CN111166876B - Immunopotentiator combination, encoding nucleic acid and application thereof - Google Patents

Abstract

The invention provides an immunopotentiator combination, which comprises Bcl2, CD40L, IL15 and IL15 receptor alpha (IL 15 Ra). 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 and SEQ ID NO 4. Vaccine compositions comprising the compositions and routes of delivery of the compositions are also provided. The immunopotentiator compositions of the present invention can provide protective immunity from pathogen infection and can be used for the prevention and/or treatment of a variety of tumors.

Description

Immunopotentiator combination, coding nucleic acid and application thereof

Technical Field

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

Background

The immune response of the body is first to capture antigen by Antigen Presenting Cells (APC), processed and processed to present antigen information to lymphocytes, and then to initiate a series of specific immune responses. Dendritic Cells (DC) are the APC considered to be the strongest in function at present, and the most important characteristic of the DC is that the DC can stimulate the proliferation and the activation of Naive T cells (nasal T cells) and is a central link for starting, regulating and maintaining specific immune response. In the anti-tumor immunity of the body, the cellular immunity mediated by T cells plays an important role.

Researches find that the tumor patients have the characteristics of reduced DC number and functional defects, and the number and the function of tumor tissues and DC infiltration around the tumor tissues have close relation with the occurrence, the development, the metastasis and the prognosis of tumors. The tumor densely infiltrated by the DC has high differentiation degree and better prognosis; whereas tumors that are mildly infiltrated by DCs are often associated with low differentiation and malignant progression. Tumor cells have high-level Fas expression, can induce apoptosis of lymphocytes expressed by FasL, and can secrete immunosuppressive cytokines such as TGF-beta, IL-10 and the like, so that the antigen presenting capability is reduced, and immune attack is avoided.

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 theory: antigen presenting cells in the patient, which fail to correctly recognize the tumor antigen, present it to T lymphocytes, and elicit a tumor-specific immune response. In recent years, increasing the number of antigen presenting cells, and improving the ability of antigen presenting cells, especially DC cells, to take up, transport, present antigen, and stimulate T cells, are a major focus in current tumor immunization research.

Numerous studies have shown that CD40-CD 40L-mediated signaling can induce APC activation. The latter plays an important role in the activation of tumor-specific immune killer T cells: provides an antigen stimulating signal and a second signal, and the two signals act synergistically to start immune cascade reaction and exert the tumor immune function. Meanwhile, the research finds that the CD40L can also maintain the activation and survival of DC cells and the expansion of CD8+ T cells, and can antagonize the inhibitory effect of IL-10 cytokines on the differentiation, maturation and function of the DC cells.

The CD40L molecule can interact with DC surface CD40 to activate DC, and promote APC co-stimulatory molecule expression and cytokine secretion. Activated DCs are the bridge between tumor cells and tumor-specific immune killer T cells, and can present tumor antigens to and activate T cells, producing tumor-specific killer T cells. Thus, activation of DCs via the CD40-CD40L signaling molecule pathway can enhance the presentation of poorly immunogenic tumor cell antigens, inducing tumor-specific immunity.

IL-15 is a cytokine with a structure similar to that of IL-2, and is widely expressed in various cells and tissues, such as monocytes, macrophages, dc cells, fibroblasts, and the like. IL-15 activates downstream JAK1, JAK3 by binding to IL-15 receptor α, leading to phosphorylation of downstream STAT3 and STAT5 and activation of signaling pathways, inducing phosphorylation of BCL2, MAP kinase pathway, lck and syk, leading to proliferation and maturation of cells.

IL-15 is capable of regulating T cell and NK cell activation and proliferation, and maintaining the survival of memory T cells in the absence of antigen stimulation. It has been demonstrated that IL-15 inhibits apoptosis by inducing BCL2L1/BCL-x (L) in rodent lymphocytes. Similarly, il-15 was also found to inhibit T lymphocyte apoptosis in humans by inducing BCL2 and/or Bcl-xL.

Recently, researchers have constructed a DNA tumor vaccine expressing recombinant Il-15 protein using NDV as a vector. Preclinical results show that this tumor vaccine has shown potential to control melanoma growth in a mouse model. Similarly, a recombinant vaccinia virus expressing influenza A protein and Il-15 could promote cross protection of CD4+ T cells. Another recombinant Brucella DNA vaccine containing the Il-15 gene was shown to enhance CD8+ T cell immune response in mice.

