CN113185597B - Human tumor antigen capable of activating anti-tumor immune response of patient and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine and health, and particularly relates to a human tumor antigen capable of activating anti-tumor immune response of a patient and application thereof. On the one hand, the invention discloses the application of the polypeptide coded by the circFam53b as a novel tumor antigen in tumor immunotherapy. The invention also provides a method for screening tumor antigens from circRNA, which takes highly expressed circRNA molecules in tumors as new objects of tumor antigen sources, selects circRNA molecules with coding capacity as candidate primary screening circRNA, then proves the coding capacity of the target circRNA and proves the immunogenicity of polypeptides coded by the circRNA. The invention provides a new target for tumor immunotherapy, particularly tumor vaccine therapy, and provides a new method for screening tumor antigens coded by circRNA serving as tumor immunotherapy and tumor vaccine therapy targets.

Description

Human tumor antigen capable of activating anti-tumor immune response of patient and application thereof

Technical Field

The invention relates to the field of biological medicine and health, in particular to a human tumor antigen capable of activating anti-tumor immune response of a patient and application thereof.

Background

At present, malignant tumors threaten human life and health greatly. With the development of scientific research, human beings have made great progress in antitumor therapy. However, the efficacy of current antitumor therapies is still not fully satisfactory. Traditional methods for treating malignant tumors include surgical treatment, radiation therapy and chemotherapy. These treatments all suffer from their own drawbacks. Researchers are constantly searching for new treatments to enhance efficacy and reduce side effects. Among them, immunotherapy is considered to have epoch-making significance in terms of antitumor activity because of its accuracy and fewer side effects than conventional treatment methods. In recent years, tumor immunotherapy has been progressing in a breakthrough manner as immunodetection point blocking antibodies such as anti-PD-1 and anti-CTLA-4 are applied to tumor immunotherapy. Currently, immunotherapy methods such as monoclonal antibody therapy, immune checkpoint blockade therapy, adoptive T cell therapy, chimeric antigen receptor-modified T cell therapy (CAR-T cell therapy), and tumor vaccines are continuously making new breakthroughs in anti-tumor therapy.

Therapeutic tumor vaccines have also found use in the clinical treatment of malignant tumors. The tumor vaccine therapy is to introduce tumor antigen into patient body to activate the immune system of the patient and induce body's immune response, so as to reach the aim of controlling or eliminating tumor. The selection of better antigens as targets for tumor vaccine therapy has been considered as a key to improve tumor vaccine therapy. The good tumor antigen as a therapeutic target is not only the key of tumor vaccine therapy, but also one of the keys of tumor immunotherapy.

However, the number of tumor antigens that have been discovered to induce a good immune response in the body is limited.

Disclosure of Invention

In view of the shortcomings and drawbacks of the prior art, it is a primary object of the present invention to provide a human tumor antigen that activates an anti-tumor immune response in a patient.

Another object of the present invention is to provide the use of the above human tumor antigen for activating anti-tumor immune response in a patient.

It is still another object of the present invention to provide a method for in vitro screening of the above human tumor antigen that activates anti-tumor immune response in a patient.

The purpose of the invention is realized by the following technical scheme: a human tumor antigen capable of activating an anti-tumor immune response in a patient, comprising the amino acid sequence shown below: ALFRLTNRA are provided.

A nucleotide sequence encoding the human tumor antigen which activates an anti-tumor immune response in a patient.

The nucleotide sequence comprises a ribonucleic acid sequence and a deoxyribonucleic acid sequence; preferably as follows: GCCCTCTTCAGATTGACCAACCGAGCA are provided.

The human tumor antigen that activates the anti-tumor immune response in the patient may serve one or more of the following functions:

1) activating the killing activity of CD 8T cells on tumors;

2) stimulating effector function of CD 8T cells;

3) stimulation of interferon-gamma (IFN- γ) secretion by CD 8T cells;

4) stimulation of tumor necrosis factor-alpha (TNF-alpha) secretion by CD 8T cells;

5) stimulating CD 8T cells to release cytotoxic particles;

6) after dendritic cells are added, the dendritic cells activate CD 8T cells;

7) stimulating T cell antigen receptor (TCR) clustering on the plasma membrane of CD 8T cells;

8) adoptive T cells activated by the polypeptide can inhibit tumor growth;

9) can be used as polypeptide vaccine to activate human body anti-tumor immunity;

10) the required mRNA vaccine can be prepared according to the polypeptide sequence to activate the anti-tumor immunity of the human body;

11) the circFam53b can be used as vaccine for activating human anti-tumor immunity

12) Can be used for preparing Dendritic Cell (DC) vaccine to activate human body anti-tumor immunity;

13) can be used for preparing DNA vaccine to activate human body anti-tumor immunity;

14) the TCR sequence can be obtained by sequencing after the CD8 cells are activated as immunogen for TCR-T treatment.

The tumors include solid tumors and non-solid tumors with circFam53b over-expressed or with circFam53b encoding polypeptides, including but not limited to: nasal cavity and sinus malignancies, nasopharyngeal carcinoma, oral cancer, laryngeal carcinoma, salivary gland tumor, intracranial tumor, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, cardiac cancer, breast cancer, mediastinal tumor, stomach cancer, large intestine cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and peri-ampullate cancer, biliary tract cancer, small intestine cancer, kidney cancer, prostate cancer, bladder cancer, testicular malignancy, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, cutaneous malignant melanoma, osteosarcoma, ewing's sarcoma, lymphoma, multiple myeloma, leukemia, and the like.

The human tumor antigen capable of activating the anti-tumor immune response of the patient and/or the application of the nucleotide sequence in preparing the anti-tumor medicament.

An antitumor drug comprising the human tumor antigen which activates the antitumor immune response of a patient and/or the nucleotide sequence.

The anti-tumor medicine also comprises one or two of T cells and dendritic cells.

Said T cells and said dendritic cells are preferably derived from a patient; preferably from autologous patients.

