WO2023137946A1 - Ccl13的用途 - Google Patents

Ccl13的用途 Download PDF

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WO2023137946A1
WO2023137946A1 PCT/CN2022/097091 CN2022097091W WO2023137946A1 WO 2023137946 A1 WO2023137946 A1 WO 2023137946A1 CN 2022097091 W CN2022097091 W CN 2022097091W WO 2023137946 A1 WO2023137946 A1 WO 2023137946A1
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fusion protein
ccl13
nucleic acid
antigen
present
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PCT/CN2022/097091
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English (en)
French (fr)
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王晓芳
蒋建东
姚艳玲
孙忠杰
李玉环
张金兰
***
刘德芳
吴婷欣
齐海龙
谢皇帆
Original Assignee
诺未科技（北京）有限公司
中国医学科学院医药生物技术研究所
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Publication of WO2023137946A1 publication Critical patent/WO2023137946A1/zh

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Definitions

  • the present invention relates to the field of biomedicine, in particular to the application of CCL13.
  • Monocyte chemoattractant protein 4 (MCP-4/CCL13) is a member of a distinct, structurally related CC chemokine subfamily.
  • CCL13 is a major chemoattractant for eosinophils, basophils, monocytes, and T lymphocytes, and is able to induce key immunomodulatory responses through actions on epithelial, muscle, and endothelial cells.
  • CCL13 has been implicated in many chronic inflammatory diseases in which it functions as a key molecule involved in the selective recruitment of cell lineages to inflamed tissues and their subsequent activation.
  • CCL13 plays an important role in the migration of dendritic cells (DCs) to inflamed epithelial layers, and when injected in vivo together with antigens, CCL13 recruits blood monocytes or blood DC precursors, which rapidly differentiate into typical DCs and improves antitumor immune responses.
  • DCs dendritic cells
  • Tumor vaccines induce the patient's effector T cells to function by enhancing the existing anti-tumor response or activating naive T cells.
  • Antigen-specific CD8+ cytotoxic T lymphocytes (CTL) play an important role in the anti-tumor process.
  • DC cells are the only professional antigen-presenting cells that can activate na ⁇ ve CD8+ T cells.
  • the uptake, processing and cross-presentation of extracellular tumor antigens through MHC-I are crucial for the generation of effective CTLs. Therefore, delivery of tumor antigens to DCs by conjugating DC cell surface molecules is an effective tumor therapy strategy for inducing CD8+ T cell immune responses.
  • the molecules that can enhance the presentation of DC cells include XCL-1, but more DC cell surface molecules that can enhance the presentation still need to be studied.
  • the technical problem to be solved by the present invention is to provide an immune enhancement delivery system for T2 fragment and/or CCL13 targeted delivery antigen formation.
  • the present invention provides:
  • nucleic acid molecule that is substituted, deleted or added one or more nucleotides in the nucleotide sequence of the nucleic acid molecule in IV), and can encode the same or similar functional protein;
  • VI a nucleic acid molecule fully or partially complementary to V.
  • the T2 fragment consists of 31 amino acids, and its sequence is SEQ ID NO:4. Studies have shown that the artificially modified T2 fragment has the effect of enhancing immunity in the human body.
  • the CCL13 is a chemokine CCL13, which is derived from humans or other animals, and is a full-length sequence, or a partial fragment with CCL13 activity.
  • the present invention finds that CCL13 can deliver antigens to DC cells, thereby improving the presentation effect and enhancing immune response.
  • the present invention also provides a fusion protein comprising CCL13 and an antigen. Or include CCL13, antigen and T2 fragments.
  • the fusion protein sequentially includes from N-terminal to C-terminal: IgE signal peptide, CCL13, linker, antigen and T2 fragment.
  • the antigens are from viruses, pathogenic bacteria and/or tumors.
  • the CCL13 is human CCL13 sequence
  • the antigen is E6 protein of HPV16 and/or E7 protein of HPV16.
  • the fusion protein sequentially includes from N-terminal to C-terminal: IgE signal peptide, CCL13, linker, E6 protein of HPV16, E7 protein of HPV16 and T2 fragment.
  • amino acid sequence of the IgE signal peptide is shown in SEQ ID NO: 5;
  • amino acid sequence of the CCL13 is shown in SEQ ID NO: 3;
  • the linker is (G 5 S)n, wherein n is 1-10; in the embodiment of the present invention, the linker sequence is GGGGGSGGGGG.
  • amino acid sequence of the E6 protein of HPV16 is shown in SEQ ID NO: 1;
  • amino acid sequence of the E7 protein of HPV16 is shown in SEQ ID NO: 2;
  • the amino acid sequence of the T2 fragment is shown in SEQ ID NO: 4;
  • the C-terminus of the fusion protein also includes a FLAG tag sequence whose amino acid sequence is DYKDDDDK, but this sequence is only a tag for identifying protein expression and does not affect the immune effect of the sequence.
  • the present invention also provides nucleic acid encoding the fusion protein.
  • nucleotide sequence encoding the fusion protein of the present invention is shown in SEQ ID NO:9.
  • the present invention provides a nucleic acid fragment, which includes nucleic acid encoding the fusion protein of the present invention, 5'-UTR, 3'-UTR and 3'-terminal PolyA.
  • the 5'-UTR is ⁇ -globin-2
  • the 3'-UTR is 2 ⁇ -globin
  • the length of the 3'-end PolyA is 120bp.
  • the structure of the nucleic acid fragment is 5'UTR-CCL13-E6E7-3'UTR-A(120).
  • the present invention also provides a transcription unit encoding the fusion protein.
  • the transcription unit includes a promoter and nucleic acid encoding the fusion protein.
  • the transcription unit further includes a terminator.
  • the promoter is a CMV or CMV/R promoter.
  • the present invention also provides an expression vector, which includes a backbone vector and a nucleic acid encoding the fusion protein.
  • the backbone vector is selected from pVAX1 series vectors or pVR series vectors.
  • the present invention also provides a recombinant host transformed or transfected with the recombinant vector.
  • the host cells of the recombinant hosts in the present invention are bacteria or mammalian cells.
  • the preparation method of the fusion protein of the present invention comprises culturing the recombinant host of the present invention to obtain a culture containing the fusion protein.
  • the invention provides a delivery system for delivering antigenic substances such as viruses, bacteria, fungi and tumors to CCR3-positive antigen-presenting cells.
  • the system fuses the corresponding antigen molecule with chemokine CCL13, and adds a T2 tag at the end of the antigen molecule to further enhance the immunogenicity.
  • the system can be a nucleic acid carrier or a fusion protein for the prevention or treatment of diseases caused by the corresponding antigen.
  • the present invention provides an application of an antigen delivery system in the preparation of a preventive or therapeutic vaccine, the delivery system comprising: a ligand CCL13 combined with CCR3.
  • fusion protein nucleic acid, expression vector, host, fusion protein prepared by the preparation method of the present invention, and/or the culture containing the fusion protein obtained by the preparation method in the preparation of products for preventing and treating diseases.
  • the present invention also provides a product for preventing and treating diseases, which includes: the fusion protein, nucleic acid, expression vector, host, the fusion protein prepared by the preparation method, and/or the culture containing the fusion protein obtained by the preparation method.
  • the present invention also provides a method for preventing and treating diseases, which is to administer the products for preventing and treating diseases described in the present invention.
  • the prevention and treatment include prevention and/or treatment, specifically including increasing the antibody level in serum, preventing tumor formation, inhibiting tumor growth, and improving the immune response ability of the body to tumor.
  • the diseases include diseases caused by viruses and/or pathogenic bacteria.
  • the disease is a tumor.
  • the products for preventing and treating diseases include medicines and/or vaccines.
  • the vaccine is DNA vaccine, recombinant protein vaccine or mRNA vaccine.
  • the administration methods include oral administration, injection and/or electroporation.
  • CC family chemokine members can be fused with antigen molecules to enhance immunogenicity; only some of them can be fused with antigen molecules to enhance the immunogenicity of antigens, initiate stronger immune responses, and provide options for the preparation of more effective vaccines.
  • relevant studies have pointed out that the fusion of 4-1BBL-S, 4-1BBL-Fc, CD80-Fc, etc. with antigen molecules can greatly enhance the immunogenicity of antigens, but the fusion of GM-CSF, mlL-23, IL-15SAG1, etc. with antigen molecules does not have the effect of enhancing immunogenicity.
