Antigen peptide library for inducing tumor specific immune response and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an antigen peptide library for inducing tumor specific immune response, the antigen peptide library comprises eight antigen peptides, and the amino acid sequences of the eight antigen peptides are respectively shown as SEQ ID NO.1-SEQ ID NO.8. The invention further discloses a preparation method of the antigen peptide library. The antigen peptide has high immunogenicity and can effectively stimulate and induce generation of specific cytotoxic T lymphocytes when being loaded on antigen presenting cells, in-vitro killing experiments show that CTL cells induced and activated by loading new antigen DC have a high tumor killing effect, and the cell killing activity of the CTL cells is 90% or above. When model mice are treated by adoptive T cell back-transfusion of CTL cells induced by the new antigen peptide library, the tumor volume can be effectively reduced, the survival rate of the tumor can be remarkably increased, and good treatment potential is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an antigen peptide library for inducing tumor-specific immune response and application thereof. Background Art

[0002] The anti-tumor response rate of immune checkpoint inhibitors (ICIs) in cancer treatment is relatively low. However, researchers have discovered and proven that immunotherapy of tumor neoantigens can activate the body's own immunity to kill tumors with few side effects and has good therapeutic 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 therapy based on neoantigen peptides. Neoantigens are tumor-specific antigens (TSA), which are not expressed in normal tissues, are not easy to produce immune tolerance, 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 transcription 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 is 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 new antigen peptides has far-reaching significance for tumor research and clinical treatment. It not only provides new targets for tumor immunotherapy, but also promotes the development of personalized and precision treatment, and helps to improve the safety and effectiveness of treatment.

[0005] Liver cancer is a common malignant tumor with high morbidity and mortality. The present invention discloses a new antigen peptide library of Hepa1-6 tumor cell line and its application, wherein the antigen peptide library has a high affinity with MHC I molecules on DC cells and can effectively stimulate and induce the production of specific cytotoxic T lymphocytes. The antigen of the present invention is easy to synthesize on a large scale and can be used for in vivo and in vitro scientific research on tumors. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the present invention provides an antigen peptide library for inducing tumor-specific immune response and its application. The antigen peptide library has a high affinity with MHC I molecules on DC cells, can effectively stimulate and induce the production of specific cytotoxic T lymphocytes (CTLs), can be used for immune clearance of tumor cells 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 solution: The first object of the present invention is to provide an antigen peptide library for inducing a tumor-specific immune response, wherein the antigen peptide library comprises the following 8 antigen peptides: Antigenic peptide 1: the amino acid sequence is shown in SEQ ID NO.1; Antigenic peptide 2: the amino acid sequence is shown in SEQ ID NO.2; Antigenic peptide 3: the amino acid sequence is shown in SEQ ID NO.3; Antigenic peptide 4: the amino acid sequence is shown in SEQ ID NO.4; Antigenic peptide 5: the amino acid sequence is shown in SEQ ID NO.5; Antigenic peptide 6: the amino acid sequence is shown in SEQ ID NO.6; Antigenic peptide 7: the amino acid sequence is shown in SEQ ID NO.7; Antigenic peptide 8: The amino acid sequence is shown in SEQ ID NO.8.

[0008] The second object of the present invention is to provide a nucleic acid molecule encoding the antigen peptide library, and the nucleic acid molecule is capable of expressing the antigen peptide.

[0009] The third object of the present invention is to provide an expression vector comprising the antigen peptide library or a nucleic acid molecule encoding the antigen peptide library; the expression vector can express the target protein in a prokaryotic or eukaryotic host cell; the vector can be a plasmid vector or a viral vector.

[0010] The fourth object of the present invention is to provide a prokaryotic or eukaryotic host cell comprising the expression vector, wherein the prokaryotic cell is an engineered bacterium.

[0011] The fifth object of the present invention is to provide an antigen presenting cell loaded with the epitope polypeptide of the antigen peptide library.

[0012] The sixth object of the present invention is to provide an application of an antigen peptide library for inducing tumor-specific immune response, specifically using the antigen peptide library to induce specific cytotoxic T cells in vitro.

