CN118615431A - Application of an antigen epitope in the preparation of a product for treating tumors - Google Patents

Abstract

The present invention discloses an application of an antigen epitope in the preparation of a product for treating tumors. The present invention introduces point mutations at the RNA level to generate new antigen epitopes for tumor antigens, so that the mutated antigen epitopes are not restricted by negative screening and can be used as targets for tumor immune products, such as tumor vaccines, cell therapy, etc., for effective treatment and/or prevention of tumors.

Description

Application of an antigen epitope in the preparation of a product for treating tumors

Technical Field

The invention belongs to the field of biotechnology, and specifically relates to an application of an antigen epitope in preparing a product for treating tumors.

Background Art

Malignant tumors are a huge threat to modern human health. Since the 20th century, people have begun to use the immune system to specifically eliminate the occurrence of tumors. Along with genetic changes, these mainly include synonymous mutations, non-synonymous mutations, insertion or deletion mutations, etc. These genetic changes may produce new antigens. Tumor cells with new antigens are captured by nearby antigen-presenting cells after death. After phagocytosis and processing, they present new antigens and activate T cells in nearby draining lymph nodes. The latter reach the tumor microenvironment through the circulatory system, recognize the new antigens on the surface of tumor cells, and then kill the tumor.

Currently, immunotherapies are being developed for neoantigens, including personalized neoantigen vaccines, immune checkpoint inhibitors (ICB), and adoptive cell transfer (ACT) of antigen-specific T cells. In 2015, an article on tumor neoantigen vaccines was first published in Science, confirming that neoantigen DC vaccines can effectively activate antigen-specific T cells. In 2017, a study on peptide vaccines and RNA vaccines targeting melanoma neoantigens was published in Nature. T cell responses to some vaccine neoepitopes were detected in 13 patients who received multi-epitope RNA vaccines. Biopsy reports from two patients confirmed the vaccine-induced infiltration of neoantigen-specific T cells and specific killing of autologous melanoma cells. In addition to tumor vaccines developed against neoantigens, adoptive cell transfer therapy (ACT) targeting neoantigens has also received attention. In 2021, researchers at the University of Pennsylvania School of Medicine developed a TCR-T cell immunotherapy targeting the KRAS G12V neoantigen for the treatment of difficult-to-treat cancers driven by KRAS mutations, including lung cancer, colorectal cancer, and pancreatic lung cancer. The first clinical trial of this therapy is expected to start in 2022, proving that neoantigens are a very promising clinical target based on immunotherapy.

However, differences in tumors and individual differences among patients also lead to highly personalized production of tumor neoantigens, and neoantigens are difficult to share and use. "Immunoediting" also greatly affects the availability of neoantigens. Nature reported the results of follow-up studies on pancreatic ductal adenocarcinoma (PDAC), revealing the existence of immunoediting in human tumors. And there is evidence that immunoediting may reduce the responsiveness of some tumors, so immunoediting is likely to reduce neoantigen-specific immune responses. The whole genome sequencing and RNA sequencing required for the neoantigen prediction process, and the production of personalized vaccines also make neoantigen therapy expensive, limiting its promotion. The inaccuracy of neoantigen prediction has caused limitations in the development of this treatment. In 2020, the Tumor Neoantigen Selection Alliance (TESLA), composed of 25 neoantigen prediction teams, simultaneously predicted neoantigens in samples from 6 tumor patients. After sorting the antigenic peptides predicted by all teams, the top 608 were selected for immunogenicity testing. The results showed that only 37 were immunogenic, and the average accuracy of immunogenicity prediction was only 6%.

In summary, the defects of neoantigen prediction technology, low expression ratio in some patients, high cost of personalized production, and low immunogenicity of natural neoantigens carried by tumors due to immune editing all limit its potential for widespread application.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes an application of antigen epitope.

The present invention also provides a biological material containing the antigen epitope.

The invention also provides an anti-tumor combination product.