  • the present invention utilizes the chemotactic binding ability of CCL13 and immune cell surface receptors such as DC cells to transport and cross-present different antigen proteins to the surface of DC cells, thereby improving the efficiency of phagocytosis, processing and presentation of various antigen proteins by DC cells, and improving the effect of preventing and treating related diseases.
  • the T2 sequence in the present invention has been tested to have a very strong immune enhancement effect, and can further stimulate humoral and cellular immune responses in the process of promoting antigen presentation, and finally achieve the effect of inhibiting the growth of related tumors.
  • Figure 1 shows the analysis of the ability of CCL13 to chemoattract various professional antigen-presenting cells
  • Figure 2 shows the vector map of encoding fusion protein nucleotides; wherein A in Figure 2 shows pVR-CCL13-E6E7-T2, B in Figure 2 shows pVR-CCL13-E6E7, and C in Figure 2 shows the plasmid map of pVR-E6E7;
  • Fig. 3 shows the expression situation of detecting three kinds of plasmid target genes of pVR-CCL13-E6E7-T2, pVR-CCL13-E6E7 and pVR-E6E7, utilizes Western blot technique (Westernblot) to detect the expression situation of the fusion protein coded nucleotide with Flag tag at the C terminal;
  • Fig. 4 shows the timeline of the prophylactic immunization of mice with different fusion genes for later testing of specific T cell responses
  • Figure 5 shows the results of flow cytometric detection of specific T cell responses after immunization of mice with different fusion genes
  • Figure 6 shows that different fusion genes, mRNA and protein vaccines perform humoral immunity and tumor inoculation timeline on mice respectively;
  • Fig. 7 shows the diagrams of the tumor measurement results of immunization in each group, wherein a shows the tumor measurement results of female mice immunization; b shows the tumor measurement results of male mice immunization;
  • Figure 8 shows that different fusion genes, mRNA and protein vaccines are respectively used for therapeutic immunization and tumor inoculation time axis in mice;
  • Figure 9 shows the volume of tumor cells treated in each group.
  • the present invention provides the use of CCL13, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.
  • the CCL13 of the present invention is an important chemokine in the human body, which belongs to a member of the CC chemokines family (CC chemokines), and can be specifically expressed at a high level in human macrophages and T cells.
  • CC chemokines CC chemokines family
  • the research of the present invention shows that CCL13 can be used to deliver substances to professional antigen-presenting cells, especially DC cells, thereby enhancing the presentation effect of DC cells.
  • the CCL13 can be human-derived fragments, or other animal-derived fragments, such as mouse-derived, rabbit-derived, monkey-derived, pig-derived, etc. It can be a complete CCL13, or a fragment or mutant with CCL13 activity, which is not limited in this application.
  • human CCL13 was used as the experimental object to verify the improvement of the antigen presentation effect of CCL13.
  • the amino acid sequence of the human source CCL13 is:
  • the T2 sequence is transformed from a short peptide (T4 phageheadfibritin) at the C-terminus of T4 phage fibrin, and the source of the species belongs to an exogenous sequence, which is completely absent in the human body, and there will be no problem of killing other human proteins after enhanced immunity. It has been reported that this sequence can promote the trimerization of certain proteins under certain conditions.
  • the T2 segment as an artificially modified polypeptide sequence, has an immune enhancing effect in the human body.
  • the T2 fragment consists of 31 amino acids, and its sequence is PGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG (SEQ ID NO: 4).
  • the number of antigens in the fusion protein of the present invention is at least one. For example, it may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more.
  • experiments were conducted on the fusion effect of one or two antigens, and all showed good results.
  • the antigens in the present invention are from viruses, pathogenic bacteria and/or tumors.
  • the antigens may be proteins from viruses, pathogenic bacteria and/or tumors.
  • the antigen is from capsid protein or non-structural protein of virus, membrane protein of pathogenic bacteria, flagellin or surface antigen of tumor. It may be a complete fragment of or an antigenic determinant thereof. It may contain only one antigenic determinant, may also be composed of multiple antigenic determinants in series, or may be one antigenic determinant repeated in series twice or more.
  • the virus includes but is not limited to at least one of HPV virus, EBV, HCV, HIV, HBV, VZV or coronavirus;
  • the tumor includes but not limited to at least one of liver cancer, cervical cancer, ovarian cancer, lung cancer, head and neck cancer, prostate cancer, breast cancer, blood tumor, ovarian cancer, and colorectal cancer.
  • the antigen is E2, E5, E6 and/or E7 protein of HPV virus or a mutant epitope thereof.
  • HPV viruses include HPV viruses of various subtypes, for example, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56 and/or HPV58 types.
  • the antigen is LMP1, LMP2, EBNA1 of Epstein-Barr virus, or a mutant epitope thereof.
  • the antigen is S protein, N protein, E protein, M protein or epitopes thereof of coronavirus. Described coronavirus is SARS virus, MERS virus and/or COVID-19.
  • the antigen is GPC3 protein and/or AFP protein of liver cancer.
  • the antigen is PSA, PSMA, PSCA, PAP and/or STEAP1 of prostate cancer.
  • the antigen is a dominant epitope of Her2/neu and/or BCAR3 of breast cancer.
  • the antigen is MAGE-A3, ISR2, NY-ESO-1, Melan A, gp100, Tyrosinase, TRP1 and/or TRP2 of melanoma.
  • the antigen is Immunoglobulin idiotype, Immunoglobulin ⁇ -chain and/or Immunoglobulin ⁇ -chain of blood cancer.
  • the antigen is AIM2, HT001, TAF1B, Micoryx and/or TGF ⁇ RII of colorectal cancer.
  • the antigen is folate receptor- ⁇ of ovarian cancer.
  • the antigen is P53, IDH1/2, BAGE, GAGE1, GAGE2, CAG3, RAGE, CEA, CDK4, CASP-8, ras, bcr/abl and/or MUC-1 of various proto-oncogenes, tumor suppressor genes and/or tumor-specific antigens.
  • the present invention utilizes the chemotactic binding ability of CCL13 and immune cell surface receptors such as DC cells to transport and cross-present the above-mentioned antigenic proteins to the surface of DC cells, thereby improving the efficiency of phagocytosis, processing and presentation of various antigenic proteins by DC cells, and improving the effect of preventing and treating related diseases.
  • CCL13 chemotactic binding ability of CCL13 and immune cell surface receptors such as DC cells to transport and cross-present the above-mentioned antigenic proteins to the surface of DC cells, thereby improving the efficiency of phagocytosis, processing and presentation of various antigenic proteins by DC cells, and improving the effect of preventing and treating related diseases.
  • the presentation efficiency of multiple antigens including HPV16 E6 or E7 proteins among the above proteins has been verified to be improved by CCL13.
  • CCL13 taking the E6 and E7 proteins of HPV16 as examples, it is proved that CCL13 can improve the efficiency of antigen protein presentation, and other proteins fused with CCL13
  • a linker is added between the fragments, wherein the linker between CCL13 and HPV virus antigen protein is GGGGGSGGGGG.
  • Different antigens can be linked by (G 5 S)n and/or AGA.
  • a signal peptide that promotes the secretion of the fusion protein to the extracellular space is added to the N-terminus of CCL13.
  • the signal peptide is an IgE signal peptide.
  • its amino acid sequence is MDWTWILFLVAAATRVHS (SEQ ID NO: 5)
  • a tag is added to the C-terminus of the fusion protein.
  • the tags are selected from recombinant protein purification tags well known in the art.
  • the label is DYKDDDDK.
  • the structure of the fusion protein includes from N-terminus to C-terminus: IgE signal peptide, human CCL13 protein sequence, linker sequence (GGGGGSGGGGG), E6E7 protein sequence, T2 protein sequence, and Flag tag sequence. Specifically, its amino acid sequence is as SEQ ID NO: 15.
  • the nucleic acid encoding protein of the present invention may be DNA, RNA, cDNA or PNA.
  • the nucleic acid is in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • the DNA can be single-stranded or double-stranded.
  • a nucleic acid can include nucleotide sequences that have different functions, such as coding regions and non-coding regions such as regulatory sequences (eg, promoters or transcription terminators).
  • Nucleic acids can be linear or circular in topology.
  • a nucleic acid can be, for example, part of a vector, such as an expression or cloning vector, or a fragment.
  • the nucleic acids may be obtained directly from natural sources, or may be prepared with the aid of recombinant, enzymatic or chemical techniques.
  • the RNA form is mRNA or the like obtained by gene transcription.
  • the DNA sequence for expressing the fusion protein is optimized, and these optimizations include but are not limited to: codon usage bias, elimination of secondary structures (such as hairpin structures) that are not conducive to expression, changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.