In recent years, there have been a number of reports of tumor treatment using antigen-loaded DC vaccines, and from the data reported so far, DC vaccines appear to represent a new and very promising approach for improved tumor immunotherapy. However, the use of DC vaccines alone generally does not lead to the desired improvement in immunotherapeutic effects and does not lead to satisfactory clinical results.

Disclosure of Invention

Therefore, the aim of the present method is to provide a safe and effective means for DC tumor vaccines, compositions enhancing DC vaccine function, in particular for the treatment of tumors and/or infectious diseases.

In particular, the object of the present invention is solved by providing a vaccine/immunomodulator combination comprising 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, as a vaccine, and BCL2, CD40L, IL-15 and IL-15Ra as modulators. In addition, the vaccine/modulator combination may be in the form of a mixture comprising nucleic acids or polypeptides of the combination, or a viral vector expressing the vaccine/modulator combination, or other forms including the vaccine/inhibitor component. 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, staphylococci, neisseria, escherichia coli, shigella, leishmania, respiratory syncytial virus, parainfluenza, adenovirus, varicella, flavivirus, mycobacterium tuberculosis, and malaria, among others.

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 consent 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 melanoma cells. 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, which are produced by mutation, are called neoantigens (neoantigen). These antigens are all recognized by cytotoxic T lymphocytes and cells presenting the antigen can be killed by T lymphocytes.

<xnotran> , : TDO2, MAGEC1, HMOX1, WT1, LY6H, AIM2, IDO1, CHI3L1, IL13RA2, LCK, GFAP, KIF20A, CNTN2, MUC16, PEG10, TNC, SOX11, IGF2BP3, S100A8, AKAP4, TTK, CHI3L2, PTHLH, CDC45, PMEL, TOP2A, PTTG1, NRCAM, HMMR, MUC4, LY6K, SOX10, FOSL1, PRAME, FOLR1, BIRC5, KIF2C, ITGAV, ART4, PROM1, CT83, S100A9, PPIB, S100A12, STAT1, EPCAM, ROR1, MLANA, KAAG1, KLK3, NT5E, PTPRZ1, SPAG4, MET, RGS1, CSPG4, PDCD1LG2, MUC1, CD274, PSCA, WDHD1, FABP7, PLIN2, PTGER2, HAVCR2, TPD52, ABCC3, TSSK6, ERBB2, CCDC110, TERT, CTAG2, SEC61G, ADORA2A, AURKB, ACPP, EPHX1, PTGS1, EZH2, PTGS2, PLK4, DDX43, PA2G4, PAX8, IDH1, SFMBT1, EPHA2, NAPSA, LPGAT1, NUF2, SPAG5, STAT3, MELK, ST8SIA1, EBAG9, KIFC3, CEACAM5, RPL19, SYCP2, DSE, ANKRD30A, TRAPPC1, RGS5, MGAT5, KRT19, B3GALNT1, CAGE1, AGER, ACRBP, LAG3, NELFA, RAB38, CCND1, SART1, UBE2V1, SLAMF7, KCNMA1, MUM1, HSPH1, GUCY1A3, AKAP13, SQSTM1, BCAN, CCNB1, TP53, SUGT1, AURKA, RAN, LY6D, NLGN4X, SART3, PRKDC, FOXP3, HBEGF, PIK3R1, SLC1A3, PCNA, KIF1C, BSG, ATP2A3, SPAG9, RPSA, NFYC, LRRC8A, IQGAP1, LY6E, TRIOBP, ART1, BAGE, BIRC7, CA9, CCDC54, DCT, IDO2, MAGED4, SOX2, SYCP1, TYR, 5T4, BAGE2, BORIS, CALR3, CSAG2, CSH1, </xnotran> CTAG1A, CTAG1B, CTAGE1, E6, FMR1NB, GAGE1, GAGE2A, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE8, GRDX, HNPRL, IGHD, MAGEA1, MAGEA10, MAGEA12, MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA9, MAGEB1, MAGEB2, MAGEC2, PAGE4, PAP, SAGE1, SPANXB1, SPO11, SSX1, SSX2, SSX4, brachyury/TFT, TTR, TYRP1, VSIR, XAGE1B, XAGE1E, ENAH, AKR1B10, GPC3, AFP, FLVCR1, FGF19, PSPH, ABL2, CCT3, SMYD3, TMEM106C, ZNF260, EPCAM, ICK, PHF20L1, ANXA3, ZNF623, CASK, FAM122B, IRS1, OTUD6B, TMEM68, VASH2, FGF3, TERF1, TSHZ2, TTC13, UTP14A.

