Ewing sarcoma antigenic peptide and use thereof

Abstract

The application belongs to the technical field of immunobiology, and particularly relates to a Ewing's sarcoma antigen peptide and application thereof. The Ewing's sarcoma antigen peptide provided by the application is based on the etiological gene fusion mutation of Ewing's sarcoma, high-frequency fusion gene mutations in the human population are detected by using second-generation sequencing data, and the optimal Ewing's sarcoma antigen peptide obtained by further analysis and prediction. The Ewing's sarcoma antigen peptide is a fusion gene neoantigen, the overlap rate of the Ewing's sarcoma antigen peptide with patients can be as high as 100%, and the Ewing's sarcoma antigen peptide not only has high bioinformatics frequency, but also has high affinity with HLA molecules, and has the potential of specific recognition of tumor cells. In addition, it is verified by immunogenicity experiments that the Ewing's sarcoma antigen peptide has good immunogenicity and immunoprotective power, and the Ewing's sarcoma antigen peptide has great market value for the development of a vaccine 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 Ewing's sarcoma ALDH18A1 gene expression, and can inhibit Ewing's sarcoma EWS-FLI1 fusion gene and downstream target genes EZH2, ID2, PTPL1, CCND1 and VEGFA expression, and can become a potential target therapy candidate small molecule. The ALDH18A1 small molecule inhibitor YG1702 has potential clinical value for finding a new Ewing's sarcoma treatment target and improving Ewing's sarcoma patient combined treatment effect.

[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 regulatory 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 only contains 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 present 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 present 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 present application is as follows:

[0008] The present 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.

[0009] 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.

[0010] The present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned Ewing's sarcoma antigen peptide, and the nucleic acid molecule is double-stranded DNA.

[0011] In addition, the present application also provides an mRNA comprising an mRNA encoding the above-mentioned Ewing's sarcoma antigen peptide.

[0012] 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.

[0013] 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.

[0014] Further, the present application also provides an mRNA vaccine, which comprises a lipid nanoparticle and the above-mentioned mRNA.

[0015] Further, the administration mode of the vaccine preparation comprises intramuscular injection, intradermal injection, intravenous injection, arterial injection or administration through a mucosal route.

[0016] In addition, the present application also provides a pharmaceutical composition comprising the above mRNA and a pharmaceutically acceptable carrier.

[0017] In addition, the present application also provides the use of the Ewing 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 sarcoma.

[0018] Currently, the fusion gene is only used as a marker for diagnosing Ewing sarcoma, and the neoantigen sequence induced by the DNA point mutation has a low patient overlap rate, and the candidate peptide obtained in the Ewing sarcoma has a patient overlap rate of <20%, so it is impossible to extract a 'universal' treatment sequence, 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 of >500 nM, which limits its immunogenicity and clinical application.

[0019] In order to solve the above problems, the present application first proposes a fusion gene mutation based on the etiology of Ewing 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 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 sarcoma antigen peptide 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 the Ewing sarcoma sample is detected, and all match the natural TCR clone, which has'real presentation + targetable' double evidence. In addition, the tumor immunogenicity verification shows that the Ewing sarcoma antigen peptide has strong immunogenicity, and has great value for the development of vaccines for preventing and / or treating Ewing sarcoma.

[0020] In summary, compared with the prior art, the present application obtains the optimal Ewing 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 sarcoma antigen peptide has good immunogenicity and immunoprotective power, can induce the body to form specific T cells, strengthen the immune response, maintain long-term immune memory, can form a public neoantigen library for Ewing sarcoma immunotherapy, and has great drug value for the development of vaccines for preventing and / or treating Ewing sarcoma. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1Flow chart of systematic screening and high-frequency verification of Ewing sarcoma neoantigen peptides.

[0022] Figure 2 Figure of IFN-γ spot number generated by Ewing sarcoma antigen peptides. DETAILED DESCRIPTION

[0023] The present application is further illustrated by the following description of specific embodiments, but this is not intended to be limiting of the present application. Based on the basic ideas of the present application, those skilled in the art can make various modifications or improvements, as long as they do not deviate from the basic ideas of the present application, and they are within the scope of the present application. The materials involved in the present application are all food-grade, and can be obtained by commercial or conventional technical means in the art.

