Patent Publication Number: US-2021162041-A1

Title: Helper epitope peptide and application thereof

Description:
INCORPORATION OF SEQUENCE LISTING 
     This application contains a sequence listing submitted in Computer Readable Form (CRF). The CFR file containing the sequence listing entitled “PA440-0007_seq.txt”, which was created on Nov. 2, 2020, and is 7,224 bytes in size. The information in the sequence listing is incorporated herein by reference in its entirety. 
     TECHNICAL FIELD 
     The invention relates to a helper epitope peptide and its application, which belongs to the technical field of biomedicine. 
     BACKGROUND OF THE INVENTION 
     Tumor vaccine is one of the most effective and economical cancer treatments. A limited number of vaccine injections can bring long-term anti-tumor immune response. However, in clinical application, the therapeutic effect of tumor vaccine has been not ideal; the reason is not only the low antigenicity of tumor itself, but also the immune tolerance of the body to tumor antigens may be an important factor. Recent studies have shown that immune tolerance is mainly due to the elimination of antigen-specific CD4+ T cells in vivo, which has little to do with the elimination of CD8+ T cells or B cells. Therefore, recruiting CD4+ T cells independent of autoantigen and breaking the immune tolerance of CD4+ T cells may be a key step to stimulate the therapeutic potential of tumor vaccine. 
     CD4+ T cells are the switch of the immune response in vivo and can regulate the strength of the immune response. The self-tolerance mechanism weaken the immune response to self-antigens maintaining homeostasis; breaking self-tolerance would bring a strong immune response to self-antigens, but at the same time, it can also induce the risk of autoimmune diseases. Therefore, in order to achieve the best effect of treatment, we should try to break the immune tolerance on the basis of minimizing the damage of autoimmune diseases, so as to maximize the effect of tumor immunotherapy. 
     PG⋅Schultz et al. found that the introduction of unnatural amino acids in some natural proteins can form new MHC-II molecular-restricted CD4 epitopes and improve their immunogenicity. The new epitope is completely exogenous, so it will not cause autoimmune diseases. However, it doesn&#39;t mean that all-natural proteins or natural peptides can improve immunogenicity by introducing the unnatural amino acid, which requires researchers conducting targeted research. In present invention, we has conducted a large number of studies with the pan HLA DR-binding epitope (PADRE) as the research object, in order to obtain a universal helper epitope peptide. 
     A Chinese invention patent with Patent No. CN201110303946.1 and REF NO. CN102370979B discloses a method for constructing an autologous vaccine against human TNF-α molecules, in which a PADRE that sequence is AKFVAAWTLKA is used. 
     Patent Application Number CN201611207485.7; Published C.N Application Number CN106749674A disclosed a new asthma polypeptide vaccine and its preparation method. This patent involves a fusion polypeptide containing PADRE polypeptide sequencing aK-Cha-VAaWTLKAa. A (i.e., D-alanine) and Cha (i.e., L-cyclohexyl alanine). 
     However, the existing technologies represented by the above technical solutions do not yet have generic helper epitopes derived from PADRE polypeptides. 
     The Invention Contents 
     The main purpose of the present invention is to overcome the problems existing in the existing technology, to provide a helper epitope peptide, which has universality and can enhance the immunogenicity of antigens or antigen epitopes; In addition, uses involving the epitope peptide are provided. 
     To achieve the above main purposes, the technical scheme of the invention is as follows: 
     A helper epitope characterized by the substitution of one or two amino acid residues in the sequence of SEQ ID NO:1 by 4-nitrophenylalanine. 
     Preferably, the sequence of the helper epitope peptide is a sequence selected from SEQ ID NO:2 to SEQ ID NO:20. 
     The invention also provides: 
     The purpose of the helper epitope described above is to enhance the immunogenicity of antigens or epitopes containing amino acid residues; Or, the use is for the preparation or construction of a vaccine. 
     Products containing the helper epitopes described above are drugs, drug compositions, biochips, vaccines, or vaccine compositions. The vaccine or vaccine composition comprises a tumor vaccine or vaccine composition. 
     The invention provides a kind of fusion antigen comprising the said helper epitopes attached to antigens or epitopes. The attached antigen or epitope contains an amino acid residue, and the helper epitope peptide is attached to the amino acid residue of the antigen or epitope. 
     Preferably, the helper epitope peptide is attached to the amino acid residue of antigens or epitopes by connecting peptides, and the sequence of the connecting peptide is GPSL. 
     Preferably, the attached antigen or epitope is one of the listing proteins:HER2, PD-L1, PD-1, EGFR, CD20, CD66e, CD227, VEGFR, IL-2R, CTLA-4, PSMA, Toll-1, GTA-4, NY-ESO-1, FR, CA125, Epcam-CD3, P53, Mesothelin, WT1, Aβ protein, or one of the proteins with a sequence selected from SEQ ID NO: 40 to SEQ ID NO:43. 
     Preferably, the fusion antigen is a polypeptide with a sequence selected from SEQ ID NO:21 to SEQ ID NO:39, or from SEQ ID NO: 44 to SEQ ID NO:47. 
     The invention provides a vaccine or vaccine composition comprising the fusion antigens described above. 
     Inventors in constant practice find that based on the helper T epitope peptide PADRE (PADRE sequence is AKFVAAWTLKAAA), replacing one or two amino acid residues with 4-nitrobenzene alanine (aka: p-nitrophenyl alanine) can significantly enhance the immunogenicity of existing antigen or epitope and break CD4 +  T cell immune tolerance and the helper epitope can be in general use. 
     Compared with the current technology, the helper epitope peptide of this invention can universally enhance the immunogenicity of existing antigens (such as HER2, PD-L1, etc.) or antigen epitopes (such as B cell epitopes, etc.) and increase the titer of specific antibodies. The helper epitope peptide is completely exogenous and can break the immune tolerance. Meanwhile, it will not cause autoimmune diseases and its physiological toxicity is low. The helper epitope peptide has the potential to assist in activating the CTL, and can assist in the construction of personalized vaccines in the clinic to treat and prevent tumors. The helper epitope peptide has excellent ability to assist existing antigens or epitopes in producing antibodies or activating the CTL, and provides ideas and a preliminary basis for constructing efficient and durable vaccines. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG. 1  to  FIG. 19  are schematic illustrations of experiment 1 to 19 of example 2 respectively. 
         FIG. 20  to  FIG. 22  are schematic illustrations of experiment 21 to 23 of example 2 respectively. 
         FIG. 23  is a schematic illustration of the results in the experiment 20 of example 2. 
         FIG. 24  is a schematic illustration of the results in the experiment 24 of Example 2. 
     