  • the prevention mentioned in the present invention refers to the administration of the drug described in the present invention before the occurrence of tumor can reduce the risk of tumor occurrence.
  • the treatment described in the present invention refers to the administration of the drug described in the present invention after tumor occurrence, which can inhibit tumor growth, reduce tumor volume or delay tumor growth rate.
  • the mouse transplanted tumor cell TC-1 was used as the experimental object to verify the effect of the fusion protein vaccine.
  • the present invention also provides the transcription unit of the fusion protein, the transcription unit refers to the DNA sequence from the promoter to the terminator. Regulatory fragments can also be included on both sides or between the promoter and terminator, and the regulatory fragments can include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence, such as enhancers of promoters, poly(A) signals, and the like.
  • the transcription unit provided by the present invention includes a CMV or CMV/R promoter, a CMV enhancer and a nucleic acid fragment encoding a fusion protein.
  • the recombinant vector in the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule that includes a desired coding sequence and an appropriate nucleic acid sequence that is necessary for the expression of an operably linked coding gene in a specific host organism.
  • Nucleic acid sequences necessary for expression in prokaryotic cells include a promoter, optionally including an operator sequence, a ribosome binding site and possibly other sequences.
  • Prokaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • plasmid and “vector” are sometimes used interchangeably because plasmid is currently the most commonly used form of vector.
  • the present invention is intended to include such other forms of expression vectors, which serve equivalent functions, that are or will become known in the art, including, but not limited to: plasmids, phage particles, viral vectors and/or simply potential genomic inserts.
  • the nucleic acid encoding the fusion protein provided by the present invention can be constructed in various eukaryotic expression vectors.
  • its backbone vector can be a pVAX1 series vector, or a pVR series vector (see Chinese patent ZL 202110624820.8).
  • the host cell of the present invention is a prokaryotic or eukaryotic host containing a nucleic acid vector and/or a target gene.
  • Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of replicating the protein-encoding vector or expressing the desired protein.
  • the preparation method of the fusion protein adopts the method of inducing the expression of the recombinant host, and the culture can be cultured bacteria, cell bodies, culture fluid, or substances obtained from the above cultures through extraction and/or purification.
  • the products for preventing and treating diseases provided by the present invention include: the fusion protein, nucleic acid, expression vector, host, the fusion protein prepared by the preparation method, and/or the culture containing the fusion protein obtained by the preparation method.
  • the products for preventing and treating diseases include medicines and/or vaccines.
  • the vaccine also includes pharmaceutically acceptable carriers, excipients and/or adjuvants.
  • the medicine also includes pharmaceutically acceptable auxiliary materials.
  • the prevention in the present invention refers to administering the product for preventing and treating the disease in the present invention before the occurrence of the disease, so as to reduce the risk of the occurrence of the disease.
  • the treatment of the present invention refers to the administration of the product for prevention and treatment of the present invention after the occurrence of the disease, which can improve the disease, inhibit the development of the disease, and restore the patient to health.
  • using the product of the present invention to prevent and treat tumors can increase the level of antibodies in serum, inhibit tumor growth, reduce tumor volume or delay tumor growth.
  • the mouse transplanted tumor cell TC-1 was used as the experimental object to verify the effect of the fusion protein vaccine and obtain good results.
  • amino acid sequences of the fragments involved and the encoded nucleic acid fragments are shown in Table 1:
  • reagent consumables used in the present invention are all common commercial products and can be purchased in the market. Below in conjunction with embodiment, further set forth the present invention:
  • Monocytes, T cells, eosinophils, and basophils were isolated from mouse bone marrow and peripheral blood, respectively.
  • the bone marrow mononuclear cells were added with M-CSF, GM-CSF, and IL4 to induce differentiation into macrophages and DC cells, and then chemotaxis experiments were performed.
  • the upper chamber of the chemotaxis chamber (transwell chamber with carbonate membrane: 5 ⁇ m; Costar, Cat: 3422) was placed into the above-mentioned isolated or differentiated cells, and the number of cells added was 1 ⁇ 10 6 /100 ⁇ l/well based on the previous work in the laboratory.
  • CCL13 factor E.coli was used to purify the recombinant mouse CCL13 protein. According to the previous work in the laboratory, 200ng/ml was the optimal chemotactic efficiency dose. After 4 hours, the cells in the lower chemotaxis chamber were collected, and the chemotaxis ability of CCL13 to various immune cells was analyzed by flow cytometry. The results showed that: CCL13 could effectively recruit various immune cells from the upper chamber to the lower chamber (P ⁇ 0.001) ( Figure 1).
  • Embodiment 2 fusion gene vaccine design and the construction of mammalian expression plasmid
  • Construct the pVR-E6E7 plasmid connect the IgE signal peptide before the E6 and E7 protein sequences of the human papillomavirus subtype HPV16, and the amino acid sequence is MDWTWILFLVAAATRVHS; connect the Flag tag consisting of 8 amino acids of DYKDDDDK thereafter.
  • the finally obtained fusion protein includes: gE signal peptide, E6 protein sequence, E7 protein sequence, and Flag tag sequence from N-terminus ⁇ C-terminus. As shown in A in Figure 2.
  • the amino acid sequence of the fusion protein was optimized for the codon expression preference of mammalian cells, and the fusion gene sequence was determined to be SEQ ID NO: 10.
  • the fusion gene sequence was gene-synthesized, and then constructed as a whole into the corresponding multiple cloning site region of the pVR plasmid vector, so that it could express the fusion protein in the correct codon translation sequence.
  • the final constructed plasmid was named pVR-CCL13-E6E7 plasmid. As shown in B in Figure 2.
  • the final constructed fusion gene includes: IgE signal peptide, human CCL13 protein sequence, linker sequence (GGGGGSGGGGG), E6 protein sequence, E7 protein sequence, and Flag tag sequence from N-terminal to C-terminal.
  • the amino acid sequence of the fusion protein was optimized for the codon expression preference of mammalian cells, and its fusion gene sequence was determined to be SEQ ID NO: 10.
  • the fusion gene sequence was gene-synthesized, and then constructed as a whole into the corresponding multi-cloning site region of the pVR plasmid vector, so that it could express the fusion protein in the correct codon translation sequence.
  • the final constructed plasmid was named pVR-CCL13-E6E7 plasmid. As shown in B in Figure 2.
  • the fusion gene from N-terminal to C-terminal finally includes: IgE signal peptide, human CCL13 protein sequence, linker sequence (GGGGGSGGGGG), E6 protein sequence, E7 protein sequence, T2 protein sequence, and Flag tag sequence. That is, first construct the fusion protein CCL13-E6E7-T2, connect the IgE signal peptide at the N-terminus of the fusion protein, the amino acid sequence is MDWTWILFLVAAATRVHS; connect the Flag tag sequence at the C-terminus of the fusion protein, and the amino acid sequence is DYKDDDDK.
  • the amino acid sequence of the fusion protein was optimized for the codon expression preference of mammalian cells, and its fusion gene sequence was determined to be SEQ ID NO: 8.
  • the fusion gene sequence was gene-synthesized, and then constructed as a whole into the corresponding multiple cloning site region of the pVR plasmid vector, so that it could express the fusion protein in the correct codon translation sequence.
  • the final constructed plasmid was named pVR-CCL13-E6E7-T2 plasmid. As shown in C in Figure 2.
  • the plasmid patterns constructed in the experiments described in this example are actually: pVR-CCL13-antigen-T2, pVR-CCL13-antigen and their control plasmid pVR-antigen.
  • the E6E7 fusion protein of HPV16 subtype was used as the antigen.
  • Recombinant plasmids pVR-E6E7, pVR-CCL13-E6E7, and pVR-CCL13-E6E7-T2 were used to transfect HEK293T cells by PEI transfection method (the transfection method of vectors containing other antigens was the same).
  • the prepared PEI transfection reagent was added to the diluted DNA, the ratio of PEI ( ⁇ g):total DNA ( ⁇ g) was 3:1. Mix well for 10s-15s, let stand at room temperature for 15 minutes, slowly add the above solution dropwise to the cells with a pipette, and incubate in the incubator for 48 hours. The above solution was slowly added dropwise to the cells with a pipette, and incubated in an incubator for 48 h. Add the mixed NP40 lysate containing protease inhibitors. The cells were scraped off with a cell scraper, transferred to a 1.5mL centrifuge tube, and placed on a shaker at 4°C for 20min to lyse.
