WO2019210782A1 - 辅助性表位肽及其应用 - Google Patents

辅助性表位肽及其应用 Download PDF

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WO2019210782A1
WO2019210782A1 PCT/CN2019/083484 CN2019083484W WO2019210782A1 WO 2019210782 A1 WO2019210782 A1 WO 2019210782A1 CN 2019083484 W CN2019083484 W CN 2019083484W WO 2019210782 A1 WO2019210782 A1 WO 2019210782A1
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seq
antigen
epitope
group
padre
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PCT/CN2019/083484
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French (fr)
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姚文兵
田浤
徐艺
何羽
高向东
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中国药科大学
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Priority to US17/052,360 priority Critical patent/US20210162041A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins

Definitions

  • the invention relates to a helper epitope peptide and an application thereof, and belongs to the technical field of biomedicine.
  • Tumor vaccine is one of the most effective and economical cancer treatment options, and a limited number of vaccine injections can bring long-term anti-tumor immune response.
  • the therapeutic effect of tumor vaccines has been unsatisfactory. The reason is that in addition to the low autoantigenicity of the tumor, the body's immune tolerance to tumor antigens may also be an important factor.
  • immune tolerance is mainly due to the elimination of antigen-specific CD4 + T cells in vivo, but not to whether CD8 + T cells or B cells are eliminated. Therefore, recruiting CD4 + T cells that are independent of their own antigens and breaking the immune tolerance status of CD4 + T cells may be a key step in stimulating the therapeutic potential of tumor vaccines.
  • CD4 + T cells are the switch of the immune response in the human body and can regulate the strength of the immune response.
  • the tolerance mechanism can attenuate the immune response against its own antigen, thereby maintaining homeostasis; on the other hand, breaking tolerance can bring a strong immune response against its own antigen, but at the same time it also induces autoimmune diseases. risk. Therefore, in order to achieve the best results in treatment, we should try to break the immune tolerance on the basis of minimizing the damage of autoimmune diseases, so as to maximize the immunotherapy effect of the tumor.
  • the Chinese invention patent of CN201110303946.1 and the authorization publication number CN102370979B disclose a method for constructing an autologous vaccine against human TNF- ⁇ molecule, wherein the PADRE amino acid sequence AKFVAAWTLKA is used.
  • Chinese Patent Application No. CN201611207485.7, Application No. CN106749674A discloses a novel asthma polypeptide vaccine and a preparation method thereof, and relates to a fusion polypeptide comprising the PADRE polypeptide aK-Cha-VAaWTLKAa, wherein a represents D-alanine, Cha Represents L-cyclohexylalanine.
  • the main object of the present invention is to overcome the problems of the prior art and to provide a helper epitope peptide which has versatility and can enhance the immunogenicity of an antigen or an epitope; in addition, it also provides an auxiliary epitope.
  • helper epitope peptide characterized in that the helper epitope peptide is obtained by replacing one or two amino acid residues of the sequence of SEQ ID NO: 1 with 4-nitrophenylalanine.
  • sequence of the helper epitope peptide is one of SEQ ID NO: 2 - SEQ ID NO: 20.
  • the invention also provides:
  • helper epitope peptide as described above for enhancing the immunogenicity of an antigen or antigenic epitope comprising an amino acid residue; or the use is for preparation or Construct a vaccine.
  • a product comprising a helper epitope peptide as hereinbefore described, which is a drug, a pharmaceutical composition, a biochip, a vaccine, or a vaccine composition.
  • the vaccine or vaccine composition includes a tumor vaccine or vaccine composition.
  • the invention also provides:
  • a fusion antigen characterized by being linked to an antigen or an epitope by a helper epitope peptide as described above; said antigen or antigen epitope comprising an amino acid residue, said helper epitope peptide and antigen or antigenic epitope The amino acid residues of the position are linked.
  • the helper epitope peptide is linked to the amino acid residue of an antigen or antigenic epitope via a linker peptide, the linker peptide sequence being GPSL.
  • the antigen or antigenic epitope is HER2, PD-L1, PD-1, EGFR, CD20, CD66e, CD227, VEGFR, IL-2R, CTLA-4, PSMA, TOLL-1, GATA-4, NY - one of ESO-1, FR ⁇ , CA125, EpCAM-CD3, P53, Mesothelin, WT1, A ⁇ protein, or one of SEQ ID NO: 40-SEQ ID NO: 43.
  • the fusion antigen is a polypeptide and the sequence is one of SEQ ID NO: 21 - SEQ ID NO: 39 or one of SEQ ID NO: 44 - SEQ ID NO: 47.
  • the invention also provides:
  • a vaccine or vaccine composition comprising a fusion antigen as hereinbefore described.
  • helper T-epitope PADRE PADRE sequence is AKFVAAWTLKAAA.
  • a helper epitope peptide obtained after the name: p-nitrophenylalanine can significantly enhance the immunogenicity of an existing antigen or epitope, break the CD4 + T cell immune tolerance, and the helper epitope peptide It is versatile.
  • helper epitope peptide of the present invention can universally enhance the immunogenicity of existing antigens (such as HER2 molecules, PD-L1 molecules, etc.) or antigenic epitopes (such as B cell epitopes, etc.).
  • existing antigens such as HER2 molecules, PD-L1 molecules, etc.
  • antigenic epitopes such as B cell epitopes, etc.
  • the helper epitope peptide is completely exogenous, and does not cause an autoimmune disease or the like while breaking the immune tolerance, and has low physiological toxicity;
  • the auxiliary epitope Peptides have the potential to assist in the activation of CTL effects, and can be clinically assisted in the construction of personalized vaccines for the prevention and treatment of tumors;
  • the helper epitope peptides have excellent ability to assist existing antigens or epitopes to produce antibodies or produce CTL effects. It provides ideas and preliminary basis for the construction of efficient and durable vaccines.