    
    
     DETAILED DESCRIPTION 
     Hereinafter, this invention will be further described in detail with reference to the attached figures and the embodiments. However, the present invention is not limited to the examples given. 
     Example 1: Construction of Helper Epitope Peptides 
     Based on the helper T epitope peptide PADRE of the sequence SEQ ID NO:1, one amino acid or two amino acid residues are replaced with 4-nitrophenylalanine, and the resulting sequence is shown in the following table: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Serial number 
                 Sequence 
                 Remark 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 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 
               
               
                   
               
               
                 Note: 
               
               
                 X in the above sequences represent 4-nitrophenylalanine. 
               
            
           
         
       
     
     Example 2: Verify the Effect of the Helper Epitope 
     The helper epitopes selected from example 1 were combined with different antigen molecules to construct the individual fusion antigen. Then the ability of the fusion antigen molecules to induce antibody production or activate the CTL was verified. 
     The protocol is as follows:
     (1) The helper epitope was connected to individual antigens or antigen epitope with the linking peptide to construct multiple fusion antigens. The sequence of the linking peptide is GPSL (i.e. Gly-Pro-Ser-Leu).   (2) The fusion antigens from (1) were emulsified adequately with complete Freund&#39;s adjuvant of the same volume. Then to immunize mice via subcutaneous injection with the emulsion at the dose of 50 ug per mouse. The strains of experimental mice including C57, Fvb and Balb/C. After 7 days and 14 days of first immunization, the fusion antigens from (1) were emulsified adequately with incomplete Freund&#39;s adjuvant of the same volume and then to immunize mice via subcutaneous injection with the emulsion at the dose of 50 ug per mouse.   (3) There are two detection methods. One is to obtain the orbital blood of immunized mice on 7, 14, 21 and 28 day respectively, centrifuge the whole blood to get the serum. Then the antibody titer is detected by indirect ELISA. The other one is: one week after the last immunization, the mice are killed to get the spleen. PBMC (peripheral blood mononuclear cells) are isolated, and the CTL-mediated cytotoxicity is detected by LDH (lactate dehydrogenase) kit.   