  • the sample was centrifuged at 12000rpm at 4°C for 20min, and the supernatant was transferred to a new centrifuge tube. Take 50 ⁇ g of the quantified protein sample into a centrifuge tube, add 4 ⁇ protein loading buffer, mix well and centrifuge, then boil at 95°C for 5 minutes. Samples were tested by Western Blot. The results showed that the plasmids could be expressed normally in mammalian cells.
  • mice Given that the fusion gene can be normally expressed in mammalian cells.
  • the plasmid dose was 25 ⁇ g, plus the negative control PBS group, a total of 4 groups, 5 mice in each group.
  • the mice were immunized according to the immunization strategy marked in the time axis of FIG. 4 , and then the mice in each group were blood tested on D14.
  • Anti-CD8 antibody and HPV16E7 antibody were diluted with 1640 medium, and 0.2 ⁇ l Anti-CD8 antibody and 1 ⁇ l HPV16 E7 (E7-tetramer) antibody were added to each 100 ⁇ l 1640 solution.
  • Add 100 ⁇ l of staining solution to each sample blow off the cell mass with a 200 ⁇ l pipette to fully stain the cells, and stain at 4°C for 1 to 2 hours. After staining, add 1ml PBS to each sample, centrifuge at 3000RPM for 5min in a high-speed refrigerated centrifuge, discard the supernatant and keep the white cell mass at the bottom.
  • the number of E7-specific T cells in the CCL13-E6E7-T2 group was significantly higher than that in the E6E7 group and the CCL13-E6E7 group.
  • the number of E7-specific T cells in the CCL13-E6E7 group was significantly higher than that in the E6E7 group.
  • chemokine CCL13 can indeed effectively induce the combination of E6E7 antigen and specific immune cells, thereby promoting the formation of T cells.
  • the addition of T2 polypeptide at the C-terminus of the antigen can significantly enhance the effect of CCL13 presenting antigen molecules.
  • fusion gene can be normally expressed in mammalian cells.
  • Another purified fusion protein is used as a protein vaccine, wherein the fusion protein corresponding to the pVR-CCL13-E6E7-T2 plasmid is denoted as CCL13-E6E7-T2.
  • RNA vaccine is as follows:
  • T7-Flash ScribeTM Transcription Kit (Cell script) was selected for in vitro transcription, and UTP was replaced with N1-Methylpseudouridine-5'-Triphosphate (Trilink Biotech) during the preparation of in vitro transcription system.
  • the reaction system is shown in the table below, and the reaction conditions are: after the first step of reaction, 35°C for 30 minutes; after the second step of reaction, 35°C for 15 minutes.
  • DOTAP ((2,3-dioleoyl-propyl)trimethylammonium chloride), DOPE (dioleoylphosphatidylethanolamine), DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) were all purchased from Shanghai Aiweite Medical Co., Ltd.
  • Nanoparticles were prepared by rotary evaporation.
  • the specific operation process of LNP-Man preparation is as follows:
  • C57BL6 (purchased from Tonglihua) 6-8 week-old female mice and male mice were randomly divided into groups as follows:
  • the right side of the mouse was depilated near the inguinal lymph node, and the mRNA and protein were directly injected.
  • Intramuscular injection of DNA vaccine supplemented by electrical pulse stimulation.
  • the injection dose of each mouse was 25 ⁇ g, and the administration frequency was once every two weeks, and a total of two injections were made.
  • One week after the last injection and before inoculation the TC-1 tumor cells with tumor-forming conditions were explored, the tumor formation time was observed, and the long diameter a and short diameter b of the tumor were measured every two days.
  • the tumor volume was calculated according to a ⁇ b ⁇ b/2, and the tumor growth curve was drawn.
  • Fusion gene DNA, mRNA and protein vaccines to treat tumors Taking protein vaccines whose antigens are HPV16 E6 and E7 proteins as an example, the verification steps for tumor intervention effects of vaccines containing other antigens are the same).