  • Fig. 23 is a view showing the results of Test Example 20 of Example 2.
  • Fig. 24 is a view showing the results of Test Example 24 of Example 2.
  • helper T-epitope peptide PADRE of the sequence SEQ ID NO: 1, one of the amino acids or two amino acid residues is replaced with 4-nitrophenylalanine, and the resulting sequence is shown in the following table:
  • AKFXAAWTXKAAA 52 AKFXAAWTLXAAA 53 AKFXAAWTLKXAA 54 AKFXAAWTLKAXA 55 AKFXAAWTLKAAX 56 AKFVXXWTLKAAA 57 AKFVXAXTLKAAA 58 AKFVXAWXLKAAA SEQ ID NO:18 59 AKFVXAWTXKAAA 60 AKFVXAWTLXAAA 61 AKFVXAWTLKXAA SEQ ID NO: 19 62 AKFVXAWTLKAXA 63 AKFVXAWTLKAAX 64 AKFVAXXTLKAAA 65 AKFVAXWXLKAAA 66 AKFVAXWTXKAAA 67 AKFVAXWTLXAAA 68 AKFVAXWTLKXAA 69 AKFVAXWTLKAXA 70 AKFVAXWTLKAAX 71 AKFVAAXLKAAA 72
  • X in each sequence is 4-nitrophenylalanine.
  • Example 1 From Example 1, a helper epitope peptide was selected and combined with different antigen molecules to construct a fusion antigen, and the ability to induce antibody production or induce a CTL effect was verified.
  • the main steps of the trial are:
  • a helper epitope peptide is linked to a different antigen or antigen epitope via a linker peptide, and the linker peptide sequence is GPSL (ie, Gly-Pro-Ser-Leu) to construct a plurality of fusion antigens.
  • GPSL ie, Gly-Pro-Ser-Leu
  • the fusion antigen obtained in (1) was separately emulsified in a 1:1 equal volume with complete Freund's adjuvant, and immunized with a dose of 50 ⁇ g of fusion antigen per mouse.
  • the experimental mouse strains included C57, Balb/. c and Fvb; after 7 and 14 days of the initial immunization, the fused antigen of (1) and the incomplete Freund's adjuvant were mixed and emulsified in an equal volume of 1:1, and each mouse was immunized by subcutaneous injection of 50 ⁇ g of the fusion antigen. .
  • the first one is: taking the whole eye of the mice for 7 days, 14 days, 21 days and 28 days of initial immunization, taking the serum and taking the serum, and detecting the antibody titer by indirect ELISA; Two kinds were: one week after the completion of immunization, the mice were sacrificed, the spleen was taken, PBMC (peripheral blood mononuclear cells) were isolated, and the CTL effect was detected by LDH (lactate dehydrogenase) kit.
  • LDH lactate dehydrogenase
  • the main implementation process of this embodiment is as follows:
  • mice 6-8 week old C57BL/6 female mice were randomly divided into 3 groups, 6 in each group, respectively, in the PBS group, and the existing antigen or antigen epitope.
  • the second step using subcutaneous immunization, immunization 3 times, one week at a time, 50 ⁇ g each time. Mix 1:1 equal volume with Freund's adjuvant.
  • the method 1 or method 2 is used for the detection.
  • Method 1 Blood was taken weekly after immunization, and serum was separated by centrifugation at 6000 rpm for 20 minutes for 4 weeks. Antibody titers were detected by indirect ELISA as follows.
  • the existing antigen or antigen epitope is diluted to 5 ⁇ g / mL with a coating solution, 100 ⁇ L per well in the enzyme label, and placed in a 37 ° C incubator for 2 h;
  • Blocking 150 ⁇ L of blocking solution was added to each well of the enzyme label and incubated at 4 ° C overnight;
  • Incubation primary antibody The collected mouse serum is diluted with antibody dilution, 100 ⁇ L per well, and then incubated at 37 ° C for 2 h;
  • Termination reaction The reaction was terminated by adding 50 ⁇ l of 2 M H 2 SO 4 stop solution to each well.
  • Method 2 One week after the completion of immunization, the mice were sacrificed, the spleen was taken, PBMC (peripheral blood mononuclear cells) were isolated, and the CTL effect was detected by the LDH (lactate dehydrogenase) kit.
  • PBMC peripheral blood mononuclear cells
  • Setting control group a total of effector group spontaneous release group, experimental group, target cell spontaneous release group, target cell maximum release group, volume-corrected control group and background control group;
  • the assay plate was incubated at 37 ° C, 5% CO 2 for 4 hours; the lysis buffer was added to the target cell maximum release group, and 10 ⁇ l of the lysis solution (10 ⁇ ) was added per 100 ⁇ l of the medium.
  • the concentration of Triton X-100 in this system is 0.8%, which can completely lyse the target cells (add the lysis solution 45 minutes before harvesting the supernatant);
  • Test number Auxiliary epitope peptide Existing antigen or epitope Fusion antigen sequence Indirect ELISA results 1 SEQ ID NO: 2 HER2 epitope SEQ ID NO: 21 figure 1 2 SEQ ID NO: 3 PD-L1 molecule SEQ ID NO: 22 figure 2 3 SEQ ID NO: 4 PD-1 extracellular domain SEQ ID NO: 23 image 3 4 SEQ ID NO: 5 EGFR SEQ ID NO:24 Figure 4 5 SEQ ID NO: 6 CD20 SEQ ID NO: 25 Figure 5 6 SEQ ID NO:7 CD66e SEQ ID NO:26 Figure 6 7 SEQ ID NO:8 CD227 extracellular region SEQ ID NO:27 Figure 7 8 SEQ ID NO: 9 VEGFR extracellular domain SEQ ID NO:28 Figure 8 9 SEQ ID NO: 10 IL-2Ra SEQ ID NO:29 Figure 9 10 SEQ ID NO: 11 CTLA-4 SEQ ID NO:30 Figure 10 11 SEQ ID NO: 12 PSMA SEQ ID NO: 31 Figure 11 12 SEQ ID NO: 13 TOLL-1
  • Figure 1 shows that the antibody titer produced by the HER2 fusion antigen group (i.e., HER2-1pPhe-PADRE) constructed in this example was significantly increased as compared with the HER2 epitope group and the HER2-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 21, ie
  • Fig. 2 shows that the antibody titer produced by the PD-L1 fusion antigen group (i.e., PD/L1-2pPhe-PADRE) constructed in this example was significantly increased as compared with the PD-L1 molecular group and the PD-L1-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 22, ie
  • Figure 3 shows that the antibody titer produced by the PD-1 fusion antigen group (i.e., PD/1-3pPhe-PADRE) constructed in this example was significantly higher than that of the PD-1 extracellular region and the PD-1-PADRE group. rise.
