A kind of tumor neoantigen polypeptide and its application
An antigenic polypeptide, a new technology, applied in antitumor drugs, cancer antigen components, vertebrate antigen components, etc., to achieve the effect of enhancing correct understanding
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Embodiment 1
[0065] This embodiment provides a tumor neoantigen polypeptide. The sequence of the polypeptide is screened by the following method:
[0066] (1) Based on the screening of high-frequency mutation sites in liver cancer from the TCGA database, the Top 10 high-frequency mutation sites were screened out
[0067] The somatic mutation data (version: 20170929) of the whole exome sequencing results of 364 cases of liver cancer were downloaded from the TCGA database (https: / / portal.gdc.cancer.gov / projects / TCGA-LIHC), and the data of at least 2 According to the missense SNV site in the case patient, the most frequent mutation site in the liver cancer patient group, taking HLA-A0201 typing as an example, was obtained, and the amino acid sequence was intercepted into a short peptide with a length of 9mer and a mutated amino acid according to the mutation information ;
[0068] (2) Prediction of tumor-specific peptides
[0069] In order to identify neoantigens that can be used as candid...
Embodiment 2
[0072] This example is used to prove that the neoantigen polypeptide has higher antigenic immunogenicity than virus-associated antigens and tumor-associated antigens.
[0073] Wherein, the virus-associated antigen used is: HBV_01, the sequence is FLLTRILT (denoted as SEQ ID NO.29); the tumor-associated antigen is: VEGFR2-775, the sequence is AMFFWLLLV (denoted as SEQ ID NO.30).
[0074] The preparation steps of CTL cells are as follows:
[0075] (1) Synthesizing the polypeptides shown in SEQ ID NO.8 and SEQ ID NO.28-30;
[0076] (2) CD in peripheral blood mononuclear cells was separated using specific magnetic beads and MS column pairs 8+ T cells and DC cells were sorted.
[0077] The specific operation process is as follows:
[0078]a. Collect 100mL of HLA-A0201 peripheral blood from healthy people, resuspend it with PBS, add an equal volume of Ficoll separation solution, and after density gradient centrifugation, use a pipette to suck out the mononuclear cell layer, trans...
Embodiment 3
[0113] This example is used to verify that the neoantigen polypeptides provided in Example 1 can present antigenic peptides to CD8 by antigen-presenting cells + T cells, and activate the corresponding antigen-specific CTL cells.
[0114] In this example, a tetramer staining experiment was performed to detect the positive rate of antigen-specific CTL cells.
[0115] Specific steps are as follows:
[0116] (1) Tetramer displacement experiment:
[0117] Use DMSO to prepare 2 mg / mL replacement target polypeptide (sequence FIYDASNQV) solution and positive peptide standard, add 50 μL of tetramer to a round-bottom 96-well plate; add 1 μL of the prepared polypeptide solution, mix with a pipette, add Peptide replacement factor 1 μL, and mix with a pipette; incubate at 25°C for 4 hours in the dark, and then store in the dark at 4°C; the tetramer replacement kit used is QuickSwitch TM , PE staining kit, purchased from MBL biological company;
[0118] (2) Detection of tetramer displacem...
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