Patent Application
20210162041
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.
The invention relates to a helper epitope peptide and its application, which belongs to the technical field of biomedicine.
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'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 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.
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.
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:
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:
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'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 H2SO4 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% CO2 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:
The results are as follows:
As
As
As
As
As
As
As
As
As
As
As
As
As
As
As
As
As
The B epitope SEQ ID NO: 43 is FLPESFDGDPASNTAPLQPE. The sequence of the fusion antigen is SEQ ID NO: 47, which is FLPESFDGDPASNTAPLQPEGPSLAKFXAAWTLKAAA.
The CTL-mediated cytotoxicity detection tests are shown in the following table:
The results shown in each figure are as follows:
The epitope SEQ ID NO: 40 is VLDNGDPL. The sequence of the fusion antigen is SEQ ID NO: 44, i.e., VLDNGDPLGPSLXKFVAAWTLKAAA.
The epitope SEQ ID NO: 41 is TGYLYISA. The sequence of the fusion antigen is SEQ ID NO: 45, i.e., TGYLYISAGPSLAXFVAAWTLKAAA.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201810408586.3 | May 2018 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/083484 | 4/19/2019 | WO | 00 |