  • the sequence of the fusion antigen is SEQ ID NO: 23, ie
  • Figure 4 shows that the antibody titer produced by the EGFR fusion antigen group (i.e., EGFR-4pPhe-PADRE) constructed in this example was significantly increased compared to the EGFR group and the EGFR-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 24, ie
  • Figure 5 shows that the antibody titer produced by the CD20 fusion antigen group (i.e., CD20-5pPhe-PADRE) constructed in this example was significantly increased as compared with the CD20 group and the CD20-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 25, ie
  • Figure 6 shows that the antibody titer produced by the CD66e fusion antigen group (i.e., CD66e-6pPhe-PADRE) constructed in this example was significantly increased as compared with the CD66e group and the CD66e-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 26, ie
  • Figure 7 shows that the antibody titer produced by the CD227 fusion antigen group (i.e., CD227-7pPhe-PADRE) constructed in this example was significantly increased as compared with the CD227 extracellular region and the CD227-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 27, ie
  • FIG. 8 shows that the antibody titer produced by the VEGFR fusion antigen set (i.e., VEGFR-8pPhe-PADRE) constructed in this example was significantly increased compared to the VEGFR extracellular domain and the VEGFR-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 28, ie
  • Fig. 9 shows that the antibody titer produced by the IL-2Ra fusion antigen group (i.e., IL/2Ra-9pPhe-PADRE) constructed in this example was significantly increased as compared with the IL-2Ra group and the IL-2Ra-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 29, ie
  • Figure 10 shows that the antibody titer produced by the CTLA-4 fusion antigen group (i.e., CTLA/4-10pPhe-PADRE) constructed in this example was significantly increased as compared with the CTLA-4 group and the CTLA-4-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 30, ie
  • Figure 11 shows that the antibody titer produced by the PSMA fusion antigen group (i.e., PSMA-11pPhe-PADRE) constructed in this example was significantly increased as compared with the PSMA group and the PSMA-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 31, ie
  • Figure 12 shows that the antibody titer produced by the TOLL-1 fusion antigen group (i.e., TOLL/1-12pPhe-PADRE) constructed in this example was significantly increased as compared with the TOLL-1 group and the TOLL-1-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 32, ie
  • Figure 13 shows that the antibody titer produced by the GATA-4 fusion antigen group (i.e., GATA/4-13pPhe-PADRE) constructed in this example was significantly increased as compared with the GATA-4 group and the GATA-4-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 33, ie
  • Figure 14 shows the NY-ESO-1 fusion antigen group constructed in this example (ie NY/ESO/1-3, 5pPhe-PADRE) compared to the NY-ESO-1 group and the NY-ESO-1-PADRE group.
  • the resulting antibody titer increased significantly.
  • the sequence of the fusion antigen is SEQ ID NO: 34, ie
  • Fig. 15 shows that the antibody titer produced by the FR- ⁇ fusion antigen group (i.e., FR ⁇ -3, 8pPhe-PADRE) constructed in the present example was significantly increased as compared with the FR- ⁇ group and the FR- ⁇ -PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 35, ie
  • Figure 16 shows that the antibody titer produced by the EPCAM fusion antigen group (i.e., EPCAM-3, 11pPhe-PADRE) constructed in this example was significantly increased as compared with the EPCAM group and the EPCAM-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 36, ie
  • Figure 17 shows that the antibody titer produced by the P53 fusion antigen group (i.e., P53-5, 8pPhe-PADRE) constructed in this example was significantly increased as compared with the P53 group and the P53-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 37, ie
  • Fig. 18 shows that the antibody titer produced by the MESOTHELIN fusion antigen group (i.e., Mesothelin-5, 11pPhe-PADRE) constructed in this example was significantly increased as compared with the MESOTHELIN group and the MESOTHELIN-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 38, ie
  • Fig. 19 shows that the antibody titer produced by the WT1 fusion antigen group (i.e., WT1-8, 11pPhe-PADRE) constructed in this example was significantly increased as compared with the WT1 group and the WT1-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 39, ie
  • Figure 23 shows the epitope fusion antigen set constructed in this example (ie, B epitope + 3pPhe-PADRE) compared to the B epitope SEQ ID NO: 43 group, B epitope SEQ ID NO: 43-PADRE group.
  • the resulting antibody titer increased significantly.
  • the B epitope SEQ ID NO: 43 is FLPESFDGDPASNTAPLQPE.
  • the sequence of the fusion antigen is SEQ ID NO: 47, FLPESFDGDPASNTAPLQPEGPSLAKFXAAWTLKAAA.
  • Figure 24 shows that the antibody titer produced by the A ⁇ protein-42 fusion antigen group (ie, A-beta protein-6pPhe-PADRE) constructed in this example was significantly increased compared with the A ⁇ protein-42 group and the A ⁇ protein-PADRE group.
  • the sequence of the fusion antigen is SEQ ID NO: 48, namely LVFFAEDVGSNKGAIIGLMVGGVVIAGPSLAKFVAXWTLKAAA.
