An antigenic peptide library for inducing tumor-specific immune responses and its applications

Abstract

The present invention belongs to the technical field of biomedicine, and particularly relates to an antigen peptide library for inducing tumor-specific immune responses. The antigen peptide library comprises 8 antigen peptides, and their amino acid sequences are respectively shown as SEQ ID NO.1 to SEQ ID NO.8. The antigen peptides of the present application have strong immunogenicity. When loaded onto antigen-presenting cells, they can effectively stimulate and induce the generation of specific cytotoxic T lymphocytes. In vitro killing experiments show that CTL cells induced and activated by loading neoantigen DC have strong tumor-killing effects, and their cell killing activities are all above 90%. Adoptive transfer of CTL cells induced by the neoantigen peptide library into model mice can effectively reduce the tumor volume and significantly improve their survival rate, showing good therapeutic potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an antigen peptide library for inducing tumor-specific immune responses and its applications. Background Art

[0002] The anti-tumor response rate of immune checkpoint inhibitors (ICIs) treatment in cancer is relatively low. However, researchers have discovered and demonstrated that the immunotherapy of tumor neoantigens has good therapeutic effects by activating the body's own immunity to kill tumors with fewer side effects.

[0003] Based on the advantages of tumor specificity and immunogenetics, neoantigens can become new targets for tumor immunotherapy, including tumor vaccines, adoptive cell transfer therapy (ACT), and combination therapies based on neoantigen peptides. Neoantigens, namely tumor specific antigens (TSAs), are not expressed in normal tissues, are not easily immunotolerant, have strong immunogenicity, and the ability to stimulate specific cytotoxic T lymphocyte (CTL) cell responses. Neoantigens can be generated from tumors through various mechanisms, such as genomic mutations, abnormal transcriptional variants, post-translational modifications, and viral open reading frames. These antigens can be presented on the cell surface by the major histocompatibility (MHC) molecules of tumor cells and recognized and cleared by T cells. The discovery and application of neoantigen peptides are an important progress in the field of cancer treatment, providing new possibilities for personalized treatment. Therefore, in the development of cancer immunotherapy, it is crucial to predict and identify tumor-specific neoantigens.

[0004] The identification of neoantigen peptides has profound significance for tumor scientific research and clinical treatment. It not only provides new targets for tumor immunotherapy but also promotes the development of personalized and precision treatment, helping to improve the safety and effectiveness of treatment.

[0005] Liver cancer is a common malignant tumor with a high incidence and high mortality. The present invention discloses a neoantigen peptide library of Hepa1-6 tumor cell line and its applications. The antigen peptide library has a high affinity with MHC I molecules on DC cells and can effectively stimulate and induce the generation of specific cytotoxic T lymphocytes. The antigens of the present invention are convenient for large-scale synthesis and can be used for in vivo and in vitro scientific research in the field of oncology. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the present invention provides an antigen peptide library for inducing tumor-specific immune responses and its applications. The antigen peptide library has a high affinity for MHC class I molecules on DC cells, can effectively stimulate and induce the generation of specific cytotoxic T lymphocytes (CTLs), can be used for the immune clearance of tumor cells associated with liver cancer-related driver gene mutations, and has good therapeutic potential.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first object of the present invention is to provide an antigen peptide library for inducing tumor-specific immune responses, and the antigen peptide library includes the following 8 antigen peptides:

[0009] Antigen peptide 1: The amino acid sequence is as shown in SEQ ID NO.1;

[0010] Antigen peptide 2: The amino acid sequence is as shown in SEQ ID NO.2;

[0011] Antigen peptide 3: The amino acid sequence is as shown in SEQ ID NO.3;

[0012] Antigen peptide 4: The amino acid sequence is as shown in SEQ ID NO.4;

[0013] Antigen peptide 5: The amino acid sequence is as shown in SEQ ID NO.5;

[0014] Antigen peptide 6: The amino acid sequence is as shown in SEQ ID NO.6;

[0015] Antigen peptide 7: The amino acid sequence is as shown in SEQ ID NO.7;

[0016] Antigen peptide 8: The amino acid sequence is as shown in SEQ ID NO.8.

