CN111166895B - Immune microenvironment regulator combination, coding nucleic acid and application thereof - Google Patents

Abstract

The invention provides an immune microenvironment modulator combination, which comprises a PD-1/PD-L1/2 channel, a CTLA-4/CD80/CD86 channel and a TGF-beta channel inhibitor. In particular, fusion proteins comprising the combination, nucleic acids encoding the proteins of the components of the composition are provided. The composition comprises amino acid sequences shown as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6. Vaccine compositions comprising the compositions and routes of delivery of the compositions are also provided. The immunomodulator composition of the present invention can provide protective immunity against pathogen infection, and can be used for prevention and/or treatment of various tumors.

Description

Immune microenvironment regulator combination, coding nucleic acid and application thereof

Technical Field

The invention relates to the field of medical products, in particular to an immune microenvironment regulator combination, a coding nucleic acid and application thereof.

Background

Traditionally, tumor cell immunotherapy has been mainly to stimulate the immune system by vaccination or adoptive cell immunotherapy, thereby eliciting an immune response. This approach is based on the assumption that: tumor cells express tumor-specific antigens and are presented by MHC complexes to the surface of tumor cells, while anti-tumor T cells are not fully activated. Therefore, to address this problem, attempts have been made primarily to increase the recognition of these antigens by stimulating key positive co-immune and innate immune pathways (such as CD28, CD40L (CD154) and various TLR receptors), or inhibiting negative immunosuppressive pathways (such as CTLA-4 receptors).

In recent years, it has become clear that the immune system does recognize tumor antigens, but despite the presence of tumor antigens, T cells are assured to remain quiescent. Based on this phenomenon, there is a hypothesis that: there are inhibitory mechanisms that limit or turn off the specificity of the anti-tumor immune response. When a negative regulatory T cell surface molecule is up-regulated in the expression of activated T cells, its activity is attenuated, resulting in less effective tumor cell killing. These inhibitory molecules are called negative costimulatory molecules due to their homology to the T cell costimulatory molecule CD 28. These proteins, also known as immunodetection point proteins, play a role in multiple pathways, such as attenuation of early activation signals, competition with positive costimulatory molecules and direct inhibition by antigen presenting cells. The members of the protein family are programmed death factor-1 (PD-1) and its ligands B7-H1/PD-L1 and B7-DC/PD-L2, and CTLA-4/CD80 or CD 86.

CD28 is a cell surface glycoprotein that is constitutively expressed on the surface of most mature T cells and thymocytes. CTLA-4 receptors are detectable only in T cells 2-3 days after activation, but are not expressed in resting T cells. The primary ligands for CD28 and CTLA-4 molecules are CD80 and CD86 molecules expressed on the surface of Antigen Presenting Cells (APCs). The biological rationale for the presence of at least two receptors (CD 28 and CTLA-4) and two ligands (CD 80 and CD 86) is currently unknown. The current research shows that CD80 inhibits the activation of T cells by binding with CTLA-4 on the surface of the T cells; meanwhile, CD80 can also provide a costimulatory signal for T cell activation by binding with CD28 on the surface of T cells, and induce the proliferation of T cells and the secretion of cytokines.

PD-1 is expressed on activated T cells, B cells, and on monocytes that are involved in mediating the balance of immune activation and tolerance. Its main role is to limit T cell activity around inflammation and to limit autoimmunity in the inflammatory response to infection. The basis for this regulation is that the ligands PD-L1 and PD-L2 of the PD-1 molecule are upregulated in response to a variety of proinflammatory factors and can bind to PD-1 molecules on activated T cells in inflamed tissues, inhibiting T cell function, thereby limiting the immune response.

In addition, it was found that PD-L1 expression is often upregulated in a variety of different tumors, which results in the development of immune escape by inhibiting local anti-tumor T cell responses by binding to PD-1 on tumor-infiltrating lymphocytes. Researches show that nearly more than half of tumor infiltration CD8 positive T cells in cervical cancer and liver cancer express PD-1 molecules, and the combination of the PD-L1 molecules expressed on the surface of tumor cells can lead to the exhaustion and apoptosis of the T cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that can affect cell growth, differentiation, apoptosis, etc. In addition, TGF-. beta.has an important immunomodulatory role. TGF-. beta.s can effectively inhibit T cell function and antigen presenting ability of DC cells. Recent studies have elucidated a direct role of TGF- β in the expression of the upregulating plasma cell-like dc (pdc) immunomodulatory enzyme indoleamine 2, 3-dioxygenase (IDO) and lead to long-term T cell tolerance. IDO inhibits the activity of effector T cells and promotes Treg differentiation and activation by catalyzing the degradation of the essential amino acid tryptophan.

Transforming growth factor-beta (TGF- β) signals by binding to the transforming growth factor-beta receptor (TGFBR) complex. The TGFBR family includes type I receptors (TGFBR 1), type II receptors (TGFBR 2) and type III receptors (TGFBR 3) depending on the molecular structural and functional characteristics. TGFBR1/2 belongs to the transmembrane type receptor serine/threonine kinase family, and contains serine/threonine kinase domain in cells. After binding of TGF- β to the TGFBR1/2 heterodimer, downstream signaling molecules are activated, which in turn activates signaling. The TGFBR3 intracellular segment does not contain active regions of kinases and is not directly involved in signaling, primarily regulating the binding of TGF- β to signaling receptors, also known as coreceptors.

The type III transforming factor-beta receptor (TGFBR 3) is a co-receptor of the classic TGF-beta signaling pathway, involved in mediating SMAD-dependent and SMAD-independent downstream signaling pathways. It was found that the expression level of TGFBR3 was significantly down-regulated compared to the patient's paracancerous normal tissue in the early stages of many tumors, such as breast cancer. There is data showing that TGFBR3 inhibits cell migration and invasion in some tumor models, indicating that TGFBR3 has the function of inhibiting tumor progression and metastasis.

Aiming at the problems, the interaction of PD-1/PD-L1 and CD80 or CD86/CTLA-4 and a TGF-beta signal path are blocked, so that the immunosuppression can be relieved, and the immunocompetence of T cells can be improved. Is helpful for the immune system to eliminate tumor cells.

Recently, several immunodetection point inhibitor drugs targeting the PD-1 receptor and its ligands PD-L1 as well as CTLA-4 have been approved for sale. The immune checkpoint inhibitor has good clinical efficacy in patients with various tumors such as melanoma, renal cancer, colorectal cancer, non-small cell lung cancer, liver cancer and the like. However, many current clinical study data indicate that clinical response rates are low using only an immunodetection point inhibitor such as the PD-1/PD-L1 antibody, and that only about 15% of patients can receive clinical benefit, such as in liver cancer patients. In more recent studies, the antitumor effect of combination with PD-1 pathway immunodetection point inhibitors has been evaluated in clinical models.

For example, studies have reported that transfection of sirnas using PD-1 ligands PD-L1 and PD-L2 into Dendritic Cells (DCs) inhibited the expression of PD-L1 and PD-L2, and dendritic cell vaccines were prepared by loading dendritic cells with minor histocompatibility antigens (MiHA). These MiHA-loaded PD-L1/L2 silenced DCs showed highly efficient activation and amplification of MiHA-specific T cell function: the function of MiHA-specific CD8+ T cells can be obviously enhanced; compared with MiHA-loaded DCs which are not silenced by PD-L1/L2, the expanded MiHA-specific CD8+ T cells are increased by 14.4 times in the in vitro stimulation process of one week, and the expanded T cells are increased by 20 times in the in vitro stimulation process of two weeks. (A.B. van der Waart et al, 2015, Cancer Immunol Immunother (2015) 64: 645-654).