According to one aspect of the present invention, a use of an antigen epitope in any one of the following (1) to (5) is provided;

(1) Preparation of products for treating and/or preventing tumors;

(2) Preparation of CAR-T target therapeutic products;

(3) Preparation of TCR-T target therapeutic products;

(4) Preparation of tumor immune products;

(5) Preparation of tumor-related vaccines;

The antigen epitope is a new antigen epitope obtained by performing site-directed mutagenesis on tumor antigens using RNA editing.

In some embodiments of the present invention, the site-directed mutagenesis is achieved by mutating A to I and/or C to U in the nucleic acid sequence of the tumor antigen RNA.

In some embodiments of the present invention, the tumor antigens include P1A, AFP and Spag9-2.

In some embodiments of the present invention, the amino acid sequence of the antigenic epitope is shown as SEQ ID NO.3, SEQ ID NO.11, SEQ ID NO.16, SEQ ID NO.17 or SEQ ID NO.18.

In some embodiments of the present invention, the tumor antigens include tumor-specific antigens and tumor-associated antigens.

In some embodiments of the present invention, the tumor-associated antigen comprises at least one of an embryonic antigen, a differentiation antigen, a cancer-testis antigen, and a viral antigen.

In some embodiments of the present invention, the tumor-specific antigen comprises at least one of a chemical-induced tumor-specific antigen, a virus-induced tumor-specific antigen, and antigenicity of a spontaneous tumor.

In some embodiments of the present invention, the tumor includes at least one of testicular cancer, rectal cancer, colorectal cancer, small intestine cancer, large intestine cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, fallopian tube cancer, kidney cancer, melanoma, brain tumor, thyroid cancer, parathyroid cancer, leukemia, lymphoma, myeloma, sarcoma, prostate cancer, bladder cancer, bile duct cancer, and gallbladder cancer.

In some embodiments of the present invention, the editing system used in the RNA editing technology includes LEAPER, λN-BoxB or MS2-MCP2.

In some embodiments of the present invention, the method further comprises a step of detecting the prepared antigen epitope, wherein the detection comprises sequence analysis of the antigen epitope.

In some embodiments of the present invention, the sequence analysis method is sequencing.

In some embodiments of the present invention, the step of performing computer-aided design and affinity analysis on new tumor-associated antigen epitopes is also included.

In some embodiments of the present invention, the design software used for computer-aided design analysis of affinity is NetMHCpan-4.1 and other auxiliary software for calculating the affinity between MHC and short peptides.

In a second aspect of the present invention, a biomaterial is provided, which can induce RNA editing to generate the above-mentioned antigenic epitope.

In some embodiments of the present invention, the biological material includes a recombinant expression vector, an expression cassette, a recombinant bacterium, a host cell or RNA.

In some embodiments of the present invention, the expression vector comprises a viral vector.

In some embodiments of the invention, the RNA comprises modified RNA or circular RNA.

In some embodiments of the present invention, the recombinant expression vector comprises an adenosine deaminase guide RNA that edits a target RNA or a construct encoding the guide RNA.

In some embodiments of the present invention, the sequence of the guide RNA expressed by the construct is not less than 16 bp, is reverse complementary to the sequence of the target site mRNA, and has a homology of not less than 85%.

In some embodiments of the present invention, the sequence of the guide RNA is shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12, SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21.

In the third aspect of the present invention, an anti-tumor combination product is proposed, wherein the anti-tumor combination product comprises the above-mentioned antigen epitope and/or biological material.

In some embodiments of the present invention, the anti-tumor combination product further comprises an immune checkpoint inhibitor (ICB).

In some embodiments of the present invention, the immune checkpoint inhibitors include CTLA-4 monoclonal antibody and PD-1/PD-L1 monoclonal antibody.

In some embodiments of the present invention, the anti-tumor combination product is a medicine, a vaccine, a health product or a food.

In some embodiments of the present invention, the drug further comprises a drug carrier and/or a pharmaceutical excipient.

In some embodiments of the present invention, the pharmaceutically acceptable vector comprises a nucleic acid vector.

In some embodiments of the present invention, the nucleic acid vector comprises at least one of a DNA vector, an RNA vector and an adeno-associated virus vector.

In some embodiments of the present invention, the vaccine includes at least one of a polypeptide vaccine, a viral vaccine, a DNA vaccine, an RNA vaccine, and a cell vaccine.

