Thymus dependent lymphocyte antigen epitope peptide of liver cancer-associated antigen AFP and application of thymus dependent lymphocyte antigen epitope peptide

Abstract

The invention discloses a thymus dependent lymphocyte antigen epitope peptide of a liver cancer associated antigen AFP and application, and belongs to the field of medical immunology and oncology. The invention discloses a thymus dependent lymphocyte antigen epitope peptide of a liver cancer associated antigen AFP. The amino acid sequence of the epitope peptide comprises any one or more of the following: KWVESIFLI; tFMNKFIYEI, fLYAPTILL is used as a template; kYIQESQAL is selected from the group consisting of the following components; the amino acid sequence is an amino acid sequence obtained by removing or replacing a single amino acid of the amino acid sequence. The epitope peptide disclosed by the invention can be specifically combined with cytotoxic thymus dependent lymphocytes to stimulate the cytotoxic thymus dependent lymphocytes to activate, proliferate and differentiate, so that an anti-tumor immune effect is exerted; the antigen peptides can be used for preparing therapeutic and prophylactic vaccines for liver cancer, can also be used for preparing quantitative detection reagents for detecting specific T cells of liver cancer related antigen AFP, and have potential application value in prevention, treatment and diagnosis of liver cancer.

Description

Technical Field

[0001] The invention relates to the fields of medical immunology and oncology, and in particular to a thymus-dependent lymphocyte antigen epitope peptide of liver cancer-related antigen AFP and application thereof. Background Art

[0002] The pathological type of primary liver cancer is mainly hepatocellular carcinoma (HCC), accounting for 85% to 90%. Traditional treatment methods and efficacy are limited and the overall prognosis is poor.

[0003] In my country, the high-risk groups for HCC mainly include those infected with hepatitis B virus (HBV) and / or hepatitis C virus (HCV), those with long-term alcoholism (alcoholic liver disease), non-alcoholic fatty hepatitis, those who eat food contaminated with aflatoxin, those with cirrhosis caused by various reasons, and those with a family history of liver cancer. At the same time, men over the age of 40 are at a higher risk. Recent studies have shown that diabetes, obesity, and smoking are also risk factors for HCC, which deserve attention.

[0004] Alpha-fetoprotein (AFP) is a common tumor-associated antigen that is overexpressed in liver cancer and has been widely studied in the diagnosis, treatment, and disease course monitoring of liver cancer. Serum AFP positivity refers to AFP ≥ 400 μg, and liver cancer is highly suspected when chronic or active hepatitis, cirrhosis, testicular or ovarian embryonic tumors, and pregnancy are excluded. Patients with a slight increase in AFP should be dynamically observed and compared with changes in liver function. About 30% of liver cancer patients have normal AFP levels, and alpha-fetoprotein isomers should be tested. This application uses the liver cancer-associated tumor antigen AFP for research.

[0005] Cytotoxic T lymphocytes (CTLs) are core cells that mediate adaptive immune responses and play a vital role in anti-infection, anti-tumor, hypersensitivity reactions and autoimmune diseases. The T cell receptors (TCRs) on their cell membranes can specifically recognize and bind to the complex of MHC class I molecules and antigenic peptides on the surface of antigen-presenting cells, i.e., MHC / antigen peptide complex molecules. CTL epitopes refer to antigenic peptides bound to MHC class I molecules. They are linear fragments or spatial conformational structures in antigen molecules that can be specifically recognized by TCRs. They are the basic antigenic units that cause immune responses and play a key role in the CTL activation process.