The tumors include solid tumors and non-solid tumors with circFam53b over-expressed or with circFam53b encoding polypeptides, including but not limited to: nasal cavity and sinus malignant tumor, nasopharyngeal carcinoma, oral cancer, laryngeal carcinoma, salivary gland tumor, intracranial tumor, thyroid cancer, tongue cancer, lung cancer, esophagus cancer, cardia cancer, breast cancer, mediastinal tumor, stomach cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampulla cancer, biliary tract cancer, small intestine malignant tumor, kidney cancer, prostate cancer, bladder cancer, testicular malignant tumor, penis cancer, cervical cancer, endometrial cancer, ovarian cancer, malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, malignant melanoma of skin, osteosarcoma, ewing's sarcoma, lymphoma, multiple myeloma, leukemia, etc.

The in vitro screening method of the human tumor antigen capable of activating the anti-tumor immune response of the patient comprises the following steps: performing circRNA high-throughput sequencing on cancer tissues and paracancer tissues; performing bioinformatics analysis on the high-throughput sequencing result, and selecting circRNA which is highly expressed in tumor tissues and has a coding function by analyzing the sequencing result of the circRNA; the result of bioinformatics analysis is determined through experiments, the polypeptide coded by the obtained circRNA is screened to be used as an action object of tumor immunotherapy, and part or all of the polypeptide coded by the circRNA is selected to be used for preparing a polypeptide vaccine, so that the human tumor antigen capable of activating the anti-tumor immune response of a patient is obtained.

The cancer tissue is preferably breast cancer tissue.

The circRNA is preferably circFam53 b.

The amino acid sequence of the polypeptide encoded by the circFam53b is as follows:

MSSCRTSWRPLGSKVWTPVEKRRCYSGGSVQRYSNGFSTMQRSSSFSLPSRANVLSSPCDQAGLHHRFGGQPCQGVPGSAPCGQAGD TWSPDLHPVGGGRLDLQRSLSCSHEQFSFVEYCPPSANSTPASTPELARRSSGLSRSRSQPCVLNDKKVGVKRRRPEEVQEQRPSLDLAKM AQKMTDGETWTGNALFRLTNRAPASGNACLKRTAHYGTGRQ。

the base sequence of the circFam53b is as follows:

the base sequence of an Open Reading Frame (ORF) of the circFam53b encoding polypeptide is as follows:

ATGTCCAGTTGCCGGACATCATGGAGGCCCTTGGGCTCCAAAGTCTGGACTCCCGTGGAAAAGAGACGCTGCTACAGCGGGGGCAGC GTCCAGCGCTATTCCAACGGCTTCAGCACCATGCAGAGGAGTTCCAGCTTCAGCCTCCCTTCCCGGGCCAACGTGCTCTCCTCACCCTGCG ACCAGGCAGGACTCCACCACCGATTTGGAGGGCAGCCCTGCCAAGGGGTGCCAGGCTCAGCCCCGTGTGGACAGGCAGGTGACACCTGGAG CCCTGACCTGCACCCCGTGGGAGGAGGCCGGCTGGACCTGCAGCGGTCCCTCTCTTGCTCACATGAGCAGTTTTCCTTTGTGGAATACTGT CCTCCCTCAGCCAACAGCACACCTGCCTCAACACCAGAGCTGGCGAGACGCTCCAGCGGCCTTTCCCGCAGCCGCTCCCAGCCGTGTGTCC TTAACGACAAGAAGGTCGGTGTTAAAAGGCGGCGCCCTGAAGAAGTGCAAGAGCAGAGGCCTTCTCTAGACCTTGCCAAGATGGCACAGAA AATGACAGATGGCGAGACCTGGACAGGAAATGCCCTCTTCAGATTGACCAACCGAGCACCAGCATCTGGGAATGCCTGCCTGAAAAGGACA GCTCACTATGGCACCGGGAGGCAGTGA。

the experiments described include, but are not limited to, the following: detecting whether the polypeptide coded by the circRNA is expressed in tissues, detecting the immunogenicity of part or all of the polypeptide coded by the circRNA, and detecting that the part or all of the polypeptide coded by the circRNA activates the anti-tumor immune activity to obtain the human tumor antigen capable of activating the anti-tumor immune response of the patient.

The detection of the presence or absence of expression of a polypeptide encoded by a circRNA in a tissue can be carried out using prior art methods comprising: preparation of Flag-carrying overexpression vector, western blot assay, mass spectrometry, and preparation of antibody immunoblot assay (western blot).

The detection of the immunogenicity of the polypeptide encoded by the circRNA can also be carried out using prior art methods comprising: enzyme linked immunospot assay (elispot), flow assay for CD 8T cytokine, and pentamer for antigen-specific T cells.

The method for detecting the activation of the antitumor immune activity of the polypeptide coded by the circRNA comprises the following steps: after establishing a patient-derived allograft PDX animal model, detecting the growth condition of the tumor by adoptively circliRNA epitope-activated T cells; the vaccine based on the circRNA epitope can be prepared, and comprises a polypeptide vaccine, a DC vaccine, an mRNA vaccine, a circRNA vaccine and a DNA vaccine to carry out clinical tests on tumor patients expressed by the polypeptide coded by the circRNA, and the immune response condition and the curative effect of the patients are observed.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the key to tumor immunotherapy is that tumor antigens are recognized by immune cells and activate anti-tumor immunity. At present, tumor antigens can be classified into exogenous tumor pathogenic microorganism antigens, endogenous tumor testis antigens, tumor-associated antigens, and new antigens generated by tumor gene mutation. Tumor testis antigens and tumor associated antigens are also expressed in small amounts in some healthy tissues and are subject to central immune tolerance. Therefore, treatments for these antigens have disadvantages such as low immunogenicity and susceptibility to side effects. The treatment of the new antigen vaccine causes the defects of high cost, long manufacturing period of the individualized vaccine and the like because different individuals need to carry out independent sequencing and individualized vaccine manufacturing. The invention discovers that the circFam53b with the coding function can code polypeptides with immunogenicity and can activate immune response in vivo. This will find a new source of antigens for tumor immunotherapy. The antigen coded by the CircFam53b has strong immunogenicity, can be expressed in different patients and even different tumors, and has wider applicability while ensuring effectiveness.

(2) In immunotherapy of highly mutationally loaded tumors, tumor neoantigens derived from mutations are considered to be key to activating anti-tumor immunity. The circRNA coded antigen can be applied to activate anti-tumor immunity in low-mutation-load breast cancer, and can provide a new basis for immunotherapy of low-mutation-load tumors.