     According to the above main steps, the main protocol is as follows: 
     The first step was to construct the fusion antigen. According to each fusion antigen, C57BL/6 female mice aged 6-8 weeks were randomly divided into three groups with 6 mice in each group. They were PBS group, existing antigen or antigen epitope group, antigen-PADRE or antigen epitope-PADRE group, and vaccine group containing fusion antigen. 
     In the second step, using fusion antigen to immunize mice via subcutaneous injection for 3 times with an interval of 50 μg each time. Mixed with Freund&#39;s adjuvant of the same volume. 
     The third step is to use method 1 or method 2 for detection. 
     Method 1: The whole blood was collected every week after immunization and centrifugated at 6000 rpm for 20 min to obtain the serum for a total of 4 weeks. Antibody titers were detected by indirect ELISA as follows: 
     (1) Coating: the existing antigen or antigen epitope was diluted to 5 μg/mL with coating solution. Then the 100 μL mixed solution was added into each well of enzyme immunoassay test strip, and incubated in 37° C. incubator for 2 h; 
     (2) use PBST to wash each well 5 times for 5 min each time; 
     (3) Sealing: 150 μL locking solution was added to each well of the enzyme immunoassay test strip and incubated at 4° C. overnight. 
     (4) Repeat step (2); 
     (5) Incubation first antibody: the collected mouse serum was diluted with antibody diluent. Then the 100 μL mixed solution was added into each well, and incubated at 37° C. for 2 h; 
     (6) Repeat step (2); 
     (7) Incubating secondary antibodies: HRP-GOAT Anti-mouse IgG was diluted with antibody dilution in the ratio of 1:10000, and 100 μL dilution was added to each well, and incubated at 37° C. for 45 min; 
     (8) Repeat step (2); 
     (9) Substrate addition: 100 μL TMB substrate reaction solution was added into each well of enzyme label, and incubated at 37° C. for 15 min in darkness. 
     (10) Termination reaction: 50 μL 2M H 2 SO 4  was added into each well to terminate the reaction. 
     (11) Color development: the absorbance value of the sample in each well was detected at 450/630 nm. 
     Method 2: one week after last immunization, mice were sacrificed, and spleen was taken; then PBMC (peripheral blood mononuclear cells) were isolated, and the CTL-mediated cytotoxicity was detected by LDH (lactate dehydrogenase) kit. 
     (1) Setting control: The control group was divided into effector cell spontaneous release group, experimental group, target cell spontaneous release group, target cell maximum release group, volume correction control group and background control group; 
     (2) The cells were centrifuged at 250 g for 4 minutes to make the effector cells fully contact with the target cells; 
     (3) The detection plate was incubated with 5% CO 2  at 37° C. for 4 hours; 10 μL of Lysis buffer was added to every 100 μL medium (10×) in the target cell maximum release group. When the concentration of Triton X-100 was 0.8%, the target cells could be completely lysed (The Lysis buffer was added 45 minutes before harvesting the supernatant) 
     (4) Centrifuge at 250 g for 4 minutes; 
     (5) Transfer 50 μL supernatant to another well plate; 
     (6) Thaw the detection buffer, take 12 mL (out of light), and quickly freeze the rest (it can be thawed in a 37° C. water bath, but not for a long time). Add 12 mL detection buffer to a bottle of substrate mixture (which can be used for two 96 well plates) and mix it upside down; after dilution, add it quickly without light; 
     (7) The diluted substrate mixture was added into 50 μL/well and incubated in dark at room temperature for 30 minutes (the unused diluted substrate mixture was stored at −20° C. for 6-8 weeks; 
     (8) Add 50 μL termination solution and remove the bubbles in the hole, and detect the absorption value (490 or 492 nm) within one hour 
     (9) Calculate % cytotoxicity if needed: 
       % Cytotoxicity=[(experimental group release-effector cell spontaneous release-target cell spontaneous release)/(maximum target cell release target cell spontaneous release)]*100% 
     The tests detected by indirect ELISA are shown in the following table: 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                 Test 
                 helper epitope 
                 existing antigen or 
                 fusion antigen 
                 indirect ELISA 
               
               
                 number 
                 peptide 
                 antigen epitope 
                 sequence 
                 result FIG. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 SEQ ID NO: 2 
                 HER2 antigen epitope 
                 SEQ ID NO: 21 
                 FIG. 1 
               
               
                 2 
                 SEQ ID NO: 3 
                 PD-L1 molecule 
                 SEQ ID NO: 22 
                 FIG. 2 
               
               
                 3 
                 SEQ ID NO: 4 
                 PD-1 extracellular 
                 SEQ ID NO: 23 
                 FIG. 3 
               
               
                   
                   
                 domain 
               
               
                 4 
                 SEQ ID NO: 5 
                 EGFR 
                 SEQ ID NO: 24 
                 FIG. 4 
               
               
                 5 
                 SEQ ID NO: 6 
                 CD20 
                 SEQ ID NO: 25 
                 FIG. 5 
               
               
                 6 
                 SEQ ID NO: 7 
                 CD66e 
                 SEQ ID NO: 26 
                 FIG. 6 
               
               
                 7 
                 SEQ ID NO: 8 
                 CD227 extracellular 
                 SEQ ID NO: 27 
                 FIG. 7 
               
               
                   
                   
                 domain 
               
               
                 8 
                 SEQ ID NO: 9 
                 VEGFR extracellular 
                 SEQ ID NO: 28 
                 FIG. 8 
               
               
                   