Similarly, a dendritic cell vaccine prepared by silencing PD-L expression of DC cells with siRNA-lipid-nanoparticle complexes targeting PD-L1/L2 and loading the DC with antigen mRNA or polypeptide can effectively increase helper T cells (Th 1 and Th2 cells) and antigen-specific CD8+ T cell responses from allogeneic stem cell transplanted tumor patients (Mieke W.H. Roeven et al, J Immunother 2015;38: 145-154).

Another study examined the effect of purkinje cells (KC) silenced PD-L1 in the liver on natural killer cells (NK cells) and CD8+ T cells. The data indicate that PD-L1 silenced purkinje cells (KC) are able to increase NK cell function and aggregation in the virus-infected liver; meanwhile, the composition can also enhance the accumulation of CD8+ T cells in virus-infected tissues, the killing capacity and cell proliferation of virus-infected cells, the virus clearing capacity and memory mediated by CD8+ T cells (Joseph S Dolina et al, Molecular Therapy-Nucleic Acids (2013) 2, e 72).

Another report examining the effect of PD-L1 inhibition on tumor growth showed that systemic use of anti-PD-L1 antibody plus melanoma peptide-loaded Dendritic Cells (DCs) was able to produce a greater number of melanoma peptide-specific CD8+ T cells, but this combination was not sufficient to retard the growth of existing B16 melanoma. Although additional body irradiation retards tumor growth, further adoptive transfer of antigen-specific CD8+ T cells is required to achieve tumor regression and long-term survival in the treated mice (pin-Thomas et al 2010.J Immunol) 1; 184 (7): 3442-9).

Recently, a study reported that down-regulation of TGFBR3 expression resulted in an immune-tolerant microenvironment. In the mouse breast cancer tumor model, the breast cancer tumor cells overexpressing TGFBR3 showed a significant growth inhibition compared to the breast cancer tumor cells lacking TGFBR3 expression, which was also observed in the melanoma tumor model. Further data show that there are more CD4+ FOXP3+ Treg cells and less CD3+ CD8+ cytotoxic T cell infiltration in the mouse mammary carcinoma model with loss of TGFBR3 expression (Brent A. Hanks et al, J Clin invest.2013, 9/3; 123 (9): 3925-.

In summary, various studies to date have shown that the use of immunodetection point inhibitors appears to represent a new and very promising approach for improved tumor immunotherapy. However, the combination of a vaccine with a single immune checkpoint inhibitor often does not result in the expected improvement of the immunotherapeutic effect, and the clinical use of immune checkpoint inhibitor combinations targeting multiple negative co-stimulatory receptors, such as anti-PD-1 antibodies with anti-CTLA-4 antibodies, or the combination of immune checkpoint inhibitors with other therapies may induce severe toxic responses.

Disclosure of Invention

It is therefore an object of the present method to provide a safe and effective means for the treatment based on immune checkpoint inhibitors, in particular based on inhibitors of the PD-1 and CD80 pathways, especially for the treatment of tumors and/or infectious diseases.

In particular, the object of the present invention is solved by providing a vaccine/immune microenvironment modulator, in particular an immune checkpoint inhibitor combination, comprising as a vaccine an RNA vaccine comprising at least one RNA or DNA (comprising at least one open reading frame encoding at least one antigen), or a DNA vaccine, more even an antigenic polypeptide vaccine, and as a modulator a PD-1 pathway inhibitor, a CTLA-4 pathway inhibitor, a transforming growth factor beta inhibitor. In addition, the vaccine/modulator combination may be in the form of a mixture containing the combination nucleic acid or polypeptide, or autologous DC cells, autologous or allogeneic PBMC cells loaded with at least one of the RNA/DNA or antigenic polypeptides described above and nucleic acids encoding the inhibitors described above (including DNA and RNA), or other forms including the vaccine/inhibitor components. In addition, the object is solved by a combination of an RNA or polypeptide vaccine and an inhibitor for use in a method for treating tumors or infectious diseases.

In the present invention, "antigen" refers to a substance that can be recognized by the immune system and is capable of eliciting an antigen-specific immune response by forming antibodies or/and antigen-specific T cells. In general, an antigen can be a protein or polypeptide that comprises at least one antigenic epitope and can be presented by the Major Histocompatibility Complex (MHC) to the surface of a T cell. In the present invention, the antigen may be a product of translation of mRNA or a product of transcription and translation of DNA.

A vaccine is typically understood to provide one or more antigens, preferably immunogens, for prophylactic or therapeutic substances. For example, a vaccine comprises the antigen, or a nucleic acid encoding the antigen, which may be DNA or RNA; or cells expressing the antigen, such as DC cells or PBMC cells. The antigen or immunogen may be derived from any material suitable for vaccination.

The antigenic nucleic acid molecules of the present invention encode immunogenic peptides of bacteria, viruses, fungi, or other pathogens including, but not limited to, human hepatitis viruses including HAV, HBV, HCV, cytomegalovirus CMV, human immunodeficiency virus HIV, EB virus, dengue virus, Human Papilloma Virus (HPV), respiratory syncytial virus, rhinoviruses, human T-lymphotropic virus type I (HTLV-1), influenza, Bovine Leukemia Virus (BLV), pertussis, polio, measles, mumps, rubella, smallpox, shingles, anthrax, tetanus, rotavirus, rabies, fowl pox, meningococcus, anthrax, encephalitis, pneumococci, streptococcus, staphylococci, neisseria, escherichia coli, shigella, leishmania, respiratory syncytial virus, parainfluenza, neisseria, and the like, Adenovirus, varicella, flavivirus, mycobacterium tuberculosis, malaria, and the like.

In one embodiment, the antigenic nucleic acid molecule encodes a tumor antigen. In this case, the tumor antigen may be expressed on the surface, cytoplasm, or nucleus of the tumor cell. The tumor antigen may also be selected from proteins that are overexpressed in tumor cells compared to normal cells. Tumor antigens can be further divided into tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). Tumor Associated Antigens (TAAs) are a class of antigenic molecules found in both tumor and normal cells, including: embryonic proteins, glycoprotein antigens, squamous cell antigens, and the like. Tumor-associated antigens are not specific to tumor cells, and normal cells can also be synthesized in minute quantities and highly expressed when tumor cells proliferate. Tumor-specific antigens refer to neoantigens that are expressed only on the surface of tumor cells and not on normal cells. Such antigens may be present in tumors of different individual consenting tissue types, e.g. melanoma specific antigens encoded by human malignant melanoma genes may be present in melanoma cells of different individuals, but not expressed by normal melanocytes. TSA can also be common to tumors of different histological types, for example, mutated Ras gene products can be commonly found in lung cancer, digestive tract tumors, etc., but because its amino acid sequence is inconsistent with the expression product of normal proto-oncogene Ras, it can be recognized by the immune system of the body to stimulate the immune response of the body. In general, such antigens resulting from mutations are referred to as neoantigens (neoantigens). These antigens are all recognized by cytotoxic T lymphocytes and cells presenting the antigen can be killed by T lymphocytes.