(3) On the animal level, a PDX animal model is established, and the T cells with the antigen epitope polypeptide coded by circFam53b are added successively to inhibit the growth of tumors, so that the peptide has potential clinical application value in tumor immunotherapy.

(4) circRNA is a circular RNA molecule formed by reverse cleavage. When the head-to-tail junction of the circRNA is within the open reading frame, the coding region will be altered relative to the parent mRNA by a frame shift-like mutation or a cleavage site mutation, and the translated polypeptide will be different from the protein translated from the parent mRNA, potentially activating an immune response. The inventor finds that part of the polypeptides encoded by circFam53b with high expression in tumor cells have immunogenicity, and the epitope derived from the polypeptides can activate anti-tumor immunity and kill the tumor cells. And high-throughput sequencing, Western Bolt, mass spectrometry, elispot experiments, flow experiments and other methods prove that the circRNA can encode polypeptides with immunogenicity, and the polypeptides can be applied to activate anti-tumor immunity. These findings exploit new sources of tumor antigens and provide new targets for tumor therapy, particularly for immunotherapy.

Drawings

FIG. 1 is a graph showing the results of detecting a polypeptide encoded by circRNA; wherein A is the relative expression amount of circFam53 detected in tumor tissues and tissues beside cancer respectively, and the result shows that the circFam53 is highly expressed in the tumor tissues; b is polypeptide coded by Flag-carrying over-expressed circFam53B detected by immunoblotting experiment (Western Blot); c is a polypeptide encoded by circFam53b that is detectable by antibodies in breast cancer tissue.

FIG. 2 is a schematic diagram showing the structure of the objective vector pHBLV-CMV-Circ-MCS-EF1-zsgreen-t2 a-puro.

FIG. 3 is a graph showing the results of verifying that a polypeptide encoded by circRNA has antigenicity; wherein A is enzyme-linked immunosorbent assay (elispot) for detecting the immunogenicity of the polypeptide encoded by the circFam53 b; b, detecting the epitope of the polypeptide coded by the circFam53B by using a flow experiment to stimulate CD 8T cells to generate a cytokine IFN-r; c, detecting the epitope of the polypeptide coded by the circFam53b by using a flow experiment to stimulate CD 8T cells to generate a cell factor TNF-a; d is flow test to detect the antigen epitope of the polypeptide coded by the circFam53b to stimulate and differentiate antigen-specific CD 8T cells.

FIG. 4 is a graph showing the results of the verification that the polypeptide encoded by the circRNA activates an anti-tumor immune response.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The invention can be realized by technical means in various aspects of molecular level, cell level and animal model, proves that the polypeptide coded by circFam53b is expressed in tumor tissues, proves the immunogenicity of the polypeptide coded by circFam53b, and proves the action and specific action mechanism of the circFam53b and the polypeptide coded by the same in anti-tumor immunity.

1. Sequencing in human breast cancer tissue and paracancerous tissue:

a. and (3) taking the breast cancer tissue and the tissue beside the breast cancer to carry out circRNA high-throughput sequencing.

b. Bioinformatics analysis was performed on the results of the high throughput sequencing. By analyzing the sequencing result of the circRNA, the circRNA which is highly expressed in the tumor tissue and has the coding function is selected. IC prediction and adaptation of neoantigens using websites such as NetMHC ₅₀ Value criteria, epitope from the polypeptide expected to be encoded by the circRNA was analyzed.

c. The target circRNA, circFam53b, was initially identified.

2. Verification of differences in circRNA expression in breast cancer tissues and paracarcinoma tissues:

a. extracting RNA from breast cancer tissues and tissues beside the breast cancer, and synthesizing related primers. The differences in expression of these circRNAs were verified by qRT-PCR.

b. The total RNA is treated by RNase R capable of hydrolyzing linear RNA, and Northern Blot is carried out on the RNA to verify that a target circRNA band still exists. Then taking RNA treated by RNase R for reverse transcription, and then carrying out fluorescence quantitative PCR to prove that the target is circRNA which can not be hydrolyzed by RNase R.

c. The sequence of the circRNA was determined by sequencing the products of the general PCR by one generation to verify the sequence at the junction of these circrnas.

3. Demonstrating the function of the circFam53 b-encoding polypeptide:

a. ORF and IRES sequences of circFAm53b and the like were predicted from the data of the databases of circBase, circRNADB and the like.

b. (ii) constructing a FLuc-IRES-RLuc dual-luciferase labelled plasmid and cloning the empty, wild-type IRES sequence and the mutant IRES sequence within the plasmid, respectively. Through a dual-luciferase report experiment, the IRES sequence of circFam53b is proved to have strong activity and the potential of encoding protein.

c. Plasmid marking the circFam53b before ORF termination code and unloaded plasmid were prepared, and the plasmid was transfected into HEK293T cells by Lipo 3000, total protein in the cells was extracted after 48 hours, and the protein marked by Flag was verified by Western Blot experiment.

d. After transfection of the same plasmid, an electrophoresis gel was obtained by Western blot, and after staining with Coomassie Brilliant blue, mass spectrometry was performed on the gel strip at the corresponding position. And (3) verifying whether the specific peptide fragment coded by the circRNA exists or not by analyzing the detection result of the mass spectrum.

e. The amino acid sequence of the polypeptide possibly encoded by the circRNA can be known from the data of the circBase, circRNADb and other databases. Antibodies specifically binding to the polypeptides were prepared, and expression of the polypeptides in tumor tissues was verified by Western Blot.