                   
                 domain 
               
               
                 9 
                 SEQ ID NO: 10 
                 IL-2Ra 
                 SEQ ID NO: 29 
                 FIG. 9 
               
               
                 10 
                 SEQ ID NO: 11 
                 CTLA-4 
                 SEQ ID NO: 30 
                 FIG. 10 
               
               
                 11 
                 SEQ ID NO: 12 
                 PSMA 
                 SEQ ID NO: 31 
                 FIG. 11 
               
               
                 12 
                 SEQ ID NO: 13 
                 TOLL-1 
                 SEQ ID NO: 32 
                 FIG. 12 
               
               
                 13 
                 SEQ ID NO: 14 
                 GATA-4 
                 SEQ ID NO: 33 
                 FIG. 13 
               
               
                 14 
                 SEQ ID NO: 15 
                 NY-ESO-1 
                 SEQ ID NO: 34 
                 FIG. 14 
               
               
                 15 
                 SEQ ID NO: 16 
                 FR-α 
                 SEQ ID NO: 35 
                 FIG. 15 
               
               
                 16 
                 SEQ ID NO: 17 
                 EPCAM 
                 SEQ ID NO: 36 
                 FIG. 16 
               
               
                 17 
                 SEQ ID NO: 18 
                 P53 
                 SEQ ID NO: 37 
                 FIG. 17 
               
               
                 18 
                 SEQ ID NO: 19 
                 Mesothelin 
                 SEQ ID NO: 38 
                 FIG. 18 
               
               
                 19 
                 SEQ ID NO: 20 
                 WT1 
                 SEQ ID NO: 39 
                 FIG. 19 
               
               
                 20 
                 SEQ ID NO: 5 
                 SEQ ID NO: 43 
                 SEQ ID NO: 47 
                 FIG. 23 
               
               
                 24 
                 SEQ ID NO: 6 
                 Aβ protein-42 
                 SEQ ID NO: 48 
                 FIG. 24 
               
               
                   
               
            
           
         
       
     
     The results are as follows: 
       FIG. 1  shows that compared with HER2 epitope group and HER2-PADRE group, the antibody titer of HER2 fusion antigen group (i.e. HER2-1pPhe PADRE) constructed in this example is significantly increased. The sequence of the fusion antigen was SEQ ID No: 21, i.e., 
     
       
         
           
               
            
               
                 TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLS 
               
               
                   
               
               
                 FLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDP 
               
               
                   
               
               
                 LNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDI 
               
               
                   
               
               
                 FHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAG 
               
               
                   
               
               
                 GCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALV 
               
               
                   
               
               
                 TYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEV 
               
               
                   
               
               
                 TAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFG 
               
               
                   
               
               
                 SLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDL 
               
               
                   
               
               
                 SVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNT 
               
               
                   
               
               
                 HLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWG 
               
               
                   
               
               
                 PGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNG 
               
               
                   
               
               
                 SVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGA 
               
               
                   
               
               
                 CQPCPINCTHSCVDLDDKGCPAEQRASPLTGPSLXKFVAAWTLKAAA. 
               
            
           
         
       
     
     As  FIG. 2  shows, compared with the PD-L1 group and the PD-L1-PADRE group, the PD-L1 fusion antigen group (i.e., PD/L1-2pPhe-PADRE) constructed in this example produced a significant increase in antibody titer. The sequence of the fusion antigen is SEQ ID NO: 22, i.e., 
     
       
         
           
               
            
               
                 MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALL 
               
               
                   
               
               
                 LLFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVA 
               
               
                   
               
               
                 NNQDKLGFEDGSVLKQFLSETEKMSPEDRAKCFEKNEATQAAHDAVAQEG 
               
               
                   
               
               
                 QCRVDDKVNEHFILENNVDGHLYELDGRMPFPVNHGASSEDTLLKDAAKV 
               
               
                   
               
               
                 CREFTEREQGEVRFSAVALCGPSLAXFVAAWTLKAAA. 
               
            
           
         
       
     
     As  FIG. 3  shows, compared with the PD-1 extracellular region group and the PD-1-PADRE group, the PD-1 fusion antigen group (i.e., PD/1-3pPhe-PADRE) constructed in this example generated a significant increase in antibody titer. The sequence of the fusion antigen is SEQ ID NO: 23, i.e., 
     
       
         
           
               
            
               
                 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRM 
               
               
                   
               
               
                 SPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT 
               
               
                   
               
               
                 YLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV 
               
               
                   
               
               
                 GPSLAKXVAAWTXKAAA. 
               
            
           
         
       
     
     As  FIG. 4  shows, compared with the EGFR group and the EGFR-PADRE group, the antibody titer generated by the EGFR fusion antigen group (i.e., EGFR-4pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 24, i.e., 
     
       
         
           
               
            
               
                 MERKERPFDVIGQLAALRRYARSLVRNSDDAEDLVHDALLRAYEKKQSFR 
               
               
                   
               
               
                 RGGNLRTWLLSIMHNAHIDRVRQARSLARRHDEAAVEAEQSLQAGQEHAV 
               
               
                   
               
               
                 RLKQVRDAFFHLSEEQREALHLVAIEDLSYQEAAMALDIPIGTLMSRISR 
               
               
                   
               
               
                 ARAQLREFEEKTPRAAHLRLIGGDGNEGNGPSLAKFXAAWTLKAAA. 
               