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 its expression product may be expressed on the cell surface or inside the cell.

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

The immune modulator combination provided by the invention comprises BCL2 protein, CD40L protein, IL-15 protein and Il-15Ra protein. Wherein the CD40L molecule has an amino acid sequence comprising SEQ ID NO: 1; an Il-15 molecule having a sequence comprising SEQ ID NO: 2; an Il-15Ra molecule having a sequence comprising SEQ ID NO: 3; a BCL2 molecule having a sequence comprising SEQ ID NO: 4.

In one embodiment, nucleic acid molecules encoding CD40L, nucleic acid molecules encoding IL-15, and nucleic acid molecules encoding IL-15Ra are presented. The compositions of the invention may be encoded by separate nucleic acid molecules, or by one or two nucleic acid molecules, and the coding regions of the components of the compositions may be encoded by the nucleic acid molecules as set forth in SEQ ID NOs: 7 (i) may be linked by a nucleic acid sequence encoding a self-cleaving polypeptide sequence.

In one embodiment, a nucleic acid molecule encoding CD40L is provided having a nucleotide sequence comprising SEQ ID NO: 6; nucleic acid molecules encoding IL-15 and IL-15Ra are provided having a nucleotide sequence comprising SEQ ID NO:5, which molecule encodes both an IL-15 and an IL-15Ra molecule; nucleic acid molecules encoding BCL2 proteins are provided having a nucleic acid sequence comprising SEQ ID NO: 8.

The vaccine/modulator compositions of the present invention can be delivered to host DC cells in vivo by methods known in the art. In one embodiment, the vaccine/modulator compositions of the present invention may be introduced by viral vectors 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 compositions of the present invention can be introduced into the subject's own DC cells by electroporation, 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 is a graph showing the survival of DC cells transfected with nucleic acid molecules encoding proteins of the present composition, which shows significantly higher survival rates than control groups.

FIG. 2 is a graph showing the phenotype of DC cells transfected with nucleic acid molecules encoding proteins of the compositions of the present invention, wherein the CD80, CD86, and CD83 on the surface of mature DC cells (mDC-survivin vs. mDC-survivin/regulator nucleic acid) are significantly upregulated, as compared to Immature Dendritic Cells (iDC), indicating significant mature DC cell characteristics; the phenotype of the DC cells transfected with the survivin/modulator nucleic acid molecule is consistent with the phenotype of DC cells transfected with survivin antigen alone, as compared to DC cells transfected with survivin antigen alone.

FIG. 3 is a graph of the effect of immune modulator combinations on T cell responses, whereby DC cells transfected with survivin/modulator nucleic acid molecules are capable of eliciting a stronger T cell response than groups transfected with survivin antigen alone, and whereby the DC cells stimulated to have a higher proportion of TNF-a and IFN-r expression.

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

A DNA sequence encoding mRNA for tumor antigen survivin protein, a DNA sequence encoding BCL2, CD40L, IL-15, and IL-15Ra in the present invention, and a subsequent in vitro transcription reaction were constructed for this example. Constructs were made by codon optimization to introduce a high GC sequence to stabilize the synthesized 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.

The second embodiment: effect of immunosuppressant composition mRNA on dendritic cell phenotype and survival

1. In vitro induction culture of DC cells

Sterile 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 putting the AIM-V culture medium into a 37 ℃ and 5% CO2 incubator for incubation so as to adhere the mononuclear cells to the wall. After 2h, nonadherent cells were removed, adherent cells were added to iDC medium (GM-CSF was added to AIM-V medium to a final concentration of 800U/mL, IL-4 was added to 500U/mL), and cultured in a 5% CO2 incubator at 37 ℃ 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 thereto, and after resuspension of the cells, the cells were added to a flask and cultured for 8 to 18 hours to induce maturation of the DC cells.