  • Test number Auxiliary epitope peptide Existing antigen or epitope Fusion antigen sequence CTL effect test result chart twenty one SEQ ID NO: 2 SEQ ID NO:40 SEQ ID NO: 44 Figure 20 twenty two SEQ ID NO: 3 SEQ ID NO:41 SEQ ID NO:45 Figure 21 twenty three SEQ ID NO: 4 SEQ ID NO:42 SEQ ID NO:46 Figure 22
  • Figure 20 shows that the epitope fusion antigen group (i.e., epitope 1+1p-PADRE) constructed in this example was induced in comparison with the epitope SEQ ID NO: 40 group and the epitope SEQ ID NO: 40-PADRE group. The resulting CTL effect is significantly enhanced.
  • epitope fusion antigen group i.e., epitope 1+1p-PADRE
  • the epitope SEQ ID NO: 40 is VLDNGDPL.
  • the sequence of the fusion antigen is SEQ ID NO: 44, VLDNGDPLGPSLXKFVAAWTLKAAA.
  • Figure 21 shows that the epitope fusion antigen set (i.e., epitope 2+2p-PADRE) constructed in this example was induced in comparison with the epitope SEQ ID NO: 41 group and the epitope SEQ ID NO: 41-PADRE group. The resulting CTL effect is significantly enhanced.
  • epitope fusion antigen set i.e., epitope 2+2p-PADRE
  • the epitope SEQ ID NO: 41 is TGYLYISA.
  • the sequence of the fusion antigen is SEQ ID NO: 45, TGYLYISAGPSLAXFVAAWTLKAAA.
  • Figure 22 shows that the epitope fusion antigen set (i.e., epitope 3+3p-PADRE) constructed in this example was induced in comparison with the epitope SEQ ID NO: 42 group and the epitope SEQ ID NO: 42-PADRE group. The resulting CTL effect is significantly enhanced.
  • epitope fusion antigen set i.e., epitope 3+3p-PADRE
  • the epitope SEQ ID NO: 42 is VLDNGDPLGPSLTGYLYISA.
  • the sequence of the fusion antigen is SEQ ID NO: 46, VLDNGDPLGPSLTGYLYISAGPSLAKXVAAWTLKAAA.
  • the present embodiment actually verified the ability of the fusion antigen obtained by linking the remaining helper epitope peptides in Example 1 to the existing antigen or antigen epitope to induce antibody production or induce CTL effect, which is limited by the space.
  • the test results are not listed here. The results showed that all of the helper epitope peptides of Example 1 have excellent ability to assist existing antigens or epitopes to produce antibodies or to produce CTL effects.

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Abstract

本发明提供了辅助性表位肽，该辅助性表位肽是通过将辅助性T细胞表位PADRE中的一个或两个氨基酸残基替换为4-硝基苯丙氨酸得到的。还提供了该辅助性表位肽用于增强抗原或抗原表位的免疫原性或用于制备或构建疫苗的用途，以及所述辅助性表位肽与抗原或抗原表位连接而成的融合抗原。

Description

辅助性表位肽及其应用 技术领域
本发明涉及一种辅助性表位肽及其应用，属于生物医药技术领域。
背景技术
肿瘤疫苗是一种最有效、最经济的癌症治疗方案，有限次数的疫苗注射即可带来长期的抗肿瘤免疫反应。而在临床应用中，肿瘤疫苗的治疗效果一直不太理想，其原因除了肿瘤自身抗原性较低以外，机体对于肿瘤抗原的免疫耐受可能也是重要的因素。最新的研究表明，免疫耐受主要是因为体内抗原特异性的CD4 +T细胞被消除了，而和CD8 +T细胞或者B细胞是否被消除没有太大关系。因此，招募不依赖自身抗原的CD4 +T细胞，打破CD4 +T细胞的免疫耐受状态，可能是激发肿瘤疫苗治疗潜力的关键步骤。
CD4 +T细胞是人体内免疫反应的开关，能调节免疫反应的强弱。一方面，耐受机制能减弱针对自身抗原的免疫反应，从而维持体内稳态；另一方面，打破耐受能带来针对自身抗原的强烈免疫反应，但与此同时也有诱发自身免疫性疾病的风险。所以，为了使治疗达到最好的效果，我们应该在最小化自身免疫疾病损害的基础上，尽量打破免疫耐受，从而使肿瘤的免疫治疗效果最大化。