[0013] The seventh object of the present invention is to provide a composition comprising the antigen peptide library and an adjuvant.

[0014] The eighth object of the present invention is to provide the use of the antigen peptide library or a composition comprising the antigen peptide library in the preparation of a product for preventing and / or treating isogenic mutated liver cancer.

[0015] The ninth object of the present invention is to provide a method for preparing cytotoxic T cells in vitro using the antigen peptide library.

[0016] Specifically, the present application provides 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 (same as the steps for preparing DC vaccines): S1: Mouse bone marrow with adjusted cell density was placed in a culture medium containing recombinant mouse GM-CSF and IL-4 for induction culture; 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 cells that adhere to the wall, add recombinant mouse GM-CSF and IL-4, and add the new antigen peptide, place them in a cell culture incubator to culture DC to allow the DC to take up the antigen, add the maturation inducers IFNγ and LPS, and induce the culture for 16-24 hours; S4: Collect mature DCs and wash them twice with PBS; 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.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1) This application screened and obtained a new antigen peptide library through high-throughput gene sequencing and data analysis. The Hepa1-6 new antigen peptide library of the present invention refers to antigen peptides expressed by tumor cells that can activate T cells. The animal experiments provided in this application verified that the antigen peptides have strong immunogenicity.

[0018] 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 activated by DC loaded with new antigens have stronger tumor killing effects than T cells induced by non-specific activation, and their cell killing activity is 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 targeting new antigens is significantly increased. Adoptive T cell infusion 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are the results of the immunogenicity experiment of screening and verifying the candidate antigen peptides in C57BL / 6 mice, where A is the result of Elispot verification of the immunogenicity of Hepa1-6 tumor neoantigen peptides; B is the result of the statistical analysis of the Hepa1-6 immunogenic peptides verified by Elispot.

[0020] Figure 2 In the method of "mouse DC induction and maturation" in the specific embodiment of the present invention, after collecting mature DCs, the morphology of DCs was observed and recorded under a microscope (microscope image), wherein A is the morphology of DCs cells under a 200x microscope, B is the morphology of DCs cells under a 400x microscope, and C is the expression level of CD11c, CD86, CD80 and CD40 of DC detected by flow cytometry.

[0021] Figure 3 In the in vitro method for inducing new antigen peptide-specific CTLs according to a specific embodiment of the present invention, after collecting T cells, flow cytometry is used to measure the proportion of T cell subpopulations.

[0022] Figure 4 This is the cytotoxic effect of T cells prepared in a specific embodiment of the present invention on tumor cells.

[0023] Figure 5 This is the tumor neoantigen-specific T cell identification result obtained in a specific embodiment of the present invention.

[0024] Figure 6 These are the results of the efficacy evaluation of the infusion of antigen peptide-specific T cells into the subcutaneous liver cancer tumor model mice, where A represents the effect on tumor volume and B represents the effect on the survival rate of mice. DETAILED DESCRIPTION

[0025] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0026] The embodiments described below 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 for protection, but merely represents the selected embodiments of the present application, and the embodiments are used to illustrate the present invention, rather than to limit the present invention. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present application.

[0027] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0029] Studies have shown that transcriptome mutations and proteome mutations of tumor cells are the main sources of atypical new antigens. First, the present invention uses RNA-seq sequencing data and proteome mass spectrometry data of the Hepa1-6 tumor cell line, and uses variant detection tools and machine learning algorithms to obtain atypical mutant peptides; secondly, the binding affinity and stability of mutant peptides to HLA molecules are predicted; finally, a combination of multiple methods is used to optimize and select candidate atypical new antigens. Detailed information on the new antigen peptides is shown in Table 1. The present invention also uses DC cells to take up new antigen libraries, and then co-cultures with T cells to specifically activate new antigen CTLs, and amplify and culture in vitro.

[0030] The Hepa1-6 new antigen peptide library provided by the present invention is shown in Table 1 below.

