Ewing's sarcoma antigen peptide and application thereof

Abstract

The invention belongs to the technical field of immunobiology, and particularly relates to an Ewing's sarcoma antigen peptide and application thereof. The Ewing's sarcoma antigen peptide provided by the invention is an optimal Ewing's sarcoma antigen peptide obtained by detecting high-frequency fusion gene mutation in people by utilizing next-generation sequencing data on the basis of Ewing's sarcoma pathogenesis-gene fusion mutation and further analyzing and predicting. The Ewing's sarcoma antigen peptide is a fusion gene new antigen, the overlapping rate of the Ewing's sarcoma antigen peptide and a patient can reach up to 100%, and meanwhile, the Ewing's sarcoma antigen peptide not only has bioinformatics high frequency, but also has high affinity with HLA molecules, and has the potential of specifically recognizing tumor cells. Besides, an immunogenicity experiment verifies that the Ewing's sarcoma antigen peptide has good immunogenicity and immune protection capacity, and the Ewing's sarcoma antigen peptide has great market value when being used for preparing vaccines for preventing and / or treating Ewing's sarcoma.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immunobiology, and particularly relates to an Ewing's sarcoma antigen peptide and application thereof. BACKGROUND

[0002] At present, the traditional treatment of Ewing's sarcoma still takes “surgery + high-intensity chemotherapy + radiotherapy” as the core, but has great toxic and side reactions. Patent document CN116712431A discloses application of YG1702 in preparation of a drug for resisting Ewing's sarcoma. The YG1702 can effectively inhibit Ewing's sarcoma proliferation, self-renewal and in-vivo tumorigenicity by inhibiting expression of Ewing's sarcoma ALDH18A1 gene, and can inhibit expression of Ewing's sarcoma EWS-FLI1 fusion gene and downstream target genes EZH2, ID2, PTPL1, CCND1 and VEGFA, and can become a potential target therapy candidate small molecule. The small molecule inhibitor YG1702 of the ALDH18A1 has potential clinical value for finding a new Ewing's sarcoma treatment target and improving combined treatment effect for Ewing's sarcoma patients.

[0003] Patent document CN118576611A discloses use of a drug combination product in preparation of a drug for treating Ewing's sarcoma. The drug combination product is a drug combination product of a chemotherapeutic agent and a multi-receptor tyrosine kinase inhibitor, and the drug combination product has an effect of treating Ewing's sarcoma.

[0004] At present, main strategies of tumor immunotherapy include: ① monoclonal antibody; ② immune-regulating factor; ③ adoptive cell therapy; and ④ tumor-specific vaccine active immunization. Among them, the vaccine based on specific antigens has advantages of individualization, easy quality control and low toxicity, and meanwhile, Ewing's sarcoma is generated by fixed several fusion genes to produce tumor-specific neoantigens, which provides an ideal target for immune intervention. Therefore, the tumor-specific vaccine active immunization is regarded as a potential means to break the bottleneck of Ewing's sarcoma metastasis / recurrence.

[0005] With popularization of “omics” technology, a new generation of products such as nucleic acid vaccine, polypeptide vaccine and gene engineering vaccine rapidly rise. Among them, the polypeptide vaccine contains only 8-30 amino acids, can accurately deliver abnormal splicing epitopes induced by fusion genes, and has: ① extremely high safety; ② can induce specific CD8⁺ T cell response; ③ short synthesis cycle, can be prepared in large quantities in GMP level; ④ easy to be combined with TLR agonists, immune checkpoint inhibitors and the like to strengthen immunogenicity. Therefore, it is an urgent problem to be solved at present to research and develop a specific antigen vaccine for Ewing's sarcoma. SUMMARY

[0006] In order to solve the defects of the prior art, the application first proposes an Ewing's sarcoma antigen peptide and a vaccine containing the antigen peptide. The Ewing's sarcoma antigen peptide provided by the application is a fusion gene neoantigen, which not only has high bioinformatics frequency, but also has high affinity with HLA molecules and has the potential to specifically recognize tumor cells. In addition, the Ewing's sarcoma antigen peptide has good immunogenicity and immunoprotective ability, and can be used for Ewing's sarcoma prevention and / or treatment.

[0007] In order to achieve the above-mentioned target, the technical scheme of the application is as follows: The application provides an Ewing's sarcoma antigen peptide, and the amino acid sequence of the antigen peptide is at least one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7.

[0008] Further, the amino acid sequence of the antigen peptide is one or a combination of two of SEQ ID NO. 4 and SEQ ID NO. 6. Preferably, the amino acid sequence of the antigen peptide is SEQ ID NO. 4.

