Spacer peptide beneficial to antigen presentation

Abstract

The invention relates to a spacer peptide which plays a role in spacing different antigens in a vaccine, and can be efficiently sheared by a specific enzyme and release the antigens in vivo, so that the antigens are effectively presented by antigen presenting cells. The invention also relates to application of the synthetic nucleic acid molecule, the spacer peptide or the nucleic acid molecule, and a nucleic acid cancer vaccine.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a spacer peptide and its use. Background Art

[0002] A spacer peptide is a short peptide that plays a role in separating different antigens in a vaccine. A multi-epitope vaccine, also known as a cocktail vaccine, is a vaccine that simultaneously carries multiple antigen-related and auxiliary epitopes. Compared with traditional vaccines, multi-epitope vaccines have many advantages: they can be recognized and bound by MHC molecules with various genetic backgrounds, thus achieving efficient presentation; they have unique advantages in cellular immunity and can effectively cope with the variation of pathogenic microorganisms and many adverse factors in the immune response.

[0003] Generally speaking, multi-epitope vaccines are prepared by using recombinant DNA technology to concatenate multiple DNA sequence fragments encoding antigenic epitopes and then recombinantly inserting them into a vector, including: recombinant protein vaccines, nucleic acid vaccines, vector vaccines, etc.; the simplest method for designing a multi-epitope vaccine is to connect different epitopes of the multi-epitope vaccine through a spacer sequence, and this design can enable different epitopes to function relatively independently. Among them, therapeutic tumor vaccines are one of the important members of multi-epitope vaccines, and most of the currently studied therapeutic tumor vaccines adopt the design idea of using spacer peptides to separate different antigens.

[0004] Since personalized tumor mRNA vaccines usually require the efficient expression of more than a dozen or even dozens of neoantigen fragments in sequence design, it is crucial to select a suitable spacer peptide for the design of personalized tumor mRNA vaccines, that is: 1. It can avoid generating a binding junction peptide that binds to MHC molecules; 2. It can avoid proteasome processing to generate a binding junction peptide; 3. It can be effectively translated and processed by the proteasome.

[0005] Currently, the more commonly used spacer peptides include: (GGGS) n 、(GSA) n, AAY, GPGPG, EAAAK, RDKR, REKR, etc. For example, researchers used the furin recognition sequences (REKR, RDKR) to prepare tumor vaccines for connecting antigen peptides of different epitopes and achieved certain effects. However, in the research and development of personalized tumor mRNA vaccines, these spacer peptides have not achieved satisfactory results. Tumor vaccines, especially neoantigen tumor vaccines, have more diverse requirements for spacer peptides. For example, flexible or moderately rigid spacer peptides are needed to ensure the independent exposure of different epitopes and avoid steric hindrance resulting in some antigens not being effectively cleaved in vivo; selecting spacer peptides with appropriate length and biological activity can promote the effective cleavage of antigen fragments by proteasomes and optimize antigen presentation; in addition, if the spacer peptide is not properly selected or contains potential immunogenic epitopes, it may compete with the target neoantigen and affect T cell activation. Finally, it is also necessary to consider optimizing the translation efficiency of the spacer peptide to avoid the generation of additional RNA secondary structures that affect translation. Therefore, it is necessary to specifically design suitable spacer peptides according to the characteristics of mRNA vaccines transfected into cells, and researchers still need to develop spacer peptides that can achieve better antigen presentation effects. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to achieve better antigen presentation effects.

[0007] Accordingly, the technical solution adopted by the present invention to solve its technical problems is to provide a spacer peptide that can effectively improve the immune effect of neoantigen vaccines in vivo. For example, the antigen peptide translated in dendritic cells (DCs) corresponding to the sequence is more likely to be cleaved preferentially at the spacer peptide, which is beneficial for better processing and presentation of each epitope peptide.

[0008] In a first aspect, the present application relates to a spacer peptide, the amino acid sequence of which is shown in SEQ ID NO.1: PLKW.

[0009] In addition, the present application relates to a synthetic nucleic acid molecule that encodes the spacer peptide of the present application.

[0010] In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2: 5’-CCGCTGAAGTGG-3’.