PG·Schultz等人发现，在部分天然的蛋白质中部分位点引入非天然氨基酸可以形成新的MHC-Ⅱ分子限制的CD4表位，提高其免疫原性。同时，因为新的表位完全是外源性的，所以不会引发自身免疫疾病。然而，并不是所有的天然蛋白质或天然多肽引入任意非天然氨基酸，都能提高免疫原性，这就需要研究者来进行针对性研究。本发明的发明人所在课题组以辅助性T表位肽PADRE为研究对象进行了大量研究，以期获得通用性的辅助性表位肽。
经检索发现，专利号CN201110303946.1、授权公告号CN102370979B的中国发明专利，公开了一种针对人TNF-α分子的自体疫苗的构建方法，其中用到了PADRE氨基酸序列AKFVAAWTLKA。
申请号CN201611207485.7、申请公布号CN106749674A的中国发明专利申请，公开了新型哮喘多肽疫苗及其制备方法，涉及含有PADRE多肽aK-Cha-VAaWTLKAa的融合多肽，其中a表示D-丙氨酸，Cha表示L-环己基丙氨酸。
然而，以上述技术方案为代表的现有技术中尚不存在以PADRE多肽为基础进行改造获得的通用性辅助性表位肽。
发明内容
本发明的主要目的是：克服现有技术存在的问题，提供一种辅助性表位肽，具有通用性，能增强抗原或抗原表位的免疫原性；此外，还提供涉及该辅助性表位肽的用途。
为实现上述主要目的，本发明的技术方案如下：
一种辅助性表位肽，其特征是，该辅助性表位肽是通过将序列SEQ ID NO:1中的一个或两个氨基酸残基替换为4-硝基苯丙氨酸得到的。
优选地，该辅助性表位肽的序列为SEQ ID NO:2-SEQ ID NO:20之一。
本发明还提供：
前文所述辅助性表位肽的用途，所述用途为用于增强抗原或抗原表位的免疫原性，所述抗原或抗原表位含有氨基酸残基；或者，所述用途为用于制备或构建疫苗。
含有前文所述辅助性表位肽的产品，所述产品为药物、药物组合物、生物芯片、疫苗、或疫苗组合物。所述疫苗或疫苗组合物包括肿瘤疫苗或疫苗组合物。
本发明还提供：
融合抗原，其特征是，由前文所述辅助性表位肽与抗原或抗原表位连接而成；所述抗原或抗原表位含有氨基酸残基，所述辅助性表位肽与抗原或抗原表位的氨基酸残基相连。
优选地，所述辅助性表位肽经连接肽与抗原或抗原表位的氨基酸残基相连，所述连接肽序列为GPSL。
优选地，所述抗原或抗原表位为HER2、PD-L1、PD-1、EGFR、CD20、CD66e、CD227、VEGFR、IL-2R、CTLA-4、PSMA、TOLL-1、GATA-4、NY-ESO-1、FRα、CA125、EpCAM-CD3、P53、Mesothelin、WT1、Aβ蛋白之一，或者为SEQ ID NO:40-SEQ ID NO:43之一。
优选地，所述融合抗原为多肽，且其序列为SEQ ID NO:21-SEQ ID NO:39之一、或为SEQ ID NO:44-SEQ ID NO:47之一。
本发明还提供：
含有前文所述融合抗原的疫苗或疫苗组合物。
发明人在深入地反复实践研究中发现，以辅助性T表位肽PADRE为基础(PADRE序列为AKFVAAWTLKAAA)，将其中的一个或两个氨基酸残基替换为4-硝基苯丙氨酸(又名：对硝基苯丙氨酸)后所得的辅助性表位肽，能显著增强已有抗原或抗原表位的免疫原性，打破CD4 +T细胞免疫耐受，而且该辅助性表位肽具有通用性。
与现有技术相比，本发明的辅助性表位肽能通用性地增强现有抗原(如HER2分子、PD-L1分子等)或抗原表位(如B细胞表位等)的免疫原性，提高特异性抗体的滴度；该辅助性表位肽完全是外源性的，在打破免疫耐受的同时，不会引起自身免疫性疾病等反应，生理毒性较低；该辅助性表位肽具有潜在的辅助激活CTL效应的能力，在临床上可以辅助构建个性化疫苗，治疗预防肿瘤；该辅助性表位肽具有优异的辅助已有抗原或抗原表位产生抗体或产生CTL效应的能力，对于构建高效、持久的疫苗提供了思路和前期基础。
附图说明
图1至图19分别为实施例2的试验例1至19的结果示意图。
图20至图22分别为实施例2的试验例21至23的结果示意图。
图23为实施例2的试验例20的结果示意图。
图24为实施例2的试验例24的结果示意图。
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。
实施例1、构建辅助性表位肽
以序列SEQ ID NO:1的辅助性T表位肽PADRE为基础，将其中的一个氨基酸或两个氨基酸残基替换为4-硝基苯丙氨酸，所得序列如下表所示：
序号 序列 备注
1 XKFVAAWTLKAAA SEQ ID NO:2
2 AXFVAAWTLKAAA SEQ ID NO:3
3 AKXVAAWTLKAAA SEQ ID NO:4
4 AKFXAAWTLKAAA SEQ ID NO:5
5 AKFVXAWTLKAAA SEQ ID NO:6
6 AKFVAXWTLKAAA SEQ ID NO:7
7 AKFVAAXTLKAAA SEQ ID NO:8
8 AKFVAAWXLKAAA SEQ ID NO:9
9 AKFVAAWTXKAAA SEQ ID NO:10
10 AKFVAAWTLXAAA SEQ ID NO:11
11 AKFVAAWTLKXAA SEQ ID NO:12
12 AKFVAAWTLKAXA SEQ ID NO:13
13 AKFVAAWTLKAAX SEQ ID NO:14
14 XXFVAAWTLKAAA  
15 XKXVAAWTLKAAA  
16 XKFXAAWTLKAAA  
17 XKFVXAWTLKAAA  
18 XKFVAXWTLKAAA  
19 XKFVAAXTLKAAA  
20 XKFVAAWXLKAAA  
21 XKFVAAWTXKAAA  
22 XKFVAAWTLXAAA  
23 XKFVAAWTLKXAA  
24 XKFVAAWTLKAXA  
25 XKFVAAWTLKAAX  
26 AXXVAAWTLKAAA  
27 AXFXAAWTLKAAA  
28 AXFVXAWTLKAAA  
29 AXFVAXWTLKAAA  
30 AXFVAAXTLKAAA  
31 AXFVAAWXLKAAA  
32 AXFVAAWTXKAAA  
33 AXFVAAWTLXAAA  
34 AXFVAAWTLKXAA  
35 AXFVAAWTLKAXA  
36 AXFVAAWTLKAAX  
37 AKXXAAWTLKAAA  
38 AKXVXAWTLKAAA SEQ ID NO:15
39 AKXVAXWTLKAAA  
40 AKXVAAXTLKAAA  
41 AKXVAAWXLKAAA SEQ ID NO:16
42 AKXVAAWTXKAAA  
43 AKXVAAWTLXAAA  
44 AKXVAAWTLKXAA SEQ ID NO:17
45 AKXVAAWTLKAXA  
46 AKXVAAWTLKAAX  