            
           
         
       
     
     As  FIG. 5  shows, compared with the CD20 group and the CD20-PADRE group, the CD20 fusion antigen group (i.e., CD20-5pPhe-PADRE) constructed in this example produced a significant increase in antibody titer. The sequence of the fusion antigen is SEQ ID NO: 25, i.e., 
     
       
         
           
               
            
               
                 MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESK 
               
               
                   
               
               
                 TLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSL 
               
               
                   
               
               
                 LAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKME 
               
               
                   
               
               
                 SLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIF 
               
               
                   
               
               
                 AFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLT 
               
               
                   
               
               
                 ETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSPGPS 
               
               
                   
               
               
                 LAKFVXAWTLKAAA. 
               
            
           
         
       
     
     As  FIG. 6  shows, compared with the CD66e group and the CD66e-PADRE group, the CD66e fusion antigen group constructed in this example (i.e., CD66e-6pPhe-PADRE) produced a significant increase in antibody titer. The sequence of the fusion antigen is SEQ ID NO: 26, i.e., 
     
       
         
           
               
            
               
                 KLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVI 
               
               
                   
               
               
                 GTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEA 
               
               
                   
               
               
                 TGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQS 
               
               
                   
               
               
                 LPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVL 
               
               
                   
               
               
                 YGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQEL 
               
               
                   
               
               
                 FIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNP 
               
               
                   
               
               
                 VEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVT 
               
               
                   
               
               
                 RNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLS 
               
               
                   
               
               
                 LSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSAS 
               
               
                   
               
               
                 GHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLW 
               
               
                   
               
               
                 WVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDP 
               
               
                   
               
               
                 VTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQ 
               
               
                   
               
               
                 QHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLS 
               
               
                   
               
               
                 AGPSLAKFVAXWTLKAAA. 
               
            
           
         
       
     
     As  FIG. 7  shows, compared with the CD227 extracellular region group and the CD227-PADRE group, the CD227 fusion antigen group constructed in this example (i.e., CD227-7pPhe-PADRE) produced a significant increase in antibody titer. The sequence of the fusion antigen is SEQ ID NO: 27, i.e., 
     
       
         
           
               
            
               
                 SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTT 
               
               
                   
               
               
                 QGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPD 
               
               
                   
               
               
                 NKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP 
               
               
                   
               
               
                 PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD 
               
               
                   
               
               
                 TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP 
               
               
                   
               
               
                 PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD 
               
               
                   
               
               
                 TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP 
               
               
                   
               
               
                 PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD 
               
               
                   
               
               
                 TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP 
               
               
                   
               
               
                 PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD 
               
               
                   
               
               
                 TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP 
               
               
                   
               
               
                 PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD 
               
               
                   
               
               
                 TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP 
               
               
                   
               
               
                 PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD 
               
               
                   
               
               
                 TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP 
               
               
                   
               
               
                 PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD 
               
               
                   
               
               
                 TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP 
               
               
                   
               
               
                 PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD 
               
               
                   
               
               
                 TRPAPGSTAPPAHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLV 
               
               
                   
               
               
                 HNGTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVP 
               
               
                   
               
               
                 PLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDI 
               
               
                   
               
               
                 SEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQ 
               
               
                   
               
               
                 YKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGGPSLAKFVAAXTLKA 
               
               
                   
               
               
                 AA. 
               
            
           
         
       
     
     As  FIG. 8  shows, compared with the VEGFR extracellular region group and the VEGFR-PADRE group, the antibody titers produced by the VEGFR fusion antigen group (i.e., VEGFR-8pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 28, i.e., 
     
       
         
           
               
            
               
                 ASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSG 
               
               
                   
               
               
                 SEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQ 
               
               
                   
               
               
                 DYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKR 
               
               
                   
               
               
                 FVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVV 
               
               
                   
               
               
                 GYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQH 
               
               
                   
               
               
                 KKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNS 
               
               
                   
               
               
                 TFVRVHEKPFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGI 
               
               
                   
               
               
                 PLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYV 
               
               
                   
               
               
                 PPQIGEKSLISPVDSYQYGTTQTLTCTVYAIPPPHHIHWYWQLEEECANE 
               
               
                   
               
               
                 PSQAVSVTNPYPCEEWRSVEDFQGGNKIEVNKNQFALIEGKNKTVSTLVI 
               
               
                   
               
               
                 QAANVSALYKCEAVNKVGRGERVISFHVTRGPEITLQPDMQPTEQESVSL 
               
               
                   
               
               
                 WCTADRSTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSN 
               
               
                   
               
               
                 STNDILIMELKNASLQDQGDYVCLAQDRKTKKRHCVVRQLTVLERVAPTI 
               
               
                   
               
               
                 TGNLENQTTSIGESIEVSCTASGNPPPQIMWFKDNETLVEDSGIVLKDGN 
               
               
                   
               
               
                 RNLTIRRVRKEDEGLYTCQACSVLGCAKVEAFFIIEGAQEKTNLEGPSLA 
               
               
                   
               
               
                 KEVAAWXLKAAA. 
               