Preferred tumor antigens according to the invention are selected from the list consisting of TDO, MAGEC, HMOX, WT, LY6, AIM, IDO, CHI3L, IL13RA, LCK, GFAP, KIF20, CNTN, MUC, PEG, TNC, SOX, IGF2BP, S100A, AKAP, TTK, CHI3L, PTHLH, CDC, PMEL, TOP2, PTTG, NRCAM, HMMR, MUC, LY6, SOX, FOSL, PRAME, FOLR, BIRC, KIF2, ITGAV, ART, PROM, CT, S100A, PPIB, S100A, STAT, EPCAM, MLANA, KAROAG, KLK, NT5, PTKLPRZ, SPAG, MET, RGS, CSPG, PDCD1LG, PSC, CD274, PSCA, HD, BP, TSHAVANA, TSROAG, TSKLK, ATKP, EPCR, EPZC, EPSCH, ATCK, ACSCH, TAC, S100A, TAC, S, TAC, S, TAC, S, TAC, S, PTGS, PLK, DDX, PA2G, PAX, IDH, SFMBT, EPHA, NAPSA, LPGAT, NUF, SPAG, STAT, MELK, ST8SIA, EBAG, KIFC, CEACAM, RPL, SYCP, DSE, ANKRD30, TRAPPC, RGS, MGAT, KRT, B3GALNT, CAGE, AGER, ACRBP, LAG, NELFA, RAB, CCND, SART, UBE2V, SLAMF, KCNMA, MUM, HSPH, GUCY1A, AKAP, SQSTM, BCAN, CCNB, TP, SUGT, AURKA, RAN, 6, NLGN4, SART, PRKDC, FOXP, EGF, PIK3R, SLC1A, PCNA, KIF1, BSG, ATP2A, NFAG, NFART, NFAR 8, PALC, TYRC, TRIRC, SARC, SACK 5, SARC, SACK 1, SARC, SACK 5, SARC, SACK 1A, SACK 1, SACK 1, SACK 1, SACK 1, SACK, SAG, SACK, CTAG1, CTAGE, E, FMR1NB, GAGE, GAGE2, GAGE, GAGE, GAGE, GAGE, GAGE, GRDX, HNPRL, IGHD, MAGEA, MAGEA, MAGEA, MAGEA, MAGEA, MAGEA, MAGEA, MAGEA, MAGEA, MAGEB, MAGEB, MAGEC, PAGE, PAP, SAGE, SPANXB, SPO, SSX, SSX, SSX, Brachyury/TFT, TTR, TYRP, VSIR, XAGE1, XAGE1, ENAH, AKR1B, GPC, AFP, FLIR, FGF, PSPH, ABL, CCT, SMYD, TMEM106, ZNF260, EPCAM, ICK, PHF20L, ANXA, ZNF, CASK, FAM122, IRS, VCR 6, TMSH, VAOTF, TTTSF, TTTSZ, TTUTP 14.

Furthermore, tumor antigens may also include individual tumor-specific neoantigens which are produced by genetic mutations in tumor cells. The mutated gene may be any gene in a cell, and the expression product thereof may be expressed on the cell surface or inside the cell.

In order to prevent RNA instability and degradation in multiple pathways, various optimizations of nucleic acid molecules can be performed based on the known multiple natural degradation pathways of RNA. For example, the terminal structure is crucial for the stability of mRNA. For example, at the 5 ' end of a naturally occurring mRNA, a modified guanosine nucleotide is present, referred to as a 5 ' cap structure, and the 3 ' end has an adenosine nucleotide (i.e., poly-A tail) structure of about 200-300 bases in length, and 5 ' and 3 ' UTR sequences, such as those of human beta-globin.

The immune microenvironment regulator combination provided by the invention comprises a PD-1/PD-L1 inhibitor and a TGF pathway inhibitor. . In one embodiment, it is a soluble PD-1 molecule, a soluble CD80 molecule and a soluble TGFBR3 molecule.

In one embodiment, the soluble PD-1 molecule is human sPD-1-Fc having an amino acid sequence comprising SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO: 4; the soluble CD80 molecule is human sCD80-Fc having a heavy chain comprising SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO: 4; the soluble TGFBR3 molecule is human sTGFBR3-Fc having an amino acid sequence comprising SEQ ID NO: 5. SEQ ID NO:3 and SEQ ID NO: 4. In one embodiment, the soluble PD-1 and CD80 molecules are a fusion protein having an amino acid sequence comprising SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO: 4; the soluble TGFBR3 molecule is human sTGFBR3-Fc having an amino acid sequence comprising SEQ ID NO: 5. SEQ ID NO:3 and SEQ ID NO: 4.

In another aspect, the invention provides nucleic acid molecules encoding immune microenvironment modulators of the invention. In one embodiment, the nucleic acid molecule encodes sPD-1-Fc and has a sequence comprising SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: 15, or a sequence of seq id no; the nucleic acid molecule encodes sCD80-Fc and has a sequence comprising SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: 16; the nucleic acid molecule encodes sCD80-Fc and has a sequence comprising SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: 17, and (ii) a sequence of (d); in one embodiment, the nucleic acid molecule encodes a PD-1/CD80 fusion protein and has an amino acid sequence comprising SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof. In one embodiment, one nucleic acid molecule encodes both sPD-1-Fc and sCD80-Fc molecules, which are linked to the coding frames of sPD-1-Fc and sCD80-Fc by a ribosome entry site (IRES) and comprise a sequence having the sequence given in SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: 20 or SEQ ID NO: 21, and (b) 21.

In the present invention, the soluble protein comprises an Fc domain. Fc domains include native human or other animal Fc and Fc variants and full-length or fragments of molecules, including, for example, IgA, IgD, IgG, IgM, IgE and subtypes thereof such as IgG1, IgG2, IgG3, IgG4, and the like. The IgG domain of the invention may be an antibody of any isotype, including IgA, IgD, IgG, IgM, IgE and their subtypes such as IgG1, IgG2, IgG3, IgG4, IgA1 and the like as previously described. In a particular embodiment, the domain of Fc used is IgG 1.

The vaccine/modulator compositions of the present invention may be delivered to host cells in vivo by methods known in the art. In one embodiment, the vaccine/modulator compositions of the present invention can be introduced in vivo by a viral vector such as adenovirus (AdV), adeno-associated virus (AAV), retrovirus, lentivirus, herpes simplex virus, and the like. In addition, the vaccine/modulator compositions of the present invention may also be introduced by transfection of liposomal nanoparticles into host cells. In one embodiment, the vaccine/modulator composition of the present invention can be introduced into the subject's own DC cells by electroporation, or introduced into the subject's body using the DC cells as a vector. In one embodiment, the vaccine/modulator composition of the present invention may be introduced into autologous PBMC cells or allogeneic PBMC cells of a subject by electroporation, and the autologous PBMC cells or allogeneic PBMC cells are used as vectors for introduction into the subject.

Drawings

FIG. 1 shows soluble sPD-1, sCD80-Fc, sPD-1-CD80, sTGFBR3-Fc expression levels

FIG. 2 is a phenotype of DC cells transfected with soluble protein nucleic acid molecules, wherein the surface of mature DC cells (mDC-PP 65 and mDC-PP 65/group of soluble protein nucleic acid molecules) are significantly upregulated in CD80, CD86, and CD83, as compared to Immature Dendritic Cells (iDC), showing significant characteristics of mature DC cells; compared with DC cells transfected with pp65 antigen only, the phenotype of DC cells transfected with pp 65/soluble protein is consistent with that of DC cells transfected with pp65 antigen only.

FIG. 3 is a graph of the effect of immune modulator combinations on T cell responses, with higher proportions of TNF-a and IFN-r expression in DC cells transfected with pp 65/soluble protein compared to the pp65 antigen alone.

Detailed Description

The following detailed description of specific embodiments of the invention, which are intended to be illustrative only, and the invention is described in further detail. These examples should not be construed as limiting the invention thereto.

The first embodiment is as follows: preparation of DNA and mRNA encoding antigens and immunodetection Point inhibitors

1. Preparation of DNA and mRNA constructs

The DNA sequences for the mRNA encoding the CMV virus pp65 protein, for the mRNA encoding the sPD-1-Fc, sCD80-Fc, sPD-1-CD80 and sTGFBR3-Fc fusion proteins of the invention were constructed for this example and used for the subsequent in vitro transcription reactions. Constructs were made by codon-optimization to introduce high GC sequences to stabilize the synthetic mRNA, followed by a 3' UTR sequence of human-derived β -globin, followed by a segment of polyadenylic acid (including but not limited to the 64 adenosine or longer poly-a-sequence used in this example).