Verification of the immunogenicity of the polypeptide encoded by CircFam53 b:

a. the affinity of a specific polypeptide sequence of the polypeptide encoded by the circFam53b with HLA class I molecules is predicted through a NetMHC website. According to the prediction result of NetMHC, the epitope of the polypeptide coded by the circRNA is selected according to the affinity and is studied.

b. Chemically synthesizing polypeptide containing the polypeptide epitope coded by the circRNA.

c. PBMC (peripheral blood mononuclear cells) and T cells from breast cancer patients were isolated. PBMCs were cultured in DMEM medium supplemented with 1% diabody (penicillin and streptomycin) and 10% human serum of AB blood group and induced by GM-CSF (granulocyte-macrophage colony stimulating factor) and IL-4(20ng/mL) at 50ng/mL, and synthetic polypeptides were added after 6 days, cytokines and polypeptides were replaced every three days, and cultured T cells were cultured at 1:1 (number/number), 9 days later were seeded into plates of an elispot kit with INF-r antibodies embedded. And removing the supernatant after 24 hours, washing with PBS for 5 times, adding primary antibody, incubating for 2 hours, washing with PBS for 5 times, adding secondary antibody, incubating for 1 hour, adding TMB color development solution, and washing with deionized water after spots appear. And observing the formation condition of INF-r spots in the culture plate by an enzyme-linked immunosorbent spot detector.

PBMC were cultured in DMEM medium supplemented with 1% diabody and 10% human serum of AB blood group and induced by 50ng/mL GM-CSF and IL-4(20ng/mL), and cultured T cells were taken at 1X 10 ⁶ The cells were co-cultured with PBMC at a ratio of 1:1, the cytokines and polypeptides were replaced every three days and stimulated with 20. mu.g/ml of synthetic polypeptide, and after 9 days Brefeldin A was added to inhibit cytokine secretion. After 24 hours, the treated cells were collected and fixed by staining with CD3, CD8 and CD4 molecules, and then were subjected to membrane rupture with a membrane rupture agent, followed by staining with IFN-. gamma.TNF-. alpha.and IL-2, respectively. And (4) carrying out flow cytometry detection, and analyzing the activation condition and cytokine production condition of CD4+ and CD8+ T cells.

e. The purpose of this example was to detect that the polypeptide encoded by circFam53b stimulates the production of antigen-specific T cells. PBMC (peripheral blood mononuclear cells) and T cells in peripheral blood of breast cancer patients were isolated from lymphocyte isolates. The CD 8T cells obtained by magnetic bead sorting are resuspended in DMEM complete medium containing 10% v/v AB blood group human serum, IL-2(20ng/mL) with corresponding concentration is added, and the mixture is cultured in a CO2 incubator at 37 ℃ to obtain cultured T cells. The remaining monocytes were resuspended in DMEM complete medium containing 10% v/v human serum of AB blood group and appropriate concentrations of GM-CSF (50ng/mL) and IL-4(20ng/mL) were added, the cytokines were changed every 3 days, and cultured in a CO2 incubator at 37 ℃ for 6 days to obtain DC cells. The DC cells and T cells were co-cultured in a 24-well plate at a ratio of 1:1 and the epitope polypeptide encoded by circFam53b (concentration 20. mu.g/mL, 100. mu.L per well) was added and co-cultured for 3 weeks. After the culture is finished, the cells are collected, cell death and cell death are carried out, antibodies of CD3 and CD8 are subjected to surface labeling, then flow staining is carried out by using an MHC class I molecule pentamer customized by Proimmune, and finally the proportion of pentamer-positive antigen-specific CD8+ T cells in the CD 8-positive cells is detected by flow detection.

5. The polypeptide coded by the circFam53b can activate T cell anti-tumor immunity:

a. a healthy NOD/SCID mouse is taken, breast cancer patient tissues are taken and implanted into a mouse breast fat pad, and a PDX mouse model is established. The groups were randomly divided into a blank control group (no treatment), a negative control group (adoptive patient T cells and dendritic cells), and an experimental group (adoptive patient T cells and dendritic cells plus the polypeptide encoded by circFam53 b).

Starting experimental treatment when tumor tissues are accessible after the PDX model is successfully established. The blank control group was not subjected to any treatment; the positive control group is adoptively used for the T cells and the dendritic cells of the patient; the experimental group was adoptively cultured on patient T cells and dendritic cells and plus the polypeptide encoded by circFam53b, and the tumor growth of the mice was observed and recorded.

c. Tumor tissues were sectioned and observed for T cell infiltration.

Example 1 demonstrates that circFam53b is highly expressed in tumor tissue and encodes a polypeptide

The test method comprises the following steps:

(1) and extracting the RNA of the breast cancer tissue and the paracarcinoma tissue and carrying out high-throughput sequencing. Analyzing the sequencing result, and determining a target molecule, namely circFam53 b. After grinding by liquid nitrogenThe RNA of the breast cancer tissue and the paracarcinoma tissue is extracted by using Trizol reagent, and the expression amount of circFam53b is verified by carrying out fluorescent quantitative PCR. The primers are as follows: the upstream primer F1: 5'-ACGACAAGAAGGTCGGTGTT-3', downstream primer R1: 5'-CCATCTGTCATTTTCTGTGCCA-3' are provided. The reaction condition is TAKARA TB Green ^TM Premix Ex Taq ^TM II, the experiment conditions recommended by the fluorescent quantitative PCR reagent kit. The results are shown in fig. 1A, suggesting that circFam53b is highly expressed in tumor tissues, significantly higher than normal tissues. The circFam53b was analyzed for ORF and IRES sequences by circBase, circRNADB, etc. databases. circFam53b was found to have IRES sequences and ORFs, suggesting that circFam53b has the potential to encode polypeptides.

(2) Demonstrating the function of circFam53b encoding a polypeptide: firstly, the activity of the IRES sequence of the circRNA is verified, a Luc-IRES-RLuc dual-luciferase labeled plasmid is constructed, the vector is psiCHECK-2, and a no-load wild-type IRES sequence and a mutant IRES sequence are respectively cloned in the plasmid, wherein the IRES is cloned between the Luc and the RLuc of the vector psiCHECK-2. Wherein the sequence of the wild-type IRES is: CCTGAAAAGGACAGCTCACTATGGCACCGGGAGGCAGTGACCGCCTGCGCTGTGACCAGTCTGATCAAAGACCTCAGCATCAGCGACCACA ACGGGAACCCCTCAGCACCCCCTAGCAAGCGCCAGTGCCGCTCACTGTCCTTCTCCGA are provided. The sequence of the mutant IRES is: CCTGTTTTGGACAGCTTACTATCCTGCCGGGACCCAGTGACCTTTTGCGCTGTGAGGTGTCTGATCGGGGACCTCAGCATCTTTGCACACA ACGGGAATTTTTCAGCATTTAACTGCCAGCGCCAGTGCCGCTCATTGTCCTTCTCACG are provided. Through a dual-luciferase report experiment, if the result shows that the fluorescence activity of the wild type plasmid RLuc/Luc is higher than that of the unloaded group and the mutant type, the IRES sequence activity of the circRNA is proved to be strong, and the protein coding potential is realized.

