatment of cancer:

1) A polypeptide according to claim 1;

2) The nucleic acid molecule of claim 2;

3) The recombinant expression vector of claim 3;

4) The expression cassette of claim 3;

5) The transgenic cell line of claim 3;

6) The recombinant bacterium of claim 3;

7) The recombinant viral vector of claim 3;

8) The vaccine of claim 4 or 5;

9) An epitope-induced specific CTL effector cell of claim 6.