2. In vitro transcription

The corresponding DNA plasmid prepared according to example 1 was first linearized using the speI endonuclease and mRNA prepared by in vitro transcription using T7 RNA polymerase using the linearized plasmid as template. The prepared mRNA was then purified by lithium chloride precipitation.

Example two: verification of expression level of immunosuppressant composition mRNA in dendritic cells and influence on dendritic cell phenotype

1. In vitro induction culture of DC cells

Aseptically extracting 50ml of healthy human venous blood, separating peripheral blood mononuclear cells by using lymphocyte separation liquid in an ultraclean workbench, adding the mononuclear cells into an AIM-V culture medium, and placing the AIM-V culture medium into a 37 ℃ and 5% CO2 culture box for incubation so as to adhere the mononuclear cells to the wall. After 2h, nonadherent cells were removed and adherent cells were cultured in iDC medium (GM-CSF was added to AIM-V medium to a final concentration of 800U/mL and IL-4 was added to 500U/mL) at 37 ℃ in a 5% CO2 incubator for 6 days. Half of the cell culture medium was transferred to a centrifuge tube, and 500g of the medium was centrifuged to collect cells, the supernatant was removed, and an equal volume of fresh mDC medium (the formulation of the fresh mDC medium: 1600U/mL GM-CSF and 1000U/mL IL-4, TNF-a (5 ng/mL), IL-1 beta (5 ng/mL), IL-6 (150 ng/mL) and prostaglandin E2 (PGE 2) (1 ug/mL)) was added to resuspend the cells, which were then added to a flask and cultured for 8-18 hours to induce DC cell maturation.

2. Transfection of DC cells with immunosuppressant compositions

On the day of transfection, DC cells were digested into cell suspensions using non-enzymatic cell digestion reagents, centrifuged, washed twice with PBS, and resuspended in PBS to adjust cell density to 25-30X 106 DCs/ml. The DC cells were mixed with soluble protein (sPD-1-Fc, sCD80-Fc, sTGFBR3-Fc or PD-1-CD 80) mRNA at a rate of 4ug mRNA per 106 DC cells transfected, the cell-mRNA mixture was added to the electric rotor, and the antigen mRNA was transfected into the DC cells using an ECM630 electric rotor. The cells after electroporation were resuspended in cytokine-free AIM-V medium, adjusted to a cell density of 1X 106 DCs/ml, seeded into 96-well cell culture plates at a volume of 200. mu.l per well, and cultured in a 5% CO2 cell culture chamber at 37 ℃. GFP mRNA was transfected into DC cells under the same conditions as the control group.

3. Determination of transfection efficiency

After 24 hours of transfection, the proportion of DC cells expressing green fluorescent protein in all DC cells was analyzed by flow cytometry, and as shown in FIG. 1, the transfection efficiency of DC cells was more than 50% after 24 hours of transfection.

4. Soluble protein expression assay

After transfection, cell supernatants were collected at 24-hour intervals, and the concentrations of each soluble protein in the supernatants were measured using ELISA kits. The results of the detection are shown in FIG. 1. As a result, it can be seen that the mRNA encoding the soluble protein described in the present invention can express a higher level of the target protein in DC cells.

5. Identification of DC cell phenotype

Using direct immunofluorescence labeling, transfected DC cells were centrifuged, FACS buffer (2% FBS in PBS) was used to resuspend the DC cells at a cell concentration of 1X 106cells/ml, 100ul of transfected DC cell suspension was added to a flow cell tube, and 5ul of the corresponding antibodies CD80, CD83, CD86, and the corresponding isotype control, respectively, were added. Staining at 4 ℃ for 30min in the dark. 3ml of FACS Buffer was added to each tube to wash the cells, the supernatant was discarded, 500ul of FACS Buffer was added, and expression of CD80, CD83, and CD86 was detected by flow analysis. As shown in fig. 2, the cell surface molecules CD80, CD83, CD86 of the DC-immunomodulator composition were stably expressed without significant difference compared to untransfected DC cells.

Example three: effect of immunomodulator compositions on T cell response

1. In vitro induction culture of DC cells

Aseptically extracting 50ml of healthy human venous blood, separating peripheral blood mononuclear cells by using lymphocyte separation liquid in an ultraclean workbench, adding the mononuclear cells into an AIM-V culture medium, and placing the AIM-V culture medium into a 37 ℃ and 5% CO2 culture box for incubation so as to adhere the mononuclear cells to the wall. After 2h, nonadherent cells were removed and adherent cells were cultured in iDC medium (GM-CSF was added to AIM-V medium to a final concentration of 800U/mL and IL-4 was added to 500U/mL) at 37 ℃ in a 5% CO2 incubator for 6 days. Half of the cell culture medium was transferred to a centrifuge tube, and 500g of the medium was centrifuged to collect cells, the supernatant was removed, and an equal volume of fresh mDC medium (the formulation of the fresh mDC medium: 1600U/mL GM-CSF and 1000U/mL IL-4, TNF-a (5 ng/mL), IL-1 beta (5 ng/mL), IL-6 (150 ng/mL) and prostaglandin E2 (PGE 2) (1 ug/mL)) was added to resuspend the cells, which were then added to a flask and cultured for 8-18 hours to induce DC cell maturation.

2. Transfection of DC cells with immunosuppressant compositions

On the day of transfection, DC cells were digested into cell suspensions using non-enzymatic cell digestion reagents, centrifuged, washed twice with PBS, and resuspended in PBS to adjust cell density to 25-30X 106 DCs/ml. The DC cells and the antigenic mRNA were combined with soluble protein (sPD-1-Fc, sCD80-Fc, sTGFBR3-Fc or PD-1-CD 80) mRNA in a ratio of 4ug of mRNA transfected per 106 DC cells, the cell-mRNA mixture was added to an electric rotor, and the antigenic mRNA was transfected into the DC cells using an ECM630 electric transducer. The cells after electroporation were resuspended in a cytokine-free 1640 medium, the cell density was adjusted to 2X 105 DCs/ml, and the cells were cultured in a 5% CO2 cell culture chamber at 37 ℃ for 6 hours.

3. MNC cells recovered overnight were inoculated at a concentration of 2' 106/ml into 96-well plates, and activation of T lymphocytes was performed. The test grouping case is: a MNC blank control group, a MNC + antigen mRNA-DC vaccine group, a MNC + antigen/M immunomodulator composition mRNA-DC vaccine group and a MNC + PMA/Ionomycin positive control group; according to grouping conditions, DC cells loaded with corresponding mRNA are added into different holes, and the ratio of MNC to DC =10: 1; the concentration of Anti-CD3/Anti-CD28 in the positive control is 1 mu g/ml or the concentration of PMA/Ionomycin is 50ng/ml and 1 ug/ml; culturing at 37 ℃ for 10-12 days.

4. Adding 2 mu M monensin or 3 mu g/ml Brefeldin A into the cell culture solution 5-8h before collecting cells, and fully and uniformly mixing; (Monensin and Brefeldin A should not exceed 12h in cytosol as blockers of protein transport).

5. The cells were transferred to a flow tube, stained with fluorescently labeled antibodies to CD3, CD4, and CD8, fixed and permeabilized, and stained intracellularly with fluorescently labeled antibodies to TNF-a and IFN-r.