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Abstract

利用CCL13与DC细胞等免疫细胞表面受体的趋化结合能力将不同的抗原蛋白转运到DC细胞表面，提高了各种抗原蛋白被DC细胞吞噬、加工、呈递的效率，改善了其防治相关疾病的效果。还在抗原中添加了T2序列可以增强免疫效果。

Description

CCL13的用途
本申请要求于2022年01月24日提交中国专利局、申请号为202210076816.7、发明名称为“CCL5的用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药领域，尤其涉及CCL13的用途。
背景技术
单核细胞趋化蛋白4(MCP-4/CCL13)是一种独特的、结构相关的CC趋化因子亚家族的成员。CCL13是嗜酸性粒细胞、嗜碱性粒细胞、单核细胞和T淋巴细胞的主要趋化剂，并能够通过对上皮细胞、肌肉细胞和内皮细胞的作用诱导关键的免疫调节反应。CCL13与许多慢性炎症性疾病有关，在这些疾病中，它作为关键分子发挥作用，参与将细胞谱系选择性募集到炎症组织及其随后的激活。此外，CCL13在树突状细胞(Dendritic cells，DC)迁移到发炎的上皮层中发挥着重要作用，当与抗原一起在体内注射时，CCL13会募集血液单核细胞或血液DC前体，这些前体会迅速分化为典型的DC，并改善抗肿瘤免疫反应。
肿瘤疫苗通过免疫增强已有的抗肿瘤反应或启动初始T细胞来诱导患者的效应T细胞发挥功能，抗原特异性CD8+细胞毒性T淋巴细胞(CTL)在抗肿瘤过程中发挥重要作用。DC细胞是唯一能激活初始CD8+T细胞的专职抗原呈递细胞，对细胞外肿瘤抗原通过MHC-I进行摄取、加工和交叉提呈，对产生有效的CTL至关重要。因此，通过偶联DC细胞表面分子将肿瘤抗原递送至DC细胞是有效诱导CD8+T细胞免疫应答的肿瘤治疗策略。
研究得出的能够对DC细胞起到递呈加强作用的分子包括XCL-1，不过仍然需要研究得出更多的能够发挥加强递呈作用的DC细胞表面分子。
发明内容
有鉴于此，本发明要解决的技术问题在于提供T2片段和/或CCL13靶向递送抗原形成的免疫增强递送***。
本发明提供了：
如I)～VI)中的至少一种在提高抗原递呈效果中的应用；
I)、如SEQ ID NO：4所示氨基酸序列的T2片段；
II)、趋化因子CCL13；
III)、与I)或II)具有80％以上同源性，且具有相同或相似功能的片段；
IV)、编码I)或II)的核酸分子；
V)、在IV)的所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子；
VI)、与V)完全互补或部分互补的核酸分子。
本发明中，T2片段由31个氨基酸组成，其序列为SEQ ID NO：4。研究表明，经过人工改造后的T2片段具有增强人体内免疫的效果。
本发明中，所述CCL13为趋化因子CCL13，其来自于人类或其他动物，其为全长序列，或具有CCL13活性的部分片段。本发明发现，CCL13能够递送抗原到DC细胞，从而提高递呈效果，增强免疫反应。
本发明还提供了一种融合蛋白，其包括CCL13和抗原。或包括CCL13、抗原和T2片段。
本发明实施例，所述融合蛋白自N端→C端依次包括：IgE信号肽、CCL13、linker、抗原和T2片段。
本发明中，所述抗原来自病毒、病原菌和/或肿瘤。
本发明中，所述CCL13为人源CCL13序列，所述抗原为HPV16的E6蛋白和/或HPV16的E7蛋白。
一些实施例中，所述融合蛋白自N端→C端依次包括：IgE信号肽、CCL13、linker、HPV16的E6蛋白、HPV16的E7蛋白和T2片段。
一些具体实施例中，所述IgE信号肽的氨基酸序列如SEQ ID NO：5所示；
所述CCL13的氨基酸序列如SEQ ID NO：3所示；
所述linker为(G 5S)n，其中n为1～10；本发明实施例中，所述linker序列为GGGGGSGGGGG。
所述HPV16的E6蛋白的氨基酸序列如SEQ ID NO：1所示；
所述HPV16的E7蛋白的氨基酸序列如SEQ ID NO：2所示；
所述T2片段的氨基酸序列如SEQ ID NO：4所示；
一些具体实施例中，所述融合蛋白的C端还包括FLAG标签序列，其氨基酸序列为DYKDDDDK，但该序列只为鉴定蛋白表达的标签不影响序列的免疫效果。
本发明还提供了编码所述融合蛋白的核酸。
本发明所述的编码融合蛋白的核苷酸序列如SEQ ID NO：9所示。
本发明提供了一种核酸片段，其包括编码本发明所述融合蛋白的核酸、5’-UTR、3’-UTR和3’-端PolyA。其中，所述5’-UTR为β-globin-2，所述3’-UTR为2β-globin，所述3’-端PolyA的长度为120bp。所述核酸片段的结构为5’UTR-CCL13-E6E7-3’UTR-A(120)。
本发明还提供了含有编码所述融合蛋白的转录单元。
所述转录单元中包括启动子和编码所述融合蛋白的核酸。
一些实施例中，所述转录单元还包括终止子。
一些具体实施例中，所述启动子为CMV或者CMV/R启动子。
本发明还提供了表达载体，其包括骨架载体和编码所述融合蛋白的核酸。
本发明中，所述骨架载体选自pVAX1系列载体、或pVR系列载体。
本发明还提供了转化或转染所述重组载体的重组宿主。
本发明所述重组宿主的宿主细胞为细菌或哺乳动物细胞。
本发明所述融合蛋白的制备方法，其包括培养本发明所述重组宿主，获得含有所述融合蛋白的培养物。
本发明提供了一种将病毒、细菌、真菌、肿瘤等抗原物质输送到CCR3阳性抗原提呈细胞中的递送***。该***由趋化因子CCL13融合相应抗原分子，并在抗原分子末端添加T2标签以进一步增强免疫原性，该***可以是核酸载体或者是融合蛋白形式用于相应抗原所引起疾病的预防或治疗。
本发明提供了一种抗原递送***在制备预防或治疗性疫苗中的用途，所述输送***包括：与CCR3结合的配体CCL13。
本发明所述的融合蛋白、核酸、表达载体、宿主、权所述制备方法制得的融合蛋白，和/或所述制备方法制得的含有所述融合蛋白的培养物在制备防治疾病的产品中的应用。
本发明还提供了一种防治疾病的产品，其包括：所述的融合蛋白、核酸、表达载 体、宿主、所述制备方法制得的融合蛋白，和/或所述制备方法制得的含有所述融合蛋白的培养物。
本发明还提供了一种防治疾病的方法，其为给予本发明所述的防治疾病的产品。
本发明中，所述防治包括预防和/或治疗，具体包括提高血清中抗体水平、预防肿瘤的形成、抑制肿瘤生长，提高机体对肿瘤的免疫反应能力。
本发明中，所述疾病包括由病毒和/或病原菌引起的疾病。或者所述疾病为肿瘤。
本发明中，所述防治疾病的产品包括药物和/或疫苗。本发明中，所述疫苗为DNA疫苗、重组蛋白疫苗或mRNA疫苗。
本发明中，所述给予的方式包括口服、注射和/或电转。
相关研究和多方实验都显示出并非所有CC家族趋化因子成员都可以与抗原分子融合来增强免疫原性；只有部分会与抗原分子相融合去增强抗原的免疫原性，启动更强的免疫应答，为更强效力的疫苗制备提供选择。例如相关研究指出4-1BBL-S、4-1BBL-Fc、CD80-Fc等与抗原分子相融合会较大程度增强抗原的免疫原性，但是GM-CSF、mlL-23、IL-15SAG1等与抗原分子相融合不具有增强免疫原性的作用。
本发明利用CCL13与DC细胞等免疫细胞表面受体的趋化结合能力将不同的抗原蛋白转运交叉呈递到DC细胞表面，提高了各种抗原蛋白被DC细胞吞噬、加工、呈递的效率，改善了其防治相关疾病的效果。本发明中的T2序列经实验测定其具有非常强的免疫增强效果，可以在促进抗原呈递的过程中进一步激发体液和细胞免疫反应，最终达到抑制相关肿瘤生长的效果。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图：
图1示CCL13趋化各类专职抗原提呈细胞能力分析；
图2示编码融合蛋白核苷酸的载体图谱；其中图2中的A示pVR-CCL13-E6E7-T2、图2中的B示pVR-CCL13-E6E7、图2中的C示pVR-E6E7的质粒图谱；
图3示检测pVR-CCL13-E6E7-T2、pVR-CCL13-E6E7和pVR-E6E7的三种质粒目的基因的表达情况，利用蛋白免疫印迹技术(Westernblot)检测C端带有Flag标签的融合蛋白编码核苷酸的表达情况；
图4示不同融合基因对小鼠进行预防型免疫用于后期测试特异性T细胞反应的时间轴；
图5示不同融合基因对小鼠进行免疫后特异性T细胞反应的流式检测结果；
图6示不同融合基因、mRNA和蛋白疫苗分别对小鼠进行体液免疫及接瘤时间轴；
图7示各组免疫的量瘤检测结果图，其中a示雌鼠免疫的量瘤检测结果；b示雄鼠免疫的量瘤检测结果；