2. Transfection of DC cells with immunosuppressant compositions

On the day of transfection, DC cells were digested into cell suspension with nonenzymatic cell-digesting agents, centrifuged, washed twice with PBS, resuspended in PBS, and adjusted to a cell density of 25-30X 10 ⁶ DCs/ml. According to each 10 ⁶ Transfection of DC cells to 4ug of mRNA, mixing of DC cellsAnd the modulator nucleic acid molecules (BCL 2, CD40L and IL-15-IRES-IL15 Ra) mRNA described herein, the cell-mRNA mixture was added to an electric rotor, and the antigen mRNA was transfected into DC cells using an ECM630 electrotransfer. The cells after the electroporation were resuspended in cytokine-free AIM-V medium and the cell density was adjusted to 1X 10 ⁶ DCs/ml were seeded into 96 well cell culture plates at a volume of 200ul per well, placed at 37 ℃ in 5% CO ₂ And continuing culturing in the cell culture box. GFP mRNA was transfected into DC cells under the same conditions as the control group. The number of DC cells in the plates was recorded daily for 5 consecutive days.

3. Determination of transfection efficiency

24 hours after transfection, the proportion of DC cells expressing green fluorescent protein to all DC cells was analyzed by flow cytometry.

4. Identification of DC cell phenotype

Using direct immunofluorescence labeling, transfected DC cells were centrifuged and the cells were resuspended in FACS buffer (2% FBS in PBS) at a cell concentration of 1X 10 ⁶ cells/ml, 100ul of transfected DC cell suspension was added to the flow cell tube, and 5ul of the corresponding antibodies CD80, CD83, CD86, and isotype control were added. Staining for 30min at 4 ℃ in 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 the expression of CD80, CD83, and CD86 was detected by flow analysis.

As shown in FIG. 1, DC cells transfected with the immunomodulator composition exhibited better cell viability than the untransfected DC cell control.

As shown in FIG. 2, there was no significant difference in the stable expression of the cell surface molecules CD80, CD83, CD86 in DC cells transfected with the immunomodulator composition compared to the untransfected DC cell control.

Example three: effect of immunomodulator compositions on T cell response

1. In vitro induction culture of DC cells

Sterile 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 putting the AIM-V culture medium into a 37 ℃ and 5% CO2 incubator for incubation so as to adhere the mononuclear cells to the wall. After 2h, nonadherent cells were removed, adherent cells were added to iDC medium (GM-CSF was added to AIM-V medium to a final concentration of 800U/mL, IL-4 was added to 500U/mL), and cultured in a 5% CO2 incubator at 37 ℃ 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 thereto, and after resuspension of the cells, the cells were added to a flask and cultured for 8 to 18 hours to induce maturation of the DC cells.

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, resuspended in PBS, and adjusted to a cell density of 25-30X 10 ⁶ DCs/ml. According to each 10 ⁶ Transfection of DC cells with 4ug of mRNA ratio, mixing DC cells with antigen mRNA and the regulator composition of the invention (Bcl 2, CD40L, IL15, and IL15 Ra) mRNA combination, adding the cell-mRNA mixture to an electric rotor, and transfecting the antigen mRNA into the DC cells using an ECM630 electric rotor. The cells after the electroporation were resuspended in a cytokine-free 1640 medium, and the cell density was adjusted to 2X10 ⁵ DCs/ml were placed in a 5% CO2 cell incubator at 37 ℃ for further 6 hours.

3. Recovering overnight MNC cells at 2x10 ⁶ The cells were seeded in 96-well plates at a concentration of/ml for T lymphocyte activation. The test grouping case is: a MNC blank control group, a MNC + antigen mRNA-DC vaccine group, a MNC + antigen/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 MNC is DC = 10; 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 1ug/ml; culturing at 37 ℃ for 10 to 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, 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, the use of either antigen mRNA alone or antigen/immunomodulator composition mRNA can elicit an anti-tumor specific immune response in T lymphocytes. In the control group transfected with mRNA for survivin antigen alone, the proportion of IFN-r positive T lymphocytes was 0.35%, whereas in the group transfected with mRNA for the antigen/immunomodulator composition, the proportion of IFN-r positive T lymphocytes was 1.96% which was 5.60 times that of the control group; in the control group transfected with mRNA for survivin antigen alone, the proportion of T lymphocytes positive to TNF-a was 1.86%, whereas in the group transfected with mRNA for the antigen/immunomodulator composition, the proportion of T lymphocytes positive to TNF-a was 3.26%, which was 1.75 times that of the control group, and was significantly different. The experimental result shows that the immunomodulator composition can obviously enhance the antigen presenting and activating capacities of DC cells on T lymphocytes, better stimulate the T lymphocytes and generate stronger anti-tumor specific immune response.