6. The ratio of TNF-a + and IFN-r + cells in lymphocytes was measured by flow cytometry.

As shown in FIG. 3, DC cells loaded with antigen mRNA, antigen mRNA plus an immunomodulator mRNA, or antigen/immunomodulator composition mRNA, all elicited an anti-tumor specific immune response in T lymphocytes after co-culture with T cells. Only in the group to which the immunomodulator composition was added (STGFBR 3+ PD1+ CD 80), the T lymphocytes had a stronger anti-tumor specific immune response. In the control group (pp 65 group), the proportion of cells secreting tumor necrosis factor (TNF-a) was 0.45%, whereas in the STGFBR3+ PD1+ CD80 group, the proportion was 2.28% which was 5.07 times that of the control group; whereas, when only a single modulator or a group of two modulators was transfected, the proportion of cells secreting tumor necrosis factor (TNF-a) was not significantly different from the control. Similarly, in the control group (pp 65 group), the proportion of cells secreting interferon gamma (IFN-r) was 0.42%, whereas in the STGFBR3+ PD1+ CD80 group, the proportion was 5.1% which was 12.14 times that of the control group; other groups transfected with either a single modulator or two modulators did not show significant differences in the proportion of tumor necrosis factor (TNF-a) secreting cells compared to the control. The data fully demonstrate that the immunomodulator combination provided by the invention can significantly enhance the antigen presenting capability of DC cells, stimulate the T lymphocytes and improve the anti-tumor specific immune response of the T lymphocytes.

Sequence listing

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<141> 2018-11-13

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Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln

165

<210> 2

<211> 242

<212> PRT

<213> Homo sapiens

<400> 2

Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr

1 5 10 15

Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys

20 25 30

Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu

35 40 45

Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile

50 55 60

Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp

65 70 75 80

Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr

85 90 95

Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly

100 105 110

Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg

115 120 125

Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr

130 135 140

Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile

145 150 155 160

Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu

165 170 175

Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp

180 185 190

Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met

195 200 205

Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg

210 215 220

Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro

225 230 235 240

Asp Asn

<210> 3

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 4

<211> 217

<212> PRT

<213> Homo sapiens

<400> 4

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215

<210> 5

<211> 646

<212> PRT

<213> Homo sapiens

<400> 5

Met Thr Ser His Tyr Val Ile Ala Ile Phe Ala Leu Met Ser Ser Cys

1 5 10 15

Leu Ala Thr Ala Gly Pro Glu Pro Gly Ala Leu Cys Glu Leu Ser Pro

20 25 30

Val Ser Ala Ser His Pro Val Gln Ala Leu Met Glu Ser Phe Thr Val

35 40 45

Leu Ser Gly Cys Ala Ser Arg Gly Thr Thr Gly Leu Pro Gln Glu Val

50 55 60

His Val Leu Asn Leu Arg Thr Ala Gly Gln Gly Pro Gly Gln Leu Gln

65 70 75 80

Arg Glu Val Thr Leu His Leu Asn Pro Ile Ser Ser Val His Ile His

85 90 95

His Lys Ser Val Val Phe Leu Leu Asn Ser Pro His Pro Leu Val Trp

100 105 110

His Leu Lys Thr Glu Arg Leu Ala Thr Gly Val Ser Arg Leu Phe Leu

115 120 125

Val Ser Glu Gly Ser Val Val Gln Phe Ser Ser Ala Asn Phe Ser Leu

130 135 140

Thr Ala Glu Thr Glu Glu Arg Asn Phe Pro His Gly Asn Glu His Leu

145 150 155 160

Leu Asn Trp Ala Arg Lys Glu Tyr Gly Ala Val Thr Ser Phe Thr Glu

165 170 175

Leu Lys Ile Ala Arg Asn Ile Tyr Ile Lys Val Gly Glu Asp Gln Val

180 185 190

Phe Pro Pro Lys Cys Asn Ile Gly Lys Asn Phe Leu Ser Leu Asn Tyr

195 200 205

Leu Ala Glu Tyr Leu Gln Pro Lys Ala Ala Glu Gly Cys Val Met Ser

210 215 220

Ser Gln Pro Gln Asn Glu Glu Val His Ile Ile Glu Leu Ile Thr Pro

225 230 235 240

Asn Ser Asn Pro Tyr Ser Ala Phe Gln Val Asp Ile Thr Ile Asp Ile

245 250 255

Arg Pro Ser Gln Glu Asp Leu Glu Val Val Lys Asn Leu Ile Leu Ile

260 265 270

Leu Lys Cys Lys Lys Ser Val Asn Trp Val Ile Lys Ser Phe Asp Val

275 280 285

Lys Gly Ser Leu Lys Ile Ile Ala Pro Asn Ser Ile Gly Phe Gly Lys

290 295 300

Glu Ser Glu Arg Ser Met Thr Met Thr Lys Ser Ile Arg Asp Asp Ile

305 310 315 320

Pro Ser Thr Gln Gly Asn Leu Val Lys Trp Ala Leu Asp Asn Gly Tyr

325 330 335

Ser Pro Ile Thr Ser Tyr Thr Met Ala Pro Val Ala Asn Arg Phe His

340 345 350

Leu Arg Leu Glu Asn Asn Ala Glu Glu Met Gly Asp Glu Glu Val His

355 360 365

Thr Ile Pro Pro Glu Leu Arg Ile Leu Leu Asp Pro Gly Ala Leu Pro

370 375 380

Ala Leu Gln Asn Pro Pro Ile Arg Gly Gly Glu Gly Gln Asn Gly Gly

385 390 395 400

Leu Pro Phe Pro Phe Pro Asp Ile Ser Arg Arg Val Trp Asn Glu Glu

405 410 415

Gly Glu Asp Gly Leu Pro Arg Pro Lys Asp Pro Val Ile Pro Ser Ile

420 425 430

Gln Leu Phe Pro Gly Leu Arg Glu Pro Glu Glu Val Gln Gly Ser Val

435 440 445

Asp Ile Ala Leu Ser Val Lys Cys Asp Asn Glu Lys Met Ile Val Ala

450 455 460

Val Glu Lys Asp Ser Phe Gln Ala Ser Gly Tyr Ser Gly Met Asp Val

465 470 475 480

Thr Leu Leu Asp Pro Thr Cys Lys Ala Lys Met Asn Gly Thr His Phe

485 490 495

Val Leu Glu Ser Pro Leu Asn Gly Cys Gly Thr Arg Pro Arg Trp Ser

500 505 510

Ala Leu Asp Gly Val Val Tyr Tyr Asn Ser Ile Val Ile Gln Val Pro

515 520 525

Ala Leu Gly Asp Ser Ser Gly Trp Pro Asp Gly Tyr Glu Asp Leu Glu

530 535 540

Ser Gly Asp Asn Gly Phe Pro Gly Asp Met Asp Glu Gly Asp Ala Ser

545 550 555 560

Leu Phe Thr Arg Pro Glu Ile Val Val Phe Asn Cys Ser Leu Gln Gln

565 570 575

Val Arg Asn Pro Ser Ser Phe Gln Glu Gln Pro His Gly Asn Ile Thr

580 585 590

Phe Asn Met Glu Leu Tyr Asn Thr Asp Leu Phe Leu Val Pro Ser Gln

595 600 605

Gly Val Phe Ser Val Pro Glu Asn Gly His Val Tyr Val Glu Val Ser

610 615 620

Val Thr Lys Ala Glu Gln Glu Leu Gly Phe Ala Ile Gln Thr Cys Phe

625 630 635 640

Ile Ser Pro Tyr Ser Asn

645

<210> 6

<211> 622

<212> PRT

<213> Homo sapiens

<400> 6

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Glu Pro Lys Ser Cys Asp Lys Thr His