            
           
         
       
     
     As  FIG. 9  shows, compared with the IL-2Ra group and the IL-2Ra-PADRE group, the antibody titers produced by the IL-2Ra fusion antigen group (i.e., IL/2Ra-9pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 29, i.e., 
     
       
         
           
               
            
               
                 ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNS 
               
               
                   
               
               
                 SHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASL 
               
               
                   
               
               
                 PGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMT 
               
               
                   
               
               
                 HGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQ 
               
               
                   
               
               
                 IQTEMAATMETSIFTTEYQGPSLAKFVAAWTXKAAA. 
               
            
           
         
       
     
     As  FIG. 10  shows, compared with the CTLA-4 group and the CTLA-4-PADRE group, the antibody titers produced by the CTLA-4 fusion antigen group (i.e., CTLA/4-10pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 30, i.e., 
     
       
         
           
               
            
               
                 KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVC 
               
               
                   
               
               
                 AATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMY 
               
               
                   
               
               
                 PPPYYLGIGNGTQIYVIDPEPCPDSDGPSLAKFVAAWTLXAAA. 
               
            
           
         
       
     
     As  FIG. 11  shows, compared with the PSMA group and the PSMA-PADRE group, the antibody titers produced by the PSMA fusion antigen group (i.e., PSMA-11pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 31, i.e., 
     
       
         
           
               
            
               
                 QGQAQQQAYDRGITIFSPDGRLYQVEYAREAVKRGTASIGVRTPEGVVLA 
               
               
                   
               
               
                 ADKRSRSPLMEPTSVEKIHKADDHIGIASAGHVADARQLIDFARRQSQVN 
               
               
                   
               
               
                 RLRYGEPIGIETLTKEVTDHIQQYTQVGGARPFGVALLIGGVENGTPRLY 
               
               
                   
               
               
                 ETDPSGTPYEWKAVSIGADRGDHQEHLEENFRDDLTLDEGIELALEAIAS 
               
               
                   
               
               
                 TSDEGTAPDGVDVATVSAETERFVELSNDEIESYLEANDLLATEDDEQTE 
               
               
                   
               
               
                 EGPSLAKFVAAWTLKXAA. 
               
            
           
         
       
     
     As  FIG. 12  shows, compared with the TOLL-1 group and the TOLL-1-PADRE group, the antibody titers produced by the TOLL-1 fusion antigen group (i.e., TOLL/1-12pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 32, i.e., 
     
       
         
           
               
            
               
                 SEFLVDRSKNGLIHVPKDLSQKTTILNISQNYISELWTSDILSLSKLRIL 
               
               
                   
               
               
                 IISHNRIQYLDISVFKFNQELEYLDLSHNKLVKISCHPTVNLKHLDLSFN 
               
               
                   
               
               
                 AFDALPICKEFGNMSQLKFLGLSTTHLEKSSVLPIAHLNISKVLLVLGET 
               
               
                   
               
               
                 YGEKEDPEGLQDFNTESLHIVFPTNKEFHFILDVSVKTVANLELSNIKCV 
               
               
                   
               
               
                 LEDNKCSYFLSILAKLQTNPKLSNLTLNNIETTWNSFIRILQLVWHTTVW 
               
               
                   
               
               
                 YFSISNVKLQGQLDFRDFDYSGTSLKALSIHQVVSDVFGFPQSYIYEIFS 
               
               
                   
               
               
                 NMNIKNFTVSGTRMVHMLCPSKISPFLHLDFSNNLLTDTVFENCGHLTEL 
               
               
                   
               
               
                 ETLILQMNQLKELSKIAEMTTQMKSLQQLDISQNSVSYDEKKGDCSWTKS 
               
               
                   
               
               
                 LLSLNMSSNILTDTIFRCLPPRIKVLDLHSNKIKSIPKQVVKLEALQELN 
               
               
                   
               
               
                 VAFNSLTDLPGCGSFSSLSVLIIDHNSVSHPSADFFQSCQKMRSIKAGDN 
               
               
                   
               
               
                 PFQCTCELGEFVKNIDQVSSEVLEGWPDSYKCDYPESYRGTLLKDFHMSE 
               
               
                   
               
               
                 LSCNITGPSLAKFVAAWTLKAXA. 
               