[0006] The MHC system refers to the major histocompatibility complex (MHC), which is a group of tightly linked genes in the vertebrate genome that encode and express MHC class I and class II protein molecules. HLA (human leukocyte antigen) is the human MHC system, responsible for presenting antigenic peptides (T cell epitope peptides) to T cells and initiating the body's specific immune response. Among them, HLA-A, B, C and other class I molecules present antigenic peptides to activate CD8 + T cells, differentiated into CTLs, are the main effector cells that target and kill tumor cells. However, HLA alleles are highly polymorphic in the population. HLA molecules are not exactly the same between different individuals, and the antigen peptide sequences that each HLA molecule can present are also very different, causing different strengths of T cell immune responses. Therefore, the same tumor antigen can cause different strengths of specific immune responses in different individuals. For human liver cancer, HLA class I molecules are mainly responsible for presenting endogenous liver cancer-related antigens to CD8 + CTL, activated CTLs secrete perforin and granzyme to cause apoptosis of tumor cells expressing tumor-associated antigens. + The number and function of T cells can accurately reflect the specific immune function status of liver cancer patients against liver cancer-related antigens. Due to the different HLA molecular types of different people, their processing, handling and presentation capabilities for different liver cancer antigens are also different, thus causing liver cancer-related antigen-specific T cell immune responses of different intensities. According to the different HLA molecular types of liver cancer patients, the antigen peptides of liver cancer-related antigens presented by them are selected to dynamically monitor the CD8 + The number and reactivity of T cells are of great significance for monitoring the disease progression, formulating diagnosis and treatment plans, observing efficacy, and judging the prognosis of liver cancer patients, and are important technical means to achieve precision medical treatment of liver cancer. At the same time, using the antigenic peptides of liver cancer-related antigens that these HLA-A molecules bind with high affinity, peptide vaccines or gene vaccines can also be prepared to prevent and treat liver cancer.

[0007] However, there are still very few AFP-specific T cell epitopes that have been identified and presented by various HLA molecules and can stimulate the body to induce T cell responses. This limits the AFP-specific T cell testing of liver cancer patients carrying different HLA alleles, the role of liver cancer AFP-specific T cells in the occurrence and development of liver cancer, and the personalized testing and precision immunotherapy based on individual differences in HLA genes and the differences in the liver cancer AFP antigen peptides they present. Summary of the invention

[0008] In view of the deficiencies of the prior art, the present invention proposes a thymus-dependent lymphocyte antigen epitope peptide of liver cancer-related antigen AFP and its application.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] The first aspect of the present invention relates to a thymus-dependent lymphocyte antigen epitope peptide of liver cancer-related antigen AFP, wherein the amino acid sequence of the epitope peptide comprises any one or more of the following:

[0011] KWVESIFLI;

[0012] TFMNKFIYEI;

[0013] FLYAPTILL;

[0014] KYIQESQAL;

[0015] The amino acid sequence above is an amino acid sequence after a single amino acid is removed or replaced.

[0016] The second aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of the liver cancer-related antigen AFP in the preparation of liver cancer vaccines.

[0017] The third aspect of the present invention relates to a nucleic acid molecule encoding the thymus-dependent lymphocyte antigen epitope peptide of the liver cancer-associated antigen AFP.

[0018] The fourth aspect of the present invention relates to a vector containing the above-mentioned nucleic acid molecule.

[0019] The fifth aspect of the present invention relates to a vaccine for treating or preventing liver cancer, comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0020] The sixth aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of the liver cancer-related antigen AFP in the preparation of a drug for treating or preventing liver cancer.

[0021] The seventh aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of liver cancer-associated antigen AFP in a kit for preparing liver cancer-associated antigen AFP T cells.

[0022] Optionally, the kit includes: the detection reagent is an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen polymer fluorescent staining or a flow cytometry reagent.

[0023] The eighth aspect of the present invention relates to a monoclonal antibody capable of specifically binding to the above-mentioned epitope peptide.

[0024] The ninth aspect of the present invention relates to a drug for treating liver cancer, comprising the above-mentioned monoclonal antibody.

[0025] More specifically, the present invention provides a novel thymus-dependent lymphocyte antigen epitope peptide of liver cancer-related antigen AFP, the amino acid sequence of which is any one of the epitope peptide sequences shown below:

[0026]

[0027] As can be seen from the above table, the above sequence is the antigen peptide sequence of alpha-fetoprotein (AFP), and it binds with high affinity to HLA2402, HLA-0207, HLA-3303, HLA-0203, HLA-0301, HLA-1101, HLA-0201, HLA-3001, HLA-B5101, HLA-B5201, HLA-B1301, HLA-B1302, HLA-B1501, HLA-C0401, HLA-C0702, HLA-C1402 and other molecules, forming "HLA / antigen peptide" complex molecules on the surface of antigen presenting cells, and binding to AFP antigen peptide-specific CD8 + T cell clones bind to stimulate their activation, proliferation and differentiation, and exert anti-liver cancer immune effector effects.