165 170 175

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

180 185 190

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

195 200 205

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

210 215 220

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

225 230 235 240

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

245 250 255

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

260 265 270

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

275 280 285

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

290 295 300

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

305 310 315 320

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

325 330 335

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

340 345 350

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

355 360 365

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

370 375 380

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu

385 390 395 400

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Val Ile

405 410 415

His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys Gly His

420 425 430

Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys

435 440 445

Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn Ile Trp

450 455 460

Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser

465 470 475 480

Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys

485 490 495

Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala

500 505 510

Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser

515 520 525

Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr

530 535 540

Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu

545 550 555 560

Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu Thr Glu

565 570 575

Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr Asn His

580 585 590

Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn Gln Thr

595 600 605

Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

610 615 620

<210> 7

<211> 501

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

augcagaucc cacaggcgcc cuggccaguc gucugggcgg ugcuacaacu gggcuggcgg 60

ccaggauggu ucuuagacuc cccagacagg cccuggaacc cccccaccuu cuccccagcc 120

cugcucgugg ugaccgaagg ggacaacgcc accuucaccu gcagcuucuc caacacaucg 180

gagagcuucg ugcuaaacug guaccgcaug agccccagca accagacgga caagcuggcc 240

gccuuccccg aggaccgcag ccagcccggc caggacugcc gcuuccgugu cacacaacug 300

cccaacgggc gugacuucca caugagcgug gucagggccc ggcgcaauga cagcggcacc 360

uaccucugug gggccaucuc ccuggccccc aaggcgcaga ucaaagagag ccugcgggca 420

gagcucaggg ugacagagag aagggcagaa gugcccacag cccaccccag ccccucaccc 480

aggccagccg gccaguucca a 501

<210> 8

<211> 726

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

augggccaca cacggaggca gggaacauca ccauccaagu guccauaccu caauuucuuu 60

cagcucuugg ugcuggcugg ucuuucucac uucuguucag guguuaucca cgugaccaag 120

gaagugaaag aaguggcaac gcuguccugu ggucacaaug uuucuguuga agagcuggca 180

caaacucgca ucuacuggca aaaggagaag aaaauggugc ugacuaugau gucuggggac 240

augaauauau ggcccgagua caagaaccgg accaucuuug auaucacuaa uaaccucucc 300

auugugaucc uggcucugcg cccaucugac gagggcacau acgagugugu uguucugaag 360

uaugaaaaag acgcuuucaa gcgggaacac cuggcugaag ugacguuauc agucaaagcu 420

gacuucccua caccuaguau aucugacuuu gaaauuccaa cuucuaauau uagaaggaua 480

auuugcucaa ccucuggagg uuuuccagag ccucaccucu ccugguugga aaauggagaa 540

gaauuaaaug ccaucaacac aacaguuucc caagauccug aaacugagcu cuaugcuguu 600

agcagcaaac uggauuucaa uaugacaacc aaccacagcu ucaugugucu caucaaguau 660

ggacauuuaa gagugaauca gaccuucaac uggaauacaa ccaagcaaga gcauuuuccu 720

gauaac 726

<210> 9

<211> 45

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gagcccaaau cuugugacaa aacucacaca ugcccaccgu gccca 45

<210> 10

<211> 651

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gcaccugaac uccugggggg accgucaguc uuccucuucc ccccaaaacc caaggacacc 60

cucaugaucu cccggacccc ugaggucaca ugcguggugg uggacgugag ccacgaagac 120

ccugagguca aguucaacug guacguggac ggcguggagg ugcauaaugc caagacaaag 180

ccgcgggagg agcaguacaa cagcacguac cgugugguca gcguccucac cguccugcac 240

caggacuggc ugaauggcaa ggaguacaag ugcaaggucu ccaacaaagc ccucccagcc 300

cccaucgaga aaaccaucuc caaagccaaa gggcagcccc gagaaccaca gguguacacc 360

cugcccccau cccgggauga gcugaccaag aaccagguca gccugaccug ccuggucaaa 420

ggcuucuauc ccagcgacau cgccguggag ugggagagca augggcagcc ggagaacaac 480

uacaagacca cgccucccgu gcuggacucc gacggcuccu ucuuccucua cagcaagcuc 540

accguggaca agagcaggug gcagcagggg aacgucuucu caugcuccgu gaugcaugag 600

gcucugcaca accacuacac gcagaagagc cucucccugu cuccggguaa a 651

<210> 11

<211> 2004

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gcaccugaac uccugggggg accgucaguc uuccucuucc ccccaaaacc caaggacacc 60

cucaugaucu cccggacccc ugaggucaca ugcguggugg uggacgugag ccacgaagac 120

acagcaggac cagagccagg cgcccugugu gaacucagcc caguguccgc uucucaucca 180

gugcaggccc ugauggagag cuucacagug cugagcggcu gcgccagcag aggcacaaca 240

ggacugccuc aggaggugca cgugcugaac cugagaaccg caggacaggg accaggacag 300

cugcagaggg aagugacccu gcaccugaac cccaucagca gcgugcacau ccaccacaag 360

agcguggugu uccugcugaa cagcccucac ccacuggucu ggcaccugaa gaccgagaga 420

cuggcuacag gcguguccag acuguuccug guguccgaag gcagcguggu gcaguuuagc 480

agcgcuaacu ucagccugac cgccgaaacc gaggagagaa acuuccccca cggcaacgag 540

caccugcuga auugggccag gaaggaguac ggagccguga ccagcuucac cgagcugaag 600

aucgcccgga acaucuacau caaggucggc gaggaccagg uguucccacc caagugcaac 660

aucggcaaga acuuccugag ccugaacuac cuggccgagu aucugcagcc uaaagccgca 720

gagggcugcg ugaugucuag ccagccccag aacgaggagg ugcacaucau cgagcugauc 780

acccccaaca gcaaccccua cagcgccuuc cagguggaca ucaccaucga cauccggccu 840

agccaggagg aucuggaggu cgugaagaac cugauccuga uccucaagug caagaagagc 900

gugaauuggg ucaucaagag cuucgacgug aagggcagcc ugaagaucau cgcccccaac 960

agcaucggcu uuggcaaaga gagcgagcgg agcaugacca ugaccaagag cauccgggac 1020

gacauccccu cuacacaggg caaccucguc aagugggcac uggauaacgg cuacagcccu 1080

aucaccagcu acaccauggc cccaguggcc aacagauucc accugcggcu ggagaacaac 1140

gccgaagaga ugggcgacga ggaagugcac accaucccuc ccgagcugag aauccugcug 1200

gaccccggcg cccugccagc ucugcagaau ccuccuauua gaggcggcga gggacagaac 1260

ggaggacugc cuuucccuuu ccccgacauc agcaggagag uguggaacga ggagggcgaa 1320

gacggacugc cuagaccuaa ggaccccgug aucccuagca uccagcuguu cccaggccug 1380

agagagccag aggaagugca gggaagcgug gacaucgcuc ugagcgucaa gugcgacaac 1440

gagaagauga ucguggccgu ggagaaggac agcuuccagg cuagcggaua cagcggaaug 1500

gacgugaccc ugcuggaccc uacuugcaag gccaagauga acggcaccca cuucgugcug 1560

gagucccccc ugaacgguug cggcacaaga ccuaggugga gcgcucugga cggaguggug 1620

uacuacaacu ccaucgugau ccaggugccc gcucugggag auucuagcgg uuggccagac 1680

ggcuacgagg aucuggagag cggagacaac ggcuucccag gcgauaugga cgagggagac 1740

gcuucucugu ucaccaggcc cgagaucgug guguucaauu gcagccugca gcagguccgc 1800

aacccuucua gcuuccagga gcagccucac ggcaacauca ccuucaacau ggagcuguac 1860

aacaccgacc uguuccuggu gccaucacag ggaguguuca gcgugcccga gaacggacac 1920

guguacgugg agguguccgu gaccaaggca gaacaggagc ugggcuucgc cauccagacu 1980

ugcuucauca gccccuacag caac 2004

<210> 13

<211> 60

<212> RNA

<213> Xenopus laevis bufonid toad smooth (Xenopus laevis)