[0031] Table 1. Hepa1-6 immunogenic peptide sequences Antigenic peptide number Gene Wild-type peptide Mutant Peptide Mutation type HLA typing score TP02 DxX ASVLNKWQ ASVLNKWEM Missense mutation H-2-Db 1.74 TP03 Nfkbia VTYQGYSPYQL VTYQAYSPYQL Missense mutation H-2-Kb 2.27 TP05 TTW 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 CxC QIYIFYQ IYIFYQTL Missense mutation H-2-Kb 10.72 TP27 Rbbp5 KSIGNLVKI KSIANLVKI Missense mutation H-2-Db 11.85 Example 1 Analysis and prediction of new antigenic peptides The brief workflow of neoantigen prediction can be summarized as the following steps: ① mutation identification, ② HLA typing, ③ neoantigen screening and prioritization based on HLA binding affinity. DNA was extracted from Hepa1-6 tumor cell line cells, and genomic data and transcriptome data were obtained by exome sequencing and transcriptome sequencing. The raw data of high-throughput sequencing were compared based on the reference genome to obtain mutation data. Based on these data, the HLA genotype typing software was used to obtain the HLA genotype; after obtaining the above-mentioned neoantigen-related data, the HLA-neoantigen binding prediction software was used to analyze whether the mutant polypeptide sequence obtained above can effectively bind to the HLA molecule. 22 mutant polypeptides were screened out as alternatives from the alternative mutant polypeptides. The top 22 mutant polypeptides were used as alternative tumor neoantigens (see Table 2). The polypeptides in Table 2 were prepared by chemical synthesis, and the prepared polypeptides were stored for future use.

[0032] Table 2. Alternative peptide sequences for Hepa1-6 immunogenicity Peptide number Gene Wild-type peptide Mutant Peptide HLA typing Mutation type score TP02 DxX ASVLNKWQ ASVLNKWEM H-2-Db Missense mutation 1.74 TP03 Nfkbia VTYQGYSPYQL VTYQAYSPYQL H-2-Kb Missense mutation 2.27 TP05 TTW 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 WQF WQFNFRFNL H-2-Kb Missense mutation 9.07 TP20 Ddhd1 RDMLNSSAM RGMLNSSAM H-2-Db Missense mutation 10.01 TP22 CxC QIYIFYQ IYIFYQTL H-2-Kb Missense mutation 10.72 TP25 CxC 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 Example 2 Verification of the immunogenicity of candidate antigenic peptides in animals In order to identify and screen the immunogenicity of candidate peptides, female C57BL / 6 mice (6-8 weeks old) were subjected to immunogenicity tests by subcutaneous injection of the peptide pool on the ventral side on days 0, 5 and 10. The 22 candidate neoantigen peptides were divided into 4 peptide pools. The peptides contained in peptide pool 1 were numbered as follows: TP02, TP03, TP05-TP07; the peptides contained in peptide pool 2 were numbered as follows: TP08-TP12; the peptides contained in peptide pool 3 were numbered as follows: TP13-TP16, TP18, TP19; the peptides contained in peptide pool 4 were numbered as follows: TP20, TP22, TP25, TP27, TP28, TP30.

[0033] On the 17th day, the mice were killed and spleen cells were obtained for enzyme-linked immunosorbent assay (Elispot). The Elispot assay was performed strictly according to the instructions provided by the mouse IFN-γ ELISpot kit (BD Biosciences). The main steps were as follows: 5×10 5 Splenocytes were seeded into each well, and peptide alone, peptide dilutions in PBS, and ConA (Sigma-Aldrich) were added as negative and positive controls, respectively. 37°C, 5% CO 2 The cells were stored for 24 hours, incubated with biotinylated anti-mouse IFN-γ detection antibody and streptavidin, and the color was developed, and the number of IFN-γ spots was determined using an automated ELISPOT plate reader (AID iSpot).

[0034] Depend on Figure 1The results showed that the number of IFN-γ spots produced by polypeptides TP02, TP03, TP05, TP06, TP12, TP18, TP22, and TP27 was statistically different, indicating that they had strong immunogenicity. The detailed information of the above polypeptides is shown in Table 1.