[0028] The thymus-dependent lymphocyte antigen epitope peptide sequence of liver cancer-related antigen AFP can be used to prepare liver cancer polypeptide vaccine or gene vaccine: polypeptide vaccine preparation: one or more polypeptides are artificially synthesized according to the polypeptide sequence of the present invention, mixed with adjuvant to form a soluble preparation, or loaded with biological nanomaterials to form a nano polypeptide vaccine, injected into the liver cancer patient, stimulate the activation and proliferation of AFP-specific T cells of liver cancer patients, and enhance their tumor-killing cell activity, thereby preparing a liver cancer polypeptide vaccine. Gene vaccine preparation: according to the polypeptide sequence of the present invention, a recombinant DNA gene fragment, recombinant plasmid or recombinant virus vector of one or more polypeptides is constructed, injected into the liver cancer patient, so that the recombinant gene expresses one or more polypeptides in vivo, stimulates the activation and proliferation of AFP-specific T cells of liver cancer patients, and enhances their tumor-killing cell activity, thereby preparing a liver cancer gene vaccine.

[0029] The thymus-dependent lymphocyte antigen epitope peptide sequence of liver cancer-related antigen AFP can be used to prepare a detection preparation or kit for detecting liver cancer-related antigen AFP-specific T cells: according to the polypeptide sequence of the present invention, one or more polypeptides are artificially synthesized, and in the enzyme-linked immunospot method, intracellular cytokine fluorescence staining method and enzyme-linked immunosorbent assay, they are used as antigen preparations, mixed cultured with peripheral blood mononuclear cells (PBMCs) of patients, stimulate AFP-specific T cells to activate, proliferate and secrete cytokines, and then detect the synthesis amount of the cytokine through other combined reagents, thereby reflecting the number and reactivity of AFP-specific T cells; these polypeptides can also be used to prepare human leukocyte antigen and polypeptide complexes (peptide-HLA complex) and multimers thereof through genetic engineering technology and protein engineering technology, and then further prepared into fluorescein-labeled preparations, and the number of AFP-specific T cells in the patient's peripheral blood mononuclear cell population is detected by flow cytometry. The related kit refers to a liver cancer-related antigen AFP-specific T cell detection kit assembled from the above preparations and other reagents commonly used in different detection methods.

[0030] The thymus-dependent lymphocyte antigen epitope peptide sequence of the liver cancer-related antigen AFP can be used to prepare drugs for treating liver cancer: the above-mentioned polypeptide vaccine or gene vaccine based on the polypeptide sequence of the present invention is combined with other immunotherapy preparations or chemotherapeutic preparations to prepare clinical drugs for treating liver cancer.

[0031] The present invention uses six online epitope prediction databases to virtually predict the AFP-specific antigen epitope peptide sequences of 13 HLA-A molecules, 15 HLA-B molecules, and 14 HLA-C molecules restricted liver cancer-related antigens, and obtains a set of epitope peptide sequences that can be respectively bound to HLA-A0201, HLA-A1101, HLA-A2402, HLA-A3101, HLA-A0206, HLA-A0207, HLA-A3 303. HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A0101, HLA-A2601, HLA-B4601, H LA-B4001, HLA-B5801, HLA-B1502, HLA-B5101, HLA-B1301, HLA-B1302, HLA-B1501, HLA-B54 01. HLA-B4006, HLA-B3501, HLA-B4403, HLA-B5201, HLA-B4801, HLA-B0702, HLA-C0102, HL A-C0602, HLA-C0702, HLA-C0801, HLA-C0304, HLA-C0302, HLA-C0303, HLA-C0401, HLA-C14 02, HLA-C1202, HLA-C1502, HLA-C1403, HLA-C1203, and HLA-C0701 molecules showed high affinity binding to the liver cancer-related antigen AFP epitope peptide sequence, and then its immunogenicity was verified by intracellular cytokine staining (ICS) functional experiments, providing specific antigen peptides for the preparation of liver cancer treatment and preventive vaccines and the development of liver cancer AFP-specific T cell detection reagents.

[0032] 1. Select the alpha-fetoprotein (AFP) amino acid sequence as the targeting sequence;

[0033] 2. Select six epitope prediction databases with high accuracy and widely recognized by researchers: SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC and EPIJEN to predict the AFP antigen epitope peptide sequences restricted by the above 13 HLA-A molecules, 15 HLA-B molecules and 14 HLA-C molecules;

[0034] 3. According to certain prediction criteria, the prediction results of six online epitope prediction websites were integrated and analyzed to obtain candidate antigen peptide sequences with relatively consistent prediction results from the six websites.