            
           
         
       
     
     As  FIG. 13  shows, compared with the GATA-4 group and the GATA-4-PADRE group, the antibody titers produced by the GATA-4 fusion antigen group (i.e., GATA/4-13pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 33, i.e., 
     
       
         
           
               
            
               
                 MYQSLAMAANHGPPPGAYEAGGPGAFMHGAGAASSPVYVPTPRVPSSVLG 
               
               
                   
               
               
                 LSYLQGGGAGSASGGASGGSSGGAASGAGPGTQQGSPGWSQAGADGAAYT 
               
               
                   
               
               
                 PPPVSPRFSFPGTTGSLAAAAAAAAAREAAAYSSGGGAAGAGLAGREQYG 
               
               
                   
               
               
                 RAGFAGSYSSPYPAYMADVGASWAAAAAASAGPFDSPVLHSLPGRANPAA 
               
               
                   
               
               
                 RHPNLDMFDDFSEGRECVNCGAMSTPLWRRDGTGHYLCNACGLYHKMNGI 
               
               
                   
               
               
                 NRPLIKPQRRLSASRRVGLSCANCQTTTTTLWRRNAEGEPVCNACGLYMK 
               
               
                   
               
               
                 LHGVPRPLAMRKEGIQTRKRKPKNLNKSKTPAAPSGSESLPPASGASSNS 
               
               
                   
               
               
                 SNATTSSSEEMRPIKTEPGLSSHYGHSSSVSQTFSVSAMSGHGPSIHPVL 
               
               
                   
               
               
                 SALKLSPQGYASPVSQSPQTSSKQDSWNSLVLADSHGDIITAGPSLAKFV 
               
               
                   
               
               
                 AAWTLKAAX. 
               
            
           
         
       
     
     As  FIG. 14  shows, compared with the NY-ESO-1 group and the NY-ESO-1-PADRE group, the antibody titers produced by the NY-ESO-1 fusion antigen group (i.e., NY/ESO/1-3, 5pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 34, i.e., 
     
       
         
           
               
            
               
                 MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGA 
               
               
                   
               
               
                 ARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPM 
               
               
                   
               
               
                 EAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSIS 
               
               
                   
               
               
                 SCLQQLSLLMWITQCFLPVFLAQPPSGQRRGPSLAKXVXAWTLKAAA. 
               
            
           
         
       
     
     As  FIG. 15  shows, compared with the FR-α group and the FR-α-PADRE group, the antibody titers produced by the FR-α fusion antigen group (i.e., FRα-3, 8pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 35, i.e., 
     
       
         
           
               
            
               
                 RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEA 
               
               
                   
               
               
                 HKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSW 
               
               
                   
               
               
                 RKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGENKCAVGAA 
               
               
                   
               
               
                 CQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEE 
               
               
                   
               
               
                 VARFYAAAMSGPSLAKXVAAWXLKAAA. 
               
            
           
         
       
     
     As  FIG. 16  shows, compared with the EPCAM group and the EPCAM-PADRE group, the antibody titers produced by the EPCAM fusion antigen group (i.e., EPCAM-3, 11pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 36, i.e., 
     
       
         
           
               
            
               
                 QEECVCENYKLAVNCFVNNNRQCQCTSVGAQNTVICSKLAAKCLVMKAEM 
               
               
                   
               
               
                 NGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTAG 
               
               
                   
               
               
                 VRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDSKSLRTALQKEITTR 
               
               
                   
               
               
                 YQLDPKFITSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGE 
               
               
                   
               
               
                 SLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKGPSLAKXV 
               
               
                   
               
               
                 AAWTLKXAA. 
               
            
           
         
       
     
     As  FIG. 17  shows, compared with the P53 group and the P53-PADRE group, the antibody titers produced by the P53 fusion antigen group (i.e., P53-5, 8pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 37, i.e., 
     
       
         
           
               
            
               
                 MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDI 
               
               
                   
               
               
                 EQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQ 
               
               
                   
               
               
                 KTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDST 
               
               
                   
               
               
                 PPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGN 
               
               
                   
               
               
                 LRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRP 
               
               
                   
               
               
                 ILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELP 
               
               
                   
               
               
                 PGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALEL 
               
               
                   
               
               
                 KDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMEKTEGPDSDGPSLAKE 
               
               
                   
               
               
                 VXAWXLKAAA. 
               
            
           
         
       
     
     As  FIG. 18  shows, compared with the MESOTHELIN group and the MESOTHELIN-PADRE group, the antibody titers produced by the MESOTHELIN fusion antigen group (i.e., Mesothelin-5, 11pPhe-PADRE) constructed in this example increased significantly. The sequence of the fusion antigen is SEQ ID NO: 38, i.e., 
     
       
         
           
               
            
               
                 LAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELA 
               
               
                   
               
               
                 VALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQA 
               
               
                   
               
               
                 CTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALG 
               
               
                   
               
               
                 GLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGP 
               
               
                   
               
               
                 PSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPER 
               
               
                   
               
               
                 TILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQM 
               
               
                   
               
               
                 DRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRK 
               
               
                   
               
               
                 WNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKD 
               
               
                   
               
               
                 TLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYP 
               
               
                   
               
               
                 KARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRT 
               
               
                   
               
               
                 DAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGL 
               
               
                   
               
               
                 QGGIPNGYLVLDLSMQEALSGPSLAKFVXAWTLKXAA. 
               
