CN111944018B

CN111944018B - Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof

Info

Publication number: CN111944018B
Application number: CN202010889660.5A
Authority: CN
Inventors: 李光磊; 苏彦景; 唐超
Original assignee: Shenzhen Letu Biomedical Co ltd
Current assignee: Shenzhen Letu Biomedical Co ltd
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2022-12-23
Anticipated expiration: 2040-08-28
Also published as: CN111944018A

Abstract

The application discloses an antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocytes and application thereof. The amino acid sequence of the antigen polypeptide for inducing the liver cancer specific cytotoxic T lymphocyte comprises a sequence shown in Seq ID No. 1. The antigen polypeptide of the application can specifically and efficiently induce the hepatoma specific cytotoxic T lymphocyte, and the induced hepatoma specific cytotoxic T lymphocyte is superior to CTL induced by other existing antigen polypeptides in immunogenicity, positive rate of antigen-loaded cytotoxic T lymphocyte cells and killing efficiency of the antigen-loaded cytotoxic T lymphocyte cells in cell in vitro experiments. The antigen polypeptide is adopted to induce the cytotoxic T lymphocyte which can efficiently and specifically kill the liver cancer tumor cells, the preparation method is efficient and convenient, compared with other immune cell therapies, the antigen polypeptide has the characteristics of low cost, high yield and simple process flow, and a new scheme and a new way are provided for clinical application of liver cancer immune cell therapy.

Description

Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof

Technical Field

The application relates to the field of liver cancer immune cell therapy, in particular to an antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocytes and application thereof.

Background

Immune cell therapy is the only method which has the possibility of completely eliminating cancer cells in the prior art, makes up the defects of the traditional therapy, and is considered as a treatment means with the most promising development prospect in the twenty-first century tumor comprehensive treatment mode.

Cytotoxic T lymphocytes (abbreviated CTLs) are the most important effector cells of the body against tumors and are widely used in immune cell therapy. Antigen loading is an essential process in the preparation process of CTL, and in CTL industrialization and clinical application, the problems of specificity and effectiveness of loaded antigen polypeptide must be solved.

In the development of the CTL therapy aiming at liver cancer at present, although relevant documents report that the liver cancer is associated with positive polypeptide sequences of viruses (HBV and HCV); and the antigen polypeptide sequence with high liver cancer expression. However, there are no reports on the strong and weak immunogenicity of these antigens, and even more, there are no reports on whether these antigens can be loaded by CTLs via antigen-presenting cells such as DC, and the strong and weak killing ability of CTLs loaded with these polypeptides of liver cancer antigens.

Disclosure of Invention

The application aims to provide a novel antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocytes and application thereof.

The following technical scheme is adopted in the application:

the first aspect of the application discloses an antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte, the amino acid sequence of the antigen polypeptide comprises the sequence shown in Seq ID No.1,

Seq ID No.1：FLXLVDATV

wherein, X is selected from any one of alanine, leucine, isoleucine, methionine, cysteine and threonine.

It is noted that the antigenic polypeptide of the present application can efficiently induce liver cancer specific CTLs, and the obtained antigen-loaded liver cancer specific CTLs have strong immunogenicity, high positive rate, and strong in vitro killing ability and efficiency. In particular, in one implementation of the present application, X is leucine, such that the antigenic polypeptide has strong immunogenicity, high positive rate, strong in vitro killing ability and efficiency. As regards X as alanine, isoleucine, methionine, cysteine or threonine, the corresponding antigenic polypeptide has, in the experimental analysis, the same specificity and function as the antigenic polypeptide in which X is leucine.

It should be noted that the key point of the present application is the antigen polypeptide with sequence shown in Seq ID No.1, and it is understood that on the basis of not affecting the performance of the antigen polypeptide in inducing liver cancer specific CTL and induced CTL, amino acid sequences with other functions or functions may be added at both ends of the antigen polypeptide with sequence shown in Seq ID No.1, and are not specifically limited herein.

In a second aspect of the present application, there is disclosed a nucleic acid sequence, which may be DNA or RNA, encoding an antigenic polypeptide of the present application.

It is noted that the nucleic acid sequences of the present application are capable of encoding the antigenic polypeptides of the present application; it is understood that the specific nucleic acid sequence can be determined by referring to the amino acid sequence of the sequence shown in Seq ID No.1 with reference to the genetic code, and is not particularly limited herein.

In one embodiment of the application, the nucleic acid sequence comprises a sequence which is degenerate as described in any one of the sequences shown in Seq ID No.2 to Seq ID No.7,

Seq ID No.2：5’-TTCCTGGCACTAGTCGATGCAACAGTA-3’

Seq ID No.3：5’-TTCCTGCTGCTAGTCGATGCAACAGTA-3’

Seq ID No.4：5’-TTCCTGATCCTAGTCGATGCAACAGTA-3’

Seq ID No.5：5’-TTCCTGATGCTAGTCGATGCAACAGTA-3’

Seq ID No.6：5’-TTCCTGTGTCTAGTCGATGCAACAGTA-3’

Seq ID No.7：5’-TTCCTGACACTAGTCGATGCAACAGTA-3’。

wherein, the sequences shown in Seq ID No.2 to Seq ID No.7 correspond to the antigen polypeptide which codes the application and X is alanine, leucine, isoleucine, methionine, cysteine, threonine.

It will be understood that degenerate sequences in accordance with the present application are nucleic acid sequences which, by virtue of the degeneracy of the degenerate codons, are likewise capable of encoding the amino acid sequence shown in Seq ID No.1 but differ from the sequences shown in Seq ID No.2 to Seq ID No. 7.

It is to be noted that the nucleic acid sequences of the present application are critical to be capable of encoding the antigenic polypeptides of the present application; it is understood that other functional or functional sequences may be added to the nucleic acid sequences of the present application without affecting the encoding or expression of the antigenic polypeptides of the present application, and are not specifically limited herein.

In a third aspect of the present application, a gene expression vector is disclosed, wherein the gene expression vector is inserted with a foreign gene capable of encoding and expressing the antigenic polypeptide of the present application.

It is understood that the key of the gene expression vector of the present application is that the foreign gene can encode and express the antigen polypeptide of the present application, and as for the framework structure of the gene expression vector, such as promoter, terminator and the like, reference can be made to the existing gene expression vector, which is not specifically limited herein.

In one implementation of the present application, the exogenous gene of the gene expression vector comprises a nucleic acid sequence of the present application.

In a fourth aspect of the present application, a recombinant cell comprising the gene expression vector of the present application is disclosed.

It is noted that the recombinant cells of the present application are capable of encoding and expressing the antigenic polypeptides of the present application by transducing or transfecting the gene expression vectors of the present application; the antigen polypeptide can be obtained by culturing the recombinant cells, extracting and purifying proteins. It is understood that the key to the recombinant cell of the present application is that it contains the gene expression vector of the present application, and the specific cell to be used is not particularly limited, depending on the framework structure of the gene expression vector.