[0035] 4. The immunogenicity of liver cancer-related antigen peptide AFP was verified through IFN-γICS cell functional experiment.

[0036] 5. The peptide competition binding experiment of HLA-A / B / C molecules was carried out by using the HMy2.CIR cell line expressing specific HLA molecules to analyze the binding force between positive epitope peptides and specific HLA-A / B / C molecules.

[0037] 6. The immunogenicity of positive epitope peptides in stimulating specific T cell activation and proliferation in vivo was verified by vaccinating HLA transgenic mice.

[0038] The present invention is an antigenic peptide sequence in alpha-fetoprotein (AFP) that can bind to HLA2402, HLA-0207, HLA-3303, HLA-0203, HLA-0301, HLA-1101, HLA-0201, HLA-3001, HLA-B5101, HLA-B5201, HLA-B1301, HLA-B1302, HLA-B1501, HLA-C0401, HLA-C0702, and HLA-C1402 molecules with high affinity and has immunogenicity; it also relates to a liver cancer polypeptide vaccine and a gene vaccine based on the above antigenic peptides, as well as a reagent and method for detecting liver cancer AFP-specific T cells based on the above antigenic peptides.

[0039] Compared with the prior art, the advantages of the present invention are as follows:

[0040] The HLA2402, HLA-0207, HLA-3303, HLA-0203, HLA-0301, HLA-1101, HLA-0201, HLA-3001, HLA-B5101, HLA-B5201, HLA-B1301, HLA-B1302, HLA-B1501, HLA-C0401, HLA-C0702, HLA-C1402 molecule-restricted liver cancer-associated antigen AFP-specific antigen epitope peptides obtained by online virtual prediction and functional experimental verification have not been reported before. These HLA-A / B / C molecules have not been reported to have restricted liver cancer-associated antigen peptides before. Therefore, these new antigen epitope peptide sequences will provide the key antigen components, namely, antigen epitope peptide sequences, required for the development of therapeutic and preventive peptide vaccines and gene vaccines for liver cancer, and the design of reagents and methods for detecting AFP-specific T cells for liver cancer. At the same time, these antigen epitope peptides also provide key antigen components for personalized testing and precision medicine for liver cancer patients with these specific HLA-A / B / C alleles. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Epitope peptides stimulate CD8 in peptide-PBMCs co-culture experiments + Flow cytometry detection of T cell activation; Figure 1 A is the CD3 + / CD8 + Flow cytometry analysis of IFN-γ secretion in T cell population after stimulation with specific epitope peptides; B is the CD3 + / CD8 + Statistical chart of the proportion of cells in the T cell population that secrete IFN-γ after being stimulated by specific epitope peptides. No. 1: epitope peptide No. 1; No. 2: epitope peptide No. 2; No. 3: epitope peptide No. 3; No. 4: epitope peptide No. 4

[0042] Figure 2 , Figure 3 Peptide competition binding assay to analyze the binding affinity of four AFP-positive T cell epitopes to specific HLA-A molecules. NC, negative control; 1: epitope peptide 1; 2: epitope peptide 2; 3: epitope peptide 3; 4: epitope peptide 4; The blue solid line and red solid line show the cell fluorescence peaks at 5μM and 15μM concentrations of the tested peptide, respectively, while the gray filled line represents the maximum fluorescence intensity of the cells when only FITC-reference peptide is present. The light green solid line in the NC graph represents the negative control background fluorescence of the cells in the peptide-free wells.

[0043] Figure 4 It provides a technical roadmap for epitope peptide prediction and screening, immunogenicity verification, peptide competition binding experiments, and epitope peptide immunization of transgenic mice.