[0017] The second object of the present invention is to provide a nucleic acid molecule encoding the antigen peptide library, and the nucleic acid molecule can express the antigen peptide.

[0018] The third object of the present invention is to provide an expression vector containing the antigen peptide library or the nucleic acid molecule encoding the antigen peptide library; the expression vector can express the target protein in prokaryotic or eukaryotic host cells; the vector can be a plasmid vector or a viral vector.

[0019] The fourth object of the present invention is to provide a prokaryotic or eukaryotic host cell containing the expression vector, wherein the prokaryotic cell is an engineered bacterium.

[0020] The fifth object of the present invention is to provide an antigen-presenting cell loaded with the epitope polypeptide of the antigen peptide library.

[0021] The sixth object of the present invention is to provide an application of an antigen peptide library for inducing a tumor-specific immune response, specifically for inducing the generation of specific cytotoxic T cells in vitro with the antigen peptide library.

[0022] The seventh object of the present invention is to provide a composition comprising the antigen peptide library and an adjuvant.

[0023] The eighth object of the present invention is to provide an application of the antigen peptide library or the composition comprising the antigen peptide library in the preparation of a product for preventing and / or treating hepatocellular carcinoma with the same gene mutation.

[0024] The ninth object of the present invention is to provide a method for preparing cytotoxic T cells in vitro using the antigen peptide library.

[0025] Specifically, the present application provides a method for in vitro amplification and culture of CTL cells induced and activated by DC loading with tumor neoantigens, comprising the following steps:

[0026] 1) In vitro induction of DC cells and antigen uptake (the same as the steps for preparing DC vaccines):

[0027] S1: Mouse bone marrow with adjusted cell density is placed in a culture medium containing recombinant mouse GM-CSF and IL-4 for induced culture;

[0028] S2: After culturing for a period of time, fresh culture medium is supplemented, and the cytokines GM-CSF and IL-4 are supplemented;

[0029] S3: Suspended cells and loosely adherent cells are collected, recombinant mouse GM-CSF and IL-4 are added, and at the same time, neoantigen polypeptides are added. The cells are placed in a cell culture incubator for culture to allow DC to uptake the antigen. IFNγ and LPS as maturation inducers are added, and the induced culture is carried out for 16 - 24 h;

[0030] S4: Mature DC are collected and washed 2 times with PBS;

[0031] 2) Co-culture of antigen-loaded DC and T cells:

[0032] S5: Antigen-loaded DC and splenocytes are placed in a complete 1640 culture medium containing IL-2 and IL-7 for co-culture;

[0033] S6: Fresh culture medium is supplemented, and at the same time, IL-2, IL-7 and IL-15 are supplemented;

[0034] S7: After culturing for a period of time, transfer to a larger culture flask for scale-up culture, and add 1640 complete medium containing IL-2, IL-7, and IL-15 quantitatively every 2 days for cytotoxic T cell expansion culture.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] 1) Through high-throughput gene sequencing and data analysis, a new antigen peptide library is screened and obtained in this application. The new Hepa1-6 antigen peptide library of the present invention refers to antigen peptides expressed by tumor cells that can activate T cells. Animal experiments provided in this application verify that the antigen peptides have strong immunogenicity.

[0037] 2) The antigen peptides of the present invention are loaded onto antigen-presenting cells (APCs), which can effectively stimulate and induce the production of specific cytotoxic T lymphocytes (CTLs). In vitro killing experiments show that CTL cells induced and activated by loading new antigen DCs have stronger tumor killing effects compared to T cells induced and amplified by non-specific activation. Their cell killing activities are all above 90%; CTL cells induced by the new antigen peptide library can effectively expand new antigen-specific T cells, and the proportion of TCR-T cells against new antigens is significantly increased. Adoptive transfer of CTL cells induced by the new antigen peptide library to treat model mice can effectively inhibit tumor growth and significantly improve their survival rate. Description of the Drawings

[0038] Figure 1 Results of the immunogenicity experiment for screening and verifying alternative antigen peptides in C57BL / 6 mice. Among them, A is the result diagram of Elispot verifying the immunogenicity of Hepa1-6 tumor neoantigen polypeptides; B is the result diagram of statistical analysis of the immunogenic peptides verified by Elispot for Hepa1-6.