In a fifth aspect of the present application, there is disclosed an Antigen Presenting Cell (APC) for inducing hepatoma-specific cytotoxic T lymphocytes, which is capable of presenting the antigenic polypeptide of the present application to CD 8-positive T cells and activating to produce corresponding specific cytotoxic T lymphocytes.

In one implementation of the present application, the antigen presenting cells that induce hepatoma-specific cytotoxic T lymphocytes are obtained by stimulating the antigen presenting cells with the antigen polypeptide of the present application, or the nucleic acid sequence of the present application is introduced into the antigen presenting cells so that the antigen presenting cells can express the antigen polypeptide of the present application, thereby obtaining the ability to induce hepatoma-specific cytotoxic T lymphocytes.

In general, an APC having the ability to induce CTL is obtained by directly stimulating culture with an antigenic polypeptide, or an APC may be formed by introducing a nucleic acid sequence expressing the antigenic polypeptide into the APC and expressing the antigenic polypeptide by the APC. In one embodiment of the present application, the antigen presenting cells obtained by direct stimulation culture of the synthesized antigen polypeptide are used for inducing hepatoma-specific cytotoxic T lymphocytes.

In a sixth aspect of the present application, there is disclosed an antigenic polypeptide of the present application or a liver cancer-specific cytotoxic T lymphocyte induced by an antigen presenting cell of the present application which induces a liver cancer-specific cytotoxic T lymphocyte.

It can be understood that the liver cancer specific CTL is obtained by induction of the antigen polypeptide or APC, so that the liver cancer specific CTL has the advantages of strong immunogenicity, high positive rate, high in-vitro killing capacity and efficiency and the like, and can better meet the clinical application of treatment, prevention or detection of liver cancer.

A seventh aspect of the present application discloses a liver cancer specific T cell antigen receptor T cell (TCR-T) comprising a liver cancer antigen TCR sequence obtained by single cell TCR full length sequencing of the liver cancer specific cytotoxic T lymphocytes of the present application.

In one implementation of the present application, the liver cancer-specific T cell antigen receptor T cell is formed by transducing a liver cancer antigen TCR sequence to a T cell via a gene transduction vector.

It can be understood that after obtaining the TCR sequence of the liver cancer antigen by the single cell TCR full-length sequencing, the PCR amplification is generally performed by using a TCR specific primer to obtain the TCR sequence of the liver cancer antigen, and then the PCR amplification product, i.e., the TCR sequence of the liver cancer antigen, is transduced into T cells by a gene transduction vector. Wherein, the TCR specific primer or the primer design can refer to the existing PCR amplification technology; the gene transduction vector may also refer to existing TCR-T technology, such as lentiviral vectors, etc., and is not particularly limited thereto.

The eighth aspect of the present application discloses the use of the antigenic polypeptide of the present application, or the nucleic acid sequence of the present application, or the gene expression vector of the present application, or the recombinant cell of the present application, or the antigen presenting cell of the present application that induces hepatoma-specific cytotoxic T lymphocytes, or the hepatoma-specific cytotoxic T lymphocytes of the present application, or the hepatoma-specific T cell antigen receptor T cells of the present application, in the preparation of a medicament for treating or preventing hepatoma or in the preparation of a reagent for detecting or screening hepatoma.

It is understood that the antigenic polypeptides, nucleic acid sequences, APC, CTL and TCR-T of the present application all have liver cancer specificity, and therefore, in addition to being used for preparing a medicament for treating or preventing liver cancer, the specificity detection or screening of liver cancer can be realized through the specificity of the antigenic polypeptides, nucleic acid sequences, APC, CTL and TCR-T. As for the gene expression vector and the recombinant cell, the gene expression vector and the recombinant cell can be used for preparing the antigen polypeptide of the application, and therefore, the gene expression vector and the recombinant cell can also be indirectly used for preparing a medicament for treating or preventing liver cancer.

The ninth aspect of the present application discloses a kit for preparing liver cancer-specific cytotoxic T lymphocytes, which contains at least one component of components (a) to (f);

(a) An antigenic polypeptide of the present application;

(b) A nucleic acid sequence of the present application;

(c) A gene expression vector of the present application;

(d) A recombinant cell of the present application;

(e) Antigen presenting cells of the present application that induce hepatoma-specific cytotoxic T lymphocytes;

(f) Exosomes capable of presenting the antigenic polypeptides of the present application to CD8 positive T cells and activating the production of corresponding specific cytotoxic T lymphocytes.

The kit for preparing the liver cancer specific CTL can prepare the liver cancer specific CTL with strong immunogenicity, high positive rate, high in-vitro killing capacity and high efficiency, and can better meet the use requirements of liver cancer research and clinical application. It is understood that the kit for preparing liver cancer-specific CTLs of the present application may optionally contain other key reagents for preparing liver cancer-specific CTLs, and of course, these reagents may also be obtained directly by commercial purchase, and are not specifically limited herein.

It is further to be noted that exosomes as defined herein refer to, for example, intracellular vesicles or similar functional or structural substances which present the antigenic polypeptides of the present application on their surface, thereby activating the production of corresponding specific cytotoxic T lymphocytes. The exosomes of the present application can be prepared, for example, by the methods described in JPH11-510507 and WO 99/03499. The exosomes of the present application are capable of presenting the antigenic polypeptides of the present application and, therefore, can also be used to prepare the liver cancer-specific CTLs of the present application.

The tenth aspect of the present application discloses a pharmaceutical composition for treating or preventing liver cancer, which comprises a pharmaceutically acceptable carrier and at least one component of the components (a) to (h);

(a) An antigenic polypeptide of the present application;

(b) A nucleic acid sequence of the present application;

(c) A gene expression vector of the present application;

(d) A recombinant cell of the present application;

(f) Exosomes capable of presenting the antigenic polypeptides of the present application to CD8 positive T cells and activating the production of corresponding specific cytotoxic T lymphocytes;

(g) The liver cancer-specific cytotoxic T lymphocytes of the present application;

(h) The liver cancer specific T cell antigen receptor T cells of the present application.

The pharmaceutically acceptable carrier includes, but is not limited to, a solution of the antigenic polypeptide, the nucleic acid sequence, the APC, the exosomes, the TCR-T or the CTL, and a diluent, an excipient, a solvent, an encapsulating material and the like of the pharmaceutical composition.

The application of the pharmaceutical composition for treating or preventing liver cancer, wherein the liver cancer specific cytotoxic T lymphocytes have the advantages of strong immunogenicity, high positive rate, high in-vitro killing capacity and efficiency and the like, and have the effect of treating or preventing liver cancer. The TCR-T can be used for target development in liver cancer immune cell therapy. The antigenic polypeptides, nucleic acid sequences, exosomes and antigen presenting cells of the present application are capable of inducing an immune response against liver cancer, thereby producing an effect of treating or preventing liver cancer. The gene expression vector and the recombinant cell can express the antigen polypeptide, so that the antigen polypeptide has similar action and effect under the condition of expressing the antigen polypeptide.