(3) It was again demonstrated whether circRNA could directly encode the polypeptide:

1) preparing a plasmid of Flag-marked circRNA and an unloaded plasmid, wherein the unloaded plasmid is pHBLV-CMV-MCS-EF1-zsgreen-t2a-puro of Hanheng organisms, and the target vector is as follows: pHBLV-CMV-Circ-MCS-EF1-zsgreen-t2a-puro as shown in FIG. 2.

The target vector is prepared by a gene synthesis company, and is obtained by directly synthesizing a target sequence (shown in the following) and seamlessly cloning the target sequence to the XhoI enzyme-digested and linearized vector pHBLV-CMV-MCS-EF1-zsgreen-t2a-puro through HB infusion (TM).

CCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGC TTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGAAAAGTGCTGAGATTACAGGCGTGAGCCACCACCCCCGGCCCAC TTTTTGTAAAGGTACGTACTAATGACTTTTTTTTTATACTTCAGAAAATGACAGATGGCGAGACCTGGACAGGAAATGCCCTCTTCAGATT GACCAACCGAGCACCAGCATCTGGGAATGCCTGCCTGAAAAGGACAGCTCACTATGGCACCGGGAGGCAGGACTACAAGGATGACGATGAC AAGGATTACAAAGACGACGATGATAAGGACTATAAGGATGATGACGACAAATGACCGCCTGCGCTGTGACCAGTCTGATCAAAGACCTCAG CATCAGCGACCACAACGGGAACCCCTCAGCACCCCCTAGCAAGCGCCAGTGCCGCTCACTGTCCTTCTCCGATGAGATGTCCAGTTGCCGG ACATCATGGAGGCCCTTGGGCTCCAAAGTCTGGACTCCCGTGGAAAAGAGACGCTGCTACAGCGGGGGCAGCGTCCAGCGCTATTCCAACG GCTTCAGCACCATGCAGAGGAGTTCCAGCTTCAGCCTCCCTTCCCGGGCCAACGTGCTCTCCTCACCCTGCGACCAGGCAGGACTCCACCA CCGATTTGGAGGGCAGCCCTGCCAAGGGGTGCCAGGCTCAGCCCCGTGTGGACAGGCAGGTGACACCTGGAGCCCTGACCTGCACCCCGTG GGAGGAGGCCGGCTGGACCTGCAGCGGTCCCTCTCTTGCTCACATGAGCAGTTTTCCTTTGTGGAATACTGTCCTCCCTCAGCCAACAGCA CACCTGCCTCAACACCAGAGCTGGCGAGACGCTCCAGCGGCCTTTCCCGCAGCCGCTCCCAGCCGTGTGTCCTTAACGACAAGAAGGTCGG TGTTAAAAGGCGGCGCCCTGAAGAAGTGCAAGAGCAGAGGCCTTCTCTAGACCTTGCCAAGATGGCACAGGTAAGAAGCAAGGAAAAGAAT TAGGCTCGGCACGGTAGCTCACACCTGTAATCCCAGCATAGCTTAAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAG CCATCTGTTGTTTGCCCCTCCCCCGTGCCT。

2) Sequencing the obtained positive clones;

3) sequencing and aligning results:

the sequencing result shows that: and (5) obtaining the target vector if the sequencing result is consistent with the target sequence.

4) Plasmid extraction

After sequencing is successful, arranging bacterial liquid amplification according to project requirements, and carrying out plasmid extraction and purification, wherein the plasmid extraction scheme is subject to the instruction of the extraction kit. The extracted plasmids were used to transfect cells after QC validation was required.

5) The plasmid was transfected into HEK293T cells by Lipo 3000, total protein in the cells was extracted after 48 hours, and the protein labeled with Flag was confirmed by Western Blot experiment. The anti-Flag tag antibody is applied in Western Blot experiment, and the ORF of the target circRNA is proved to have the function of encoding polypeptide (shown in FIG. 1B). By specific antibody, the antibody is a custom antibody, and the immunogen sequence is as follows: VQEQRPSLDLAKMAQKMTDGETWTGNALFRLTNRAPASGNACLKRTAHYGTGRQ, antibody manufactured by Nanjing Kinshire Biotechnology Ltd. After extracting proteins from breast cancer tissues, Western Blot experiments prove that the polypeptide encoded by circFam53b is expressed in breast cancer tissues (as shown in fig. 1C).

The purpose of this example was to demonstrate the encoding capacity of circFam53b and to demonstrate that the polypeptide encoded by circFam53b is expressed in tumor tissue.

Example 2 the polypeptide encoded by circFam53b is immunogenic.

The affinity of the specific polypeptide sequence of the polypeptide encoded by the circFam53b with HLA class I molecules is predicted through IEDB and NetMHC websites. According to the prediction results of IEDB and NetMHC, the epitope of the polypeptide coded by the circRNA is selected according to the degree of affinity and is researched. IEDB predicts the polypeptide RANK <3, as a preferred epitope. The purpose of this example was to verify that the polypeptide encoded by circFam53b is immunogenic.