[0035] Example 3 In vitro induction method and evaluation of new antigen peptide-specific CTL (1) CTL induction: Hepa1-6 cells (1.8×10 6 / 100μL). On the 7th day, the spleen of the mice was collected and prepared into a single cell suspension, and the red blood cells were lysed, washed, counted and cultured. The cells were divided into two parts, one of which was expanded and cultured according to the addition of CD3, CD28 and IL-2 (marked as non-specific activated T cell group) without adding polypeptide, and the other was induced to expand CTL according to the specific stimulation method of the new antigen peptide library in the present invention.

[0036] The steps of inducing and culturing CTL in the present invention are as follows: 1) Obtain bone marrow cells from C57BL / 6 mice: C57BL / 6 mice were killed by cervical dislocation, and disinfected in 70% ethanol for 5 minutes. The obtained mouse bone marrow cells were added with 5-8 mL of red blood cell lysis buffer to lyse the red blood cells for 5 minutes. Centrifuge at 300g for 5 minutes, discard the supernatant, and then add 5 mL of PBS to wash once. Centrifuge at 300g for 5 minutes, discard the supernatant, add 1 mL of RPMI1640 complete medium to resuspend, and count the cells. Add RPMI1640 complete medium to adjust the cell concentration to 1 x 10 6 / mL.

[0037] 2) Mouse DC induction and maturation (same as the preparation method of DC vaccine) Day 0: Add recombinant mouse GM-CSF (20 ng / mL) and IL-4 (10 ng / ml) to the mouse bone marrow cells with adjusted cell density and place in a 37°C, 5% CO 2 Incubator culture, this is the 0th day of culture; Day 3: add 5 ml of culture medium and cytokines GM-CSF (20 ng / ml) and IL-4 (10 ng / ml); Day 5: Gently pipette the culture medium to collect the suspended cells and the cells that adhered to the wall, centrifuge at 300g for 5 minutes, discard the supernatant; resuspend the cells in RPMI 1640 complete culture medium and count them, and adjust the cell concentration to 1 x 10 6 / mL, and recombinant mouse GM-CSF (20 ng / ml) and IL-4 (10ng / ml) were added, and the new antigen peptides were added at the same time, and the final concentration of each peptide was 2μg / mL; Place in a cell culture incubator and culture for 7 h (DC uptake of antigens), then add maturation inducers IFNγ (500 U / mL) and LPS (100 ng / mL) and induce culture for 20 h; Collect mature DCs: centrifuge at 300 g for 5 min, discard the supernatant, add 5 mL PBS to wash the cells twice, discard the supernatant, add 1 mL RPMI 1640 complete medium, and count the cells.

[0038] 3) Co-culture of neoantigen-activated mouse splenocytes with neoantigen-loaded DCs Day 0: Equal volumes of induced mature DCs were co-cultured with mouse spleen cells (DC:spleen cells = 1:16), and the seeding density of mouse spleen cells was 4×10 6 cells / mL, IL-7 was added to a final concentration of 5 ng / mL, and IL-2 was added to a final concentration of 500 U / mL; Day 3: Add 1-fold fresh culture medium, and add cytokines IL-2 with a final concentration of 500 U / mL, and IL-7 and IL-15 with a final concentration of 5 ng / mL; Day 5: Transfer cells to a larger culture flask and adjust the cell density to 2 × 10 5 cells / mL, supplemented with fresh medium, and supplemented with factors IL-2 at a final concentration of 500U / mL, and IL-7 and IL-15 at a final concentration of 5ng / mL; Day 7: Change half of the cell culture medium, add fresh culture medium and adjust the cell density to 2×10 5 cells / mL, 10 ng / mL IL-7, 10 ng / mL IL-15, and 500 U / mL IL-2 were added, and cultured for another 3 days; Day 10: Collect cells for functional testing and add fresh culture medium to adjust the cell density to 2×10 5 cells / mL, 10 ng / mL IL-7, 10 ng / mL IL-15, and 500 U / mL IL-2 were added, and cultured for another 3 days; Day 13: Add fresh culture medium to adjust the cell density to 2 × 10 5 cells / mL, 10 ng / mL IL-7, 10 ng / mL IL-15, and 500 U / mL IL-2 were added, and cultured for another 2 days; Day 15: T cells were collected.