According to some embodiments of the present invention, at least the following beneficial effects are achieved: the scheme of the present invention edits currently existing tumor antigen epitopes to avoid inaccuracy in new antigen prediction and reduce the time and economic cost of prediction; tumor-related antigen epitopes that are shared and expressed in tumor cells but not expressed in normal tissues are selected, and the artificially introduced new antigens have tumor specificity and sharing; an RNA editing system that is safer and more controllable than DNA editing is used, and the editing targets mRNA rather than genomic DNA, and the editing results are reversible and controllable; at the same time, current research on shared antigen epitopes is limited by the highly reactive T cell negative screening of this type of antigen, and the present invention introduces point mutations into this type of antigen, and the mutated antigen epitopes are not limited by negative screening; immune editing may reduce the responsiveness of current new antigen treatments, while the scheme of the present invention can flexibly introduce new antigens at any time during the occurrence and development of tumors, thereby enhancing the responsiveness of immunotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

FIG1 is a flow chart of the design and detection of mutant peptide REISN produced by RNA editing in Example 1 of the present invention;

FIG2 is a map of the pLKO-P1A-P2A-EGFP lentiviral vector in Example 2 of the present invention;

FIG3 is a graph showing the editing efficiency results of the P1A guide RNAs designed by the three systems of LEAPER, λN-BoxB or MS2-MCP2 in Example 2 of the present invention;

FIG4 is a diagram showing the result of successful RNA editing of the P1A epitope in Example 2 of the present invention in CT26 cells;

FIG5 is a map of the Dox-inducible ADAR lentiviral vector in Example 2 of the present invention;

FIG6 is a diagram showing the verification results of the Dox-inducible RNA edited cell line in Example 2 of the present invention;

FIG7 is a graph showing the detection results of the efficiency of RNA editing induced in tumors in Example 2 of the present invention;

FIG8 is a flow chart of the immunotherapy of the REISN vaccine in Example 2 of the present invention;

FIG9 is a graph showing the effect of the REISN vaccine on tumor proliferation in Example 2 of the present invention;

FIG10 is a graph showing the detection results of the efficiency of RNA editing induced in tumors in Example 3 of the present invention;

FIG11 is a graph showing the effect of the REISN vaccine on tumor proliferation in Example 3 of the present invention;

FIG. 12 is a graph showing the editing efficiency detection results of the AFP guide RNAs designed by the three systems of LEAPER, λN-BoxB or MS2-MCP2 in Example 4 of the present invention.

DETAILED DESCRIPTION

The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

Example 1

This embodiment provides a method for preparing an antigen epitope, the principle of which is shown in FIG1 , and the specific process is as follows:

(1) For tumor antigen genes, NetMHCpan-4.1 software was used to predict short peptides with high binding affinity to the corresponding MHC in the gene;

(2) among the adenine nucleotides in the nucleotide sequence of the short peptide screened in (1), find the mutant peptides that can change the amino acid code after mutation to guanine and whose binding affinity to MHC is predicted to be not less than 1000 nM by NetMHCpan-4.1 software, perform site-directed RNA editing point mutation, and prepare new antigen epitopes (mutant peptides);

(3) Detection of new antigenic epitopes, including:

1) Guide RNA design and verification: Design guide RNA based on the RNA editing system (MS2-MCP2, λN-BoxB or LEAPER) and verify the RNA editing efficiency of the target by in vitro transfection of mammalian cells.

2) Verification of the anti-tumor effect of vaccines prepared based on new antigenic epitopes.

Example 2

This example uses the mouse cancer testis antigen P1A as a target to test the preparation method of the antigen epitope in Example 1. The method includes the following steps:

1. Design of REISN site

Taking the mouse cancer testis antigen target P1A as the target, the sequence of the antigen epitope predicted by NetMHCpan-4.1 is shown in SEQ ID NO.1, and the corresponding RNA sequence is shown in SEQ ID NO.2. Among the adenine nucleotides on the peptide segment, the REISN short peptide (sequence: LPCLGWLVF) that can change the amino acid code after mutation to guanine and has a binding affinity with MHC of more than 1000nM is found. After inducing RNA editing, a tumor neoantigen (RNA Editing-Induced Site-Specific Neoantigen, REISN) is generated, the sequence is shown in SEQ ID NO.3, and the corresponding RNA sequence is shown in SEQ ID NO.4. The new antigen epitope predicted by NetMHCpan-4.1 has a binding affinity of 437.34nM with H-2-Ld, and the specific sequence is shown in Table 1.