The test method comprises the following steps:

PBMC (peripheral blood mononuclear cells) and T cells in peripheral blood (from Guangdong province) of breast cancer patients were isolated from lymphocyte isolates. The PBMC cells sorted with magnetic beads were resuspended in DMEM complete medium containing 1% diabody (penicillin and streptomycin), 10% (v/v) AB blood group human serum, appropriate concentrations of GM-CSF (50ng/mL in medium) and IL-4(20ng/mL in medium) were added, the cytokines were changed every 3 days, 37 ℃ with CO ₂ The cells were cultured in an incubator for 6 days to obtain DC cells. The CD 8T cells obtained by magnetic bead sorting were resuspended in DMEM complete medium containing 10% v/v AB blood group human serum and IL-2(20ng/mL concentration in the medium) was added at a corresponding concentration at 37 ℃ and CO ₂ Culturing in an incubator for 6 days to obtain cultured T cells. Co-culturing DC cells and cultured T cells according to the number ratio of 1:1, wherein the concentration of the epitope polypeptide in the cell mixed solution is 20mu.g/mL, were mixed and cultured in 1mL of medium, and cytokines (GM-CSF at a final concentration of 50ng/mL and IL-4 at a final concentration of 20ng/mL, with the replacement of cytokines being performed by adding the epitope polypeptide at a final concentration of 20. mu.g/mL based on DMEM complete medium containing 10% (v/v) of human serum from the AB blood group) were replaced every 3 days. After 9 days, co-culturing in an IFN-gamma ELISpot 96 pore plate coated with human IFN-gamma antibody produced by Mabtech company, adding an antigenic epitope polypeptide coded by circFam53b (namely ALFRLTNRA, the concentration of the antigenic epitope polypeptide in cell mixed liquor is 20 mu g/mL, the antigenic epitope polypeptide is predicted to have high affinity with HLA-A2: 01 through databases such as IEDB, NetMCH and the like), discarding supernatant after 24 hours, washing with PBS for 5 times, adding a biotin detection antibody for incubation for 2 hours, washing with PBS for 5 times, adding an HRP labeled secondary antibody for incubation for 1 hour, finally adding an HRP TMB color developing solution, and washing with deionized water after spots appear. And observing the formation condition of INF-r spots in the culture plate by an enzyme-linked immunosorbent spot detector.

PBMC (peripheral blood mononuclear cells) and T cells in peripheral blood (from Guangdong province) of breast cancer patients were isolated from lymphocyte isolates. The PBMC cells obtained by magnetic bead sorting were resuspended in DMEM complete medium containing 1% double antibody and 10% (v/v) AB blood group human serum, GM-CSF (50ng/mL in medium) and IL-4(20ng/mL in medium) were added at appropriate concentrations, cytokines were replaced every 3 days, 37 ℃ and CO was added at 37 ℃. (in terms of concentration) ₂ The cells were cultured in an incubator for 6 days to obtain DC cells. CD 8T cells obtained by magnetic bead sorting were resuspended in DMEM complete medium containing 10% v/v human serum of blood group AB, IL-2(20ng/mL concentration in the medium) was added at a corresponding concentration, 37 ℃ and CO ₂ Culturing in an incubator for 6 days to obtain cultured T cells. DC cells and cultured T cells (1X 10) ⁶ Individual) were co-cultured in 24-well plates at a quantitative ratio of 1:1 and the epitope polypeptide encoded by circFam53b was added. The concentration of the epitope polypeptide in the cell mixture was 20. mu.g/mL, and after culturing in 1mL of the medium for 9 days, the cytokines (GM-CSF at a final concentration of 50ng/mL and IL-4 at a final concentration of 20ng/mL, and in the case of changing the cytokines, the epitope polypeptide was added at a final concentration of 20. mu.g/mL based on the DMEM complete medium containing 10% (v/v) of human serum from AB blood group, followed by addition of Brefeldin A (concentration 5. mu.g/mL in the cell mixture) was added to each well 1mL of DMEM complete medium containing 10% (v/v) AB blood group human serum and cultured for 6 hours to inhibit cytokine secretion. Collecting cells after the culture is finished, carrying out cell death and survival, carrying out surface marking on CD3 and CD8 antibodies, then fixing the cells for 30 minutes at room temperature by using a Sermefei IC fixing solution, perforating a cell membrane by using a Sermefei film breaking agent, adding corresponding proteins in anti-IFN-gamma, TNF-alpha, Granzyme B and antibody marking cells, and finally detecting the expression levels of the corresponding proteins such as IFN-gamma, TNF-alpha and Granzyme B in CD8 positive cells by flow detection.

The results of this part of the experiment are shown in fig. 3, which suggests that the epitope polypeptide (i.e. ALFRLTNRA) encoded by circFam53b can activate the immune response of human CD8+ T cells.

Example 3 the polypeptide encoded by circFam53b stimulates the production of antigen-specific T cells

The purpose of this example was to detect that the polypeptide encoded by circFam53b stimulates the production of antigen-specific T cells. PBMC (peripheral blood mononuclear cells) and T cells in peripheral blood of breast cancer patients were isolated by lymphocyte separation. CD 8T cells obtained by magnetic bead sorting were resuspended in DMEM complete medium containing 10% v/v AB blood group human serum and IL-2(20ng/mL) was added at the corresponding concentration, at 37 ℃ and CO ₂ Culturing in an incubator for 6 days to obtain cultured T cells. The remaining monocytes were resuspended in DMEM complete medium containing 10% v/v human serum of AB blood group and appropriate concentrations of GM-CSF (50ng/mL) and IL-4(20ng/mL) were added, the cytokines were changed every 3 days at 37 deg.C with CO ₂ The cells were cultured in an incubator for 6 days to obtain DC cells. The DC cells and the cultured T cells were co-cultured in a 24-well plate at a ratio of 1:1 and the epitope polypeptide encoded by circFam53b (concentration 20. mu.g/mL, 100. mu.L per well) was added and co-cultured for 3 weeks. After the culture is finished, the cells are collected, cell death and cell death are carried out, antibodies of CD3 and CD8 are subjected to surface labeling, then flow staining is carried out by using an MHC class I molecule pentamer customized by Proimmune, and finally, the proportion of pentamer-positive antigen-specific CD8+ T cells in the CD8 positive cells is detected through flow detection.

The results shown in figure 3D suggest that the results of this section of the experiment suggest that the epitope polypeptide encoded by circFam53b (i.e., ALFRLTNRA) can stimulate the production of antigen-specific CD8+ T cells.