[0011] In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.3: 5’-CCCTTAAAGTGG-3’.

[0012] In a second aspect, the use of the spacer peptide or nucleic acid molecule of the present application in the preparation of vaccines is provided.

[0013] In some embodiments, the vaccines include: Microbial vector-based vaccines, Viral vector vaccines, Cell-based vaccines, Nucleic acid vaccines, Peptide-based vaccines.

[0014] In some embodiments, the nucleic acid vaccines include nucleic acid infectious disease vaccines, nucleic acid autoimmune disease vaccines, and nucleic acid cancer vaccines.

[0015] In some embodiments, the nucleic acid infectious disease vaccines include: pneumonia vaccines, rotavirus vaccines, varicella vaccines, hand, foot and mouth disease vaccines, influenza vaccines, COVID-19 vaccines, rabies virus vaccines, respiratory syncytial virus vaccines, varicella-zoster virus vaccines.

[0016] In some embodiments, the cancers or tumors targeted by the nucleic acid cancer vaccines include, but are not limited to, sarcomas, malignancies, metastatic cancers, or any disease or disorder characterized by uncontrolled cell growth such that it would be considered cancerous. The cancer can be primary or metastatic cancer.

[0017] In some embodiments, the cancers can include, but are not limited to, biliary tract cancer, bladder cancer, brain cancer including glioblastoma and medulloblastoma; breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological malignancies (including acute lymphocytic and myeloid leukemia), multiple myeloma, liver cancer, lung cancer, lymphoma, neuroblastoma, oral cancer, including squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, choriocarcinoma, stromal tumors and germ cell tumors, thyroid cancer, and kidney cancer.

[0018] In some embodiments, the autoimmune diseases targeted by the nucleic acid autoimmune disease vaccines include: systemic lupus erythematosus (SLE), rheumatoid arthritis, psoriatic arthritis, myasthenia gravis, psoriasis, vitiligo, multiple sclerosis, neuromyelitis optica, Crohn's disease, lupus nephritis.

[0019] In some specific embodiments, the nucleic acid cancer vaccine comprises: one or more nucleic acids, each of the one or more nucleic acids having one or more open reading frames encoding 5 - 130, 20 - 40, 30 - 35, or 34 peptide epitopes. The peptide epitopes can be selected from portions of personalized cancer antigens and / or tumor - associated antigens (TAAs), tumor - specific antigens, and wherein each of the peptide epitopes has the same or different lengths.

[0020] In some specific embodiments, the minimum length of any peptide epitope is 8 amino acids; in some embodiments, the maximum length of any peptide epitope is 31 amino acids. In some embodiments, the minimum length of any or all peptide epitopes is 13 amino acids. In some embodiments, the maximum length of any or all peptide epitopes is 35 amino acids. In some embodiments, the length of any or all peptide epitopes is 25 amino acids.

[0021] In some specific embodiments, each of the one or more nucleic acids encodes 3 - 130 peptide epitopes, such as 3 - 10 peptide epitopes, 5 - 10 peptide epitopes, 10 - 20 peptide epitopes, 20 - 30 peptide epitopes, 30 - 40 peptide epitopes, 40 - 50 peptide epitopes, 50 - 60 peptide epitopes, 60 - 70 peptide epitopes, 70 - 80 peptide epitopes, 80 - 90 peptide epitopes, 90 - 100 peptide epitopes, 100 - 110 peptide epitopes, 110 - 120 peptide epitopes, or 120 - 130 peptide epitopes.

[0022] In some more specific embodiments, each of the one or more nucleic acids encodes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 peptide epitopes.

[0023] In some specific embodiments, each of the peptide epitopes is encoded by a separate open reading frame. In some embodiments, the peptide epitopes are in the form of a multi - linked cancer antigen composed of 3 - 130 peptide epitopes. In some embodiments, the cancer vaccine composition comprises an mRNA having an open reading frame encoding 15 peptide epitopes.

[0024] In some embodiments, antigen sequences (e.g., peptide epitopes) are preferably separated by spacer peptides.

[0025] In some embodiments, all antigen sequences except the terminal antigen sequences are arranged in antigen subunits, each subunit consisting of an antigen sequence and a spacer peptide. Since the antigen sequences are separated by spacer peptides, each antigen is presented to the immune system in an optimal manner, enabling optimal immune effects.