Table 1

2. REISN guide RNA design and validation

The guide RNA design for the P1A site shown in SEQ ID NO.2 was based on and compared with three systems:

(1) Design of guide RNA based on MS2-MCP2 system: The MS2-MCP2 system consists of two parts: exogenous ADAR1 and guide RNA (the sequence is shown in SEQ ID NO.5 in Table 2).

The two plasmids pcDNA3.1(-)-U6-MCP-ADAR1-TAG and pUC57-arP1A-MS2 were constructed. Both the pcDNA3.1(-)-U6-MCP-ADAR1-TAG plasmid and the arP1A-MS2 gene were synthesized by Nanjing GenScript Company.

The pcDNA3.1(-)-U6-MCP-ADAR1-TAG plasmid is obtained by inserting the U6-MCP-ADAR1-TAG sequence (sequence shown in SEQ ID NO. 24) into the pcDNA3.1 vector between EcoRI and BamHI.

pUC57-arP1A-MS2 is obtained by inserting the U6-arP1A-MS2 fragment (sequence shown in SEQ ID NO. 25) into the EcoRI and HindIII restriction sites in the MCS region of the pUC57 vector. The sequence was verified to be correct and complete.

(2) Guide RNA design based on the LEAPER system: The LEAPER editing system consists of a guide RNA (the sequence is shown in SEQ ID NO.6 in Table 2), which contains a domain that can recruit endogenous ADARs.

The LEAPER editing system consists of a guide RNA (i.e., ar16, the sequence of which is shown in SEQ ID NO.6) to construct the pLKO-p1a-ar16 plasmid. The plasmid clones the U6 promoter and the synthetic ar16 fragment (the sequence of which is shown in SEQ ID NO.6) into the pLKO.1puro (Addgene #8453) backbone between XbaI and SalI by homologous recombination.

(3) Design of guide RNA based on the λN-BoxB system: The λN-BoxB editing system consists of exogenous ADAR2 and guide RNA (the sequence is shown in SEQ ID NO.7 in Table 2), and the pUC57-arP1A-BOXB and pcDNA3.1(-)-U6-arP1A-BOXB-ADAR2 plasmids were constructed and synthesized by Nanjing GenScript Co., Ltd.

Among them, pcDNA3.1(-)-U6-arP1A-BOXB-ADAR2 is obtained by homologous recombination of pcDNA3.1(-)-myc-HisA, inserting the fragment of λN-ADAR2 (sequence as shown in SEQ ID NO.26) into the CMV promoter of the backbone, and then inserting arP1A-BoxB (sequence as shown in SEQ ID NO.7 in Table 2) into the U6 promoter. pUC57-arP1A-BoxB is obtained by cloning the U6 promoter and guide RNA (sequence as shown in SEQ ID NO.7 in Table 2) into the EcoRI and HindIII restriction sites of the pUC57 backbone. Sequencing verifies that the sequence is correct and complete.

(4) Verification of in vitro editing efficiency: Hela cells were plated in 12-well plates and plasmids of guide RNA (respectively, plasmids in (1)-(3), plasmids based on the MS2-MCP2 system: 2500 ng pUC57-arP1A-MS2 and 250 ng pcDNA3.1(-)-U6-MS2-ADAR1-TAG; plasmids based on the BoxB system: 2500 ng pUC57-arP1A-BoxB and 250 ng pcDNA3.1(-)-U6-LN-ADAR2-TAG; plasmids based on the LEAPER system: 250 ng pLKO-p1a-ar16) and 125 ng of P1A overexpression plasmid (pLKO-P1A-P2A-EGFP, sequence as shown in SEQ ID NO: 1) were added. NO.27) (as shown in FIG2) were co-transfected with lipo8000 (Biyuntian), and the cells were lysed 48 hours later to extract RNA. cDNA was obtained after RT-PCR, and PCR was performed using P1A primers (s) (P1A-1F, P1A-RTR2 in Table 3). The PCR products were sent to Sanger sequencing to verify the editing efficiency. The verification results are shown in FIG3 . The editing efficiency of the MS2-MCP2 system was the best, so subsequent experiments were performed using this system. Using the same method, the effectiveness of the MS2-MCP2 system in inducing RNA editing was verified in mouse CT26 cells. The editing effect is shown in FIG4 . The results show that the scheme of the present invention can effectively induce the occurrence of RNA editing.