An eleventh aspect of the present application discloses a kit for inducing an immune response against liver cancer, which contains at least one component of components (a) to (h);

(a) An antigenic polypeptide of the present application;

(b) A nucleic acid sequence of the present application;

(c) A gene expression vector of the present application;

(d) A recombinant cell of the present application;

It is noted that the antigenic polypeptides, nucleic acid sequences, antigen presenting cells, exosomes, liver cancer specific cytotoxic T lymphocytes and TCR-ts of the present application are capable of specifically eliciting an immune response against liver cancer; therefore, it can be used as a corresponding agent, such as "vaccine" and the like. The above components may be used alone or in combination, and are not particularly limited herein.

The beneficial effect of this application lies in:

the antigen polypeptide can specifically and efficiently induce the hepatoma carcinoma cell cytotoxic T lymphocyte, and the induced hepatoma carcinoma specific cell cytotoxic T lymphocyte is superior to CTL induced by other existing antigen polypeptides in immunogenicity, positive rate of antigen-loaded cell cytotoxic T lymphocyte and killing efficiency of the cell in vitro experiments. The antigen polypeptide is adopted to efficiently induce the cytotoxic T lymphocyte which specifically kills the liver cancer tumor cells, the preparation method is efficient and convenient, compared with other immune cell therapies, the antigen polypeptide has the characteristics of low cost, high yield and simple process flow, and a new scheme and a new way are provided for clinical application of liver cancer immune cell therapy.

Drawings

FIG. 1 is a schematic diagram showing the induction of liver cancer-specific CTL by the antigenic polypeptide in the present example;

FIG. 2 is a graph showing the results of the immunogenicity test of the antigenic polypeptides in the examples of the present application;

FIG. 3 is a graph showing the flow assay fluorescence results of the antigen-loaded CTL positivity test in the examples of the present application;

FIG. 4 is a graph showing the standard displacement efficiency of the antigen-loaded CTL positive rate test in the examples of the present application;

FIG. 5 is a graph showing the results of flow cytometry analysis of CTL positive cell rates induced by antigenic polypeptides in the examples of the present application;

FIG. 6 shows the results of the test for the killing rate of CTL induced by the antigenic polypeptide in the examples of the present application.

Detailed Description

At present, although there are related researches and reports on antigenic polypeptides of liver cancer, the existing researches and reports are basically limited to screening and expression performance research of positive polypeptide sequences; the immunogenicity of antigenic polypeptides has not been investigated, nor has the ability of CTLs to be loaded with such antigenic polypeptides been investigated or reported, as well as the killing ability of CTLs loaded with such antigenic polypeptides.

The research of the application considers that although the existing research has screened positive antigen polypeptide, the positive antigen polypeptide has high expression performance; however, if the immunogenicity is weak, or the CTL cannot be loaded, or the killing ability of the CTL loaded with the antigen polypeptide is weak, these factors directly influence the CTL treatment effect and cost of the liver cancer.

Based on the research and the recognition, the application develops a new antigen polypeptide capable of efficiently inducing the liver cancer specific CTL, and the positive rate of the induced liver cancer specific CTL in immunogenicity, antigen-loaded cytotoxic T lymphocyte cells and killing efficiency of the induced liver cancer specific CTL in-vitro experiments are superior to those of the CTL induced by other existing antigen polypeptides.

The antigen polypeptide is adopted to efficiently induce the cytotoxic T lymphocyte with specificity for killing the liver cancer tumor cells, and the preparation method is efficient and convenient. Compared with other immune cell therapies, the method for preparing the liver cancer specific CTL by adopting the antigen polypeptide has lower cost than that of using a plasmid/virus vector to express the transfection antigen to cells; in addition, the CTL has the performance of efficiently killing the liver cancer tumor cells, so that the return dose of the CTL can be reduced, and the CTL culture cost is reduced. The process flow for preparing the liver cancer specific CTL is simple, and particularly, the process flow for preparing the CTL is more simple compared with the process flow for constructing a plasmid library and a virus cell library in the cell therapy methods such as TCR-T, CAR-T.

In addition, CTL carrying the liver cancer antigen polypeptide screened by the invention is screened out positively by a flow cytometer, after collection, the TCR sequence corresponding to the liver cancer antigen is easily obtained only by high-throughput single-cell TCR full-length sequencing and TCR specific primer amplification, and the TCR sequence has corresponding guiding significance for the subsequent preparation of TCR-T and can promote the target development of the TCR-T in liver cancer immune cell therapy.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in different instances or may be replaced by other kits, materials, methods. In some instances, certain operations related to the present application have not been shown or described in detail in this specification in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that the related operations will be fully understood from the description in the specification and the general knowledge of the art. In the following examples, the reagents and instruments used are conventional products available on the market, not indicated by manufacturers.

The numbers in the present application, such as "Seq ID No.", "first aspect", "second aspect", "third aspect", "(a)", "(b)", "(c)" and the like, are used only to distinguish the described objects, and do not have any order or technical meaning.

Examples

This example develops a new Antigen polypeptide inducing liver cancer specific cytotoxic T lymphocytes, the development process is shown in FIG. 1, which comprises obtaining Whole exon sequencing from the TCGA database of liver cancer patients, performing Tumor neoantigen and polypeptide prediction (Tumor neoantigen), antigen binding affinity analysis (Antigen binding affinity prediction) with MHC class I molecules, and screening out the new target polypeptide with strong affinity; then, antigen-presenting cells are stimulated, APC cells stimulated by cytokines (IL-4, GM-CSF, IFN-gamma and LPS) present antigens to CD8+ T cells (Antigen presenting), and after maturation is stimulated by corresponding cytokines (IL-21, IL-2, IL-7 and IL-15), CTLs secrete cytokines (IFN-gamma) to kill target cells or tumor cells to thereby treat the tumor patient.

The antigen polypeptide and CTL induced by the antigen polypeptide in the embodiment have immunogenicity, positive rate of antigen-loaded cytotoxic T lymphocyte cells and killing efficiency in vitro cell experiments, which are superior to CTL induced by other antigen polypeptides in the prior art, and detailed experiments are as follows:

1. tumor antigen polypeptide screening

1.1 screening of tumor neoantigen Polypeptides

According to six bioinformatics software of PSSMHCpan, netMHC-4.0, netMHCpan-3.0, netMHCpan-4.0, pickPocket and SMM, a plurality of human high-frequency mutation sites in the HLA-A0201-typed liver cancer population are predicted, and polypeptide-MHC affinity prediction is carried out on the mutation sites corresponding to the several sites. In this example, 364 cases of somatic mutation data (version 20170929) were downloaded from TCGA database (https:// port.gdc.cancer.gov) for sequencing of the whole exon of hepatocarcinoma. Screening missense SNV sites reproduced on at least 2 patients to obtain the highest frequency 10 mutation sites in a liver cancer patient population taking HLA-A0201 as an example, and intercepting an amino acid sequence into a short peptide with the length of 9 mers and containing variant amino acids according to variant information.

And generating a short peptide sequence which contains variant amino acids and is 9 mers in length according to the variation information from the 10 high-frequency mutation sites obtained by screening. In order to identify neoantigens that can be used as candidate markers for developing anti-tumor vaccines and to optimize prediction accuracy, this example incorporates six software items PSSMHCpan, netMHC-4.0, netMHCpan-3.0, netMHCpan-4.0, pickPocket and SMM into an affinity prediction protocol for detection of candidate neoantigens in TCGA liver cancer samples. Finally, candidate neoantigens meeting the following conditions were selected: the conjugate IC50<500nM was predicted by at least 3 softwares and the minimum of the corresponding IC50 values of several softwares predicted to be conjugate was taken as the final prediction and IC50<50nM.