[0044] Figure 5 Epitope peptides induced specific CD8 in HLA-A2 / DR1 transgenic mice + T cell response

[0045] A mixed polypeptide vaccine was prepared by combining 20 HLA-A0201-restricted positive epitope peptides with the adjuvant poly(I:C) and immunizing HLA-A2 / DR1 transgenic mice on days 0, 7, and 21, respectively. Seven days after the last immunization, the mouse spleen cells were taken and restimulated with PBS and each positive epitope peptide for 10 h, and then BFA / Monesion (to block cytokine release) was added to continue culturing for 6 h. Then, IFN-γ intracellular staining and flow cytometry analysis were performed to detect IFN-γ. + / CD3 + / CD8 + The frequency of T cells. Figure 5 A is a representative flow cytometry analysis; B is the IFN-γ + / CD3 + / CD8 + T cell frequency statistics. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] In some embodiments of the present invention, HLA-A0201, HLA-A1101, HLA-A2402, HLA-A3101, HLA-A0206, HLA-A0207, HLA-A3303, HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A0101, and HLA-A2601 molecules-restricted liver cancer-associated antigen AFP T cell epitopes are disclosed, and the sequences are screened and identified by the following steps:

[0048] 1Online virtual prediction of dominant T cell epitope peptides of 13 HLA-A molecules restricted liver cancer-associated antigen AFP

[0049] The liver cancer-related antigen protein: alpha-fetoprotein (AFP) was selected, and its amino acid sequence was obtained by searching the UniProt global protein resource database (the specific sequence refers to SEQ ID NO.1). Then, six commonly used epitope peptide prediction databases, including SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC, and EPIJEN, were used to predict HLA-B4601, HLA-B4001, HLA-B5801, HLA-B1502, HLA-B5101, HLA-B1301, HLA-B1302, HLA-B1501, HLA-B5401, HLA-B4006, HLA-B3501, HLA-B4403, HLA-B5201, and HLA-B The T cell epitope peptides for AFP antigen protein restricted by HLA-4801, HLA-B0702, HLA-C0102, HLA-C0602, HLA-C0702, HLA-C0801, HLA-C0304, HLA-C0302, HLA-C0303, HLA-C0401, HLA-C1402, HLA-C1202, HLA-C1502, HLA-C1403, HLA-C1203, and HLA-C0701 molecules were virtually predicted, wherein the SVMHC database includes two prediction algorithms, MHCPEP model and SYFEPITHI model; the IEDB database includes three methods, ANN, SMM, and ARB, and the ANN and SMM methods with better statistical significance are selected in this embodiment. The websites of these epitope peptide prediction databases are shown in Table 1.

[0050] The antigen binding groove of HLA class I molecules is closed at both ends, and the length of the antigen peptide accepted is 8-11 amino acid residues, among which 9 and 10 amino acids are the most common. Therefore, this embodiment mainly selects polypeptides with a length of 9 and 10 amino acids as the research object. The amino acid sequence of AFP is input into the corresponding amino acid sequence input box of the prediction database website, and then the length of the epitope peptide is selected as 9 and 10 amino acids respectively, and then a specific HLA-A / B / C molecule is selected to perform online virtual prediction of the T cell epitope peptide of the liver cancer-related antigen AFP.

[0051] Table 1 Epitope peptide prediction database website

[0052]

[0053] For each HLA-A / B / C molecule, the peptides predicted by different databases are arranged from high to low according to the scores, and then the epitope peptides that meet the scoring criteria of at least 4 prediction methods are selected as candidate epitope peptides. For each HLA-A / B / C molecule, 1-5 peptides with the highest scores (highest affinity) are selected from the candidate epitope peptides as epitope peptides to be identified. For the above 42 HLA-A / B / C molecules, a total of 112 dominant T cell epitope peptides to be identified were screened and predicted.

[0054] 2. Isolation of PBMC from peripheral blood of patients with liver cancer

[0055] Take fresh anticoagulated whole blood stored at room temperature and dilute it appropriately with sterile PBS; add 1 times the volume of original blood human lymphocyte separation solution (Dakoway Biotechnology, Shenzhen) to a 15 mL centrifuge tube; slowly spread the diluted blood on the separation solution and centrifuge at room temperature for 20 min at 2000 rpm; aspirate the mononuclear cell (PBMCs) layer and wash it twice by centrifugation; resuspend it with serum-free medium (Dakoway Biotechnology, Shenzhen), count the cells, and adjust the cell concentration to 3×10 6 / mL for future use.