Table 2

3. Establishment of a model for inducing REISN production in mouse tumors.

The model of inducing REISN production in mouse tumors was established as follows:

(1) Construction of Dox-inducible P1A RNA-edited cell line:

1) Construction of ADAR lentiviral vector, the vector map is shown in Figure 5. The specific construction method is as follows: pCW57-MCS1-P2A-MCS2 (Addgene, #89180) plasmid was transformed, and the ADAR1-MCP gene (sequence shown in SEQ ID NO.24) was cloned by Gibson cloning (Clone IIOne Step Cloning Kit, Vazyme, C112) method, and inserted into the NheI and MluI restriction sites downstream of the tight TRE promoter to obtain the Dox-inducible PCW-ADAR1-PURO (Dox-inducible ADAR) lentiviral vector.

2) To construct a guide RNA lentiviral expression vector, the pLKO.1puro vector (Addgene, #8453) plasmid was modified, and arP1A (sequence shown in SEQ ID NO.5, which was synthesized by Qingke Biotechnology Co., Ltd.) was cloned between the AgeI and EcoRI restriction sites downstream of the U6 promoter; and the puro antibiotic tag was replaced with BSD (product: blasticidin Sdeaminase) to obtain the guide RNA expression vector pLKO.1-arP1A-MS2-BLAST.

3) Since CT26 cells do not express P1A, an overexpression lentiviral vector pLKO-P1A-P2A-EGFP was designed (the P1A-P2A-EGFP sequence (sequence shown in SEQ ID NO.28) was cloned into the pLKO.1puro vector (Addgene, #8453) between NheI and SalI) (the map is shown in Figure 2). After transfecting 293T cells with the packaging plasmid, the supernatant containing the lentivirus was used to infect CT26 cells, and the positive cells were sorted out by the fluorescence channel FITC, which were CT26-P1A cells.

After transfecting 293T cells with the lentiviral vectors obtained in 1) and 2) together with the packaging plasmid, the supernatant containing the lentivirus was harvested. The above 500μl Dox-inducible ADAR and arP1A lentiviral supernatant was used to infect CT26-P1A cells together with 10μg/mL protamine. After 48h, Puromycin (puromycin) with a final concentration of 12μg/mL and Blastidin with a final concentration of 6μg/ml were added for screening to obtain the stable cell line CT26-P1A-REISN. After the cell line was induced with 0.5ug/mL doxycyline, RNA was extracted and RT-PCR was performed (PCR conditions: 95℃, 3min; 95℃, 15s; 60℃15s; 72℃, 1min; cycles 40; 72℃, 5min; 4℃). PCR primers are shown in Table 3. Sanger sequencing. The results are shown in Figure 6. The left and right sides of Figure 6 show the results without adding dox and with adding dox, respectively, proving that arP1A can successfully achieve A-to-I RNA editing in vitro through dox induction.

Balb/C mice (n=3) were subcutaneously injected with 5×105CT26-P1A-REISN cells. After the tumor size grew to a certain size (70-100mm3), they were fed with a feed containing doxycycline (0.2% doxycycline) to induce the expression of REISN after RNA editing. Tumor tissues were collected 10 days later, and total RNA was extracted according to the kit (after DNase digestion), reverse transcribed into cDNA by RT-PCR (further DNase digestion), and the target site was amplified by PCR with corresponding primers. The PCR product was used for Sanger sequencing. The left and right sides of Figure 7 are the sequencing results of tumor tissues in normal mouse diet and dox-added mouse diet, respectively, proving that arP1A can successfully achieve A-to-I RNA editing in vivo through dox induction. The efficiency of RNA editing induced in the tumor was about 38.2% (the green peak represents A before editing, the black peak represents G after editing, and the peak height was measured by QSV analysis software, and the editing efficiency % = G/(A+G)).