Six software combination methods are used for predicting the binding capacity of the generated short peptide and MHC, IC50<500nM represents affinity, IC50<50nM represents high affinity, and the lower the IC50 score, the higher the affinity. Sorting according to the IC50 score from low to high, selecting the short peptide with the IC50 score of less than 50nM in the example, and finally obtaining 2 high-frequency mutant polypeptides with the strongest affinity and synthesizing the polypeptides for subsequent experiments. The details of the 2 candidate neoantigen polypeptides synthesized in this example are shown in Table 1.

TABLE 1 sequence information of the screened antigenic polypeptides

Serial number	HLA typing	Name of Gene	Amino acid sequence	Length of polypeptide	Seq ID No.
						1	HLA-A0201	TGIF2LX	FLLLVDATV	9	8
2	HLA-A0201	OR2G2	ILLGFSDYLQL	11	9

Wherein, the nucleic acid sequence of the TGIF2LX antigen polypeptide is the sequence shown in Seq ID No.3,

Seq ID No.3：5’-TTCCTGCTGCTAGTCGATGCAACAGTA-3’。

1.2 Virus-associated antigen screening

In order to compare the immunogenicity of the predicted neoantigenic polypeptide with the viral-associated antigenic polypeptide, positive HBV-associated polypeptide sequences were obtained according to the reference, and the polypeptide information is shown in Table 2.

TABLE 2 information on the synthesis of HBV, HCV-related viral polypeptides

Serial number	Name (R)	Amino acid sequence	Length of polypeptide	Seq ID No.
					1	HBV_01	FLLTRILT	8	10

1.3 tumor-associated antigen screening

Meanwhile, in order to compare the immunogenicity of the predicted new antigen polypeptide with the immunogenicity of the tumor highly-expressed related antigen polypeptide, the present example also obtains the liver cancer highly-expressed related polypeptide sequences according to the reference literature, in the present example, 6 tumor highly-expressed related polypeptide sequences are obtained by reference, the polypeptide sequence with the best effect is selected as the control group test of the present example, and the polypeptide information is shown in table 3.

TABLE 3 Synthesis of information on liver cancer highly expressed related Polypeptides

Serial number	Name (R)	Amino acid sequence	Length of polypeptide	Seq ID No.
					1	VEGFR2-775	AMFFWLLLV	9	11

Tumor-associated antigen screening references are as follows:

Tada Y,Yoshikawa T,Shimomura M,Sawada Y,Sakai M,Shirakawa H,Nobuoka D,Nakatsura T:Analysis of cytotoxic T lymphocytes from a patient with hepatocellular carcinoma who showed a clinical response to vaccination with a glypican3derived peptide.Int J Oncol 2013,43(4):1019-1026.

Kan Z,Zheng H,Liu X,Li S,Barber TD,Gong Z,Gao H,Hao K,Willard MD,Xu J et al:Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma.Genome Res 2013,23(9):1422-1433.

Loffler MW,Mohr C,Bichmann L,Freudenmann LK,Walzer M,Schroeder CM,Trautwein N,Hilke FJ,Zinser RS,Muhlenbruch L et al:Multi-omics discovery of exome-derived neoantigens in hepatocellular carcinoma.Genome Med 2019,11(1):28.

Schulze K,Nault JC,Villanueva A:Genetic profiling of hepatocellular carcinoma using next-generation sequencing.J Hepatol 2016,65(5):1031-1042.

Meyerson M,Gabriel S,Getz G:Advances in understanding cancer genomes through second-generation sequencing.Nat Rev Genet 2010,11(10):685-696.

2. immunogenicity assays

After synthesis of polypeptides having the sequences shown in tables 1, 2 and 3, by entrustochem bioengineering (shanghai) gmbh PBMC cells were purchased, in this example from mitsunotong biotechnology limited, product number: PER-F, and sorting CD8 positive T cells and DC cells by using specific magnetic beads and an MS column. Wherein, the specificity magnetic bead specifically is, the magnetic bead and the magnetic force frame of thermo fisher, the magnetic bead name: dynabeads CD8, cat No.: 11147D; magnetic bead name: dynabeads FlowComp Human CD14 Kit, cat No.: 11367D; magnetic bead separator, name: dynaMag-2Magnet, cat #: 12321D; specificity refers to magnetic beads positive for the CD8 target and magnetic beads positive for DC cells (CD 14+ magnetic beads). After sorting, IL-4 and GM-CSF cytokines are added to culture DC cells, and corresponding cytokines IL-4, GM-CSF, LPS and IFN-gamma are added to stimulate maturation for 24h, and then the maturation is stimulated by the target polypeptides synthesized above respectively. The DC cells were co-cultured with CD 8-positive T cells for one week, specifically, IL-21 for 72 hours, followed by culture with IL-2, IL-7, and IL-15. And collecting and carrying out EliSpot assay detection after CD8 positive T cells are stimulated to mature, observing whether the polypeptide has immunogenicity or not through the condition that secreted IFN-gamma generates purple spots, and carrying out AID reading on the EliSpot plate. The specific experimental steps are as follows:

2.1 sorting of DCs and T cells

(1) Collecting 50-100mL of HLA-A0201 typed healthy human Peripheral blood, resuspending with PBS, adding Ficoll separating medium with corresponding volume, centrifuging with density gradient, sucking out mononuclear cell layer withbase:Sub>A pipette, transferring tobase:Sub>A centrifuge tube, resuspending with sterile PBS, centrifuging, washing repeatedly for one time, removing redundant Ficoll separating medium and platelets to obtain Peripheral Blood Mononuclear Cells (PBMCs), and freezing for later use;

(2) Take 50X 10 ⁶ Peripheral blood mononuclear cells, resuscitatedWashing with 1640 culture medium, centrifuging at 300g rotation speed for 5min, counting, and incubating at 37 deg.C for 6-12h with 1640 culture medium and 10% FBS;

(3) After incubation, the cells were centrifuged at 300g for 5min and resuspended in a MACS buffer and counted. Adding corresponding CD14 magnetic beads and MACS buffer according to the quantity, incubating for 15min at 4 ℃, and shaking once every 5min; wherein the addition amount of the magnetic beads is 20 μ L/10 ⁷ cells, MACS buffer was added in an amount of 80. Mu.L/10 ⁷ cells；

(4) Taking out and washing with 20mL of MACS buffer for one time, centrifuging at 300g for 10min, resuspending with MACS, passing through MC column, rinsing MC column with 1mL of MACS once, filtering to obtain CD14 negative cells, washing old tube with 1mL of MACS once after cell suspension filtration, rinsing column with 1mL of MACS twice, taking off MC column, rinsing CD14 positive cells (DC cells) with piston, and counting 1.722 × 10 ⁷ Half of the cells are cultured in GM-CSF medium containing IL-4+86ng/mL as the result of the concentration of FBS +10ng/mL by CG-DC +5, and half of the cells are frozen in CS10 frozen stock solution;

(5) Counting the CD14 negative cells, adding corresponding volumes of CD8 magnetic beads and MACS buffer, incubating for 15min at 4 ℃, and shaking once every 5min; wherein the dosage of CD8 magnetic beads is 20 μ L/10 ⁷ cells, MACS buffer was added in an amount of 80. Mu.L/10 ⁷ cells；

(6) Taking out, washing with 20mL of MACS buffer, centrifuging at 300g for 10min, resuspending with MACS, passing through MC column, rinsing MC column with 1mL of MACS, filtering to obtain CD8 negative cells, washing the old tube with 1mL of MACS after cell suspension filtration, rinsing column with 1mL of MACS twice, rinsing CD8 positive cells (T cells) with piston, and counting to 3.42 × 10 ⁶ And freezing and storing.