图8示不同融合基因、mRNA和蛋白疫苗分别对小鼠进行治疗型免疫和接瘤时间轴；
图9示各组处理的细胞瘤体积。
具体实施方式
本发明提供了CCL13的用途，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
本发明所述CCL13是一种人体内重要的趋化因子，它属于CC趋化因子家族(CC chemokines)中的一员，其可以特异性的在人体巨噬细胞和T细胞中高水平表达。本发明研究表明CCL13能够用于递送物质到专职抗原提呈细胞，特别是DC细胞，从而增强DC细胞的递呈效果。
本发明中，所述CCL13可为人源片段，也可为其他动物来源的片段，例如鼠源，兔源、猴源、猪源等，其可为完整的CCL13，也可为其中具有CCL13活性的片段或突变体，本申请对此不做限定。本申请实施例中，以人源CCL13为实验对象，验证CCL13对抗原的递呈效果的提高。所述人源CCL13的氨基酸序列为：
Figure PCTCN2022097091-appb-000001
Figure PCTCN2022097091-appb-000002
本发明所述融合蛋白中，所述T2序列是由T4噬菌体纤维蛋白C末端的一段短肽(T4 phageheadfibritin)改造而来，种属来源属于外源序列，该序列在人体内完全没有，不会出现增强免疫后对人体其他蛋白造成杀伤的问题。有报道认为该序列可以在某些情况下促进某些蛋白的三聚体化。本发明中发现T2这段作为经过人工改造后的多肽序列具有人体内的免疫增强效果。本发明中，T2片段由31个氨基酸组成，其序列为PGSGYIPEAPRDGQAYVRK DGEWVLLSTFLG(SEQ ID NO：4)。
本发明所述融合蛋白中的抗原的个数至少为1个。例如，可为1个、2个、3个、4个、5个、6个、7个、8个、9个或10个及以上。在本发明的研究中，对1个或2个抗原的融合效果进行实验，都表现出良好的效果。
本发明所述抗原来自病毒、病原菌和/或肿瘤。在本发明中，所述抗原可为来自病毒、病原菌和/或肿瘤的蛋白。一些实施例中，所述抗原来自病毒的衣壳蛋白或非结构蛋白、病原菌的膜蛋白、鞭毛蛋白或肿瘤的表面抗原。其可为的完整片段，也可为其抗原决定簇。其可仅含有一个抗原决定簇，也可以由多个抗原决定簇串联而成，也可以为一个抗原决定簇重复串联两次或以上。
本发明中，所述病毒包括但不限于HPV病毒、EBV、HCV、HIV、HBV、VZV或冠状病毒中至少一种；
本发明中，所述肿瘤包括但不限于肝癌、***、卵巢癌、肺癌、头颈癌、***癌、乳腺癌、血液肿瘤、卵巢癌、结直肠癌中至少一种。
一些实施例中，所述抗原为HPV病毒的E2、E5、E6和/或E7蛋白或其突变表位。所述HPV病毒包括各种亚型的HPV病毒，例如，HPV6、HPV11、HPV16、HPV18、HPV31、HPV33、HPV35、HPV39、HPV45、HPV51、HPV52、HPV56和/或HPV58型。
一些实施例中，所述抗原为EB病毒的LMP1、LMP2、EBNA1、或其突变表位。
一些实施例中，所述抗原为冠状病毒的S蛋白、N蛋白、E蛋白、M蛋白或其表位。所述冠状病毒为SARS病毒、MERS病毒和/或COVID_19。
一些实施例中，所述抗原为肝癌的GPC3蛋白和/或AFP蛋白。
一些实施例中，所述抗原为***癌的PSA，PSMA，PSCA，PAP和/或STEAP1。
一些实施例中，所述抗原为乳腺癌的Her2/neu和/或BCAR3的优势表位。
一些实施例中，所述抗原为黑色素瘤的MAGE-A3，ISR2，NY-ESO-1，Melan A，gp100，Tyrosinase，TRP1和/或TRP2。
一些实施例中，所述抗原为血癌的Immunoglobulin idiotype，Immunoglobulinκ-chain和/或Immunoglobulinλ-chain。
一些实施例中，所述抗原为结直肠癌的AIM2，HT001，TAF1B，Micoryx和/或TGFβRII。
一些实施例中，所述抗原为卵巢癌的folate receptor-α。
一些实施例中，所述抗原为多种原癌基因、抑癌基因和/或肿瘤特异性抗原的P53， IDH1/2，BAGE，GAGE1，GAGE2，CAG3，RAGE，CEA，CDK4，CASP-8，ras，bcr/abl和/或MUC-1。
本发明利用CCL13与DC细胞等免疫细胞表面受体的趋化结合能力将上述的抗原蛋白转运交叉呈递到DC细胞表面，从而提高了各种抗原蛋白被DC细胞吞噬、加工、呈递的效率，改善了其防治相关疾病的效果。在此前的预实验中，上述蛋白中包括HPV16的E6或E7的蛋白在内的多个抗原的递呈效率已经被验证能够被CCL13提高。本发明实施例中，以HPV16的E6和E7蛋白为案例，证明CCL13对抗原蛋白递呈效率的提高效果，其他蛋白与CCL13融合后也会起到良好的效果。
本发明中为了保证融合蛋白中各功能片段不受空间位阻影响而顺利地折叠，在片段间添加linker，其中，CCL13与HPV病毒抗原蛋白之间的linker为GGGGGSGGGGG。不同抗原之间可以通过(G 5S)n和/或AGA相连。
在本发明中，为了提高融合蛋白的表达效果，在CCL13的N端添加促使融合蛋白分泌到胞外的信号肽。一些实施例中，所述信号肽为IgE信号肽。具体的，其氨基酸序列为MDWTWILFLVAAATRVHS(SEQ ID NO：5)
在本发明中，为了方便融合蛋白的纯化，在融合蛋白的C端添加标签。所述标签选自本领域熟知的重组蛋白纯化标签。一些实施例中，所述标签为DYKDDDDK。
一些具体实施例中，所述融合蛋白的结构为自N端→C端依次包括：IgE信号肽、人源CCL13蛋白序列、linker序列(GGGGGSGGGGG)、E6E7蛋白序列、T2蛋白序列、Flag标签序列。具体的，其氨基酸序列如SEQ ID NO：15。
本发明所述的编码蛋白的核酸可以是DNA、RNA、cDNA或PNA。在本发明实施例中，所述核酸为DNA或RNA形式。所述DNA形式包括cDNA、基因组DNA或人工合成的DNA。所述DNA可以是单链的或是双链的。核酸可以包括具有不同功能的核苷酸序列，如编码区和非编码区如调控序列(例如启动子或转录终止子)。核酸在拓扑学上可以是线性或环状的。核酸可以是例如载体(如表达或克隆载体)的一部分，或一个片段。所述核酸可直接从天然来源获得，或者可由重组、酶法或化学技术辅助制备。所述RNA形式为由基因转录获得的mRNA等。
在本发明中，对表达融合蛋白的DNA序列进行了优化，这些优化包括但不限于：密码子使用偏好性，消除不利于表达的二级结构(如发夹结构)，改变GC含量，CpG二核苷酸含量，mRNA的二级结构，隐蔽剪接位点，早期多聚腺苷化位点，内部核糖体进入位点和结合位点，负CpG岛，RNA不稳定区，重复序列(直接重复、反向重复等)和可能影响克隆的限制性位点。
本发明所述的预防是指在肿瘤发生前给予本发明所述的药物能够起到降低肿瘤发生风险的作用。本发明所述的治疗是指在肿瘤发生之后给予本发明所述的药物，能够抑制肿瘤生长，降低肿瘤体积或延缓肿瘤的生长速度。本发明实施例中，以小鼠移植瘤细胞TC-1为实验对象，验证融合蛋白疫苗的效果。
本发明中还提供了融合蛋白的转录单元，所述转录单元是指启动子开始至终止子结束的DNA序列。启动子和终止子两侧或之间还可包括调控片段，所述调控片段可 以包括与核酸序列可操作地连接的启动子、增强子、转录终止信号、多腺苷酸化序列、复制起点、核酸限制性位点、和同源重组位点，例如启动子的增强子，poly(A)信号等。本发明提供的转录单元中，包括CMV或CMV/R启动子、CMV增强子和编码融合蛋白的核酸片段。
本发明所述重组载体，是指重组的核酸载体，是一种重组DNA分子，其包含期望的编码序列和对可操作连接的编码基因在具体宿主生物内的表达所必不可少的合适的核酸序列。对原核细胞中的表达必需的核酸序列包括启动子，任选包括操纵基因序列，核糖体结合位点及可能的其它序列。已知原核细胞利用启动子，增强子以及终止和多腺苷酸化信号。一经转化进入合适的宿主，载体可以独立于宿主基因组进行复制和发挥作用，或者，在一些情况下，自己整合进入基因组。在本说明书中，“质粒”和“载体”有时可以交换通用，因为质粒是当前最普遍使用的载体形式。然而，本发明意图包括表达载体的这样的其它形式，其发挥等价作用，其在本领域是已知的或将变为已知的，包括但不限于：质粒，噬菌体颗粒，病毒载体和/或仅为潜在的基因组***物。具体实施例中，编码本发明提供的融合蛋白的核酸可构建于各种真核表达载体中。例如，其骨架载体可以是pVAX1系列载体、或pVR系列载体(参见中国专利ZL 202110624820.8)。
本发明所述的宿主细胞，为含有核酸载体和/或目标基因的原核或真核宿主。用使用重组DNA技术构建的载体转化或转染宿主细胞。这样的转化宿主细胞有能力复制编码蛋白质的载体或表达期望蛋白质。
本发明实施例中，所述融合蛋白的制备方法采用诱导重组宿主表达的方式，所述培养物可以为培养获得的菌体、细胞体、培养液、或者有上述培养物中经提取和/或纯化获得的物质。