47 AKFXXAWTLKAAA  
48 AKFXAXWTLKAAA  
49 AKFXAAXTLKAAA  
50 AKFXAAWXLKAAA  
51 AKFXAAWTXKAAA  
52 AKFXAAWTLXAAA  
53 AKFXAAWTLKXAA  
54 AKFXAAWTLKAXA  
55 AKFXAAWTLKAAX  
56 AKFVXXWTLKAAA  
57 AKFVXAXTLKAAA  
58 AKFVXAWXLKAAA SEQ ID NO:18
59 AKFVXAWTXKAAA  
60 AKFVXAWTLXAAA  
61 AKFVXAWTLKXAA SEQ ID NO:19
62 AKFVXAWTLKAXA  
63 AKFVXAWTLKAAX  
64 AKFVAXXTLKAAA  
65 AKFVAXWXLKAAA  
66 AKFVAXWTXKAAA  
67 AKFVAXWTLXAAA  
68 AKFVAXWTLKXAA  
69 AKFVAXWTLKAXA  
70 AKFVAXWTLKAAX  
71 AKFVAAXXLKAAA  
72 AKFVAAXTXKAAA  
73 AKFVAAXTLXAAA  
74 AKFVAAXTLKXAA  
75 AKFVAAXTLKAXA  
76 AKFVAAXTLKAAX  
77 AKFVAAWXXKAAA  
78 AKFVAAWXLXAAA  
79 AKFVAAWXLKXAA SEQ ID NO:20
80 AKFVAAWXLKAXA  
81 AKFVAAWXLKAAX  
82 AKFVAAWTXXAAA  
83 AKFVAAWTXKXAA  
84 AKFVAAWTXKAXA  
85 AKFVAAWTXKAAX  
86 AKFVAAWTLXXAA  
87 AKFVAAWTLXAXA  
88 AKFVAAWTLXAAX  
89 AKFVAAWTLKXXA  
90 AKFVAAWTLKXAX  
91 AKFVAAWTLKAXX  
注：各序列中的X为4-硝基苯丙氨酸。
实施例2、验证辅助性表位肽的实施效果
从实施例1中选取辅助性表位肽和不同的抗原分子相结合，构建融合抗原，验证其诱导产生抗体或诱导产生CTL效应的能力。该试验的主要步骤为：
(1)将辅助性表位肽经连接肽与不同的抗原或抗原表位相连，连接肽序列为GPSL(即Gly-Pro-Ser-Leu)，构建出多个融合抗原。
(2)将(1)所得融合抗原分别与完全弗氏佐剂相1：1等体积混合乳化，以每只小鼠皮下注射50μg融合抗原的剂量进行免疫，实验小鼠品系包括C57、Balb/c和Fvb；分别在初次免疫7天和14天后，将(1)所得融合抗原和不完全弗氏佐剂1：1等体积混合乳化，以每只小鼠皮下注射50μg融合抗原的剂量进行免疫。
(3)检测方法有两种，第一种为：分别取初次免疫7天、14天、21天和28天的小鼠眼眶全血，离心取血清，通过间接ELISA法检测抗体滴度；第二种为：免疫完成后一周，处死小鼠，取脾脏，分离出PBMC(外周血单核细胞)，采用LDH(乳酸脱氢酶)试剂盒检测CTL效应。
按照上述主要步骤，本实施例的主要实施过程如下：
第一步、构建融合抗原后，针对每个融合抗原，将6-8周龄C57BL/6雌性小鼠随机分为3组，每组6只，分别为PBS组，现有抗原或抗原表位组，抗原-PADRE或抗原表位-PADRE组，以及含融合抗原的疫苗组。
第二步、采用皮下免疫的方式，免疫3次，间隔一周，每次50μg。与弗氏佐剂1:1等体积混合。
第三步、采用方法1或方法2进行检测。
方法1：免疫后每周取血，6000rpm离心20min分离血清，共取4周。采用如下间接ELISA法检测抗体滴度。
(1)包被：将现有抗原或抗原表位用包被液稀释至5μg/mL，在酶标条中每孔加入100μL，置于37℃恒温箱中孵育2h；
(2)PBST清洗5次，每次5min；
(3)封闭：在酶标条中每孔加入150μL封闭液，置于4℃孵育过夜；
(4)重复步骤(2)；
(5)孵一抗：将采集的小鼠血清用抗体稀释液倍比稀释后，每孔加入100μL，置于37℃恒温孵育2h；
(6)重复步骤(2)；
(7)孵二抗：HRP-goat-anti-mouse IgG用抗体稀释液按1:10000的比例稀释，每孔加入100μL，置于37℃恒温孵育45min；
(8)重复步骤(2)；
(9)加底物：酶标条中每孔加入100μl TMB底物反应液，置于37℃恒温箱中避光孵育15min；
(10)终止反应：每孔加入50μl 2M的H 2SO 4终止液终止反应。
(11)显色：在450/630nm下，检测孔内样品的吸光值。
方法2：免疫完成后一周，处死小鼠，取脾脏，分离出PBMC(外周血单核细胞)，采用LDH(乳酸脱氢酶)试剂盒检测CTL效应。
(1)设置对照组：共分为效应细胞自发释放组、实验组、靶细胞自发释放组、靶细胞最大释放组、体积校正对照组和背景对照组；
(2)250g离心4分钟，以使效应细胞与靶细胞充分接触；
(3)37℃，5％CO 2孵育检测板4小时；靶细胞最大释放组中加入裂解液，每100μl培养基加10μl裂解溶液(10×)。该体系中Triton X-100的浓度为0.8％，可使靶细胞完全裂解(收获上清前45分钟加入裂解溶液)；
(4)250g离心4分钟；
(5)转移50μl上清至另一孔板；
(6)解冻检测缓冲液，取12ml(避光)，将剩余的迅速冻存(可以使用37℃水浴解冻，但不可放置过长时间)。将12ml检测缓冲液添加到一瓶底物混合液(可用于两个96孔板)中，倒置混匀；稀释后，避光、迅速添加；
(7)50μl/孔添加稀释的底物混合液，室温避光孵育30分钟(未使用的稀释后底物混合物液放于-20℃保存6-8周；
(8)添加50μl终止溶液，将孔中含有的气泡去除，一小时内检测吸收值(490或492nm)；
(9)结果统计，细胞杀伤率(％)＝[(实验组释放-效应细胞自发释放-靶细胞自发释放)/(靶细胞最大释放-靶细胞自发释放)]×100％。
采用间接ELISA法检测的试验例如下表所示：
试验序号 辅助性表位肽 现有抗原或抗原表位 融合抗原序列 间接ELISA法结果图
1 SEQ ID NO:2 HER2抗原表位 SEQ ID NO:21 图1
2 SEQ ID NO:3 PD-L1分子 SEQ ID NO:22 图2
3 SEQ ID NO:4 PD-1胞外区 SEQ ID NO:23 图3
4 SEQ ID NO:5 EGFR SEQ ID NO:24 图4
5 SEQ ID NO:6 CD20 SEQ ID NO:25 图5
6 SEQ ID NO:7 CD66e SEQ ID NO:26 图6
7 SEQ ID NO:8 CD227胞外区 SEQ ID NO:27 图7