<400> 13

aagcuucuug uucuuuuugc agaagcucag aauaaacgcu caacuuuggc agaucugaac 60

<210> 12

<211> 88

<212> RNA

<213> Homo sapiens

<400> 12

gcuggagccu cgguagccgu uccuccugcc cgcugggccu cccaacgggc ccuccucccc 60

uccuugcacc ggcccuuccu ggucuuug 88

<210> 20

<211> 2005

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gagaccggcc ucgagcagcu gaagcuugca ugccugcagg ucgacucuag agccaccaug 60

agggcccugu gggugcuggg ccucugcugc guccugcuga ccuucggguc ggucagagcu 120

gacgaugaag uugaugugau ggagagccgg ggccggcggu gccccgaaau gaucagcgug 180

cugggaccaa uuuccggcca ugugcucaaa gccguguuua gccggggaga cacucccguc 240

cugccccaug aaaccagacu gcuccagacc ggcauccacg ugagagugag ccaacccucc 300

cugauccucg ucagccaaua cacccccgac agcacacccu gccaucgggg cgacaaucag 360

cugcaggugc agcacacuua uuucaccgga agcgaggugg agaacgucag cgugaacgug 420

cacaacccca ccgggcggag cauuugcccc ucccaggagc ccauguccau cuacguguac 480

gcccugcccc ugaagaugcu gaacauccca uccaucaacg ugcaccacua uccuuccgcc 540

gccgaacgga agcaucggca ccuccccguc gccgacgccg ugauccacgc cagcggcaag 600

caaauguggc aggcccggcu gaccgugagc ggccuggccu ggacccggca gcaaaaccag 660

uggaaggagc cugacgugua cuacacaagc gccuucgucu ucccaaccaa ggacgucgca 720

cugcggcacg uggugugcgc ccacgagcuc gucuguucca uggaaaacac cagagccacc 780

aaaaugcagg ugauuggcga ccaguacgug aagguguacc uggaguccuu cugcgaggac 840

gugcccuccg ggaagcucuu caugcacgug acccugggca gcgacgugga ggaggaccuc 900

accaugaccc ggaaccccca gcccuuuaug cggccccacg agcggaacgg cuucaccguc 960

cuguguccca agaacaugau caucaagccc ggcaagauca gccacaucau gcuggacgug 1020

gcuuucacca gccacgagca cuucgggcug cucugcccca aguccauccc cggccucagc 1080

aucagcggca aucuccucau gaacggccag cagaucuucc uggaggugca agccauccgg 1140

gagaccgucg agcugcggca guacgauccc guggccgccc uguucuucuu cgacaucgac 1200

cugcuccugc agcggggccc ccaauacagc gagcacccca ccuucaccag ccaguaccgg 1260

auucagggca agcucgagua ccggcacacc ugggaccggc acgacgaggg cgccgcccag 1320

ggagacgacg acguguggac cagcggcucc gacagcgacg aggagcucgu gacaacugag 1380

cggaagaccc cccgggucac cggcgggggc gccauggcca gcgccagcac cuccgccggc 1440

cggaaacgga agagcgccag cagcgcuacc gccugcaccg ccggcgugau gacacggggg 1500

cggcugaagg ccgagagcac cguggccccc gaggaggaca ccgacgaaga cuccgacaac 1560

gaaauccaca accccgccgu cuucaccugg ccccccuggc aagccggcau ccucgcccgg 1620

aaccuggucc ccauggucgc caccgugcag gggcaaaauc ugaaguacca ggaguucuuu 1680

ugggacgcaa acgacaucua ccggaucuuc gccgagcugg agggcgugug gcagcccgcc 1740

gcacagccca agagacggcg gcaccggcag gacgcccugc ccggcccaug caucgccagc 1800

acacccaaga agcauagagg ccugaucccc aucgcugugg guggugcccu ggcggggcug 1860

guccucaucg uccucaucgc cuaccucguc ggcaggaaga ggagucacgc aggcuaccag 1920

acuaucuagg aauucuuaau uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaaaaaaa aaaaaaaaaa aaaaa 2005

<210> 14

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca agagcccaaa tcttgtgaca aaactcacac atgcccaccg 540

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 600

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 660

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 720

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 780

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 840

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 900

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 960

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1020

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1080

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1140

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 1200

<210> 15

<211> 1425

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgggccaca cacggaggca gggaacatca ccatccaagt gtccatacct caatttcttt 60

cagctcttgg tgctggctgg tctttctcac ttctgttcag gtgttatcca cgtgaccaag 120

gaagtgaaag aagtggcaac gctgtcctgt ggtcacaatg tttctgttga agagctggca 180

caaactcgca tctactggca aaaggagaag aaaatggtgc tgactatgat gtctggggac 240

atgaatatat ggcccgagta caagaaccgg accatctttg atatcactaa taacctctcc 300

attgtgatcc tggctctgcg cccatctgac gagggcacat acgagtgtgt tgttctgaag 360

tatgaaaaag acgctttcaa gcgggaacac ctggctgaag tgacgttatc agtcaaagct 420

gacttcccta cacctagtat atctgacttt gaaattccaa cttctaatat tagaaggata 480

atttgctcaa cctctggagg ttttccagag cctcacctct cctggttgga aaatggagaa 540

gaattaaatg ccatcaacac aacagtttcc caagatcctg aaactgagct ctatgctgtt 600

agcagcaaac tggatttcaa tatgacaacc aaccacagct tcatgtgtct catcaagtat 660

ggacatttaa gagtgaatca gaccttcaac tggaatacaa ccaagcaaga gcattttcct 720

gataacgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 780

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 960

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1140

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1200

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1320

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380

aaccactaca cgcagaagag cctctccctg tctccgggta aatga 1425

<210> 16

<211> 2637

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgacttccc attatgtgat tgccatcttt gccctgatga gctcctgttt agccactgca 60