[0039] Figure 2 In the method of "mouse DC induction and maturation" in the specific embodiment of the present invention, after collecting mature DCs, observe and record the morphology of DCs under a microscope (microscope diagram). Among them, A is the morphology of DCs cells under a 200-fold microscope, B is the morphology of DCs cells under a 400-fold microscope, and C is the expression levels of CD11c, CD86, CD80, and CD40 of DCs detected by flow cytometry.

[0040] Figure 3 In the method for in vitro induction of new antigen peptide-specific CTLs in the specific embodiment of the present invention, after collecting T cells, the proportion of T cell subsets is measured by flow cytometry.

[0041] Figure 4 Cytotoxic effect of the T cells prepared in the specific embodiment of the present invention on tumor cells.

[0042] Figure 5 Identification results of tumor neoantigen-specific T cells obtained from the specific embodiments of the present invention.

[0043] Figure 6 Evaluation results of the efficacy of antigen peptide-specific T cell reinfusion in a subcutaneous liver cancer tumor model mice. Among them, A is the effect on tumor volume, and B is the effect on the survival rate of mice. Specific embodiments

[0044] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0045] The following described embodiments are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application claimed, but merely represents the selected embodiments of the present application. The embodiments are used to illustrate the present invention, rather than to limit the present invention. All other embodiments obtained by those skilled in the art based on the embodiments in the present application without creative efforts belong to the scope of protection of the present application.

[0046] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of conflict, this specification prevails.

[0047] Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0048] Research shows that: transcriptional and proteomic mutations of tumor cells are the main sources of non-classical neoantigens. First, the present invention uses RNA-seq sequencing data and proteomic mass spectrometry data of Hepa1-6 tumor cell line, and uses variant detection tools and machine learning algorithms to obtain non-classical mutant peptides; secondly, predict the binding affinity and stability of mutant peptides with HLA molecules; finally, use a combination of multiple methods to optimize and select candidate non-classical neoantigens. The detailed information of the neoantigen peptides is shown in Table 1. The present invention also uses DC cells to uptake the neoantigen library, and then co-cultures with T cells to specifically activate neoantigen CTL, and amplifies and cultures in vitro.

[0049] The Hepa1-6 neoantigen peptide library provided by the present invention is shown in Table 1 below.

[0050] Table 1. Polypeptide sequences of Hepa1-6 immunogenicity

[0051] Antigen peptide number Gene Wild-type polypeptide Mutant polypeptide Mutation type HLA typing Score TP02 Ddx1 ASVLNKWQM ASVLNKWEM Missense mutation H-2-Db 1.74 TP03 Nfkbia VTYQGYSPYQL VTYQAYSPYQL Missense mutation H-2-Kb 2.27 TP05 Stt3b SAIAFSNV SAVAFSNV Missense mutation H-2-Kb 2.69 TP06 Vti1b VGLVYYKFF VSLVYYKFF Missense mutation H-2-Kb 2.76 TP12 Tmem259 MAVTYSRL MAVSYSRL Missense mutation H-2-Kb 4.66 TP18 Ap3d1 IIKLFGAL ISKLFGAL Missense mutation H-2-Kb 8.41 TP22 Cxcr6 IYIFYQKL IYIFYQTL Missense mutation H-2-Kb 10.72 TP27 Rbbp5 KSIGNLVKI KSIANLVKI Missense mutation H-2-Db 11.85