[0026] In some specific embodiments, the nucleic acid vaccine includes: RNA vaccines and DNA vaccines.

[0027] In some other specific embodiments, the RNA vaccine is preferably an mRNA vaccine, more preferably a multi-epitope mRNA vaccine, more preferably a neoantigen mRNA tumor (cancer) vaccine, and most preferably a DC-based neoantigen mRNA tumor (cancer) vaccine.

[0028] In some embodiments, the mRNA vaccine further includes a 5'-UTR. The 5'-UTR refers to the region immediately upstream of the start codon of the mRNA that does not encode a protein or peptide.

[0029] In some embodiments, the mRNA vaccine further includes a 5' cap. Exemplary 5' caps include Cap0, Cap1, or Cap2.

[0030] In some embodiments, the mRNA vaccine further includes a 3'-UTR. The 3'-UTR refers to the region located at the 3' end of the mRNA, downstream of the stop codon in the protein-coding region, and is transcribed but not translated into a protein or peptide.

[0031] In some embodiments, the mRNA vaccine further includes a polyA tail. The polyA tail refers to a sequence of adenosine residues that is usually located at the 3' end of an RNA molecule. Exemplary polyA tails include those having at least 20 and at most 500, such as 30, 40, 50, 60, 80, 100, 120, 150, 200, 300, 400 A nucleotides.

[0032] In some other specific embodiments, the total length of the neoantigen mRNA anti-tumor vaccine encodes a total protein length of 50 - 100 amino acids, 100 - 200 amino acids, 200 - 300 amino acids, 300 - 400 amino acids, 400 - 500 amino acids, 500 - 600 amino acids, 600 - 700 amino acids, 700 - 800 amino acids, 800 - 900 amino acids, 900 - 1000 amino acids, 1000 - 1100 amino acids, or 1100 - 1200 amino acids.

[0033] In a specific embodiment, the vaccine is a multi-epitope vaccine.

[0034] In a specific embodiment, the vaccine is a neoantigen tumor vaccine.

[0035] In a third aspect, the present application relates to the use of a plurality of spacer peptides or nucleic acid molecules encoding them in combination for the preparation of a vaccine, wherein the amino acid sequence of at least one of the spacer peptides is as shown in SEQ ID NO.1.

[0036] In some embodiments, the spacer peptide may further be selected from one or more of the group consisting of: GPGPG, EAAAK, AAY, GGGS, KK, GGSGGGGSG, GGS, GGFG, GS, and RDKR.

[0037] In a fourth aspect, the present application relates to a nucleic acid cancer vaccine, wherein the vaccine encodes 3 - 40 peptide epitopes and a spacer peptide between adjacent peptide epitopes, and the amino acid sequence of at least one spacer peptide is as shown in SEQ ID NO.1.

[0038] In some embodiments, the spacer peptide may further be optionally selected from one or more of the group consisting of: GPGPG, EAAAK, AAY, GGGS, KK, GGSGGGGSG, GGS, GGFG, GS, and RDKR.

[0039] In some embodiments, the nucleic acid cancer vaccine includes an mRNA vaccine encoding a plurality of peptide epitope antigens, and the plurality of peptide epitope antigens are arranged such that a spacer peptide is provided between adjacent peptide epitopes, or are directly linked to each other in the case where there is no spacer peptide between some adjacent peptide epitopes.

[0040] In some embodiments, the epitope is a predictive epitope.

[0041] In some embodiments, the epitope is a non - predictive epitope, which includes identified or experimentally confirmed T - cell epitopes or B - cell epitopes.

[0042] In some embodiments, the antigen includes, but is not limited to: infectious disease virus antigens, tumor antigens, and autoimmune disease antigens.

[0043] In some embodiments, the tumor antigen includes: tumor - associated antigen (TAA) and tumor - specific antigen (TSA). In some embodiments, the tumor - specific antigen (TSA) includes tumor neoantigen, also known as tumor neoantigen.