本发明提供的防治疾病的产品包括：所述的融合蛋白、核酸、表达载体、宿主、所述制备方法制得的融合蛋白，和/或所述制备方法制得的含有所述融合蛋白的培养物。本发明中，所述防治疾病的产品包括药物和/或疫苗。所述疫苗中还包括药学上可接受的运载体、赋形剂和/或佐剂。所述药物中还包括药学上可接受的辅料。
本发明所述的预防是指在疾病发生前给予本发明所述的防治疾病的产品，从而能够起到降低疾病发生风险的作用。本发明所述的治疗是指在疾病发生之后给予本发明所述的防治疾病的产品，能够改善病症，抑制疾病发展，使患者恢复健康。例如，将本发明所述的产品用于防治肿瘤，能够提高血清中抗体水平、抑制肿瘤生长，降低肿瘤体积或延缓肿瘤的生长速度。本发明实施例中，以小鼠移植瘤细胞TC-1为实验对象，验证融合蛋白疫苗的效果并获得良好的效果。
本发明实施例中，涉及的片段的氨基酸序列和编码的核酸片段如表1：
表1本发明涉及的氨基酸序列和编码的核酸片段
SEQ ID NO.1 HPV16的E6蛋白的氨基酸序列
SEQ ID NO.2 HPV16的E7蛋白的氨基酸序列
SEQ ID NO.3 CCL13的氨基酸序列
SEQ ID NO.4 T2多肽氨基酸序列
SEQ ID NO：5 IgE信号肽
SEQ ID NO.6 质粒pVR-CCL13-E6E7-T2的核苷酸序列
SEQ ID NO.7 质粒pVR-CCL13-E6E7的核苷酸序列
SEQ ID NO.8 质粒pVR-E6E7的核苷酸序列
SEQ ID NO.9 质粒pVR-CCL13-E6E7-T2中编码融合蛋白的DNA序列
SEQ ID NO.10 质粒pVR-CCL13-E6E7中编码融合蛋白的DNA序列
SEQ ID NO.11 质粒pVR-E6E7中编码融合蛋白的DNA序列
SEQ ID NO.12 质粒pVR-CCL13-E6E7-T2中编码融合蛋白的mRNA序列
SEQ ID NO.13 质粒pVR-CCL13-E6E7中编码融合蛋白的mRNA序列
SEQ ID NO.14 质粒pVR-E6E7中编码融合蛋白的mRNA序列
SEQ ID NO.15 质粒pVR-CCL13-E6E7-T2中融合蛋白的氨基酸序列
SEQ ID NO.16 质粒pVR-CCL13-E6E7中融合蛋白的氨基酸序列
SEQ ID NO.17 质粒pVR-E6E7中融合蛋白的氨基酸序列
本发明采用的试剂耗材皆为普通市售品，皆可于市场购得。下面结合实施例，进一步阐述本发明：
实施例1
分别从小鼠骨的髓和外周血中分离出单核细胞、T细胞、嗜酸性粒细胞、嗜碱性粒细胞。其中骨髓单核细胞分别加入M-CSF和GM-CSF、IL4诱导分化为巨噬细胞和DC细胞后进行趋化实验。趋化小室(碳酸脂膜Transwell小室：5μm；Costar，Cat：3422)的上室中放入上述分离或诱导分化的细胞，根据实验室前期工作基础加入细胞数量为1×10 6/100μl/孔。同时设置自发迁移对照组以及CCL13细胞因子组，所加入的细胞数目相同，每组有五只重复。CCL13因子采用E.coli纯化重组鼠CCL13蛋白，根据实验室前期已有的工作基础，200ng/ml为最佳趋化效率剂量。4小时后收集趋化下室中的细胞，通过流式细胞仪分析CCL13对各类免疫细胞的趋化能力。结果显示：CCL13能有效的从上室中募集各类免疫细胞细胞到下室中(P＜0.001)(图1)。
实施例2融合基因疫苗设计及哺乳动物表达质粒的构建
构建pVR-E6E7质粒：在人***瘤病毒亚型HPV16的E6和E7蛋白序列前连接IgE信号肽，氨基酸序列为MDWTWILFLVAAATRVHS；在其后连接DYKDDDDK的8个氨基酸组成的Flag标签。
最终得到的融合蛋白自N端→C端依次包括：gE信号肽、E6蛋白序列、E7蛋白序列、Flag标签序列。如图2中的A所示。
将融合蛋白的氨基酸序列进行哺乳动物细胞表达偏好的密码子优化，确定其融合 基因序列为SEQ ID NO：10将该融合基因序列进行基因合成，之后将其整体构建到pVR质粒载体的对应多克隆位点区域，使其能够以正确的密码子翻译顺序表达融合蛋白。最终构建形成的质粒命名为pVR-CCL13-E6E7质粒。如图2中B所示。
构建质粒pVR-CCL13-E6E7：同样的令最终构建的融合基因自N端→C端依次包括：IgE信号肽、人源CCL13蛋白序列、linker序列(GGGGGSGGGGG)、E6蛋白序列、E7蛋白序列、Flag标签序列。
将融合蛋白的氨基酸序列进行哺乳动物细胞表达偏好的密码子优化，确定其融合基因序列为SEQ ID NO：10将该融合基因序列进行基因合成，之后将其整体构建到pVR质粒载体的对应多克隆位点区域，使其能够以正确的密码子翻译顺序表达融合蛋白。最终构建形成的质粒命名为pVR-CCL13-E6E7质粒。如图2中B所示。
构建质粒pVR-CCL13-E6E7-T2：融合基因自N端→C端最终依次包括：IgE信号肽、人源CCL13蛋白序列、linker序列(GGGGGSGGGGG)、E6蛋白序列、E7蛋白序列、T2蛋白序列、Flag标签序列。即先构建融合蛋白CCL13-E6E7-T2，在融合蛋白N末端连接IgE信号肽，氨基酸序列为MDWTWILFLVAAATRVHS；在融合蛋白C末端连接Flag标签序列，氨基酸序列为DYKDDDDK。
将融合蛋白的氨基酸序列进行哺乳动物细胞表达偏好的密码子优化，确定其融合基因序列为SEQ ID NO：8将该融合基因序列进行基因合成，之后将其整体构建到pVR质粒载体的对应多克隆位点区域，使其能够以正确的密码子翻译顺序表达融合蛋白。最终构建形成的质粒命名为pVR-CCL13-E6E7-T2质粒。如图2中的C所示。
具体的，在本实施例所阐述的实验中构建的质粒模式实际上为：pVR-CCL13-抗原-T2、pVR-CCL13-抗原及其对照质粒pVR-抗原。在本实施例的实验中抗原使用的是HPV16亚型的E6E7融合蛋白。
实施例3 Western Blot检测融合基因或蛋白疫苗胞内表达
重组质粒pVR-E6E7、pVR-CCL13-E6E7、pVR-CCL13-E6E7-T2用PEI转染法对HEK293T细胞进行转染(含有其他抗原的载体转染方式与此相同)。
转染前24h对细胞进行传代，控制转染时细胞密度为60％～80％，在离心管中加入Opti-MEM Medium和质粒，比例如表2：
表2质粒转染表
培养板类型 Opti-MEM Medium 质粒DNA
6孔板 200μL 3μg
10cm大皿 1mL 8μg
配好的PEI转染试剂加入稀释好的DNA中，PEI(μg)：总DNA(μg)为3∶1。混匀10s～15s，室温静置15min，将上述溶液用移液器缓慢逐滴加入到细胞中，在培养箱中培养48h。将上述溶液用移液器缓慢逐滴加入到细胞中，在培养箱中培养48h。加入混好的含蛋白酶抑制剂的NP40裂解液。用细胞刮将细胞刮下，转移到1.5mL离心管中，置于4℃摇床上裂解20min。样本12000rpm，4℃离心20min，将上清转移到新的离心管中。将定量好的蛋白样品取50μg于离心管中，加入4×蛋白上样缓冲液， 混匀离心后置于95℃煮5min。样本进行Western Blot检测。结果显示质粒在哺乳动物细胞中均能正常表达。
实施例4
探索CCL13趋化因子和T2多肽分别对于融合基因疫苗诱导细胞特异性T细胞反应的影响(以抗原为HPV16 E6和E7蛋白的疫苗为例，对含有其他抗原的疫苗诱导T细胞反应的步骤与此相同)：
鉴于融合基因在哺乳动物细胞可以正常表达。我们提取单独pVR-CCL13-E6E7-T2、pVR-CCL13-E6E7、pVR-E6E7质粒，利用TERESA活体基因导入仪对小鼠进行免疫质粒电转，质粒剂量25μg，加上阴性对照PBS组共4组，每组5只。按照图4的时间轴标注的免疫策略对小鼠进行免疫，之后在D14对各组小鼠进行采血测试。
取小鼠新鲜血液100μl，加入适量抗凝剂(肝素钠)，于高速冷冻上3000rpm离心5分钟；轻轻吸掉上清后振散，加入1mLRBC裂红1分钟，3000rpm离心5分钟；弃掉上清，每个样品加入1mlPBS后用旋涡振荡器混匀，在高速冷冻离心机上1500RPM离心5min，弃掉上清液保留底部白色细胞团。用1640培养基对Anti-CD8抗体、HPV16E7抗体进行稀释，每100μl1640溶液加入0.2μlAnti-CD8抗体和1μlHPV16 E7(E7-tetramer)抗体。每个样品加入100μl染色液，用200μl移液器吹散细胞团使细胞充分染色，4℃染色1～2小时。染色完成后每个样品加入1ml PBS，在高速冷冻离心机上3000RPM离心5min，弃掉上清液保留底部白色细胞团。每个样品加入200μl PBS，用200μl移液器吹散细胞团制成细胞悬液后转移至流式管中。流式染色：cd8-pe；E7-tetramer-Fitc。结果如图所示。该结果表明在相同剂量组别的比较中，E6E7组、CCL13-E6E7组、CCL13-E6E7-T2组的E7特异性T细胞数量均远远高于对照组。在相同剂量组别的比较中CCL13-E6E7-T2组的E7特异性T细胞数量明显高于E6E7组、CCL13-E6E7组。在相同剂量组别的比较中CCL13-E6E7组的E7特异性T细胞数量明显高于E6E7组。这表明趋化因子CCL13的确可以有效诱导E6E7抗原与特异性免疫细胞的结合，从而促进T细胞的形成。除此之外，在抗原C末端加入T2多肽可以显著增强CCL13递呈抗原分子的效果。