[0052] Example 1. Analysis and prediction of neoantigen peptides

[0053] The brief workflow of neoantigen prediction can be summarized as the following steps: ① Mutation identification, ② HLA typing, ③ Screening and prioritizing neoantigens based on HLA binding affinity. Genomic data and transcriptomic data were obtained by DNA extraction from Hepa1-6 tumor cell line cells and through whole-exome sequencing and transcriptome sequencing. The raw data of high-throughput sequencing were aligned based on the reference genome to obtain mutation data. Based on these data, HLA genotypes were obtained using HLA genotyping software; after obtaining the above-mentioned neoantigen-related data, HLA-neoantigen binding prediction software was used to analyze whether the obtained mutant polypeptide sequences could effectively bind to HLA molecules. Twenty-two mutant polypeptides were selected as candidates from the alternative mutant polypeptides. The top 22 mutant polypeptides were used as candidate tumor neoantigens (see Table 2). The polypeptides in Table 2 were prepared by chemical synthesis and stored for future use.

[0054] Table 2. Alternative polypeptide sequences of Hepa1-6 immunogenicity

[0055] Polypeptide number Gene Wild-type polypeptide Mutant polypeptide HLA typing Mutation type Score TP02 Ddx1 ASVLNKWQM ASVLNKWEM H-2-Db Missense mutation 1.74 TP03 Nfkbia VTYQGYSPYQL VTYQAYSPYQL H-2-Kb Missense mutation 2.27 TP05 Stt3b SAIAFSNV SAVAFSNV H-2-Kb Missense mutation 2.69 TP06 Vti1b VGLVYYKFF VSLVYYKFF H-2-Kb Missense mutation 2.76 TP08 Sppl3 VAMIAFVRL VGMIAFVRL H-2-Kb Missense mutation 3.65 TP07 Dohh GAIGNPEVL GAIGNPDVL H-2-Db Missense mutation 3.68 TP09 Unc50 VGYYIYVTFL VAYYIYVTFL H-2-Kb Missense mutation 4.10 TP10 Unc50 IAVGYYIYV IAVAYYIYV H-2-Kb Missense mutation 4.28 TP11 Riok2 SSFEVTAL SSFQVTAL H-2-Kb Missense mutation 4.56 TP12 Tmem259 MAVTYSRL MAVSYSRL H-2-Kb Missense mutation 4.66 TP13 Tmtc3 SALFNLALL SALFNLPLL H-2-Db Missense mutation 5.02 TP14 Kif20a KVYLRIRPF KVYLRIRPL H-2-Kb Missense mutation 5.34 TP15 Cyp20a1 SNFPLLLQL SNFALLLQL H-2-Kb Missense mutation 5.36 TP16 Gja1 VLFIFRILL VLFIFRIPL H-2-Kb Missense mutation 7.67 TP18 Ap3d1 IIKLFGAL ISKLFGAL H-2-Kb Missense mutation 8.41 TP19 Cnot8 WQFNFKFNL WQFNFRFNL H-2-Kb Missense mutation 9.07 TP20 Ddhd1 RDMLNSSAM RGMLNSSAM H-2-Db Missense mutation 10.01 TP22 Cxcr6 IYIFYQKL IYIFYQTL H-2-Kb Missense mutation 10.72 TP25 Cxcr6 IIYIFYQKLR IIYIFYQTLR H-2-Kb Missense mutation 11.07 TP27 Rbbp5 KSIGNLVKI KSIANLVKI H-2-Db Missense mutation 11.85 TP28 Setx TVIQFPSGL TVIQFPAGL H-2-Kb Missense mutation 12.21 TP30 Uba7 AANLYARM AAILYARM H-2-Kb Missense mutation 13.28

[0056] Example 2. Verification of the immunogenicity of alternative antigen peptides in animals

[0057] To identify and screen the immunogenicity of alternative polypeptides, female C57BL / 6 mice (6-8 weeks old) were immunized by ventral subcutaneous injection of polypeptide pools on days 0, 5, and 10 for immunogenicity tests. The above 22 alternative neoantigen polypeptides were divided into 4 polypeptide pools. Polypeptide pool 1 contained polypeptide numbers: TP02, TP03, TP05-TP07; polypeptide pool 2 contained polypeptide numbers: TP08-TP12; polypeptide pool 3 contained polypeptide numbers: TP13-TP16, TP18, TP19; polypeptide pool 4 contained polypeptide numbers: TP20, TP22, TP25, TP27, TP28, TP30.