Example 4 efficacy of adoptive circFam53 b-encoded epitope-activated T cells in a mouse model of breast cancer PDX

The purpose of this example is to further verify that the epitope-activated T cells encoded by circFam53b have tumor growth-inhibiting effect. A healthy NOD/SCID mouse (Hunan Slek Jingda laboratory animals Co., Ltd.) was taken, breast cancer patient tissues were taken, and a mouse breast fat pad was implanted to establish a PDX mouse model. The experimental treatment was started when the tumor tissue was accessible. The blank control group was not subjected to any treatment; the positive control group is adoptively treated by 2.5X 10 patients ⁶ T cells and 5X 10 ⁵ A plurality of dendritic cells; the experimental group was adoptively treated with equal amounts of autologous T cells and dendritic cells from the patient and supplemented with 20. mu.g/mL of epitope polypeptide encoded by circFam53b, the cell culture method was as in example 2, and the tumor growth of the mice was observed and recorded. The results are shown in FIG. 4, which shows that the antigenic epitope polypeptide encoded by circFam53b acts on T cells obtained by adoptive T cells to effectively inhibit the growth of tumors.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various applications, as well as variations of methods and compositions of the invention, set forth herein will be apparent to those of skill in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Sequence listing

<110> grand era memorial Hospital of Zhongshan university

<120> human tumor antigen capable of activating anti-tumor immune response of patient and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> human (Homo sapiens)

<223> human tumor antigen capable of activating anti-tumor immune response of patient

<400> 1

Ala Leu Phe Arg Leu Thr Asn Arg Ala

1 5

<210> 2

<211> 27

<212> DNA

<213> human (Homo sapiens)

<223> nucleotide sequence encoding human tumor antigen that activates anti-tumor immune response in patients

<400> 2

gccctcttca gattgaccaa ccgagca 27

<210> 3

<211> 219

<212> PRT

<213> human (Homo sapiens)

<223> circFam53 b-encoding polypeptide sequence

<400> 3

Met Ser Ser Cys Arg Thr Ser Trp Arg Pro Leu Gly Ser Lys Val Trp

1 5 10 15

Thr Pro Val Glu Lys Arg Arg Cys Tyr Ser Gly Gly Ser Val Gln Arg

20 25 30

Tyr Ser Asn Gly Phe Ser Thr Met Gln Arg Ser Ser Ser Phe Ser Leu

35 40 45

Pro Ser Arg Ala Asn Val Leu Ser Ser Pro Cys Asp Gln Ala Gly Leu

50 55 60

His His Arg Phe Gly Gly Gln Pro Cys Gln Gly Val Pro Gly Ser Ala

65 70 75 80

Pro Cys Gly Gln Ala Gly Asp Thr Trp Ser Pro Asp Leu His Pro Val

85 90 95

Gly Gly Gly Arg Leu Asp Leu Gln Arg Ser Leu Ser Cys Ser His Glu

100 105 110

Gln Phe Ser Phe Val Glu Tyr Cys Pro Pro Ser Ala Asn Ser Thr Pro

115 120 125

Ala Ser Thr Pro Glu Leu Ala Arg Arg Ser Ser Gly Leu Ser Arg Ser

130 135 140

Arg Ser Gln Pro Cys Val Leu Asn Asp Lys Lys Val Gly Val Lys Arg

145 150 155 160

Arg Arg Pro Glu Glu Val Gln Glu Gln Arg Pro Ser Leu Asp Leu Ala

165 170 175

Lys Met Ala Gln Lys Met Thr Asp Gly Glu Thr Trp Thr Gly Asn Ala

180 185 190

Leu Phe Arg Leu Thr Asn Arg Ala Pro Ala Ser Gly Asn Ala Cys Leu

195 200 205

Lys Arg Thr Ala His Tyr Gly Thr Gly Arg Gln

210 215

<210> 4

<211> 773

<212> DNA

<213> human (Homo sapiens)

<223> base sequence of circFam53b

<400> 4

aaaatgacag atggcgagac ctggacagga aatgccctct tcagattgac caaccgagca 60

ccagcatctg ggaatgcctg cctgaaaagg acagctcact atggcaccgg gaggcagtga 120

ccgcctgcgc tgtgaccagt ctgatcaaag acctcagcat cagcgaccac aacgggaacc 180

cctcagcacc ccctagcaag cgccagtgcc gctcactgtc cttctccgat gagatgtcca 240

gttgccggac atcatggagg cccttgggct ccaaagtctg gactcccgtg gaaaagagac 300

gctgctacag cgggggcagc gtccagcgct attccaacgg cttcagcacc atgcagagga 360

gttccagctt cagcctccct tcccgggcca acgtgctctc ctcaccctgc gaccaggcag 420

gactccacca ccgatttgga gggcagccct gccaaggggt gccaggctca gccccgtgtg 480

gacaggcagg tgacacctgg agccctgacc tgcaccccgt gggaggaggc cggctggacc 540

tgcagcggtc cctctcttgc tcacatgagc agttttcctt tgtggaatac tgtcctccct 600

cagccaacag cacacctgcc tcaacaccag agctggcgag acgctccagc ggcctttccc 660

gcagccgctc ccagccgtgt gtccttaacg acaagaaggt cggtgttaaa aggcggcgcc 720

ctgaagaagt gcaagagcag aggccttctc tagaccttgc caagatggca cag 773

<210> 5

<211> 660

<212> DNA

<213> human (Homo sapiens)