[0009] The application provides a nucleic acid molecule, which comprises a nucleotide sequence encoding the above-mentioned Ewing's sarcoma antigen peptide, and the nucleic acid molecule is double-stranded DNA.

[0010] In addition, the application also provides an mRNA, which comprises an mRNA encoding the above-mentioned Ewing's sarcoma antigen peptide.

[0011] Further, the nucleotide sequence of the mRNA is at least one of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14.

[0012] Further, the nucleotide sequence of the mRNA is one or a combination of two of SEQ ID NO. 11 and SEQ ID NO. 13. Preferably, the nucleotide sequence of the mRNA is SEQ ID NO. 11.

[0013] Further, the application also provides an mRNA vaccine, which comprises a lipid nanoparticle and the above-mentioned mRNA.

[0014] Further, the administration mode of the vaccine preparation comprises intramuscular injection, intradermal injection, intravenous injection, arterial injection or administration through a mucosal route. Further, the administration mode of the vaccine preparation comprises intramuscular injection, intradermal injection, intravenous injection, arterial injection or administration through a mucosal route.

[0015] In addition, the present application also provides a pharmaceutical composition comprising the above-mentioned mRNA and a pharmaceutically acceptable carrier.

[0016] In addition, the present application also provides the use of the Ewing's sarcoma antigen peptide, the nucleic acid molecule, the mRNA, the mRNA vaccine or the pharmaceutical composition in the preparation of a drug for preventing and / or treating Ewing's sarcoma.

[0017] Currently, the fusion gene is only used as a marker for diagnosing Ewing's sarcoma, and the neoantigen sequence induced by the DNA point mutation has a low patient overlap rate, and the candidate peptide obtained in Ewing's sarcoma has a patient overlap rate of <20%, which cannot refine a “universal” treatment sequence, and each peptide needs to be produced separately, and the quality control and cost increase exponentially. In addition, the DNA mutation derived neoantigen often has poor HLA (human leukocyte antigen) binding capacity, with a median IC50>500 nM, which limits its immunogenicity and clinical application.

[0018] In order to solve the above problems, the present application first proposes a fusion gene mutation based on the etiology of Ewing's sarcoma, detects high-frequency fusion gene mutations in the population using second-generation sequencing data, and further analyzes and predicts the neoantigen sequence. The Ewing's sarcoma antigen peptide is a fusion gene neoantigen, and the patient overlap rate can be as high as 100% (20 / 20). The high-frequency experiment and affinity experiment verify that the Ewing's sarcoma antigen peptide segment not only has high bioinformatics frequency, but also has high affinity with HLA molecules, and has the potential to specifically recognize tumor cells. At the same time, the immunopeptidomics-TCR database joint verification shows that among more than 30,000 immunopeptides, only in Ewing's sarcoma samples, and all matched to natural TCR clones, have “real presentation + targetable” double evidence. In addition, the tumor immunogenicity verification shows that the Ewing's sarcoma antigen peptide has strong immunogenicity, and has great value for the development of vaccines for preventing and / or treating Ewing's sarcoma.

[0019] In summary, compared with the prior art, the present application obtains the optimal Ewing's sarcoma antigen peptide, which not only has high bioinformatics frequency, but also has high affinity with HLA molecules, and has the potential to specifically recognize tumor cells. At the same time, the Ewing's sarcoma antigen peptide has good immunogenicity and immunoprotective power, can induce the body to form specific T cells, strengthen immune response, maintain immune memory for a long time, can form a public neoantigen library and be applied to Ewing's sarcoma immunotherapy, and has great drug value for the development of vaccines for preventing and / or treating Ewing's sarcoma. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1A flowchart for the systematic screening and high-frequency validation of Ewing's sarcoma neoantigen peptide.

[0021] Figure 2 This is a graph showing the number of IFN-γ spots generated by Ewing's sarcoma antigen peptide. Detailed Implementation

[0022] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the invention, but as long as they do not depart from the basic idea of ​​the invention, they are all within the scope of the invention. All materials involved in this invention are food-grade and can be obtained commercially available or through conventional techniques in the art.

[0023] Example 1: Systematic screening and high-frequency validation of Ewing's sarcoma neoantigen peptides 1. Systematic screening and high-frequency validation of Ewing's sarcoma neoantigen peptides: The flowchart for systematic screening and high-frequency validation of Ewing's sarcoma neoantigen peptides is as follows: Figure 1 As shown. The specific process is as follows: 1.1 Data Sources and Preprocessing: RNA-seq data from tumor tissues of 20 Ewing's sarcoma patients were selected from the European Genome Archive (EGA, ID: EGAD00001004493). Quality control was performed using FastQC, low-quality reads were removed using FastP, STAR alignment was performed to the hg19 reference genome, and StringTie quantitative expression (TPM) was performed.