[0058] On the 17th day, the mice were sacrificed, and mouse splenocytes were obtained for enzyme-linked immunosorbent assay (Elispot). The Elispot detection steps were strictly operated according to the instructions provided by the mouse IFN-γ ELISpot kit (BD Biosciences). The main steps were as follows: 5×10 5Spleen cells were seeded into each well, and individual polypeptides were added. The diluent of the polypeptide, PBS, and ConA (Sigma-Aldrich) were used as negative and positive controls, respectively. They were incubated at 37°C with 5% CO2 for 24 h. They were incubated and developed with biotinylated anti-mouse IFN-γ detection antibody and streptavidin, and the number of IFN-γ spots was determined using an automated ELISPOT plate reader (AID iSpot).

[0059] It can be seen from Figure 1 the results that there were statistically significant differences in the number of IFN-γ spots produced by polypeptides TP02, TP03, TP05, TP06, TP12, TP18, TP22, and TP27, indicating strong immunogenicity. The detailed information of the above polypeptides is shown in Table 1.

[0060] Example 3 Method for in vitro induction and evaluation of neoantigen peptide-specific CTL

[0061] (1) Induction of CTL: Hepa1-6 cells (1.8×10 6 / 100 μL) were implanted subcutaneously into the axilla of C57BL / 6 mice. On the 7th day, the spleens of the mice were collected and prepared into single-cell suspensions. After lysing red blood cells, they were washed, counted, and then cultured. The cells were divided into two parts. One part was amplified and cultured by adding CD3, CD28, and IL-2 (labeled as the non-specific activated T cell group) without adding polypeptides, and the other part was induced and amplified to CTL by using the specific stimulation method of the neoantigen peptide library in the present invention.

[0062] The induction and culture steps of CTL in the present invention are as follows:

[0063] 1) Obtaining bone marrow cells of C57BL / 6 mice: C57BL / 6 mice were sacrificed by cervical dislocation and immersed in 70% ethanol for 5 min for disinfection. The obtained mouse bone marrow cells were added with 5 - 8 mL of red blood cell lysate to lyse red blood cells for 5 min. Centrifuged at 300 g for 5 min, the supernatant was discarded, then added with 5 mL of PBS and washed once. Centrifuged at 300 g for 5 min, the supernatant was discarded, and 1 mL of RPMI1640 complete medium was added to resuspend the cells, and the cells were counted. The cell concentration was adjusted to 1 x 10 6 / mL by adding RPMI1640 complete medium.

[0064] 2) Induction and maturation of mouse DCs (the same as the preparation method of DC vaccine)

[0065] Day 0: The above mouse bone marrow cells with adjusted cell density were added with recombinant mouse GM-CSF (20 ng / mL) and IL-4 (10 ng / ml), and placed in an incubator at 37°C with 5% CO2 for culture. This was the 0th day of culture;

[0066] Day 3: Add 5 ml of medium, and supplement cytokines GM-CSF (20 ng / ml) and IL-4 (10 ng / ml);

[0067] Day 5: Gently pipette the culture medium, collect the suspended cells and the cells growing loosely adherent to the wall, centrifuge at 300 g for 5 min, discard the supernatant; resuspend the cells with RPMI 1640 complete medium and count, adjust the cell concentration to 1 x 10 6 / mL, and add recombinant mouse GM-CSF (20 ng / ml) and IL-4 (10 ng / ml), and at the same time add neoantigen polypeptides, and the final concentration of each polypeptide is 2 μg / mL;

[0068] Place in a cell culture incubator and culture for 7 h (DC takes up antigen), then add maturation inducer IFNγ (500 U / mL) and LPS (100 ng / mL), and induce culture for 20 h;

[0069] Collect mature DC: Centrifuge at 300 g for 5 min, discard the supernatant, add 5 mL of PBS to wash the cells twice, discard the supernatant, then add 1 mL of RPMI 1640 complete medium, and count the cells.