            
           
         
       
     
       FIG. 19  shows that compared with the WT1 group and the WT1-PADRE group, the WT1 fusion antigen group constructed in this example (i.e., WT1-8, 11pPhe-PADRE) produced a significant increase in antibody titers. The sequence of the fusion antigen is SEQ ID NO: 39, i.e., 
     
       
         
           
               
            
               
                 MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGS 
               
               
                   
               
               
                 LGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQF 
               
               
                   
               
               
                 TGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYS 
               
               
                   
               
               
                 TVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVY 
               
               
                   
               
               
                 GCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGV 
               
               
                   
               
               
                 AAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDV 
               
               
                   
               
               
                 RRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGE 
               
               
                   
               
               
                 KPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKT 
               
               
                   
               
               
                 HTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHENMHQRNMTKLQLALG 
               
               
                   
               
               
                 PSLAKFVAAWXLKXAA. 
               
            
           
         
       
     
       FIG. 23  shows that compared with the B epitope SEQ ID NO: 43 group and the B epitope SEQ ID NO: 43-PADRE group, a significant increase in antibody titers in the epitope fusion antigen group constructed in this embodiment (i.e., B epitope +3 pPhe-PADRE). 
     The B epitope SEQ ID NO: 43 is FLPESFDGDPASNTAPLQPE. The sequence of the fusion antigen is SEQ ID NO: 47, which is FLPESFDGDPASNTAPLQPEGPSLAKFXAAWTLKAAA. 
       FIG. 24  shows that compared with the Aβ protein-42 group and the Aβ protein-PADRE group, the Aβ protein-42 fusion antigen group constructed in this experiment i.e., A-beta protein-6pPhe-PADRE) produced a significant increase in antibody titers. The sequence of the fusion antigen is SEQ ID NO: 48, i.e., LVFFAEDVGSNKGAIIGLMVGGVVIAGPSLAKFVAXWTLKAAA. 
     The CTL-mediated cytotoxicity detection tests are shown in the following table: 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                   
                 CTL-mediated 
               
               
                   
                   
                   
                   
                 cytotoxicity 
               
               
                 Test 
                 Helper epitope 
                 Existing antigen 
                 Fusion antigen 
                 detection 
               
               
                 number 
                 peptide 
                 or epitope 
                 sequence 
                 result diagram 
               
               
                   
               
             
            
               
                 21 
                 SEQ ID NO: 2 
                 SEQ ID NO: 40 
                 SEQ ID NO: 44 
                 FIG. 20 
               
               
                 22 
                 SEQ ID NO: 3 
                 SEQ ID NO: 41 
                 SEQ ID NO: 45 
                 FIG. 21 
               
               
                 23 
                 SEQ ID NO: 4 
                 SEQ ID NO: 42 
                 SEQ ID NO: 46 
                 FIG. 22 
               
               
                   
               
            
           
         
       
     
     The results shown in each figure are as follows: 
       FIG. 20  shows that the CTL-mediated cytotoxicity induced by the epitope fusion antigen group (i.e., the epitope 1+ 1p-PADRE constructed in this embodiment is significantly enhanced when compared with the epitope SEQ ID NO:40 group and the epitope SEQ ID NO: 40-PADre group. 
     The epitope SEQ ID NO: 40 is VLDNGDPL. The sequence of the fusion antigen is SEQ ID NO: 44, i.e., VLDNGDPLGPSLXKFVAAWTLKAAA. 
       FIG. 21  shows that the CTL-mediated cytotoxicity induced by the epitope fusion antigen group constructed in this embodiment (i.e., the epitope 2+ 2p-PADRE) is significantly enhanced compared with the epitope SEQ ID NO:41 group and the epitope SEQ ID NO: 41-PADre group. 
     The epitope SEQ ID NO: 41 is TGYLYISA. The sequence of the fusion antigen is SEQ ID NO: 45, i.e., TGYLYISAGPSLAXFVAAWTLKAAA. 
       FIG. 22  shows that the CTL-mediated cytotoxicity induced by the epitope fusion antigen group constructed in this embodiment (i.e., the epitope 3+ 3p-PADRE) is significantly enhanced when compared with the epitope SEQ ID NO:42 group and the epitope SEQ ID NO: 42-PADre group. 
     The epitope SEQ ID NO: 42 is VLDNGDPLGPSLTGYLYISA. The sequence of the fusion antigen is SEQ ID NO: 46, i.e., VLDNGDPLGPSLTGYLYISAGPSLAKXVAAWTLKAAA. 
     In addition, this example actually verifies the ability of the fusion antigen obtained by linking the remaining helper epitope peptides with existing antigens or epitopes in Example 1 of this example to induce antibodies or activate the CTL. Due to space limitations, specific test results are not listed here. The results show that all the helper epitope peptides of Example 1 have excellent ability to assist existing antigens or epitopes to produce antibodies or CTL-mediated cytotoxicity. 
     In addition to the above-mentioned embodiments, the present invention can also have other embodiments. All technical solutions formed by equivalent replacements or equivalent transformations fall within the protection scope of the present invention.