[0039] Note: The cell growth needs to be observed regularly, mainly the changes in morphology, number and proliferation, cell debris and impurities. The culture medium should be added every 2-3 days.

[0040] (2) Experimental testing and result analysis 1) Identification of C57BL / 6 mouse DCs: "Mouse DC induction and maturation" was performed according to the above method. After collecting mature DCs cells, the morphology of DCs was observed and recorded under a microscope. DCs cells were then stained with CD11c (Biolegen, Catalog No. 117308, FITC), CD80 (Biolegen, Catalog No. 104708, PE), CD86 (Biolegen, Catalog No. 105012, APC) and CD40 (Biolegen, Catalog No. 124612, APC) and analyzed by flow cytometry.

[0041] The experimental results are as follows Figure 2 As shown, the results show that the DCs cells loaded with the new antigen of the present invention have obvious dendritic protrusions and obvious clusters under the microscope ( Figure 2 A: 200x, Figure 2 B: 400x); flow cytometry was used to detect that the DC cell surface highly expressed CD11c, CD86, CD80 and CD40 molecules ( Figure 2 C), indicating that the method successfully obtained mature DCs.

[0042] 2) Determination of T cell subsets: After DCs and T cells were co-cultured, 1×10 6 The proportion of CD3 (Biolegen, Cat. No. 100203, FITC) and CD8 (Biolegen, Cat. No. 100712, APC) double-positive cells was analyzed by flow cytometry.

[0043] The experimental results are as follows Figure 3 As shown, flow cytometry was used to measure CTL subsets on the 15th day of in vitro culture, among which the proportion of CD3 and CD8 double-positive T cells of neoantigen-specific activated CTLs was higher and significantly different from that of the non-specific activated T cell group, indicating that DC loaded with neoantigens induced specific expansion of activated CTLs.

[0044] 3) Detection of tumor cell killing activity of cytotoxic T cells activated by DC loaded with tumor neoantigens: The experiment was performed using the xCELLigence RTCA MP instrument from ACEA Biosciences, USA. Hepa1-6 tumor cells growing in the logarithmic phase in the example were collected as target cells and the density was adjusted to 1×10 550 uL of target cancer cell culture medium was added to each well of 96-well E-Plates (ACEABiosciences), and the background impedance was measured and displayed as the cell Index (CI) value was stable and at the baseline level. Then, the target cells Hepa1-6 at the above concentration were plated at 100 μL / well. After the plate was plated, the 96-well E-Plate was transferred to 37°C and 5% CO 2 On the RTCA MP instrument in the cell culture incubator. During the entire experiment, data was recorded every 15 minutes, and the monitoring was performed for 16-24 hours. When the CI value was observed to be ≥1, effector cells were added; CTLs were collected on the 15th day of amplification, and the CTLs harvested after DC-loaded tumor neoantigen-induced activation (neoantigen-specific activated CTL group) were used as the experimental group, and the cells activated and amplified by nonspecific stimulation (nonspecific activated T cell group) were used as the control group. The CTL density was adjusted to 2×10 6 The 96-well E-Plate was taken out from the RTCA MP instrument, and CTL (50 μL) was mixed with target cells at an effector-target ratio of 10:1. At the same time, the corresponding control wells with only target cells were added with an equal volume of cell culture medium. Three parallel wells were set up for each group. After adding effector cells, the 96-well E-Plate was transferred to 37°C and 15% CO 2 Continue to culture and monitor on the RTCA MP instrument in the cell culture incubator. Continue to monitor for 48 hours after adding effector cells, and determine the CI value of each well. Calculation method of cell killing activity: cell killing activity = (1 - CI value of effector target cell well / CI value of target cell well) × 100%. When the calculated killing efficiency is ≥30%, it is judged to have obvious killing efficiency.