2.2 culture of DCs and T cells

(1) D1, DC cell count 1.722 × 10 after magnetic bead sorting ⁷ One half of the cells were frozen and one half of the cells were cultured, resuspended using CG-DC +5% FBS +10ng/mL of IL-4+86ng/mL GM-CSF medium, 1 mL/well, cultured in 12-well plates for a total of 12 wells, with the number of cells in each well being 5X 10 ⁵ Culturing for 48h per well;

(2) D3, supplementation of CG-DC +5% FBS +20ng/mL per well of IL-4+176ng/mL GM-CSF medium 500. Mu.L, i.e., of medium supplemented with 1/2 of the original volume and at double cytokine concentration; continuously culturing for 48h;

(3) D5, configuring CG-DC +5% FBS +10ng/mL IL-4+86ng/mL GM-CSF +10ng/mL LPS +10ng/mL IFN-gamma culture medium, and fully changing the culture medium; specifically, centrifuging at a rotating speed of 300g for 10min, removing the original culture medium, resuspending by using the prepared culture medium, and performing mature culture for 24h; resuscitate T cells simultaneously, resuspend in FBS +10ng/mL IL-7 medium using CG-DC + 5%;

(4) D6, loading the DC cells with the corresponding target polypeptide, in this case, 10 mu g/mL,37 ℃ and CO ₂ Culturing in an incubator for 16h;

2.3 Co-culture of DCs and T cells

(5) D7, after the loading of the polypeptide is finished, co-culturing the DC cells and the T cells, wherein the number of the DC cells is more than that of the DC: t = 1:4-1:8, volume ratio 1:1, the culture density is 1.0-1.5 × 10 ⁶ cells/cm ² (ii) a At this time, 30ng/mL IL-21 was added and the culture was continued for 72h;

(6) D10, adding 10ng/mL IL-2, 10ng/mL IL-7 (10 ng/mL) and 10ng/mL IL-15 after 3 days, and culturing for 48-72h;

(7) D12 or D13, half the volume of the solution is changed, and the solution is supplemented with the cytokines of IL-2 of 10ng/mL, IL-7 of 10ng/mL and IL-15 of 10ng/mL, so as to ensure that the cell density is 1 multiplied by 10 ⁶ about/mL, culturing for 48-72h;

(8) D14 or D16, total volume exchange solution, and cytokine IL-2 10ng/mL, IL-7 10ng/mL, and IL-15 10 ng/mL.

2.4 Enzyme-linked Immunospot Assay (ELISPOT, enzyme-linked Immunospot Assay)

(1) D20, resuscitating HLA-0201 typed T2 cells (purchased from ATCC) -T02-0201 cells (IMDM +10% FBS), centrifuging at 300g rpm for 5min, and counting;

(2) D22, collecting T02-0201 cells, centrifuging for 5min at the rotating speed of 300g, counting the cells, resuspending the cells by serum-free IMDM, adding 1.5mL of EP tubes, respectively loading corresponding target polypeptides, wherein the concentration of the polypeptides is 10 mu g/mL, and taking the polypeptides as an experimental group; setting a group as a negative control group independently, namely adding DMSO into T02-0201 cells, wherein the volume of DMSO is consistent with that of polypeptide, and the temperature is 37 ℃ for 4h;

(3) After 4h, the polypeptide-loaded T02-0201 cells were harvested and resuspended in CG-DC +2% FBS medium adjusted to a cell concentration of 5X 10 ⁴ Per mL;

(4) PBS wash the elispot plate 5 times, 150. Mu.L/well, every 5min, after which IMDM +10 FBS, 100. Mu.L/well, 37 ℃,30min;

(5) Taking the T cell sample to be detected, resuspending with CG-DC +2% FBS medium, adjusting cell concentration to 2X 10 ⁵ Per mL;

(6) Taking out an elispot plate, beating liquid off a clean paper towel, adding T02 and T cells into a hole of 100 mu L/hole respectively according to the plate paving sequence, simultaneously adding a positive control group OKT3 and a negative control group, and carrying out heating at 37 ℃ for 16-20h;

(7) D23, removing cells from the plate, washing the elispot plate 5 times with PBS, 150 μ L/well, every 5min, adding the prepared 7-b6-1-ALP antibody, 100 μ L/well, incubating for 2h at 37 ℃, preparing PBS +0.5% FBS, adding antibody 200, 0.22 μm filtration; among them, the 7-b6-1-ALP antibody kit was purchased from mabtech, under the name: human IFN-. Gamma.ELISpot kit (ALP), cat #: 3420-2APW-2, antibody concentration consistent with kit instructions, 1;

(8) Removing primary antibody, washing the elispot plate with PBS for 5 times at 150 muL/hole, washing once every 5min, adding NBT/BCIP, developing in dark for 2-8min, and at 100 muL/hole;

(9) The observation was that a large amount of tap water was flushed.

(10) And (4) drying by using an oven, counting the purple spots of the pore plates by using an AID (AID identification) reader, and sorting and counting.

In the above test procedures, "D" represents the number of days, for example, "D1" represents the first day, and is the number of cells counted from DC 1.722X 10 ⁷ Half of the total number was used for the number of days counted from the start of the culture.

The AID reading plate results are shown in fig. 2, the first column is a neoantigen polypeptide screening group TGIF2LX, the second column is a neoantigen polypeptide screening group OR2G2, the third column is a virus-associated antigen polypeptide group HBV _01, and the fourth column is a tumor highly expressed antigen polypeptide group VEGFR2-775; rows 1 to 3 are 3 replicates of each experimental group, and rows 4 to 6 are 3 replicates of the negative control group.

The results in fig. 2 show that more purple spots can be observed in the neoantigen polypeptide screening group TGIF2LX, indicating that the antigen polypeptide has higher immunogenicity and is statistically significant.