[0044] In some embodiments, tumor neoantigens include: shared neoantigens and personalized neoantigens. Shared neoantigens can be expressed among different individuals or tumor types. Personalized neoantigens are specifically expressed in a particular patient's body.

[0045] In some embodiments, the shared neoantigens include: KRAS G12 mutation, KRAS G13 mutation, NY-ESO-1 mutation, MART1 mutation.

[0046] The beneficial effect of the present invention is that the spacer peptides of the present application are effective in presenting antigen peptides with different target sequences and are significantly superior to the control. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : Results of the MART1-Jurkat cell antigen presentation experiment.

[0048] Figure 2 : Results of the G12V-Jurkat cell antigen presentation experiment. DETAILED DESCRIPTION OF THE INVENTION

[0049] In some embodiments, the present application provides a nucleic acid cancer vaccine comprising one or more nucleic acids, wherein each nucleic acid encodes at least one suitable cancer antigen, such as a personalized antigen specific to a cancer subject. For example, the nucleic acid cancer vaccine may comprise nucleic acids encoding one or more cancer antigens (referred to as neoepitopes) specific to each subject. Antigens expressed in or by tumor cells are referred to as "tumor-associated antigens". Specific tumor-associated antigens may or may not also be expressed in non-cancerous cells. Many tumor mutations are well known in the art. Tumor-associated antigens that are not expressed or are rarely expressed in non-cancerous cells, or whose expression in non-cancerous cells is sufficiently reduced compared to their expression in cancerous cells and that induce an immune response after vaccination, are referred to as neoepitopes. Neoepitopes generally do not generate an immune response against healthy tissues and are not masked by protective components of the immune system.

[0050] In some embodiments, personalized vaccines based on neoepitopes are desirable because such vaccine formulations will maximize specificity against a patient's specific tumor. Mutagenic neoepitopes can be generated by: point mutations, non-synonymous mutations that result in different amino acids in a protein; read-through mutations, in which a stop codon is modified or deleted, resulting in the translation of a longer protein with a novel tumor-specific sequence at the C-terminus; splice-site mutations, which result in the inclusion of introns in the mature mRNA and thus a unique tumor-specific protein sequence; chromosomal rearrangements, which produce chimeric proteins (i.e., gene fusions) with tumor-specific sequences at the junction of two proteins; frameshift mutations or deletions, which result in new open reading frames and / or translocations with novel tumor-specific protein sequences.

[0051] Each peptide epitope can be any length reasonable for an epitope. In some embodiments, the lengths of each peptide epitope are not necessarily equal. In some embodiments, each peptide epitope in a nucleic acid cancer vaccine has a different length. In certain embodiments, at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, and up to and including all) of the peptide epitopes in a nucleic acid cancer vaccine have different lengths.

[0052] In some embodiments, the length of at least one of the peptide epitopes is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 amino acids. In other embodiments, the length of at least one of the peptide epitopes is 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer amino acids. In other embodiments, the length of at least one of the peptide epitopes is up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, or up to 10 amino acids.

[0053] In some more specific embodiments, the nucleic acid or mRNA can be particularly used to transfect antigen-presenting cells and as a tool for presenting an antigen, where the antigen to be presented corresponds to the peptide or protein expressed by the mRNA; the antigen-presenting cells can be used to stimulate T cells, particularly CD4+ and / or CD8+ T cells, in vivo or in vitro.

[0054] In certain embodiments, the antigen or its functional fragment includes, but is not limited to, tumor neoantigen, tumor-associated antigen, tumor-specific antigens, and universal tumor mutation site antigen.

[0055] In certain specific embodiments, "peptide or protein" includes peptides and proteins that act as antigens, i.e., the peptide or protein elicits an immune response in a subject, and the immune response can be prophylactic or therapeutic or partially or fully protective.

[0056] In certain specific embodiments, RNA encoding an antigen, such as a tumor-associated antigen, particularly mRNA, is administered to a mammal, especially if it is desired to treat a mammal suffering from a disease involving the antigen. The RNA, particularly mRNA, is taken up by antigen-presenting cells of the mammal (monocytes, macrophages, dendritic cells, thymic cortical epithelial cells or other cells). The antigenic translation product of the RNA, particularly mRNA, is formed and the product is displayed on the cell surface for recognition by T cells.