[0070] 3) Co-culture of neoantigen-activated mouse splenocytes and DCs loaded with neoantigen

[0071] Day 0: Co-culture an equal volume of induced and mature DC cells with mouse splenocytes (DC: splenocytes = 1:16), the seeding density of mouse splenocytes is 4×10 6 cells / mL, add IL-7 with a final concentration of 5 ng / mL and IL-2 with a final concentration of 500 U / mL;

[0072] Day 3: Supplement with 1-fold fresh medium, and at the same time supplement cytokine IL-2 with a final concentration of 500 U / mL, and IL-7 and IL-15 with final concentrations of 5 ng / mL;

[0073] Day 5: Transfer the cells to a larger culture flask, adjust the cell density to 2×10 5 cells / mL, supplement with fresh medium, and at the same time supplement factor IL-2 with a final concentration of 500 U / mL, and IL-7 and IL-15 with final concentrations of 5 ng / mL;

[0074] Day 7: Replace half of the culture medium, supplement with fresh medium and adjust the cell density to 2×10 5 cells / mL, add 10 ng / mL IL-7, 10 ng / mL IL-15 and 500 U / mL IL-2 respectively, and culture for another 3 days;

[0075] Day 10: Collect cells for functional assays, and add fresh medium to adjust the cell density to 2×10 5 cells / mL. Add 10 ng / mL IL-7, 10 ng / mL IL-15, and 500 U / mL IL-2 respectively, and culture for another 3 days;

[0076] Day 13: Add fresh medium to adjust the cell density to 2×10 5 cells / mL. Add 10 ng / mL IL-7, 10 ng / mL IL-15, and 500 U / mL IL-2 respectively, and culture for another 2 days;

[0077] Day 15: Collect T cells.

[0078] Note: Regularly observe the cell growth conditions, mainly observing the morphological changes, the number and proliferation, the cell debris and impurities. Add liquid for culture every 2 - 3 days.

[0079] (2)Experimental detection and result analysis

[0080] 1) Identification of C57BL / 6 mouse DCs: Perform "Mouse DC Induction and Maturation" according to the aforementioned method. After collecting mature DCs cells, observe and record the morphology of DCs under the microscope. Subsequently, stain the DCs cells with CD11c (Biolegen, catalog number 117308, FITC), CD80 (Biolegen, catalog number 104708, PE), CD86 (Biolegen, catalog number 105012, APC), and CD40 (Biolegen, catalog number 124612, APC), and analyze by flow cytometry.

[0081] The experimental results are as Figure 2 shown. The results show that the DCs cells loaded with neoantigens of the present invention have obvious dendritic-like protrusions and obvious cluster aggregates under the microscope ( Figure 2 A: 200x, Figure 2 B: 400x); meanwhile, flow cytometry detects that the DC cell surface highly expresses CD11c, CD86, CD80, and CD40 molecules ( Figure 2 C), indicating that the method successfully obtains mature DCs.

[0082] 2) Determination of T cell subsets: After co-culturing DCs and T cells, on the 15th day of co-culture, collect 1×10 6 cells and analyze the proportion of CD3 (Biolegen, catalog number 100203, FITC) and CD8 (Biolegen, catalog number 100712, APC) double-positive cells by flow cytometry.

[0083] The experimental results are as follows Figure 3 shown. On the 15th day of in vitro culture, CTL subsets were measured by flow cytometry. The proportion of CD3 and CD8 double-positive T cells that specifically activate CTL with neoantigens was higher, and there was a significant difference from the group of non-specifically activated T cells, indicating that CTL was specifically amplified by DC loaded with neoantigens.