3. Antigen-loaded polypeptide CTL positive rate test

In this example, the subsequent test was conducted using the more immunogenic TGIF2LX antigen polypeptide, and in order to evaluate that the selected neoantigen polypeptide can pass through antigen presenting cells such as DC, present antigen peptides to CD8 positive T cells, and activate to produce corresponding specific CTL cells, the test of positive rate of detection of antigen specific CTL cells by corresponding tetramer staining was conducted. In this example, a tetramer displacement kit (QuickSwitchTM, PE staining) from Beijing Bourmeyer Biotechnology, inc. (MBL Biotech) was purchased and tested as follows:

3.1 tetramer Displacement experiments

(1) Preparation of 2mg/mL (2 mM) replacement with the polypeptide solution of interest and the positive polypeptide standard in DMSO:

(2) Add 50. Mu.L of tetramer to round bottom 96-well plate;

(3) Adding 1 mu L of the polypeptide solution prepared in the step (1), and uniformly mixing by using a pipettor;

(4) Adding 1 mu L of polypeptide replacement factor, and uniformly mixing by using a pipettor;

(5) Incubating for 4h at room temperature in the dark;

(6) Then stored at 4 ℃ in the dark.

3.2 detection of tetramer displacement efficiency:

(1) 20 mu L of capture magnetic beads are added into the hole 1-5 of the 96-hole plate with the conical bottom;

(2) mu.L of 1 Xdetection buffer was added to

wells

2,5. Mu.L of unsubstituted tetramer to

wells

1 and 3, and the displaced tetramer was added to

wells

4 and 5;

(3) Wrapping the 96-well plate with tinfoil, placing the 96-well plate on a flat plate shaker, and oscillating at 550rpm for 45min at room temperature;

(4) After oscillation, adding 1 Xdetection buffer solution into the holes 1-5, washing 150 μ L per hole, planting the 96-well plate on a magnetic plate, standing for 5min, discarding the supernatant after standing, keeping the 96-well plate on the magnetic plate, stirring for 2 seconds by using a vortex mixer, and then taking the 96-well plate off the magnetic plate;

(5) Add 1 Xantibody-FITC (antibody specific for the polypeptide in the tetramer of the kit) 25. Mu.L each into well 2-5, add 1 Xdetection buffer into well 1;

(6) Wrapping the 96-well plate with tinfoil, placing the 96-well plate on a flat plate shaker, and oscillating at 550rpm for 45min at room temperature;

(7) After oscillation, adding 1 Xdetection buffer solution into the holes 1-5, washing 150 μ L per hole, planting the 96-well plate on a magnetic plate, standing for 5min, discarding the supernatant after standing, keeping the 96-well plate on the magnetic plate, stirring for 2 seconds by using a vortex mixer, and then taking the 96-well plate off the magnetic plate;

(8) Adding 1 Xdetection buffer solution into the hole 6, adding 5 mu L of capture magnetic beads, and mixing uniformly;

(9) All the wells were transferred to a flow tube and placed on ice, and then the fluorescent intensity of the polypeptide in the tetramer of the kit was detected by an up-flow cytometer, and the substitution efficiency of the polypeptide was calculated, and the results are shown in fig. 3, fig. 4 and table 4.

TABLE 4 replacement efficiency test results

In Table 4, FITC is the mean fluorescence intensity of the displaced prototetramer. FIG. 3 is a graph showing the results of flow-through fluorescence, wherein the first column shows the results of the unsubstituted polypeptide, the second column shows the results of the positive polypeptide, and the third column shows the results of the nascent polypeptide TGIF2 LX. Fig. 4 is a standard curve of the displacement efficiency, wherein the left graph is a specific value and the right graph is a standard curve.

The results in FIG. 3, FIG. 4 and Table 4 show that the replacement efficiency of the nascent polypeptide TGIF2LX of this example is about 95.42%; according to the standard of the kit, if the replacement rate is more than 75%, the result is positive; thus, the nascent polypeptide TGIF2LX of this example was successfully displaced and the tetramer was used for subsequent detection.

3.3 Positive Rate detection of antigen-specific cytotoxic T cells:

(1) Collection of mature effector cells 2X 10 ⁶ Centrifuging the mixture in a 1.5mL centrifuge tube at 400g for 5min;

(2) Discard the supernatant and resuspend the cells using PBS (0.5% FBS), 200. Mu.L per tube;

(3) mu.L of tetramer-PE and CD8 surface antibody-FITC (1;

(4) After the incubation was completed, 400. Mu.L each of PBS (0.5% FBS) was added to each tube for washing, and centrifuged at 400g for 5min;

(5) Adding PBS (0.5% FBS) into each tube, washing with 400 μ L, and centrifuging at 400g for 5min;

(6) 300 μ L of PBS (0.5% FBS) was added per tube, resuspended, transferred to flow tubes, placed on ice, and the cells were then examined for fluorescence intensity on an up-flow cytometer, the Q2 quadrant being positive for antigen-specific cytotoxic T cells, the results are shown in FIG. 5.

FIG. 5 is a diagram showing the results of analysis of the CTL positive cell rate induced by detecting neoantigen peptide by flow cytometry, wherein the left diagram "Control" is a Control group, and the right diagram is a neoantigen peptide TGIF2LX test group. The results in fig. 5 show that T cells stimulated with TGIF2LX antigenic polypeptide antigen in this example detected 1.21% of positive tumor specific T cells with tetramers, and a positive population in Q2 quadrant. Correspondingly, the control group was only 0.075%.

The CTL positive rate of the existing liver cancer antigen polypeptide after induction is lower than 0.5 percent, and even CTL which can be detected by flow cytometry cannot be induced. The results in FIG. 5 show that the positive rate of the liver cancer-specific cytotoxic T lymphocytes induced by the TGIF2LX antigenic polypeptide of this example is significantly higher than that of CTL induced by the existing liver cancer antigenic polypeptide.

4. Test of killing ability of liver cancer specific CTL in vitro cell

In order to evaluate the in vitro cell-killing ability of the selected neo-antigenic peptides to induce CTLs, the following experimental procedures were performed.

Selecting the newborn antigen polypeptide with immunogenicity screened in the EliSpot Assay experiment, and adopting a lactate dehydrogenase detection Kit (LDH Assay Kit) to perform a target cell killing performance test. Lactate Dehydrogenase (LDH) is one of the cytosolic enzymes of living cells and is normally impermeable to the cell membrane. When target cells T2 are attacked and damaged by effector cells CTL, the permeability of cell membranes is changed, LDH can be released into a medium, the released LDH enables oxidized coenzyme I (NAD +) to be changed into reduced coenzyme I (NADH 2) in the process of catalyzing lactic acid to generate pyruvic acid, the latter reduces iodonitronitronitrocloazodicarbonyl blue (INT) or Nitrocloazodicarbonyl Blue (NBT) through hydrogen transfer body-phenazine dimethyl sulfate (PMS) to form a colored formazan compound, a high absorption peak is formed at 490nm wavelength, OD value read by an enzyme labeling instrument is used, and the activity of the target cells can be obtained through calculation, so that the killing performance of antigen polypeptide is detected. The specific operation steps are as follows:

(1) Taking T2 cells, counting, and carrying out resuspension by serum-free IMDM, wherein an experimental group is loaded with screened newborn antigen polypeptide, a control group is loaded with DMSO with the same volume, the concentration of the polypeptide is 10 mu g/mL, the polypeptide is loaded for 4 hours at 37 ℃;