[0057] In certain specific embodiments, the antigen is displayed on the cell surface for recognition by CAR-modified T cells directed against the antigen. In one embodiment, the antigen or a product optionally produced by its processing is displayed on the cell surface in the context of MHC molecules and is thus recognized by T cells via the T cell receptor.

[0058] In certain specific embodiments, RNA expressing an antigen, particularly mRNA, is introduced into ex vivo antigen-presenting cells (such as antigen-presenting cells obtained from a patient), and the antigen-presenting cells, optionally ex vivo clonally expanded antigen-presenting cells, are transplanted back into the same patient. Any method known in the art can be used, preferably the transfected cells are reintroduced into the patient in a sterile form by intravenous, intracavitary, intraperitoneal or intratumoral administration.

[0059] In certain specific embodiments, the methods of the invention may involve antigen-presenting cells for expressing RNA encoding an antigen, particularly mRNA. To this end, the methods of the invention may involve introducing RNA encoding an antigen, particularly mRNA, into antigen-presenting cells such as dendritic cells (DC cells). For transfection of antigen-presenting cells (such as dendritic cells), a pharmaceutical composition containing RNA encoding an antigen, particularly mRNA, can be used. A delivery vehicle that targets the RNA, particularly mRNA, to dendritic cells or other antigen-presenting cells can be administered to the patient to effect transfection in vivo.

[0060] Specific embodiments are used in this application to illustrate the principles and implementation manners of the present invention. The descriptions of the above embodiments are only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0061] Example 1: Preparation of DC cells containing mRNA

[0062] 1.1 In vitro synthesis of mRNA

[0063] The in vitro synthesis of mRNA can be obtained by conventional techniques of the prior art, and the general steps are as follows:

[0064] (1) The antigen amino acid sequences are concatenated with spacer peptide sequences, codon-optimized into nucleotide sequences, and constructed into an mRNA transcription template plasmid. The mRNA adopts a consistent cap, 5'UTR sequence, 3'UTR sequence, and polyA tail. The template plasmid is transformed into competent Escherichia coli cells. After overnight culture at 37°C, monoclonal colonies are picked. After shaking the bacteria at 37°C and 220 rpm for 16 h, the plasmid is extracted using an endotoxin-free large plasmid extraction kit.

[0065] (2) The plasmid is added to the SpeI-HF restriction endonuclease reaction system and incubated at 37°C for enzyme digestion for 30 - 60 min. The DNA product purification kit is used to purify the linearized plasmid, obtaining a linearized plasmid precipitate. Sterilized injection water is added for dissolution, and the solution is centrifuged for collection. The concentration, purity, and proportion of the target band of the linearized plasmid are detected.

[0066] (3) Components such as the linearized plasmid, ATP, UTP, CTP, GTP, T7 RNA polymerase, and cap analog are added to the reaction buffer system in a certain proportion, mixed well, and incubated at 37°C for 2 h. After the reaction, DNase I is incubated at 37°C for 15 min to obtain the crude mRNA product. Lithium chloride with a final concentration of 2.5 M is added, and after standing, it is centrifuged at 4°C for 15 min using a tabletop high-speed refrigerated centrifuge to obtain the mRNA precipitate. The precipitate is washed with 70% ethanol solution and then dissolved with sterilized injection water to obtain mRNA (A146 and controls A144, A148).

[0067] Table 1: mRNA-related sequences involved in the examples

[0068]

[0069]

[0070] 1.2 DC cell culture and maturation promotion

[0071] (1) Resuscitation of iDC cells (Immature dendritic cells): Take out the iDC cells from the liquid nitrogen tank and place them in a 37°C water bath until the cryopreservation solution completely melts. Aspirate a certain volume of AIM-V medium (Thermo Fisher) into a centrifuge tube, open the lid of the cryopreservation tube, aspirate the cryopreserved suspension of iDC cells in the cryopreservation tube and add it to the medium. After mixing well, transfer the cell suspension to a centrifuge tube, cover the centrifuge tube, invert it several times to mix evenly, and centrifuge at 600 rcf at room temperature for 10 min.