[0024] Example 1, Systematic screening and high-frequency verification of Ewing sarcoma neoantigen peptides

[0025] 1. Systematic screening and high-frequency verification of Ewing sarcoma neoantigen peptides:

[0026] The flow chart of systematic screening and high-frequency verification of Ewing sarcoma neoantigen peptides is shown in FIG. 1. The specific process is as follows: Figure 1

[0027] 1.1. Data source and pretreatment:

[0028] Select 20 cases of Ewing sarcoma tumor tissue RNA-seq data from the European Genome Archive (EGA, number: EGAD00001004493). Use FastQC for quality control, fastp to remove low-quality reads, STAR to align to the hg19 reference genome, and StringTie to quantify expression (TPM).

[0029] 1.2. Fusion gene detection and annotation:

[0030] Use Arriba (v2.3) and STAR-Fusion (v1.10) to cross-verify and detect 11 fusion genes, of which EWSR1-FLI1, EWSR1-WT1, EWSR1-WT1, and EWSR1-FEV are found in all patients.

[0031] 1.3. Neoantigen prediction process:

[0032] For each fusion gene translated abnormal connection peptide segment (containing 15 aa before and after the fusion breakpoint), the following screening is performed:

[0033] Table 1

[0034]

[0035] ​1.4, High-frequency sharing peptide segment locking:

[0036] Finally, 7 high-frequency shared neoantigen peptides were locked, which were detected in all 20 patients, with a coverage rate of 87.6% for HLA-A02:01, HLA-A24:02, and HLA-B*07:02 three supertypes. The information table of the finally locked 7 high-frequency shared candidate neoantigens is shown in Table 2.

[0037] Table 2. Information table of candidate neoantigens

[0038] 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

[0039] 2. Affinity measurement verification (surface plasmon resonance, SPR):

[0040] The HLA-A*02:01 purified protein was randomly selected, and the affinity of SEQ ID NO. 1 was determined by Biacore 8K:

[0041] The measured KD = 4.3 × M The predicted value is 47 nM, which is consistent.

[0042] The positive control (CMV pp65) KD = 2.1 × M

[0043] The negative control (irrelevant peptide) has no binding signal.

[0044] 3. Experimental conclusion:

[0045] The above results show that the 7 Ewing's sarcoma antigen peptide segments 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.

[0046] Example 2, immunopeptidomics-TCR database joint verification

[0047] 1. Experimental purpose:

[0048] By cross comparison of the public immunopeptidomics database and the TCR specificity database, it is proved that SEQ ID NO. 1-7:

[0049] (1) can be naturally presented by tumor cells;

[0050] (2) can be recognized by natural TCR clonotypes;

[0051] (3) has tumor specificity and meets the dual requirements of "real existence + targetability" of therapeutic neoantigens.

[0052] 2. Database source:

[0053] 2.1, Immune peptide library:

[0054] (1) IEDB v2.28: Select human HLA-I / II antigen epitope peptides;

[0055] (2) SysteMHC Atlas v2.0: Select human mass spectrometry antigen epitope peptides;

[0056] (3) Among them, 12 cases of Ewing sarcoma (Ewing sarcoma) were labeled samples.

[0057] 2.2, TCR specificity library:

[0058] (1) VDJdb 2025-04: Select 20,000 human TCR a / b CDR3 1 sequences, with corresponding antigen peptides;

[0059] (2) McPAS-TCR-2025-04: 30,000 human pathologically related TCR records, containing tumor / infection antigens.

[0060] 3, Analysis process:

[0061] 3.1 Construct high-confidence target peptide library

[0062] (1) Directly import the final locked 7 core sequences SEQ ID NO. 1-7, 30-mer) in Example 1;

[0063] (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.

[0064] (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.