8 SEQ ID NO:9 VEGFR胞外区 SEQ ID NO:28 图8
9 SEQ ID NO:10 IL-2Ra SEQ ID NO:29 图9
10 SEQ ID NO:11 CTLA-4 SEQ ID NO:30 图10
11 SEQ ID NO:12 PSMA SEQ ID NO:31 图11
12 SEQ ID NO:13 TOLL-1 SEQ ID NO:32 图12
13 SEQ ID NO:14 GATA-4 SEQ ID NO:33 图13
14 SEQ ID NO:15 NY-ESO-1 SEQ ID NO:34 图14
15 SEQ ID NO:16 FR-α SEQ ID NO:35 图15
16 SEQ ID NO:17 EPCAM SEQ ID NO:36 图16
17 SEQ ID NO:18 P53 SEQ ID NO:37 图17
18 SEQ ID NO:19 Mesothelin SEQ ID NO:38 图18
19 SEQ ID NO:20 WT1 SEQ ID NO:39 图19
20 SEQ ID NO:5 SEQ ID NO:43 SEQ ID NO:47 图23
24 SEQ ID NO:6 Aβ蛋白-42 SEQ ID NO:48 图24
各图所表示的结果如下：
图1显示，与HER2抗原表位组、HER2-PADRE组相比，本实施例所构建的HER2融合抗原组(即HER2-1pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:21，即
Figure PCTCN2019083484-appb-000001
Figure PCTCN2019083484-appb-000002
图2显示，与PD-L1分子组、PD-L1-PADRE组相比，本实施例所构建的PD-L1融合抗原组(即PD/L1-2pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:22，即
Figure PCTCN2019083484-appb-000003
图3显示，与PD-1胞外区组、PD-1-PADRE组相比，本实施例所构建的PD-1融合抗原组(即PD/1-3pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:23，即
Figure PCTCN2019083484-appb-000004
图4显示，与EGFR组、EGFR-PADRE组相比，本实施例所构建的EGFR融合抗原组(即EGFR-4pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:24，即
Figure PCTCN2019083484-appb-000005
图5显示，与CD20组、CD20-PADRE组相比，本实施例所构建的CD20融合抗原组(即CD20-5pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:25，即
Figure PCTCN2019083484-appb-000006
图6显示，与CD66e组、CD66e-PADRE组相比，本实施例所构建的CD66e融合抗原组(即CD66e-6pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:26，即
Figure PCTCN2019083484-appb-000007
Figure PCTCN2019083484-appb-000008
图7显示，与CD227胞外区组、CD227-PADRE组相比，本实施例所构建的CD227融合抗原组(即CD227-7pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:27，即
Figure PCTCN2019083484-appb-000009
图8显示，与VEGFR胞外区组、VEGFR-PADRE组相比，本实施例所构建的VEGFR融合抗原组(即VEGFR-8pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:28，即
Figure PCTCN2019083484-appb-000010
Figure PCTCN2019083484-appb-000011
图9显示，与IL-2Ra组、IL-2Ra-PADRE组相比，本实施例所构建的IL-2Ra融合抗原组(即IL/2Ra-9pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:29，即
Figure PCTCN2019083484-appb-000012
图10显示，与CTLA-4组、CTLA-4-PADRE组相比，本实施例所构建的CTLA-4融合抗原组(即CTLA/4-10pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:30，即
Figure PCTCN2019083484-appb-000013
图11显示，与PSMA组、PSMA-PADRE组相比，本实施例所构建的PSMA融合抗原组(即PSMA-11pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:31，即
Figure PCTCN2019083484-appb-000014
图12显示，与TOLL-1组、TOLL-1-PADRE组相比，本实施例所构建的TOLL-1融合抗原组(即TOLL/1-12pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:32，即
Figure PCTCN2019083484-appb-000015
图13显示，与GATA-4组、GATA-4-PADRE组相比，本实施例所构建的GATA-4融合抗原组(即GATA/4-13pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:33，即
Figure PCTCN2019083484-appb-000016
Figure PCTCN2019083484-appb-000017
图14显示，与NY-ESO-1组、NY-ESO-1-PADRE组相比，本实施例所构建的NY-ESO-1融合抗原组(即NY/ESO/1-3、5pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:34，即
Figure PCTCN2019083484-appb-000018
图15显示，与FR-α组、FR-α-PADRE组相比，本实施例所构建的FR-α融合抗原组(即FRα-3、8pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:35，即