[0084] 3) Detection of the tumor cell killing activity of cytotoxic T cells induced and activated by DC loaded with tumor neoantigens: The experiment was detected using the xCELLigence RTCA MP instrument of ACEA Biosciences, USA. The Hepa1-6 tumor cells in the logarithmic growth phase in the examples were collected as target cells, and the density was adjusted to 1×10 5 cells / mL; 50 μL of the target cancer cell culture medium was added to each well of 96-well E-Plates (ACEABiosciences), and the background impedance was measured and the cell Index (CI) value was shown to be stable and at the baseline level. Subsequently, the above-concentration target cells Hepa1-6 were plated at 100 μL / well; after plating, the 96-well E-Plate was transferred to the RTCA MP instrument in a 37°C, 5% CO2 cell culture incubator. During the whole experiment, data was recorded every 15 minutes, monitored for 16 - 24 h, and when the CI value ≥ 1, effector cells were added; CTL on the 15th day of amplification was collected. The CTL harvested from the culture induced and activated by DC loaded with tumor neoantigens (neoantigen-specific activation of CTL group) was used as the experimental group, and the cells activated and amplified by non-specific stimulation (non-specific activation of T cell group) were used as the control group. The CTL density was adjusted to 2×10 6 cells / mL; the 96-well E-Plate was taken out from the RTCA MP instrument, and CTL (50 μL) was mixed with the target cells according to an effector-to-target ratio of 10:1. At the same time, control wells with only target cells were set up and an equal volume of cell culture medium was added. Each group was set with 3 parallel wells; after adding the effector cells, the 96-well E-Plate was transferred to the RTCA MP instrument in a 37°C, 15% CO2 cell culture incubator for continued culture and monitoring. After adding the effector cells, it was monitored for another 48 h, and the CI value of each well was measured. The calculation method of cell killing activity: cell killing activity = (1 - CI value of effector-to-target cell action well / CI value of target cell well) × 100%. When the calculated killing efficiency ≥ 30%, it was determined to have obvious killing efficiency.

[0085] The experimental results are as follows Figure 4 and Table 3 show that the CTL induced and activated by DC loaded with tumor neoantigens has a stronger tumor killing effect compared with the T cells induced and amplified by non-specific activation, and its cell killing activity is above 90%.

[0086] Table 3. Inhibition rate of T cells after adding Hepa1-6 tumor target cells to the amplification culture

[0087]

[0088] 4) Identification of neoantigen-specific T cells: Collect CTLs on the 15th day of in vitro amplification. Use T cell surface markers 4-1BB (Biolegen, catalog number 106110, APC), IFNγ (Biolegen, catalog number 505808, PE), granzyme B (GZB) (Biolegen, eBioscience™, 17-8898-82, APC) and tetramers to identify neoantigen-specific T cells. The principle of MHC tetramer staining is that MHC molecules bind to specific antigen peptides and are labeled with fluorescent substances to form tetramers. Fluorescently labeled MHC tetramers can bind to specific T cell receptors (TCRs), thereby detecting and quantifying the binding of specific antigen peptides. This technique has high sensitivity and high specificity and can accurately detect and quantify the expression level of specific antigen peptides. Prepare Hepa1-6 tumor neoantigen polypeptides (polypeptide numbers TP02 and TP27) into tetramers, named tetramer-TP02 (PE-labeled) and tetramer-TP27 (PE-labeled) respectively. Collect 1×10 6 cells. After antibody staining, analyze by flow cytometry.

[0089] The experimental results are as Figure 5 shown. The results show that compared with the non-specifically activated T cell group, the T cells of neoantigen-specific CTLs all highly express CD8 + IFNγ + 、CD3 + GZB + 、CD3 + tetramer-TP02 + and CD3 + tetramer-TP27 + , and there are significant differences, indicating that the neoantigen peptide can specifically induce and activate CTLs and proliferate in large numbers in vitro.

[0090] Example 4 Evaluation of the efficacy of antigen peptide-specific T cell adoptive transfer in tumor model mice

[0091] (1) Establishment of a subcutaneous liver cancer tumor model in mice: Subcutaneously implant Hepa1-6 tumor cells (1.8×10 6 / 100 μL) into the axilla of C57BL / 6 mice to establish a mouse liver cancer model. Monitor the body weight, tumor volume and normal physiological activity indicators of the mice weekly.