ggtccagagc ctggtgcact gtgtgaactg tcacctgtca gtgcctccca tcctgtccag 120

gccttgatgg agagcttcac tgttttgtca ggctgtgcca gcagaggcac aactgggctg 180

ccacaggagg tgcatgtcct gaatctccgc actgcgggcc aggggcctgg ccagctacag 240

agagaggtca cacttcacct gaatcccatc tcctcagtcc acatccacca caagtctgtt 300

gtgttcctgc tcaactcccc acaccccctg gtgtggcatc tgaagacaga gagacttgcc 360

actggggtct ccagactgtt tttggtgtct gagggttctg tggtccagtt ttcatcagca 420

aacttctcct tgacagcaga aacagaagaa aggaacttcc cccatggaaa tgaacatctg 480

ttaaattggg cccgaaaaga gtatggagca gttacttcat tcaccgaact caagatagca 540

agaaacattt atattaaagt gggggaagat caagtgttcc ctccaaagtg caacataggg 600

aagaattttc tctcactcaa ttaccttgct gagtaccttc aacccaaagc agcagaaggg 660

tgtgtgatgt ccagccagcc ccagaatgag gaagtacaca tcatcgagct aatcaccccc 720

aactctaacc cctacagtgc tttccaggtg gatataacaa ttgatataag accttctcaa 780

gaggatcttg aagtggtcaa aaatctcatc ctgatcttga agtgcaaaaa gtctgtcaac 840

tgggtgatca aatcttttga tgttaaggga agcctgaaaa ttattgctcc taacagtatt 900

ggctttggaa aagagagtga aagatctatg acaatgacca aatcaataag agatgacatt 960

ccttcaaccc aagggaatct ggtgaagtgg gctttggaca atggctatag tccaataact 1020

tcatacacaa tggctcctgt ggctaataga tttcatcttc ggcttgaaaa taatgcagag 1080

gagatgggag atgaggaagt ccacactatt cctcctgagc tacggatcct gctggaccct 1140

ggtgccctgc ctgccctgca gaacccgccc atccggggag gggaaggcca aaatggaggc 1200

cttccgtttc ctttcccaga tatttccagg agagtctgga atgaagaggg agaagatggg 1260

ctccctcggc caaaggaccc tgtcattccc agcatacaac tgtttcctgg tctcagagag 1320

ccagaagagg tgcaagggag cgtggatatt gccctgtctg tcaaatgtga caatgagaag 1380

atgatcgtgg ctgtagaaaa agattctttt caggccagtg gctactcggg gatggacgtc 1440

accctgttgg atcctacctg caaggccaag atgaatggca cacactttgt tttggagtct 1500

cctctgaatg gctgcggtac tcggccccgg tggtcagccc ttgatggtgt ggtctactat 1560

aactccattg tgatacaggt tccagccctt ggggacagta gtggttggcc agatggttat 1620

gaagatctgg agtcaggtga taatggattt ccgggagata tggatgaagg agatgcttcc 1680

ctgttcaccc gacctgaaat cgtggtgttt aattgcagcc ttcagcaggt gaggaacccc 1740

agcagcttcc aggaacagcc ccacggaaac atcaccttca acatggagct atacaacact 1800

gacctctttt tggtgccctc ccagggcgtc ttctctgtgc cagagaatgg acacgtttat 1860

gttgaggtat ctgttactaa ggctgaacaa gaactgggat ttgccatcca aacgtgcttt 1920

atctctccat attcgaacga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 1980

ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 2040

accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 2100

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 2160

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 2220

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 2280

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 2340

accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 2400

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 2460

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 2520

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 2580

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 2637

<210> 17

<211> 1869

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca agagcccaaa tcttgtgaca aaactcacac atgcccaccg 540

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 600

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 660

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 720

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 780

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 840

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 900

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 960

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1020

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1080

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1140

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaagag 1200

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagttatcca cgtgaccaag 1260

gaagtgaaag aagtggcaac gctgtcctgt ggtcacaatg tttctgttga agagctggca 1320

caaactcgca tctactggca aaaggagaag aaaatggtgc tgactatgat gtctggggac 1380

atgaatatat ggcccgagta caagaaccgg accatctttg atatcactaa taacctctcc 1440

attgtgatcc tggctctgcg cccatctgac gagggcacat acgagtgtgt tgttctgaag 1500

tatgaaaaag acgctttcaa gcgggaacac ctggctgaag tgacgttatc agtcaaagct 1560

gacttcccta cacctagtat atctgacttt gaaattccaa cttctaatat tagaaggata 1620

atttgctcaa cctctggagg ttttccagag cctcacctct cctggttgga aaatggagaa 1680

gaattaaatg ccatcaacac aacagtttcc caagatcctg aaactgagct ctatgctgtt 1740

agcagcaaac tggatttcaa tatgacaacc aaccacagct tcatgtgtct catcaagtat 1800

ggacatttaa gagtgaatca gaccttcaac tggaatacaa ccaagcaaga gcattttcct 1860

gataactga 1869

<210> 18

<211> 638

<212> DNA

<213> Encephalomyocarditis virus

<400> 18

cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tcatccaatt 60

ccgccccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt 120

gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct 180

ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa 240

ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg 300

tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc 360

caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg 420

agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg 480

aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc 540

tttacatgtg tttagtcgag gttaaaaaac gtctaggccc cccgaaccac ggggacgtgg 600

ttttcctttg aaaaacacga tgataatatg gccacaac 638

<210> 19

<211> 3283

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca agagcccaaa tcttgtgaca aaactcacac atgcccaccg 540

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 600

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 660

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 720

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 780

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 840

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 900

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 960

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1020

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1080

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1140

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 1200

gaattccgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatcat 1260

ccaattccgc cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt 1320

gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg 1380

aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga 1440

atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa 1500

acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc 1560

tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac 1620

gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag 1680

gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc 1740

acatgcttta catgtgttta gtcgaggtta aaaaacgtct aggccccccg aaccacgggg 1800

acgtggtttt cctttgaaaa acacgatgat aatatggcca caactgcaga tcggatccat 1860

gggccacaca cggaggcagg gaacatcacc atccaagtgt ccatacctca atttctttca 1920

gctcttggtg ctggctggtc tttctcactt ctgttcaggt gttatccacg tgaccaagga 1980

agtgaaagaa gtggcaacgc tgtcctgtgg tcacaatgtt tctgttgaag agctggcaca 2040

aactcgcatc tactggcaaa aggagaagaa aatggtgctg actatgatgt ctggggacat 2100

gaatatatgg cccgagtaca agaaccggac catctttgat atcactaata acctctccat 2160

tgtgatcctg gctctgcgcc catctgacga gggcacatac gagtgtgttg ttctgaagta 2220

tgaaaaagac gctttcaagc gggaacacct ggctgaagtg acgttatcag tcaaagctga 2280

cttccctaca cctagtatat ctgactttga aattccaact tctaatatta gaaggataat 2340

ttgctcaacc tctggaggtt ttccagagcc tcacctctcc tggttggaaa atggagaaga 2400

attaaatgcc atcaacacaa cagtttccca agatcctgaa actgagctct atgctgttag 2460

cagcaaactg gatttcaata tgacaaccaa ccacagcttc atgtgtctca tcaagtatgg 2520

acatttaaga gtgaatcaga ccttcaactg gaatacaacc aagcaagagc attttcctga 2580

taacgagccc aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact 2640

cctgggggga ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc 2700

ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa 2760

gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga 2820

gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct 2880

gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa 2940

aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc 3000

ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc 3060

cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac 3120

gcctcccgtg ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa 3180

gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa 3240

ccactacacg cagaagagcc tctccctgtc tccgggtaaa tga 3283

Claims (4)

1. An immunomodulatory composition for synergistically increasing the proportion of IFN- γ and/or TNF- α positive cells comprising soluble PD-1, soluble CD80 and soluble TGFBR3, or a nucleic acid molecule encoding said soluble PD-1, soluble CD80 and soluble TGFBR3, wherein:

(1) the soluble PD-1 consists of SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO: 4;

(2) the soluble CD80 consists of SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO: 4;

(3) the soluble TGFBR3 consists of SEQ ID NO: 5. SEQ ID NO:3 and SEQ ID NO:4, and (b) the amino acid sequence shown in the figure.

2. An immunomodulatory composition for synergistically increasing the proportion of IFN- γ and/or TNF- α positive cells comprising soluble PD-1, soluble CD80 and soluble TGFBR3 or nucleic acid molecules encoding said soluble PD-1, soluble CD80 and soluble TGFBR3 wherein said soluble PD-1 and CD80 molecules are a fusion protein consisting of the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4 and the soluble TGFBR3 consists of the amino acid sequence shown in SEQ ID NO: 5. SEQ ID NO:3 and SEQ ID NO:4, and (b) the amino acid sequence shown in the figure.

3. A vaccine composition comprising an immunomodulator composition according to any of claims 1 or 2.

4. Use of an agent for the preparation of an immunomodulating agent composition for synergistically increasing the proportion of IFN- γ and/or TNF- α positive cells, wherein said agent consists of the following (1) to (3):

(1) the amino acid sequence is SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO:4, soluble PD-1;

(2) the amino acid is SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, soluble CD 80;

(3) the amino acid is SEQ ID NO: 5. SEQ ID NO:3 and SEQ ID NO:4 soluble TGFBR 3.

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