Table 3

Primer name sequence P1A-1F Tgataacaagaagccagacaaagc(SEQ ID NO.8) P1A-RTR2 ccaggaaattagggtcgtggaag(SEQ ID NO.9)

(3) Immunotherapy of tumor-bearing mice with a new antigen vaccine targeting the REISN epitope

Balb/C mice (n=3) were subcutaneously injected with 5×105CT26-p1a-REISN cells. After the tumor grew to a certain size (70-100mm3), they were immunized with 2×107 new antigen short peptide pulsed spleen cells (after irradiation) and fed with a feed containing doxycycline (0.2% doxycycline) to induce the expression of REISN after RNA editing. One week later, the same 2×107 new antigen short peptide pulsed spleen cells were boosted with immunization. The detection process is shown in Figure 8. The size of the subcutaneous tumor was measured to evaluate the growth of the tumor.

The test results are shown in Figure 9. It can be seen from the figure that the tumor of tumor-bearing mice regressed after REISN immunotherapy, indicating that immunotherapy with the new antigen vaccine can target and kill tumor cells expressing REISN.

Example 3

This example uses the mouse Spag9-2 gene as an example to verify a method for preparing an antigen epitope. The specific process is as follows:

1. Design of REISN site

For the Spag9-2 gene of mice, NetMHCpan-4.1 software was used to predict the binding affinity of peptides of all protein sequences in the ORF of the gene to mouse MHC H-2-Kd. The short Spag9-2 peptide was screened out with a high affinity of 484.06 nM for H-2-Kd. Among the adenine nucleotides on the peptide segment, the mutant peptide Spag9-2-REISN, which could change the amino acid coding after mutation to guanine, was screened out. NetMHCpan-4.1 software was used to predict the mutant peptide and its binding affinity to H-2-Kd. The prediction results of sequence and affinity are shown in Table 4 (this implementation method predicts the MHC-I class complex).

Table 4

Protein name Peptide MHC Aff(nM) Spag9-2 VYKDQISVL (SEQ ID NO. 10) H-2-Kd 484.06 SPAG9-2–REISN VYKDQMSVL (SEQ ID NO.11) H-2-Kd 651.29

2. Design of REISN Guide RNA

(1) Design of guide RNA based on MS2-MCP2 system: The MS2-MCP2 system consists of two parts: exogenous ADAR1 and guide RNA (sequence shown in SEQ ID NO. 12 in Table 5). The construction method is the same as that in Example 2.

Table 5

3. Establishment of a model for inducing REISN production in mouse tumors.

The model of inducing REISN production in mouse tumors was established as follows:

(1) Construction of Dox-inducible Spag9-2 RNA editing cell line:

1) Construction of ADAR lentiviral vector, the vector map is shown in Figure 5. The construction method is consistent with Example 2.

2) Constructing the guide RNA lentiviral expression vector pLKO.1-arSPAG9-2-MS2-BLAST, the construction method is consistent with Example 2.

3) After transfecting 293T cells with the lentiviral vector obtained in 1) and 2) together with the packaging plasmid, the supernatant containing the lentivirus was harvested. CT26 cells were infected with 500 μl of the supernatant of the above Dox-inducible ADAR and arSPAG9-2 lentiviruses together with 10 μg/mL protamine. After 48 hours, a final concentration of 12 μg/mL Puromycin (puromycin) and a final concentration of 6 μg/ml Blastidin were added for screening to obtain a stable cell line CT26-SPAG9-2-REISN.

Balb/C mice (n=3) were subcutaneously injected with 5×105CT26-SPAG9-2-REISN cells. After the tumor size grew to a certain size (70-100mm3), they were fed with a feed containing doxycycline (0.2% doxycycline) to induce the expression of REISN after RNA editing. Tumor tissues were collected, and total RNA was extracted according to the requirements of the kit (after DNase digestion), reverse transcribed into cDNA by RT-PCR (further DNase digestion), and the target site was PCR amplified with the corresponding primers (sequences are shown in Table 6), and the PCR products were used for Sanger sequencing. The sequencing results are shown in Figure 10. It can be seen from the figure that arSpag9-2 can successfully achieve A-to-I RNA editing in vivo through dox induction. The efficiency of intratumoral induced RNA editing is about 26%.