[0024] 1.2 Fusion Gene Detection and Annotation: Cross-validation using Arriba (v2.3) and STAR-Fusion (v1.10) software identified a total of 11 fusion genes, of which EWSR1-FLI1, EWSR1-WT1, EWSR1-WT1, and EWSR1-FEV were found in all patients.

[0025] 1.3 Neoantigen Prediction Process: The following screening was performed on the aberrant linker peptides (containing 15 amino acids before and after the fusion breakpoint) after translation of each fusion gene: Table 1

[0026] 1.4 High-frequency shared peptide locking: The final locking 7 high-frequency shared neoantigen peptides were detected in all 20 patients, and the coverage rate of HLA-A02:01, HLA-A24:02 and HLA-B*07:02 three super types reached 87.6%, and the information table of the final locking 7 high-frequency shared candidate neoantigens is shown in Table 2.

[0027] Table 2 Information table of candidate neoantigens Number Fusion gene Amino acid sequence Nucleic acid sequence (5'→3') Predicted HLA-supertype coverage 1 EWSR1-WT1 GDRGGFPPRGPRGSRPSCQKKFARSDELVR (SEQ ID NO. 1) GGCGATCGCGGCGGCTTTCCGCCGCGCGGCCCGCGCGGCAGCCGCCCGAGCTGCCAGAAAAAATTTGCGCGCAGCGATGAACTGGTGCGC (SEQ ID NO. 8) A*02:01 / A*24:02 / B*07:02 2 EWSR1-WT1 LHMALSLLIQPMGSSQQPLHLQAIPLHSRL (SEQ ID NO. 2) CTGCATATGGCGCTGAGCCTGCTGATTCAGCCGATGGGCAGCAGCCAGCAGCCGCTGCATCTGCAGGCGATTCCGCTGCATAGCCGCCTG (SEQ ID NO. 9) A*02:01 / B*07:02 3 EWSR1-WT1 SQYSQQSSSYGQQSEKPYQCDFKDCERRFS (SEQ ID NO. 3) TCCCAGTACTCCCAGCAGTCCTCCTCCTACGGCCAGCAGTCCGAGAAGCCCTACCAGTGCGACTTCAAGGACTGCGAGCGGCGGTTCTCC (SEQ ID NO. 10) A*02:01 / A*24:02 4 EWSR1-FLI1 SQYSQQSSSYGQQSSLLAYNTTSHTDQSSR (SEQ ID NO. 4) TCCCAGTACTCCCAGCAGTCCTCCTCCTACGGCCAGCAGTCCTCCCTGCTGGCCTACAACACCACCTCCCACACCGACCAGTCCTCCCGG (SEQ ID NO. 11) A*02:01 / A*24:02 / B*07:02 5 EWSR1-ERG SQQSSSYGQQNPYQILGPTSSRLANPGSGQ (SEQ ID NO. 5) TCCCAGCAGTCCTCCTCCTACGGCCAGCAGAACCCCTACCAGATCCTGGGCCCCACCTCCTCCCGGCTGGCCAACCCCGGCTCCGGCCAG (SEQ ID NO. 12) A*02:01 / A*24:02 6 EWSR1-ERG SQYSQQSSSYGQQSSGQIQLWQFLLELLSD (SEQ ID NO. 6) TCCCAGTACTCCCAGCAGTCCTCCTCCTACGGCCAGCAGTCCTCCGGCCAGATCCAGCTGTGGCAGTTCCTGCTGGAGCTGCTGTCCGAC (SEQ ID NO. 13) A*02:01 / A*24:02 7 EWSR1-FEV MGSAGERGGFNKPGDPVGDGLFKDGKNPSW (SEQ ID NO. 7) ATGGGCTCCGCCGGCGAGCGGGGCGGCTTCAACAAGCCCGGCGACCCCGTGGGCGACGGCCTGTTCAAGGACGGCAAGAACCCCTCCTGG (SEQ ID NO. 14) A*02:01 / B*07:02 2. Affinity verification (surface plasmon resonance, SPR): The HLA-A*02:01 purified protein was randomly selected, and the affinity of SEQ ID NO. 1 was determined by Biacore 8K: The measured KD = 4.3 × M ( The predicted value is 47 nM, which is consistent); The positive control (CMV pp65) KD = 2.1 × M; The negative control (irrelevant peptide) has no binding signal.