[0056] 3. Identification of the immunogenicity of T cell epitope peptides of liver cancer-related antigen AFP by IFN-γICS method

[0057] Technical route such as Figure 3 As shown:

[0058] 4HLA-A / B / C allele typing

[0059] 200 μL of anticoagulated blood was collected from the patient, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Bio, Beijing). HLA-A locus-specific primers A1 and A3 were used for PCR to amplify the DNA sequences of exon 2, intron 2, exon 3, and part of introns 1 and 3 of the A locus, with a product size of 985 bp. The amplification conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s; 62°C annealing for 15 s; 72°C extension for 90 s; 35 cycles; 72°C extension for 5 min. The amplified product was identified by 1% agarose gel electrophoresis and sent to Shanghai Sunny Biotechnology Co., Ltd. for purification and bidirectional sequencing. PCR reagents were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0060] Table 2 HLA-A / B / C locus-specific PCR amplification primers

[0061]

[0062] The sequencing results of exon 2 and exon 3 were assembled into a complete HLA-A overlapping sequence (Contig) by using the Seqman software of the Lasergene program. The bases of the bidirectional sequencing were carefully checked to be complete and consistent. The bases of the heterozygotes were found and replaced with annexed bases, such as M for A and C, R for A and G, W for A and T, S for C and G, Y for C and T, and K for G and T. Finally, the sequence fragments of the amplified HLA-A alleles were determined. The Nucleotide BLAST tool was used to compare the assembled HLA-A base sequence with the exon 2 and exon 3 sequences of all HLA-A / B / C alleles in the database until a completely matched gene combination was obtained, thereby determining the HLA-A / B / C alleles.

[0063] 5. Peptide-PBMCs co-culture and IFN-γ intracellular staining experiment

[0064] PBMCs were separated by density gradient centrifugation and the concentration of PBMCs was adjusted to 3 × 10 6 / mL, inoculated into a U-bottom 96-well plate, 100 μL / well, and placed in a 37°C, 5% CO2 constant temperature incubator for standby use. According to the patient's HLA-A allele typing results, the corresponding candidate epitope peptides were co-cultured with the PBMCs in the above 96-well plate for 6 hours, and the Brefeldin ASolution / Monensin Solution (1000×) blocking agent was added for co-culture, and then IFN-γ intracellular fluorescence staining was performed, and flow cytometry was used to detect the CD8 secretion of IFN-γ. +T cell frequency; at the same time, a single culture well of PBMCs without peptide stimulation was set up as a negative control group. Compared with the negative control, IFN-γ + / CD8 + The candidate epitope peptides with a T cell frequency increased by more than 2 times were set as positive epitope peptides, that is, the epitope peptides could stimulate the memory CD8 + T cells become activated and secrete cytokines.

[0065] A total of 216 AFP samples were collected through IFN-γICS experiment. + PBMCs of HCC patients were co-cultured with relevant peptides from 112 candidate epitope peptides. Among them, the four epitope peptides in this example (sequences were KWVESIFLI, TFMNKFIYEI, FLYAPTILL, KYIQESQAL) induced strong CD8 + T cell response, IFN-γ expression in PBMCs of at least 3 HCC patients + / CD8 + The T cell frequency increased by more than 10 times, so the three candidate epitope peptides were determined to be positive epitopes and dominant epitope peptides with strong immunogenicity ( Figure 1 ).

[0066] 6. Peptide competition binding assay to analyze the affinity of candidate epitope peptides to corresponding HLA-A molecules

[0067] The 39 candidate epitopes, including the four positive epitope peptides No. 1, No. 2, No. 3, and No. 4 of this example screened in the above steps and verified by the peptide-PBMCs costimulation experiment, were assigned to the relevant epitope peptide queues of each HLA-A molecule according to the predicted HLA-A restriction and the HLA-A allele type of the patient with a positive T cell response, as peptides to be tested, and the peptide competition binding experiment was carried out with the fluorescent reference peptide of the HLA-A molecule. Finally, the epitope peptides No. 1, No. 2, No. 3, and No. 4 of this example all shifted the cell fluorescence peak to the left ( Figure 2 , Figure 3 ), indicating that these positive epitope peptides can compete with the fluorescent reference peptides for binding to the relevant HLA-A molecules on the HMy2.CIR cell membrane. These four positive epitope peptides all showed high or medium affinity binding to the corresponding HLA-A molecules (Table 3).