<223> sequence of open reading frame of circFam53b encoding polypeptide

<400> 5

atgtccagtt gccggacatc atggaggccc ttgggctcca aagtctggac tcccgtggaa 60

aagagacgct gctacagcgg gggcagcgtc cagcgctatt ccaacggctt cagcaccatg 120

cagaggagtt ccagcttcag cctcccttcc cgggccaacg tgctctcctc accctgcgac 180

caggcaggac tccaccaccg atttggaggg cagccctgcc aaggggtgcc aggctcagcc 240

ccgtgtggac aggcaggtga cacctggagc cctgacctgc accccgtggg aggaggccgg 300

ctggacctgc agcggtccct ctcttgctca catgagcagt tttcctttgt ggaatactgt 360

cctccctcag ccaacagcac acctgcctca acaccagagc tggcgagacg ctccagcggc 420

ctttcccgca gccgctccca gccgtgtgtc cttaacgaca agaaggtcgg tgttaaaagg 480

cggcgccctg aagaagtgca agagcagagg ccttctctag accttgccaa gatggcacag 540

aaaatgacag atggcgagac ctggacagga aatgccctct tcagattgac caaccgagca 600

ccagcatctg ggaatgcctg cctgaaaagg acagctcact atggcaccgg gaggcagtga 660

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> upstream primer F1

<400> 6

acgacaagaa ggtcggtgtt 20

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> downstream primer R1

<400> 7

ccatctgtca ttttctgtgc ca 22

<210> 8

<211> 149

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> wild-type IRES

<400> 8

cctgaaaagg acagctcact atggcaccgg gaggcagtga ccgcctgcgc tgtgaccagt 60

ctgatcaaag acctcagcat cagcgaccac aacgggaacc cctcagcacc ccctagcaag 120

cgccagtgcc gctcactgtc cttctccga 149

<210> 9

<211> 149

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> mutant IRES

<400> 9

cctgttttgg acagcttact atcctgccgg gacccagtga ccttttgcgc tgtgaggtgt 60

ctgatcgggg acctcagcat ctttgcacac aacgggaatt tttcagcatt taactgccag 120

cgccagtgcc gctcattgtc cttctcacg 149

<210> 10

<211> 1117

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> sequence of interest

<400> 10

ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc 60

tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg 120

gagacccaag ctggctagaa aagtgctgag attacaggcg tgagccacca cccccggccc 180

actttttgta aaggtacgta ctaatgactt tttttttata cttcagaaaa tgacagatgg 240

cgagacctgg acaggaaatg ccctcttcag attgaccaac cgagcaccag catctgggaa 300

tgcctgcctg aaaaggacag ctcactatgg caccgggagg caggactaca aggatgacga 360

tgacaaggat tacaaagacg acgatgataa ggactataag gatgatgacg acaaatgacc 420

gcctgcgctg tgaccagtct gatcaaagac ctcagcatca gcgaccacaa cgggaacccc 480

tcagcacccc ctagcaagcg ccagtgccgc tcactgtcct tctccgatga gatgtccagt 540

tgccggacat catggaggcc cttgggctcc aaagtctgga ctcccgtgga aaagagacgc 600

tgctacagcg ggggcagcgt ccagcgctat tccaacggct tcagcaccat gcagaggagt 660

tccagcttca gcctcccttc ccgggccaac gtgctctcct caccctgcga ccaggcagga 720

ctccaccacc gatttggagg gcagccctgc caaggggtgc caggctcagc cccgtgtgga 780

caggcaggtg acacctggag ccctgacctg caccccgtgg gaggaggccg gctggacctg 840

cagcggtccc tctcttgctc acatgagcag ttttcctttg tggaatactg tcctccctca 900

gccaacagca cacctgcctc aacaccagag ctggcgagac gctccagcgg cctttcccgc 960

agccgctccc agccgtgtgt ccttaacgac aagaaggtcg gtgttaaaag gcggcgccct 1020

gaagaagtgc aagagcagag gccttctcta gaccttgcca agatggcaca ggtaagaagc 1080

aaggaaaaga attaggctcg gcacggtagc tcacacctgt aatcccagca tagcttaagt 1140

ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 1200

ctcccccgtg cct 1213

<210> 11

<211> 54

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<223> immunogen

<400> 11

Val Gln Glu Gln Arg Pro Ser Leu Asp Leu Ala Lys Met Ala Gln Lys

1 5 10 15

Met Thr Asp Gly Glu Thr Trp Thr Gly Asn Ala Leu Phe Arg Leu Thr

20 25 30

Asn Arg Ala Pro Ala Ser Gly Asn Ala Cys Leu Lys Arg Thr Ala His

35 40 45

Tyr Gly Thr Gly Arg Gln

50

Claims (11)

1. A human tumor antigen that activates an anti-tumor immune response in a patient, comprising: the amino acid sequence of the human tumor antigen capable of activating the anti-tumor immune response of the patient is shown as follows: ALFRLTNRA are provided.

2. A nucleic acid molecule encoding the human tumor antigen of claim 1 that activates an anti-tumor immune response in a patient.

3. The nucleic acid molecule of claim 2, wherein: the sequence of the nucleic acid molecule is shown as follows: GCCCTCTTCAGATTGACCAACCGAGCA are provided.

4. Use of a human tumor antigen as claimed in claim 1 and/or a nucleic acid molecule as claimed in any one of claims 2 to 3 for the preparation of an anti-tumor medicament for activating an anti-tumor immune response in a patient, wherein: the tumor comprises solid tumor and non-solid tumor with circFam53b over-expression or circFam53b encoding polypeptide expression;

the base sequence of the circFam53b is shown in SEQ ID NO. 4.

5. Use according to claim 4, characterized in that: the tumor is breast cancer.

6. An antitumor agent characterized by: comprising a human tumor antigen as claimed in claim 1 which activates an anti-tumor immune response in a patient, and/or a nucleic acid molecule as claimed in any one of claims 2 to 3.

7. The antitumor agent according to claim 6, characterized in that: also included are one or both of T cells and dendritic cells.

8. The antitumor agent as claimed in claim 7, wherein: said T cells and said dendritic cells are derived from adoptive patients.

9. The antitumor agent as claimed in any one of claims 6 to 8, wherein: the tumor comprises solid tumor and non-solid tumor with circFam53b over-expression or circFam53b encoding polypeptide expression;

the base sequence of the circFam53b is shown in SEQ ID NO. 4.

10. The antitumor agent as claimed in claim 9, characterized in that: the tumor is breast cancer.

11. An in vitro method for screening human tumor antigens as claimed in claim 1 which activate anti-tumor immune response in a patient, comprising the steps of: carrying out circRNA high-throughput sequencing on a breast cancer tissue and a breast cancer adjacent tissue; performing bioinformatics analysis on the high-throughput sequencing result, and selecting circRNA which is highly expressed in the tumor tissue and has a coding function by analyzing the sequencing result of the circRNA; determining the result of bioinformatics analysis through experiments, screening the polypeptide encoded by circFam53b obtained as a target for tumor immunotherapy, and selecting part or all of the polypeptide encoded by circFam53b to prepare a polypeptide vaccine, so as to obtain the human tumor antigen capable of activating the anti-tumor immune response of the patient as claimed in claim 1;

the base sequence of the circFam53b is shown in SEQ ID NO. 4.

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