[0028] 3. Experimental conclusion: The above results show that the 7 Ewing sarcoma antigen peptide fragments provided by the application not only have high bioinformatics frequency, but also have high affinity with HLA molecules, and have the potential to specifically recognize tumor cells.

[0029] Example 2, immunopeptidomics-TCR database joint verification 1. Experimental purpose: By cross comparison of the public immunopeptidomics database and the TCR specificity database, it is proved that SEQ ID NO. 1-7: (1) can be naturally presented by tumor cells; (2) can be recognized by natural TCR clonotypes; (3) has tumor specificity and meets the dual requirements of "real existence + targetability" of therapeutic neoantigens.

[0030] 2. Database sources: 2.1. Immunopeptidomics: (1) IEDB v2.28 version: select human HLA-I / II epitope peptides; (2) SysteMHC Atlas v2.0 version: select mass spectrometry epitope peptides of human; (3) Among them, 12 Ewing sarcoma (Ewing sarcoma) labeled samples.

[0031] 2.2. TCR specificity library: (1) VDJdb 2025-04 version: 20,000 human TCR a / b CDR3 1 sequences are selected, with corresponding antigen peptides; (2) McPAS-TCR-2025-04 version: 30,000 human pathologically related TCR records, containing tumor / infection antigens.

[0032] 3. Analysis process: 3.1 Build high-confidence target peptide library (1) Directly import the final locked 7 core sequences of Example 1 SEQ ID NO. 1-7, 30-mer); (2) Take each core sequence as a template, and extend 1 aa to the N / C end according to the "fusion breakpoint centering" principle, generate 17-mer long fragments, which are used for subsequent substring matching.

[0033] (3) Write all 17-mer fragments into final_target.fa, keep the original SEQ ID+subsequence in the header for subsequent analysis and accurate matching calls.

[0034] 3.2, Artificial intelligence retrieval of immunopeptide group: (1) Merge IEDB and SysteMHC-Atlas data; (2) Use self-developed Python 3.11 script (based on pyteomics 4.6) to perform "accurate substring matching": (a) Algorithm complexity O(n), allowing 0-mismatch; (b) Only output hits with cancer=Ewing sarcoma and length≥15 aa; (3) The results are corrected by 1% FDR Percolator to ensure zero false positives.

[0035] 3.3, Deep mining of TCR specificity: (1) Take the 17-mer core of step 1 as the query to batch search VDJdb & McPAS-TCR; (2) Use Needleman-Wunsch global alignment (gap open=10, gap extend=1) to retain records with alignment score≥80 and CDR3 beta mismatch≤1 aa; (3) Remove redundant clones by TCRdist 3.0 clustering, and finally obtain unique CDR3 beta sequences and their HLA restriction information.

[0036] 3.4, Tumor specificity algorithm filtering: (1) Perform blastp on the full length of the positive peptide (UniProt 2025-03, e-value < 1 x ); (2) Calculate the maximum continuous matching length L using a self-written script. (3) If L < 8 aa, determine it as a potential self-antigen and eliminate it.

[0037] 3.5, Multi-dimensional evidence integration and output: (1) Construct a SQLite result database with fields: peptide ID, sequence, immune peptide group hit number, TCR clone number, average TPM, L value. (2) Only keep the peptide segments that meet the following conditions simultaneously: hit number > 3, TCR clone number > 1, L < 8 aa, and generate Table 3.

[0038] Table 3 High confidence joint verification results Peptide ID Amino acid sequence Immune peptide group hits (Ewing's sarcoma / total samples) Unique TCR clonotypes (CDR3beta) HLA restriction Average TPM Max self-match aa SEQ ID NO. 1 GDRGGFPPRGPRGSRPSCQKKFARSDELVR 4 / 12 CASSLGRGPQFYF A*02:01 42 6 SEQ ID NO. 4 SQYSQQSSSYGQQSSLLAYNTTSHTDQSSR 5 / 12 CASSLGQAYEQYF A*24:02 38 5 SEQ ID NO. 6 SQYSQQSSSYGQQSSGQIQLWQFLLELLSD 3 / 12 CASSLGSAYEQYF A*24:02 29 6 4, Experimental conclusion: 4.1, Peptides SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 6 were detected in >30,000 immune peptide groups only in Ewing's sarcoma samples, and matched to natural TCR clones, with "real presentation + targetable" double evidence. 4.2, SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 6 peptide segments have the longest continuous matching <8 aa with UniProt human protein group, excluding the risk of self-antigen, and have the ability to specifically recognize tumor cells.