[0068] Table 3 Peptide competition binding assay analysis of the binding affinity of 4 AFP-positive T cell epitopes to 8 dominant HLA-A molecules, 5 dominant HLA-B molecules, and 3 dominant HLA-C molecules, as well as HLA-A / B / C cross-restriction analysis

[0069]

[0070] Immunization of HLA transgenic mice with 7 epitope peptides to verify their immunogenicity in inducing specific T cell responses in vivo

[0071] 20 epitope peptides restricted by HLA-A0201 molecules (including epitope peptides No. 1, 2, 3, and 4 in this example) were made into mixed polypeptides and then mixed with adjuvant poly I:C. HLA-A0201 / DR0101 transgenic mice were randomly divided into 2 groups, 3 mice in each group. The experimental group was inoculated with peptide cocktail / poly(I:C) (epitope peptide: 10μg / single peptide, poly(I:C): 100μg / mouse) on day 0, day 7, and day 21, respectively; the adjuvant group was also inoculated with poly(I:C) (100μg / mouse) on day 0, day 7, and day 21, respectively. Four subcutaneous injections were made (neck back, tail base, bilateral groins).

[0072] On the 28th day, mice were killed and spleen cell suspension was routinely prepared. The cell concentration was adjusted to 1×10 7 / mL, inoculated 48-well cell culture plates (0.5mL / well), each mouse spleen cell inoculation well was divided into 21 groups, and PBS (negative control) and each positive epitope peptide (including epitope peptides No. 1, 2, 3, and 4 in this example, 20μg / mL / peptide) were added respectively; after culturing for 10 hours, BFA and Monensin were added to each well and incubated for another 6h; then the cells in each well were collected, blocked with FcR Blocking Reagentmouse for 15 minutes, and then co-stained with FITC-anti-mouse CD3 and PE-anti-mouse CD8a for 30min; after washing, each tube was treated with a membrane permeabilizer, and finally APC-anti-mouse IFN-γ monoclonal antibody was added for intracellular staining for 30min; after washing, CD3 was analyzed by flow cytometry + / CD8 + / IFN-γ+T cell frequency, observe the specific CD8 induced by epitope peptide + The strength of the T cell response.

[0073] The results showed that the four positive epitope peptides (peptide cocktail / polyI:C group) in this example all induced strong epitope peptide-specific CD8 + T cell response. Compared with the adjuvant group, the IFN-γ induced by these three epitope peptides + / CD8 + The T cell frequency was 16.3-60.5 times that of the adjuvant group.

[0074] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0075] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A thymus-dependent lymphocyte antigen epitope peptide of liver cancer-related antigen AFP, characterized in that: The amino acid sequence of the epitope peptide includes any one or more of the following: KWVESIFLI; TFMNKFIYEI; FLYAPTILL; KYIQESQAL; The amino acid sequence above is an amino acid sequence after a single amino acid is removed or replaced.

2. Use of the thymus-dependent lymphocyte antigen epitope peptide of the liver cancer-related antigen AFP according to claim 1 in the preparation of liver cancer vaccines.

3. A nucleic acid molecule encoding a thymus-dependent lymphocyte antigen epitope peptide of the liver cancer-associated antigen AFP according to claim 1. A vector comprising the nucleic acid molecule according to claim 3.

5. A vaccine for treating or preventing liver cancer, characterized in that: Containing the nucleic acid molecule according to claim 3 or the vector according to claim 4.

6. Use of the thymus-dependent lymphocyte antigen epitope peptide of the liver cancer-related antigen AFP according to claim 1 in the preparation of a drug for treating or preventing liver cancer.

7. Use of the thymus-dependent lymphocyte antigen epitope peptide of liver cancer-associated antigen AFP according to claim 1 in a kit for preparing liver cancer-associated antigen AFP T cells.

8. The use of the thymus-dependent lymphocyte antigen epitope peptide of liver cancer-associated antigen AFP according to claim 1 in a kit for preparing liver cancer-associated antigen AFP T cells according to claim 7, characterized in that: The kit comprises: the detection reagent is an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen polymer fluorescent staining or a flow cytometric analysis reagent.

9. A monoclonal antibody, characterized in that Capable of specifically binding to the epitope peptide according to claim 1.

10. A drug for treating liver cancer, characterized in that: Comprising the monoclonal antibody according to claim 9.

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