Table 6

(2) Immunotherapy of tumor-bearing mice with a new antigen vaccine targeting the REISN epitope

Balb/C mice (n=5) were subcutaneously injected with 5×105CT26-SPAG9-2-REISN cells. After the tumor grew to a certain size (70-100mm3), they were immunized with 1×107 new antigen short peptide pulsed spleen cells (after irradiation) and fed with a feed containing doxycycline (0.2% doxycycline) to induce the expression of REISN after RNA editing. The size of the subcutaneous tumor was measured every 3 days to evaluate the growth of the tumor.

The test results are shown in Figure 11. It can be seen from the figure that the tumor of tumor-bearing mice regressed after REISN immunotherapy, indicating that immunotherapy with the new antigen vaccine can target and kill tumor cells expressing REISN.

Example 4

This example uses the human AFP gene as an example to verify a method for preparing an antigen epitope. The specific process is as follows:

1. Design of REISN site

For the human AFP gene, the binding affinity of peptides of all protein sequences in the ORF of the gene to human HLA-A*02:01 was predicted using NetMHCpan-4.1 software, and the AFP short peptides were screened to have high affinity to HLA-A*02:01. Among the adenine nucleotides on the peptide segment, three mutant peptides (sequences shown in Table 7) that can change the amino acid coding after mutation to guanine were screened, and the mutant peptides were predicted using NetMHCpan-4.1 software and their binding affinity to HLA-A*02:01. The prediction results of the sequence and affinity are shown in Table 7 (the present implementation method predicts the MHC-I class complex).

Table 7

Protein name Peptide MHC Aff(nM) AFP FMNKFIYEI (SEQ ID NO. 15) HLA-A*02:01 2.2 AFP–REISN1 FMNKFVYEI (SEQ ID NO. 16) HLA-A*02:01 2.3 AFP–REISN2 FMNKFICEI (SEQ ID NO. 17) HLA-A*02:01 4.7 AFP–REISN3 FMNRFIYEI (SEQ ID NO. 18) HLA-A*02:01 2.2

2. Design of REISN Guide RNA

(1) Design of guide RNA based on MS2-MCP2 system: The MS2-MCP2 system consists of two parts: exogenous ADAR1 and guide RNA (as shown in SEQ ID NO. 20). Two plasmids, pcDNA3.1(-)-MCP-ADAR1-TAG and pUC57-arAFP-MS2, were constructed. Both plasmids were synthesized by Nanjing GenScript Co., Ltd. The cloning process of pcDNA3.1(-)-MCP-ADAR1-TAG is shown in Example 2.

pUC57-arAFP-MS2 is obtained by inserting the U6 promoter and arAFP-MS2 (as shown in SEQ ID NO. 20) fragment into the EcoRI and HindIII restriction sites in the MCS region of the pUC57 vector. The sequence was verified to be correct and complete.

(2) Guide RNA design based on the LEAPER system: The LEAPER editing system consists of a guide RNA (i.e., ar13, the sequence of which is shown in SEQ ID NO.19) to construct the pLKO-p1a-ar13 plasmid. The plasmid clones the U6 promoter and the synthetic ar13 fragment (the sequence of which is shown in SEQ ID NO.19) into the pLKO.1puro (Addgene #8453) backbone between XbaI and SalI by homologous recombination.

(3) Design of guide RNA based on λN-BoxB system: λN-BoxB editing system is composed of exogenous ADAR2 and guide RNA (i.e., arAFP, sequence as shown in SEQ ID NO.21), and pUC57-arAFP-BOXB and pcDNA3.1(-)-U6-BOXB-ADAR2 were constructed. The plasmids were synthesized by Nanjing GenScript Co., Ltd., and the cloning process of pcDNA3.1(-)-U6-BOXB-ADAR2 is shown in Example 2. pUC57-arAFP-BoxB is a fragment of U6 promoter and arAFP-BoxB (sequence as shown in SEQ ID NO.21) inserted between EcoRI and HindIII restriction sites in the MCS region of pUC57 backbone vector. Sequencing verified that the sequence was correct and complete.