[0072] (2)Resuspension and counting: Discard the supernatant, aspirate the DC cell medium to resuspend the cell pellet, blow and mix well, and then count. According to the test results, supplement the corresponding medium to adjust the cell density to 1.00E+06 cells / mL.

[0073] (3)Sub-packaging preparation for electroporation: After mixing the cell suspension, sub-package it into culture dishes or culture flasks, mark the corresponding numbers, and then place it in an incubator at 37.0°C with 5.0% CO2 for culture. After 24 hours of culture, collect the mDC cells (mature dendritic cell), add fresh medium, count, and use them for subsequent electroporation.

[0074] 1.3 Electroporation process and cell cryopreservation

[0075] (1)The mRNAs used for electroporation are the above-mentioned A146 and the controls A144 and A148. Except for the different sequences of the coding spacer peptide (linker) element sequences, the remaining sequences of all mRNAs are the same. The electroporation dose of each group of mRNAs is 100 μg, the total volume of the electroporation system is 300 μL. The prepared electroporation system is transferred to an electroporation cuvette and electroporated using a cell electroporator.

[0076] (2)After electroporation, dilute the cell suspension in the electroporation cuvette with medium to adjust the cell density to 1.00E+06 cells / mL. The diluted cell suspension is sub-packaged into cell plates and placed in an incubator at 37.0°C with 5.0% CO2 for culture.

[0077] (3)Collect the cells of each group after 3 hours of culture after the above electroporation, centrifuge at 600 rcf at room temperature for 5 minutes. Discard the supernatant, resuspend the cells with CS10 cell cryopreservation solution, and sub-package the cell suspension into cryotubes and store it in a liquid nitrogen tank for standby.

[0078] Example 2: Detection of the antigen presentation ability of DC cells stimulating Jurkat cells

[0079] Experimental principle: Jurkat reporter cells are a stable Jurkat cell line expressing a reporter gene. The reporter gene vector includes a specific T cell antigen receptor, a response element, and a GFP reporter gene. When antigen-presenting cells are co-incubated with Jurkat reporter cells, when the antigen-presenting surface presents a specific antigen, the specific TCR on the surface of Jurkat cells binds to the corresponding antigen, the transcription factor is activated and transported into the nucleus, promoting the expression of the downstream GFP reporter gene. The positive rate of GFP and the expression level of GFP in Jurkat cells are positively correlated with the amount of antigen presented on the surface of dendritic cells, so that the amount of antigen presented on the surface of dendritic cells can be accurately quantified.

[0080] (1) DC cell resuscitation: Take out the electrotransfected and cryopreserved cells from the liquid nitrogen tank and place them in a 37°C water bath until the cryopreservation solution is completely melted. Pipette a certain volume of AIM-V medium into a centrifuge tube, open the lid of the cryopreservation tube, aspirate and add the cryopreserved suspension of iDC cells in the cryopreservation tube into the medium, mix well and count;

[0081] (2) According to the counting results, take out the cell suspension required for the experiment and transfer it to a centrifuge tube, centrifuge at 600 rcf at room temperature for 5 min; discard the supernatant, and add the corresponding volume of 1640 complete medium to resuspend the cell pellet according to the number of experimental groups in each group. Gently pipette and mix well for later use. Supplement the remaining DC with the corresponding medium and adjust the cell density to 1*10 6 cells / mL, and place it in a 37.0°C, 5.0% CO2 incubator for continuous culture.

[0082] (3) Preparation of Jurkat cells: Prepare Jurkat cells containing MART1 (ELAGIGILTV) or KRAS-G12V (VVVGAVGVGK). According to the experimental needs, take out a certain amount of Jurkat cell suspensions of different epitope types respectively, centrifuge at 1000 rpm at room temperature for 5 min. Discard the supernatant, add 1640 complete medium to resuspend the cell pellet, and mix well and count.

[0083] (4) Co-culture seeding: After mixing the prepared DC cells and Jurkat cells respectively, take 5*10 4 cell suspensions of each and add them to a 96-well plate and mix well; in the positive control group, in addition to adding 5*10 4 of DC cells and Jurkat cells respectively to each well, 4 μg of the antigen peptide corresponding to the epitope needs to be added to each well; in the negative control group, only 1*10 5 of Jurkat cells are added to each well, and then the cells are placed in a 37.0°C, 5.0% CO2 incubator for culture.