[0039] Example 3, Ewing's sarcoma neoantigen peptide tumor immunogenicity evaluation experiment 1, Experimental method: (1) Collect peripheral blood mononuclear cells (PBMC): 4 peripheral blood samples from 4 Ewing's sarcoma cancer patients were collected from the hospital, and the collected blood was gently mixed with an equal volume of PBS buffer. Then prepare 4 centrifuge tubes containing Ficoll lymphocyte separation medium, and slowly add the diluted blood sample to it. After centrifugation, remove the upper plasma, carefully collect the middle PBMC, and then freeze the collected 4 PBMC.

[0040] (2) T cell incubation: The frozen PBMC cells were taken out, centrifuged to remove the supernatant after resuscitation. Two culture systems were obtained by resuspending the cells with culture solution containing 5% serum, and then 7 synthetic peptide segments mentioned in Example 1 and cell growth factors IL-2 and IL-7 were added to the first culture system to make the working concentration reach 50 ng / mL; then incubated at 37 ℃. The second culture system added SEQ ID NO. 1-7 synthetic peptides, and then the same operation was performed. After ten days, the cells were collected and washed to prepare for subsequent experiments.

[0041] (3) Enzyme-linked immunospot (ELISPOT) detection and counting: First, 100 μL of pre-coated IFN-γ antibody was added to each well of the ELISPOT plate and incubated at 4 ℃ overnight. The next day, 200 μL of 1640 culture solution was added and incubated at room temperature for a period of time. Then, different peptide segments in Example 1 were added to make the working concentration 5 μg / mL, and positive and negative (polypeptide diluent PBS) control groups were set. The plate was placed in a 5% incubator for incubation. After incubation, detection antibody and streptavidin were added for color development, and the number of IFN-γ spots was determined using an automatic ELISPOT plate reader (AID iSpot).

[0042] 2. Experimental results: The experimental results are shown in Figure 2 and Table 4.

[0043] 2.1, Figure 2 The number of IFN-γ spots produced by Ewing's sarcoma antigen peptides (SEQ ID NO. 1-7) is shown in the figure.

[0044] 2.2, The number of IFN-γ spots produced by Ewing's sarcoma antigen peptides (SEQ ID NO. 1-7) is shown in Table 4.

[0045] Table 4 Enzyme-linked immunospot plate spot counting table Sequence IFN-g spot number Significance (vs PBS) SEQ ID NO. 1 17 p < 0.01 SEQ ID NO. 2 27 p < 0.001 SEQ ID NO. 3 31 p < 0.001 SEQ ID NO. 4 56 p < 0.0001 SEQ ID NO. 5 28 p < 0.001 SEQ ID NO. 6 33 p < 0.001 SEQ ID NO. 7 31 p < 0.001 Positive control 118 \ Negative control 0 \ As can be seen from Table 4, the number of IFN-γ spots produced by the Ewing's sarcoma antigen peptides (SEQ ID NO. 1-7) provided by the present application has a statistical difference, which indicates that the Ewing's sarcoma antigen peptides have strong immunogenicity.

[0046] The above embodiments are only illustrative of the principles of the present application and its efficacy, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. An Ewing's sarcoma antigen peptide, characterized in that, The amino acid sequence of the antigenic peptide is at least one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.

7.

2. The Ewing's sarcoma antigen peptide as described in claim 1, characterized in that, The amino acid sequence of the antigenic peptide is one or a combination of two of SEQ ID NO.4 and SEQ ID NO.

6.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding the Ewing sarcoma antigen peptide of claim 1, and the nucleic acid molecule is double-stranded DNA.

4. An mRNA, characterized in that, The mRNA comprises an mRNA encoding the Ewing sarcoma antigen peptide as described in claim 1.

5. The mRNA as described in claim 4, characterized in that, The nucleotide sequence is at least one of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.

14.

6. The mRNA as described in claim 5, characterized in that, The nucleotide sequence is one or a combination of two of SEQ ID NO.11 and SEQ ID NO.

13.

7. An mRNA vaccine, characterized in that, The mRNA vaccine comprises lipid nanoparticles and mRNA as described in any one of claims 4 to 6.

8. The mRNA vaccine as described in claim 7, characterized in that, The vaccine preparation can be administered via intramuscular injection, intradermal injection, intravenous injection, arterial injection, or through mucosal route.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mRNA as described in any one of claims 4 to 6 and a pharmaceutically acceptable carrier.

10. The use of the Ewing sarcoma antigen peptide of claim 1, the nucleic acid molecule of claim 3, the mRNA of claim 4, the mRNA vaccine of claim 7, or the pharmaceutical composition of claim 9 in the preparation of a drug for the treatment, prevention, and / or treatment of Ewing sarcoma.

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