[0045] The experimental results are as follows Figure 4 As shown in Table 3, the results showed that CTLs activated by DC loaded with tumor neoantigens had a stronger tumor killing effect than T cells induced and expanded by nonspecific activation, and their cell killing activity was above 90%.

[0046] Table 3. Inhibition rate of T cells after Hepa1-6 tumor target cells were added to the amplification culture

[0047] 4) Identification of neoantigen-specific T cells: CTLs were collected on day 15 of in vitro expansion, and neoantigen-specific T cells were identified using T cell surface markers 4-1BB (Biolegen, Cat. No. 106110, APC), IFNγ (Biolegen, Cat. No. 505808, PE), granzyme B (GZB) (Biolegen, Cat. No. eBioscience™, 17-8898-82, APC), and tetramers. The principle of MHC tetramer staining is to form tetramers by binding MHC molecules to specific antigenic peptides and labeling them with fluorescent substances. Fluorescently labeled MHC tetramers can bind to specific T cell receptors (TCRs) to detect and quantify the binding of specific antigenic peptides. This technology has high sensitivity and specificity, and can accurately detect and quantify the expression level of specific antigenic peptides. Hepa1-6 tumor neoantigen peptides (peptide numbers TP02 and TP27) were prepared into tetramers, named tetramer-TP02 (PE-labeled) and tetramer-TP27 (PE-labeled), respectively. 1×10 6 The cells were stained with antibodies and analyzed by flow cytometry.

[0048] The experimental results are as follows Figure 5 The results showed that compared with the nonspecific activated T cell group, the T cells of the new antigen-specific CTLs highly expressed CD8 + IFNγ + 、CD3 + G JZ + 、CD3 + Tetramer-TP02 + and CD3 + Tetramer-TP27 + , and there were significant differences, indicating that the new antigen peptides can specifically induce the activation of CTLs and proliferate them in large numbers in vitro.

[0049] Example 4 Evaluation of therapeutic efficacy by infusing antigen peptide-specific T cells into tumor model mice (1) Establishment of subcutaneous liver cancer model mice: Hepa1-6 tumor cells (1.8×10 6 / 100μL) to establish a mouse liver cancer model, and monitor the body weight, tumor volume and normal physiological activity indicators of the mice every week.

[0050] (2) Mouse grouping experiment: 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-specific activated T cell group) and the neoantigen library specific expanded CTL group (neoantigen specific activated CTL group). Each group of 8 mice was treated with immunotherapy by tail vein reinfusion of tumor-specific CTL induced by the neoantigen peptide library obtained in the above example on days 0, 7, and 14, respectively. The PBS group was simultaneously reinfused with an equal volume of PBS. The general condition, activity status and tumor growth (size, volume, texture) of the tumor-bearing mice were observed every 3 days, and the length (L), width (W), and height (H) of the tumor were measured with a vernier caliper, and the volume of the tumor was calculated using the formula V = 1 / 2 length × width 2, and the mice were killed after 4 weeks.

[0051] The experimental results are as follows Figure 6 As shown: the T cell transfusion therapy obtained by the present invention can significantly reduce the tumor volume of liver cancer mice and improve their survival rate.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution 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 antigen peptides: Antigenic peptide 1: the amino acid sequence is shown in SEQ ID NO.1; Antigenic peptide 2: the amino acid sequence is shown in SEQ ID NO.2; Antigenic peptide 3: the amino acid sequence is shown in SEQ ID NO.3; Antigenic peptide 4: the amino acid sequence is shown in SEQ ID NO.4; Antigenic peptide 5: the amino acid sequence is shown in SEQ ID NO.5; Antigenic peptide 6: the amino acid sequence is shown in SEQ ID NO.6; Antigenic peptide 7: the amino acid sequence is shown in SEQ ID NO.7; Antigenic 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 antigenic peptide library of claim 1 or 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 with homogeneous mutations.

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.

Images