[0084] The specific results are as follows:

[0085] 1. The spacer peptide (RDKR) used in control group A144 is a sequence preferentially recognized and cleaved by furin. Furin is an endoprotease widely present in eukaryotes, and its cleavage of substrates has high specificity. Using RDKR as a Linker can enable precise cleavage and function of vaccine-related proteins. However, the experimental results show (see Figure 1, Table 2): Compared with the control group A144, the A146 spacer peptide (PLKW) obtained in this application has stronger DC cell antigen presentation ability and a more persistent antigen presentation time. Specifically, the positive rate of GFP expression in MART1-Jurkat cells activated by DC cells electroporated with A146 at 0 h after resuscitation was 89.15%, and the positive rate of the control A144 was 66.88%. The former was 30% more than the latter and maintained an advantage within 0 - 45 h after resuscitation. The GFP expression intensity of A146 was more than 1.6 times and nearly twice that of A144 within 0 - 45 h after resuscitation.

[0086] 2. The spacer peptide (GGFG) used in the control group A148 is a Linker widely used in antibody-drug conjugates (ADCs) and is used to connect cytotoxic drugs and antibodies, which can balance stability and cleavability. The experimental results show (see Figure 2 , Table 3): The A146 spacer peptide (PLKW) obtained in this application still has stronger cleavage ability compared with the spacer peptide (GGFG). The positive rate of GFP expression in KRAS-G12V-Jurkat cells activated by A146 at 0 h after resuscitation was 91%, and the positive rate of the control A148 was 57.71%. The former was 57% more than the latter and expanded its advantage thereafter. At 46 h after resuscitation, A146 was 540% more than A148. The GFP expression intensity of A146 was 2.4 times that of the control A148 after 0 h of resuscitation. When the antigen presentation ability of the control A148 decreased significantly at 46 h after resuscitation, A146 still maintained a high antigen presentation ability, and its GFP expression intensity was nearly 10 times that of A148.

[0087] In summary, the A146 spacer peptide obtained in this application has achieved unexpected technical effects, and its effects are independent of the antigen peptides presented. The relevant spacer peptides are effective and significantly superior to the control when presenting antigen peptides with different targets and different sequences.

[0088] Table 2: Detection results of DC antigen presentation ability (MART1)

[0089]

[0090] Table 3: Detection results of DC antigen presentation ability (G12V)

[0091] 。

Claims

1. An intervening peptide, the amino acid sequence of which is as shown in SEQ ID NO.

1.

2. A synthetic nucleic acid molecule encoding the intervening peptide according to claim 1, the sequence of the synthetic nucleic acid molecule being as shown in SEQ ID NO.

2.

3. Use of the intervening peptide according to claim 1 or the synthetic nucleic acid molecule according to claim 2 in the preparation of a vaccine.

4. The use according to claim 3, wherein the vaccine is selected from microbial vector vaccines, cell vaccines, nucleic acid vaccines or peptide-based vaccines.

5. The use according to claim 4, wherein the nucleic acid vaccine is a nucleic acid cancer vaccine.

6. The use according to claim 4 or 5, wherein the nucleic acid vaccine encodes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 peptide epitopes.

7. Use of a plurality of intervening peptides or nucleic acid molecules encoding them in combination in the preparation of a vaccine, the amino acid sequence of at least one of the plurality of intervening peptides being as shown in SEQ ID NO.

1.

8. The use according to claim 7, wherein the plurality of intervening peptides further comprises one or more intervening peptides selected from the group consisting of GPGPG, EAAAK, AAY, GGGS, KK, GGSGGGGSG, GGS, GS and REKR.

9. Use of the intervening peptide according to claim 1 or the synthetic nucleic acid molecule according to claim 2 in the preparation of a fusion protein.

10. A nucleic acid cancer vaccine, characterized in that, The nucleic acid cancer vaccine encodes 3-40 peptide epitopes and intervening peptides located between the peptide epitopes, and the amino acid sequence of at least one of the intervening peptides is as shown in SEQ ID NO.1.

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