Figure PCTCN2019083484-appb-000019
图16显示，与EPCAM组、EPCAM-PADRE组相比，本实施例所构建的EPCAM融合抗原组(即EPCAM-3、11pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:36，即
Figure PCTCN2019083484-appb-000020
图17显示，与P53组、P53-PADRE组相比，本实施例所构建的P53融合抗原组(即P53-5、8pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:37，即
Figure PCTCN2019083484-appb-000021
图18显示，与MESOTHELIN组、MESOTHELIN-PADRE组相比，本实施例所构建的MESOTHELIN融合抗原组(即Mesothelin-5、11pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:38，即
Figure PCTCN2019083484-appb-000022
Figure PCTCN2019083484-appb-000023
图19显示，与WT1组、WT1-PADRE组相比，本实施例所构建的WT1融合抗原组(即WT1-8、11pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:39，即
Figure PCTCN2019083484-appb-000024
图23显示，与B表位SEQ ID NO:43组、B表位SEQ ID NO:43-PADRE组相比，本实施例所构建的表位融合抗原组(即B表位+3pPhe-PADRE)产生的抗体滴度显著上升。
该B表位SEQ ID NO:43为FLPESFDGDPASNTAPLQPE。该融合抗原的序列为SEQ ID NO:47，即FLPESFDGDPASNTAPLQPEGPSLAKFXAAWTLKAAA。
图24显示，与Aβ蛋白-42组、Aβ蛋白-PADRE组相比，本实施例所构建的Aβ蛋白-42融合抗原组(即A-beta protein-6pPhe-PADRE)产生的抗体滴度显著上升。该融合抗原的序列为SEQ ID NO:48，即LVFFAEDVGSNKGAIIGLMVGGVVIAGPSLAKFVAXWTLKAAA。
采用CTL效应检测的试验例如下表所示：
试验序号 辅助性表位肽 现有抗原或抗原表位 融合抗原序列 CTL效应检测结果图
21 SEQ ID NO:2 SEQ ID NO:40 SEQ ID NO:44 图20
22 SEQ ID NO:3 SEQ ID NO:41 SEQ ID NO:45 图21
23 SEQ ID NO:4 SEQ ID NO:42 SEQ ID NO:46 图22
各图所表示的结果如下：
图20显示，与表位SEQ ID NO:40组、表位SEQ ID NO:40-PADRE组相比，本实施例所构建的表位融合抗原组(即表位1+1p-PADRE)所诱导产生的CTL效应明显增强。
该表位SEQ ID NO:40为VLDNGDPL。该融合抗原的序列为SEQ ID NO:44，即VLDNGDPLGPSLXKFVAAWTLKAAA。
图21显示，与表位SEQ ID NO:41组、表位SEQ ID NO:41-PADRE组相比，本实施例所构建的表位融合抗原组(即表位2+2p-PADRE)所诱导产生的CTL效应明显增强。
该表位SEQ ID NO:41为TGYLYISA。该融合抗原的序列为SEQ ID NO:45，即TGYLYISAGPSLAXFVAAWTLKAAA。
图22显示，与表位SEQ ID NO:42组、表位SEQ ID NO:42-PADRE组相比，本实施例所构建的表位融合抗原组(即表位3+3p-PADRE)所诱导产生的CTL效应明显增强。
该表位SEQ ID NO:42为VLDNGDPLGPSLTGYLYISA。该融合抗原的序列为SEQ ID  NO:46，即VLDNGDPLGPSLTGYLYISAGPSLAKXVAAWTLKAAA。
此外，本实施例实际上还验证了实施例1中其余辅助性表位肽与现有抗原或抗原表位连接所得融合抗原的诱导产生抗体或诱导产生CTL效应的能力，受篇幅所限，具体试验结果不在此处一一列出。结果表明，实施例1的所有辅助性表位肽均具有优异的辅助已有抗原或抗原表位产生抗体或产生CTL效应的能力。
除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。

Claims (10)

  1. 一种辅助性表位肽，其特征是，该辅助性表位肽是通过将序列SEQ ID NO:1中的一个或两个氨基酸残基替换为4-硝基苯丙氨酸得到的。
  2. 根据权利要求1所述的辅助性表位肽，其特征是，该辅助性表位肽的序列为SEQ ID NO:2-SEQ ID NO:20之一。
  3. 权利要求1或2所述辅助性表位肽的用途，所述用途为用于增强抗原或抗原表位的免疫原性，所述抗原或抗原表位含有氨基酸残基；或者，所述用途为用于制备或构建疫苗。
  4. 含有权利要求1或2所述辅助性表位肽的产品，所述产品为药物、药物组合物、生物芯片、疫苗、或疫苗组合物。
  5. 根据权利要求4所述的产品，其特征是，所述疫苗或疫苗组合物包括肿瘤疫苗或疫苗组合物。
  6. 融合抗原，其特征是，由权利要求1或2所述辅助性表位肽与抗原或抗原表位连接而成；所述抗原或抗原表位含有氨基酸残基，所述辅助性表位肽与抗原或抗原表位的氨基酸残基相连。
  7. 根据权利要求6所述的融合抗原，其特征是，所述辅助性表位肽经连接肽与抗原或抗原表位的氨基酸残基相连，所述连接肽序列为GPSL。
  8. 根据权利要求7所述的融合抗原，其特征是，所述抗原或抗原表位为HER2、PD-L1、PD-1、EGFR、CD20、CD66e、CD227、VEGFR、IL-2R、CTLA-4、PSMA、TOLL-1、GATA-4、NY-ESO-1、FR-α、CA125、EpCAM-CD3、P53、Mesothelin、WT1、Aβ蛋白之一，或者为SEQ ID NO:40-SEQ ID NO:43之一。
  9. 根据权利要求8所述的融合抗原，其特征是，所述融合抗原为多肽，且其序列为SEQ ID NO:21-SEQ ID NO:39之一、或为SEQ ID NO:44-SEQ ID NO:47之一。
  10. 含有权利要求6至9任一项所述融合抗原的疫苗或疫苗组合物。
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