(2) After 4h, the T2 cells were centrifuged and the T2 resuspended in CG-DC +5 FBS medium and adjusted to a concentration of 2X 10 ⁵ Per mL; wherein, CG-DC is purchased from CellGenix, name:

GMP DC Medium, good number: 20801-0500;

(3) Effector cells were taken and cultured and diluted according to the plating protocol indicated in table 5 with an effective target ratio of 5:1 (E: T = 5:1); the medium was CG-DC +5% FBS;

TABLE 5LDH plating Condition

In Table 5, "(1)" indicates the experimental group (50. Mu.L of effector cells + 50. Mu.L of target cells), "(2)" indicates the effector cells naturally released (50. Mu.L of effector cells + 50. Mu.L of medium), "(3)" indicates the target cells naturally released (50. Mu.L of target cells + 50. Mu.L of medium), "(4)" indicates the target cells maximally released (50. Mu.L of target cells + 50. Mu.L of medium + 50. Mu.L of lysate), "(5)" indicates the volume correction (110. Mu.L), "(6)" indicates the culture control (100. Mu.L).

(4) Plating with 96-well plate with pointed bottom or round bottom, and final volume of all wells is 100 μ L;

(5) 96 well plates at 37 ℃ C. 5% CO ₂ Culturing for 4 hours in an incubator;

(6) Adding 10 mu L of lysine buffer to the target cell maximum release group and the volume correction group when culturing for 3.5 h;

(7) Centrifuging a 96-well plate after incubation is finished, and centrifuging for 4min at 250 g;

(8) Taking 50 mu L/well of the supernatant to a flat-bottom 96-well plate for detection, and avoiding bubbles;

(9) Adding 50 mu L/hole of Substrate Mix, and preserving at-20 ℃ after the use;

(10) Reacting for 30min at room temperature in a dark place;

(11) Adding 50 mu L/hole Stop solution, and lightly beating and uniformly mixing to avoid bubbles;

(12) In the inspection hole, the bubble is punctured by a syringe, and the absorbed light is read on a full-wavelength reading instrument at 490nm to derive the original data.

Calculated% kill:

(13) All experimental and control groups should be subtracted with the background mean.

(14) Corrected values were used for the killing efficiency calculation, cell killing rate (%) = [ (experimental group release-effector cell spontaneous release-target cell spontaneous release)/(target cell maximum release-target cell spontaneous release) ] × 100%.

The results of killing rate are shown in FIG. 6, the abscissa "TGIF2LX" represents CTLs induced by TGIF2LX antigen polypeptide, and "DMSO" represents CTLs induced by negative control in which DMSO was equally substituted for TGIF2LX antigen polypeptide; the ordinate represents cytotoxicity, i.e. killing rate.

The results in FIG. 6 show that CTL cells induced and stimulated by the selected neoantigen polypeptide TGIF2LX have in vitro cell killing ability, and the killing efficiency is 76% when the E: T is 5:1; the background killing rate of general T cells is lower than 10%, the DMSO group is a negative control group, and the killing rate of the T cell background group without loaded polypeptide is lower than 5%. As can be seen, the liver cancer specific CTL induced by TGIF2LX in this example has strong killing ability in vitro.

The test results show that the TGIF2LX antigen polypeptide with the amino acid sequence shown in Seq ID No.8 obtained in the embodiment can efficiently induce the liver cancer specific cytotoxic T lymphocyte, and the positive rate of the induced liver cancer specific cytotoxic T lymphocyte in immunogenicity, antigen-loaded cytotoxic T lymphocyte and the killing rate of the induced liver cancer specific cytotoxic T lymphocyte in vitro experiments are superior to those of the existing antigen polypeptide and CTL induced by the antigen polypeptide, and the clinical application value is very high.

The results of the specificity and function prediction and analysis of the TGIF2LX antigen polypeptide of the present example by using protein analysis software and antigen polypeptide prediction software show that the antigen polypeptides of the sequence shown in Seq ID No.1 have the same specificity and function as the TGIF2LX antigen polypeptide of the present example, namely, can efficiently induce liver cancer specific cytotoxic T lymphocytes, and the induced liver cancer specific cytotoxic T lymphocytes are superior to the existing antigen polypeptides and CTL induced by the same in immunogenicity, positive rate of antigen-loaded cytotoxic T lymphocytes and killing rate of the cells in vitro experiments.

Seq ID No.1：FLXLVDATV

Wherein, X is selected from any one of alanine, leucine, isoleucine, methionine, cysteine and threonine. When X is leucine, this is the TGIF2LX antigenic polypeptide of this example.

Accordingly, the nucleic acid sequences of the antigenic polypeptides where X is alanine, leucine, isoleucine, methionine, cysteine and threonine are the sequences shown in Seq ID No.2 to Seq ID No.7,

Seq ID No.2：5’-TTCCTGGCACTAGTCGATGCAACAGTA-3’

Seq ID No.3：5’-TTCCTGCTGCTAGTCGATGCAACAGTA-3’

Seq ID No.4：5’-TTCCTGATCCTAGTCGATGCAACAGTA-3’

Seq ID No.5：5’-TTCCTGATGCTAGTCGATGCAACAGTA-3’

Seq ID No.6：5’-TTCCTGTGTCTAGTCGATGCAACAGTA-3’

Seq ID No.7：5’-TTCCTGACACTAGTCGATGCAACAGTA-3’。

the antigen polypeptide used in this example is an antigen polypeptide synthesized by entrusted organism company, and it is understood that, according to the conventional genetic engineering technique, the nucleic acid sequences of the sequences shown in Seq ID No.2 to Seq ID No.7 can be completely inserted into a plasmid vector having an expression function to form a gene expression vector, and then the gene expression vector is introduced into a corresponding host cell to form a recombinant cell; and culturing the recombinant cell to express the nucleic acid sequence to the antigen polypeptide of the embodiment, and extracting and purifying the polypeptide to obtain the antigen polypeptide of the embodiment.

In addition, on the basis of the liver cancer specific cytotoxic T lymphocyte prepared in the embodiment, the sequence information of a liver cancer specific TCR sequence can be obtained through high-throughput single-cell TCR full-length sequencing, a complete TCR sequence is obtained through TCR specific primer amplification, the TCR sequence is inserted into a gene transduction vector, such as lentivirus, and the TCR sequence is transduced into a T cell through a gene transduction technology, so that a liver cancer specific T cell antigen receptor T cell, namely a liver cancer specific TCR-T cell, can be obtained.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

SEQUENCE LISTING

<110> Shenzhen Letu biomedicine Limited

<120> antigenic polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof

<130> 20I30206

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> X

<222> (4)..(4)