实施例5
探究融合基因DNA、mRNA以及蛋白疫苗诱导体液免疫反应影响(以抗原为HPV16 E6和E7蛋白的蛋白形式疫苗为例，对含有其他抗原的疫苗对肿瘤干预效果的验证步骤与此相同)
鉴于融合基因在哺乳动物细胞可以正常表达。我们提取单独pVR-CCL13-E6E7-T2和pVR-E6E7质粒，利用TERESA活体基因导入仪对小鼠进行免疫质粒电转，并体外转录制备表达pVR-CCL13-E6E7-T2的mRNA疫苗与体内转染试剂in vivo-jet PEI包裹成脂质纳米颗粒作为mRNA形式疫苗(记做CCL13-E6E7-T2-mRNA)。
另纯化融合蛋白作为蛋白疫苗，其中pVR-CCL13-E6E7-T2质粒对应的融合蛋白 记做CCL13-E6E7-T2。
同种移植TC-1细胞后观察融合基因疫苗对TC-1移植瘤细胞生长的抑制情况。
首先摸索TC-1细胞的成瘤条件，选择最优的剂量，然后按照图6时间轴标注的免疫策略对小鼠进行质粒电转和mRNA肌肉注射以及蛋白皮下注射。RNA疫苗的制备过程为：
将5’UTR-CCL13-E6E7-3’UTR-A(120)分别连入克隆载体pGEM-3Zf(+)(promega)中，完成体外转录表达***的构建，命名为pGEM-CCL13-E6E7，将5’UTR-CCL13-E6E7-T2-3’UTR-A(120)分别连入克隆载体pGEM-3Zf(+)(promega)中，完成体外转录表达***的构建，命名为pGEM-CCL13-E6E7-T2，体外转录***的UTR序列如下：
5-UTR(β-globin-2)
Figure PCTCN2022097091-appb-000003
3-UTR(2β-globin)
Figure PCTCN2022097091-appb-000004
重组质粒线性化，单酶切反应体系如下表反应条件为37，3h
组成 体积(微升)
Xhol限制性内切酶 4
重组质粒 12微克
10X内切酶缓冲液 5
水 补加
总计 50
体外转录反应
选用T7-Flash ScribeTM Transcription Kit(Cell script)进行体外转录，在体外转录体系配制过程中，将UTP替换为N1-Methylpseudouridine-5’-Triphosphate(Trilink Biotech)。
反应体系如下表所示，反应条件为：第一步反应结束后，35℃，30min；第二步反应后，35℃，15min。
mRNA加帽
选用试剂盒ScriptCap TM Cap 1 Capping System(Cell script)进行操作，反应体系如下表所示：
表4
Figure PCTCN2022097091-appb-000005
Figure PCTCN2022097091-appb-000006
Cap-mRNA纯化：选用MEGAclear TM Kit Purification(Invitrogen)试剂盒进行纯化反应。
脂质体纳米颗粒LNP-Man的制备
DOTAP((2，3-二油酰基-丙基)三甲基氯化铵)、DOPE(二油酰磷脂酰乙醇胺)、DSPE-PEG2000(二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000)均购自上海艾韦特医
药科技有限公司；DSPE-PEG2000-Man购自西安昊然生物科技有限公司；
采用旋转蒸发法制备纳米颗粒。LNP-Man制备的具体操作过程如下：
1)按摩尔比DOTAP∶DOPE∶DSPE-PEG2000-Man＝50∶50∶1，依次加入圆底烧瓶中，加入6mL三氯甲烷，直至固体充分溶解；
2)水浴超声15min；
3)将圆底烧瓶装入旋转蒸发仪中，圆底烧瓶内溶解物需没过水面以下。转速为100rpm，旋转蒸发15min；
4)取下圆底烧瓶，放置通风橱内，加入8mLHEPES缓冲液，将瓶内壁上的薄膜溶解下来；
5)水浴超声30min；
6)将超声好的溶液通过0.22μm的滤膜，过滤三次，即得到所需脂质体纳米颗粒LNP-Man。
LNPs/mRNA的制备
用制备的阳离子脂质纳米材料LNP-Man与mRNA按照设定的N/P＝10∶1(摩尔比)混合，计算所需的LNPs和mRNA的体积；将LNPs与mRNA混合前分别加入等体积的10mM HEPES缓冲液；混合后的LNPs/mRNA在漩涡振荡器上震荡1min，室温静置30min。
将C57BL6(购自为通利华)6～8周龄雌性小鼠和雄性小鼠随机分组，具体分组如下：
表5动物分组
Figure PCTCN2022097091-appb-000007
将小鼠右侧靠近腹股沟***处脱毛，直接注射mRNA，蛋白。肌肉注射DNA疫苗，并辅以电脉冲刺激。每只鼠的注射剂量均为25μg，给药频率为每两周一次，共注射两次。在最后一次注射后一周接种之前摸索好成瘤条件的TC-1瘤细胞，观察肿瘤形成时间并每两天测量肿瘤的长径a和短径b，按照a×b×b/2进行肿瘤体积计算，绘制肿瘤生长曲线。结果如图7所示，注射pVR-E6E7、pVR-CCL13-E6E7-T2、CCL13-E6E7-T2-mRNA及CCL13-E6E7-T2-蛋白疫苗均能彻底防止肿瘤的生长，达到预防肿瘤生长的效果。此外，在D30天对所有实验组小鼠进行二次接瘤挑战，移植瘤依然无法成瘤。说明四种疫苗均有很好的预防肿瘤生长的效果。
实施例6
融合基因DNA、mRNA以及蛋白疫苗***效果(以抗原为HPV16 E6和E7蛋白的蛋白形式疫苗为例，对含有其他抗原的疫苗对肿瘤干预效果的验证步骤与此相同)。
鉴于四种疫苗的预防效果显著，我们进行了疫苗的治疗性效果验证。在D0天接种TC-1细胞，D4进行分组，D4、D11注射疫苗，剂量与预防组一致，方案如图8，以后每周两到三次测量肿瘤大小，计算方法同上。肿瘤生长曲线如图9。结果显示四种疫苗均能不同程度的抑制肿瘤细胞生长，且pVR-CCL13-E6E7-T2质粒、CCL13-E6E7-T2-mRNA以及CCL13-E6E7-T2蛋白组***的效果更好，能达到完全清除肿瘤的效果。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本 发明各实施例技术方案的范围。

Claims (12)

  1. 如I)～VI)中的至少一种在提高抗原递呈效果中的应用；
    I)、如SEQ ID NO：4所示氨基酸序列的T2片段；
    II)、趋化因子CCL13；
    III)、与I)或II)具有80％以上同源性，且具有相同或相似功能的片段；
    IV)、编码I)或II)的核酸分子；
    V)、在IV)的所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子；
    VI)、与V)完全互补或部分互补的核酸分子。
  2. 融合蛋白，其包括CCL13和抗原；或包括CCL13、抗原和T2片段。
  3. 根据权利要求2所述的融合蛋白，其特征在于，自N端→C端依次包括：IgE信号肽、CCL13、linker、抗原和T2片段。
  4. 根据权利要求2或3所述的融合蛋白，其特征在于，所述抗原来自病毒、病原菌和/或肿瘤；
    所述病毒包括HPV病毒、EBV、HCV、HIV、HBV、VZV或冠状病毒中至少一种；
    所述肿瘤包括肝癌、***、卵巢癌、肺癌、头颈癌、***癌、乳腺癌、血癌、卵巢癌、结直肠癌中至少一种。
  5. 编码权利要求2～4任一项所述融合蛋白的核酸。
  6. 核酸片段，其包括权利要求5所述的核酸、5’-UTR、3’-UTR和3’-端PolyA。
  7. 表达载体，其包括骨架载体和权利要求5所述的核酸，或者权利要求6所述的核酸片段。
  8. 转化或转染权利要求7所述表达载体的宿主。
  9. 权利要求2～4任一项所述融合蛋白的制备方法，其特征在于，培养权利要求8所述宿主，获得含有所述融合蛋白的培养物。
  10. 权利要求2～4任一项所述的融合蛋白、权利要求5所述的核酸、权利要求6所述的核酸片段、权利要求7所述的表达载体、权利要求8所述的宿主、权利要求9所述制备方法制得的融合蛋白，和/或权利要求9所述制备方法制得的含有所述融合蛋白的培养物在制备防治疾病的产品中的应用。
  11. 一种防治疾病的产品，其特征在于，其包括：权利要求2～4任一项所述的融合蛋白、权利要求5所述的核酸、权利要求6所述的核酸片段、权利要求7所述的表达载体、权利要求8所述的宿主、权利要求9所述制备方法制得的融合蛋白，和/或权利要求9所述制备方法制得的含有所述融合蛋白的培养物。
  12. 一种防治疾病的方法，其特征在于，给予权利要求11所述的产品。
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