[0092] (2)Grouping experiment of mice: The liver cancer mouse model was divided into 3 groups, namely the PBS group, the CD3+CD28+IL-2 in vitro expanded T cell group (non-specifically activated T cell group), and the neoantigen library-specifically expanded CTL group (neoantigen-specifically activated CTL group). There were 8 mice in each group. On days 0, 7, and 14 respectively, the tumor-specific CTL induced by the neoantigen peptide library obtained in the foregoing embodiment was intravenously infused through the tail vein for immunotherapy, and the PBS group was simultaneously infused with an equal volume of PBS. Every 3 days, the general condition, activity status of the tumor-bearing mice, and the growth of the tumor (size, volume, texture) were observed, and the length (L), width (W), and height (H) of the tumor were measured with a vernier caliper. The volume of the tumor was calculated using the formula V = 1 / 2 length × width2, and the mice were sacrificed after 4 weeks.

[0093] The experimental results are as Figure 6 shown: The T cell infusion therapy obtained by the present invention can significantly reduce the tumor volume of liver cancer mice and improve their survival rate.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An antigen peptide library for inducing tumor-specific immune response, characterized in that: The antigen peptide library includes the following 8 new antigen peptides: New antigen peptide 1: the amino acid sequence is shown in SEQ ID NO.1; New antigen peptide 2: the amino acid sequence is shown in SEQ ID NO.2; New antigen peptide 3: the amino acid sequence is shown in SEQ ID NO.3; New antigen peptide 4: the amino acid sequence is shown in SEQ ID NO.4; New antigen peptide 5: the amino acid sequence is shown in SEQ ID NO.5; New antigen peptide 6: the amino acid sequence is shown in SEQ ID NO.6; New antigen peptide 7: the amino acid sequence is shown in SEQ ID NO.7; New antigen peptide 8: The amino acid sequence is shown in SEQ ID NO.

8.

2. A nucleic acid molecule encoding the antigenic peptide library according to claim 1.

3. An expression vector comprising the nucleic acid molecule of claim 2.

4. The expression vector according to claim 3, characterized in that The vector is a plasmid vector or a viral vector.

5. A prokaryotic host cell or eukaryotic host cell comprising the expression vector according to any one of claims 3 or 4.

6. An antigen presenting cell loaded with the new antigen peptide in the antigen peptide library of claim 1.

7. The use of the antigen peptide library as claimed in claim 1, characterized in that: The antigen peptide library is used to induce specific cytotoxic T cells in vitro.

8. A composition, characterized in that It comprises the antigen peptide library according to claim 1 and an adjuvant.

9. Use of the antigen peptide library according to claim 1 or the composition according to claim 8 in the preparation of a product for preventing and / or treating liver cancer.

10. A method for in vitro proliferation and culture of CTL cells activated by DC loaded with tumor neoantigens, comprising the following steps: 1) In vitro induction of DC cells and antigen uptake S1: Mouse bone marrow cells with adjusted cell density were induced in a culture medium containing recombinant mouse GM-CSF and IL-4; S2: After a period of culture, fresh culture medium is added, and cytokines GM-CSF and IL-4 are added; S3: Collect the suspended cells and the cells that adhere to the wall, add recombinant mouse GM-CSF and IL-4, and add the antigen peptide library described in claim 1, place them in a cell culture incubator to culture DC to allow DC to take up the antigen, add maturation inducers IFNγ and LPS, and induce the culture for 16-24 hours; S4: Mature DCs were collected, washed twice with PBS, and then resuspended in complete medium; 2) Co-culture of antigen-loaded DC and T cells S5: DCs loaded with antigens were co-cultured with spleen cells in 1640 complete medium containing IL-2 and IL-7; S6: fresh culture medium was added, and IL-2, IL-7 and IL-15 were also added; S7: After a period of culture, the cells are transferred to a larger culture flask for expansion, and 1640 complete culture medium containing IL-2, IL-7 and IL-15 is quantitatively added every 2 days for cytotoxic T cell expansion culture.

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