[0065] 3.2, Immune peptide artificial intelligence search:

[0066] (1) Merge IEDB and SysteMHC-Atlas data;

[0067] (2) Use self-developed Python 3.11 script (based on pyteomics 4.6) to perform "accurate substring matching":

[0068] (a) Algorithm complexity O(n), allowing 0-mismatch;

[0069] (b) Only output hits with cancer=Ewing sarcoma and length≥15 aa;

[0070] (3) Results were corrected by 1% FDR Percolator to ensure zero false positives.

[0071] 3.3, Deep mining of TCR specificity:

[0072] (1) Take the 17-mer core of step 1 as query to batch search VDJdb & McPAS-TCR;

[0073] (2) Use Needleman-Wunsch global alignment (gap open=10, gap extend=1) to retain records with alignment score≥80 and CDR3 mismatch≤1 aa;

[0074] (3) Cluster by TCRdist 3.0 to remove redundant clones, and finally obtain unique CDR3 sequences and their HLA restriction information.

[0075] 3.4, Tumor-specificity algorithm filtering:

[0076] (1) Perform blastp (UniProt 2025-03, e-value≤1× ) on the full length of positive peptides;

[0077] (2) Calculate the maximum continuous matching length L using a self-written script;

[0078] (3) If L≥8 aa, it is determined as a potential self-antigen and removed.

[0079] 3.5, Multi-dimensional evidence integration and output:

[0080] (1) Construct SQLite result database with fields: peptide ID, sequence, immune peptide group hit number, TCR clone number, average TPM, L value;

[0081] (2) Only retain peptides that meet the following conditions simultaneously: hit number≥3, TCR clone number≥1, L<8 aa, to generate Table 3.

[0082] Table 3 High confidence joint verification results

[0083] 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

[0084] 4, Experimental conclusion:

[0085] 4.1, Peptides SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.6 were detected only in Ewing sarcoma samples among >30,000 immune peptide groups, and matched to natural TCR clones, with dual evidence of "real presentation + targetable";

[0086] 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.

[0087] Example 3, Ewing's sarcoma neo-antigen peptide tumor immunogenicity evaluation experiment

[0088] 1. Experimental method:

[0089] (1) Collect peripheral blood mononuclear cells (PBMC):

[0090] 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 4 centrifuge tubes containing Ficoll lymphocyte separation medium were prepared, and the diluted blood sample was slowly added. After centrifugation, the upper plasma was removed, and the PBMC in the middle was carefully collected, and then the 4 collected PBMC were frozen.

[0091] (2) T cell incubation:

[0092] The frozen PBMC cells were taken out, and after resuscitation, the supernatant was removed by centrifugation. Two culture systems were obtained by resuspending the cells with culture medium 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 incubate at 37°C. 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.

[0093] (3) Enzyme-linked immunospot (ELISPOT) detection and counting:

[0094] First, add 100 μL of pre-coated IFN-γ antibody to each well of the ELISPOT plate and incubate at 4°C overnight. The next day, add 200 μL of 1640 culture medium and incubate at room temperature for a period of time. Then, add different peptide segments in Example 1 to make the working concentration 5 μg / mL, and set up positive and negative (polypeptide diluent PBS) control groups. Place the plate in a 5% % incubator for incubation. After incubation, add detection antibody and streptavidin for color development, and use an automatic ELISPOT plate reader (AID iSpot) to determine the number of IFN-γ spots.

[0095] 2. Experimental results:

[0096] The experimental results are as follows Figure 2and shown in Table 4.

[0097] 2.1, Figure 2 The IFN-γ spot number generated by the Ewing's sarcoma antigen peptides (SEQ ID NO. 1-7) is shown in Table 4.

[0098] 2.2, The IFN-γ spot number generated by the Ewing's sarcoma antigen peptides (SEQ ID NO. 1-7) is shown in Table 4.

[0099] Table 4 Enzyme-linked immunospot plate spot counting table

[0100] 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 \

[0101] As can be seen from Table 4, the IFN-γ spot number generated by the Ewing's sarcoma antigen peptides (SEQ ID NO. 1-7) has statistical difference, which shows that the Ewing's sarcoma antigen peptide has strong immunogenicity.

[0102] The above examples only illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should 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 prevention and / or treatment of Ewing sarcoma.

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