<223> A or L or I or M or C or T

<400> 1

Phe Leu Leu Xaa Val Asp Ala Thr Val

1 5

<210> 2

<211> 27

<212> DNA

<213> Artificial sequence

<400> 2

ttcctggcac tagtcgatgc aacagta 27

<210> 3

<211> 27

<212> DNA

<213> Artificial sequence

<400> 3

ttcctgctgc tagtcgatgc aacagta 27

<210> 4

<211> 27

<212> DNA

<213> Artificial sequence

<400> 4

ttcctgatcc tagtcgatgc aacagta 27

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence

<400> 5

ttcctgatgc tagtcgatgc aacagta 27

<210> 6

<211> 27

<212> DNA

<213> Artificial sequence

<400> 6

ttcctgtgtc tagtcgatgc aacagta 27

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence

<400> 7

ttcctgacac tagtcgatgc aacagta 27

<210> 8

<211> 9

<212> PRT

<213> Artificial sequence

<400> 8

Phe Leu Leu Leu Val Asp Ala Thr Val

1 5

<210> 9

<211> 11

<212> PRT

<213> Artificial sequence

<400> 9

Ile Leu Leu Gly Phe Ser Asp Tyr Leu Gln Leu

1 5 10

<210> 10

<211> 8

<212> PRT

<213> Artificial sequence

<400> 10

Phe Leu Leu Thr Arg Ile Leu Thr

1 5

<210> 11

<211> 9

<212> PRT

<213> Artificial sequence

<400> 11

Ala Met Phe Phe Trp Leu Leu Leu Val

1 5

Claims

1. An antigenic polypeptide for inducing hepatoma-specific cytotoxic T lymphocytes, characterized in that: the amino acid sequence of the antigen polypeptide is a sequence shown in Seq ID No.8,

Seq ID No.8：FLLLVDATV。

2. a nucleic acid encoding the antigenic polypeptide of claim 1.

3. The nucleic acid of claim 2, wherein: the nucleic acid is shown as a sequence of Seq ID No.3,

Seq ID No.3：5’-TTCCTGCTGCTAGTCGATGCAACAGTA-3’。

4. a gene expression vector characterized by: the gene expression vector is inserted with a foreign gene capable of encoding and expressing the antigenic polypeptide of claim 1.

5. The gene expression vector of claim 4, wherein: the exogenous gene is the nucleic acid of claim 2 or 3.

6. A recombinant cell, wherein: the recombinant cell contains the gene expression vector of claim 4 or 5.

7. Use of the antigenic polypeptide of claim 1 or the nucleic acid of claim 2 or 3 for the preparation of antigen presenting cells inducing hepatoma-specific cytotoxic T lymphocytes.

8. Use of the antigenic polypeptide of claim 1 or the nucleic acid of claim 2 or 3 for the preparation of hepatoma-specific cytotoxic T lymphocytes.

9. The use of the antigenic polypeptide of claim 1, or the nucleic acid of claim 2 or 3, or the gene expression vector of claim 4 or 5, or the recombinant cell of claim 6, or an antigen presenting cell that induces a liver cancer-specific cytotoxic T lymphocyte, or a liver cancer-specific T cell antigen receptor T cell in the preparation of a medicament for treating or preventing liver cancer, or in the preparation of a reagent for detecting or screening liver cancer;

the antigen presenting cells for inducing hepatoma-specific cytotoxic T lymphocytes are obtained by stimulating the antigen presenting cells with the antigen polypeptide of claim 1, or the antigen presenting cells are obtained by introducing the nucleic acid of claim 2 or 3 into the antigen presenting cells so as to express the antigen polypeptide of claim 1, thereby obtaining the capacity of inducing hepatoma-specific cytotoxic T lymphocytes;

the liver cancer-specific cytotoxic T lymphocyte is the antigen polypeptide of claim 1 or the liver cancer-specific cytotoxic T lymphocyte induced by the antigen presenting cell for inducing the liver cancer-specific cytotoxic T lymphocyte;

the liver cancer specific T cell antigen receptor T cell contains a liver cancer antigen TCR sequence, the liver cancer antigen TCR sequence is obtained by single cell TCR full-length sequencing of the liver cancer specific cytotoxic T lymphocyte, and the liver cancer specific T cell antigen receptor T cell is formed by transducing the liver cancer antigen TCR sequence to a T cell through a gene transduction vector.

10. A kit for preparing liver cancer specific cytotoxic T lymphocytes is characterized in that: the kit comprises at least one component of the components (a) to (e);

(a) The antigenic polypeptide of claim 1;

(b) The nucleic acid of claim 2 or 3;

(c) The gene expression vector of claim 4 or 5;

(d) The recombinant cell of claim 6;

(e) Inducing an antigen presenting cell of hepatoma-specific cytotoxic T lymphocyte, wherein said antigen presenting cell of hepatoma-specific cytotoxic T lymphocyte is obtained by stimulating said antigen presenting cell with the antigen polypeptide of claim 1, or introducing the nucleic acid of claim 2 or 3 into said antigen presenting cell so that said antigen presenting cell can express the antigen polypeptide of claim 1, thereby obtaining the ability to induce hepatoma-specific cytotoxic T lymphocyte.

11. A pharmaceutical composition for treating or preventing liver cancer, characterized in that: the pharmaceutical composition comprises a pharmaceutically acceptable carrier and at least one component of the components (a) to (h);

(a) The antigenic polypeptide of claim 1;

(b) The nucleic acid of claim 2 or 3;

(c) The gene expression vector of claim 4 or 5;

(d) The recombinant cell of claim 6;

(e) Inducing antigen presenting cells of hepatoma-specific cytotoxic T lymphocytes, wherein the antigen presenting cells of hepatoma-specific cytotoxic T lymphocytes are obtained by stimulating the antigen presenting cells with the antigenic polypeptide of claim 1, or introducing the nucleic acid of claim 2 or 3 into the antigen presenting cells so that the antigen presenting cells can express the antigenic polypeptide of claim 1, thereby obtaining the ability to induce hepatoma-specific cytotoxic T lymphocytes;

(g) A hepatoma-specific cytotoxic T lymphocyte induced by the antigenic polypeptide of claim 1 or the hepatoma-specific cytotoxic T lymphocyte-inducing antigen-presenting cells;

(h) The T cell comprises a liver cancer antigen TCR sequence obtained by full-length sequencing of a single cell TCR of the liver cancer specific cytotoxic T lymphocyte, and the liver cancer specific T cell antigen receptor T cell is formed by transducing the liver cancer antigen TCR sequence to a T cell through a gene transduction carrier.

12. A kit for inducing an immune response against liver cancer, characterized by: the kit comprises at least one component of the components (a) to (h);

(a) The antigenic polypeptide of claim 1;

(b) The nucleic acid of claim 2 or 3;

(c) The gene expression vector of claim 4 or 5;

(d) The recombinant cell of claim 6;

(g) A hepatoma-specific cytotoxic T lymphocyte, said hepatoma-specific cytotoxic T lymphocyte being a hepatoma-specific cytotoxic T lymphocyte induced by the antigen polypeptide of claim 1 or the hepatoma-specific cytotoxic T lymphocyte-inducing antigen-presenting cells;

(h) The T cell comprises a liver cancer antigen TCR sequence, the liver cancer antigen TCR sequence is obtained by single cell TCR full-length sequencing of the liver cancer specific cytotoxic T lymphocyte, and the liver cancer specific T cell antigen receptor T cell is formed by transducing the liver cancer antigen TCR sequence to a T cell through a gene transduction vector.