(4) Verification of in vitro editing efficiency: Since Hela cells do not express AFP, an AFP overexpression plasmid pLKO-AFP-EGFP was constructed. The pLKO-AFP-EGFP plasmid is the AFP-EGFP sequence (sequence shown in SEQ ID NO.28) cloned between NheI and SalI of the pLKO.1puro backbone vector (Addgene, #8453). For the LEAPER editing system, Hela cells were plated in 96-well plates, and 250 ng of pLKO-AFP-ar13 plasmid and 12.5 ng of pLKO-AFP-EGFP overexpression plasmid were co-transfected using lipo8000 (Biyuntian). After 48 hours, the cells were lysed to extract RNA, and cDNA was obtained after RT-PCR. The primers of AFP (AFPRT F1CAGCCAAAGTGAAGAGGGAAGA (SEQ ID NO.22), AFPRT R1

TTTTCCCCATCCTGCAGACAAT (SEQ ID NO.23)) was used for PCR, and the PCR product was sent to Sanger sequencing to verify the editing efficiency; for MS2 and BoxB editing systems, HeLa cells were plated in 12-well plates, and 2.5μg guide RNA plasmid, 250ng ADAR plasmid and 125ng AFP overexpression plasmid (pLKO-AFP-EGFP) were transfected into the cells simultaneously with Lipo800 transfection reagent. After 48h, the cells were lysed to extract RNA, and cDNA was obtained after RT-PCR. PCR was performed using the AFP primer sequences AFPRT F1 and AFPRT R1, and the PCR product was sent to Sanger sequencing to verify the editing efficiency.

Table 8

As can be seen from Figure 12, both the MS2 and BoxB systems can effectively perform gene editing.

The gene fragments and plasmids in the embodiments of the present invention were synthesized by Nanjing GenScript Company.

The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims (10)

1. Use of an antigen epitope in any one of the following (1) to (5); (1) Preparation of products for treating and/or preventing tumors; (2) Preparation of CAR-T target therapeutic products; (3) Preparation of TCR-T target therapeutic products; (4) Preparation of tumor immune products; (5) Preparation of tumor-related vaccines; The antigen epitope is a new antigen epitope obtained by performing site-directed mutagenesis on tumor antigens using RNA editing. 2. The use according to claim 1, characterized in that the tumor antigens include P1A, AFP and Spag9-2. 3. The use according to claim 1, characterized in that the amino acid sequence of the antigen epitope is shown as SEQ ID NO.3, SEQ ID NO.11, SEQ ID NO.16, SEQ ID NO.17 or SEQ ID NO.18. 4. A biomaterial, characterized in that the biomaterial can induce RNA editing to generate the antigen epitope as described in claim 1. 5. The biomaterial according to claim 4, characterized in that the biomaterial combination comprises a recombinant expression vector, an expression cassette, a recombinant bacterium, a host cell or RNA. 6. The biomaterial according to claim 5, characterized in that the recombinant expression vector comprises a guide RNA that edits the target RNA or a construct encoding the guide RNA; preferably, the sequence of the guide RNA is shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12, SEQ ID NO.19, SEQ ID NO.20, or SEQ ID NO.21. 7 . The biomaterial according to claim 5 , wherein the RNA comprises modified RNA or circular RNA. 8. An anti-tumor combination product, characterized in that the anti-tumor combination product comprises the antigen epitope described in claim 1 and/or the biological material described in any one of claims 4-7. 9. The anti-tumor combination product according to claim 8, characterized in that the anti-tumor combination product further comprises an immune checkpoint inhibitor; preferably, the immune checkpoint inhibitor comprises CTLA-4 monoclonal antibody and PD-1/PD-L1 monoclonal antibody. 10. The anti-tumor combination product according to claim 8, characterized in that the anti-tumor combination product is a medicine, a vaccine, a health product or a food.