Sequence listing

<120> an immunomodulator composition, nucleic acid encoding same and use thereof

<141> 2018-11-12

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 261

<212> PRT

<213> Homo sapiens

<400> 1

Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly

1 5 10 15

Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu

20 25 30

Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg

35 40 45

Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val

50 55 60

Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser

65 70 75 80

Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys

85 90 95

Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu

100 105 110

Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser

115 120 125

Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly

130 135 140

Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln

145 150 155 160

Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr

165 170 175

Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser

180 185 190

Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala

195 200 205

Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His

210 215 220

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

225 230 235 240

Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe

245 250 255

Gly Leu Leu Lys Leu

260

<210> 2

<211> 162

<212> PRT

<213> Homo sapiens

<400> 2

Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr

1 5 10 15

Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His

20 25 30

Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala

35 40 45

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

50 55 60

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

65 70 75 80

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

85 90 95

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

100 105 110

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

115 120 125

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

130 135 140

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

145 150 155 160

Thr Ser

<210> 3

<211> 267

<212> PRT

<213> Homo sapiens

<400> 3

Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr

20 25 30

Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser

35 40 45

Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys

50 55 60

Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala

65 70 75 80

Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp

85 90 95

Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr

100 105 110

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

115 120 125

Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala

130 135 140

Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr

145 150 155 160

Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser

165 170 175

Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln

180 185 190

Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile

195 200 205

Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu

210 215 220

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

225 230 235 240

Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg

245 250 255

Asp Glu Asp Leu Glu Asn Cys Ser His His Leu

260 265

<210> 8

<211> 239

<212> PRT

<213> Homo sapiens

<400> 8

Met Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Arg Glu Ile Val Met

1 5 10 15

Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg Gly Tyr Glu Trp Asp Ala

20 25 30

Gly Asp Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Pro Gly Ile

35 40 45

Phe Ser Ser Gln Pro Gly His Thr Pro His Pro Ala Ala Ser Arg Asp

50 55 60

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

65 70 75 80

Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu Thr

85 90 95

Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Arg Asp Phe

100 105 110

Ala Glu Met Ser Ser Gln Leu His Leu Thr Pro Phe Thr Ala Arg Gly

115 120 125

Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg Asp Gly Val Asn Trp

130 135 140

Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu

145 150 155 160

Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp

165 170 175

Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp Ile Gln Asp Asn

180 185 190

Gly Gly Trp Asp Ala Phe Val Glu Leu Tyr Gly Pro Ser Met Arg Pro

195 200 205

Leu Phe Asp Phe Ser Trp Leu Ser Leu Lys Thr Leu Leu Ser Leu Ala

210 215 220

Leu Val Gly Ala Cys Ile Thr Leu Gly Ala Tyr Leu Gly His Lys

225 230 235

<210> 4

<211> 2316

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

agaccggccu cgagcagcug aagcuugcau gccugcaggu cgacucuaga ggauccgcca 60

ccaugagaau uucgaaacca cauuugagaa guauuuccau ccagugcuac uuguguuuac 120

uucuaaacag ucauuuucua acugaagcug gcauucaugu cuucauuuug ggcuguuuca 180

gugcagggcu uccuaaaaca gaagccaacu gggugaaugu aauaagugau uugaaaaaaa 240

uugaagaucu uauucaaucu augcauauug augcuacuuu auauacggaa agugauguuc 300

accccaguug caaaguaaca gcaaugaagu gcuuucucuu ggaguuacaa guuauuucac 360

uugaguccgg agaugcaagu auucaugaua caguagaaaa ucugaucauc cuagcaaaca 420

acaguuuguc uucuaauggg aauguaacag aaucuggaug caaagaaugu gaggaacugg 480

aggaaaaaaa uauuaaagaa uuuuugcaga guuuuguaca uauuguccaa auguucauca 540

acacuucuug agcggccgcc gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc 600

acgaccccau cauccaauuc cgcccccccc cccuaacguu acuggccgaa gccgcuugga 660

auaaggccgg ugugcguuug ucuauauguu auuuuccacc auauugccgu cuuuuggcaa 720

ugugagggcc cggaaaccug gcccugucuu cuugacgagc auuccuaggg gucuuucccc 780

ucucgccaaa ggaaugcaag gucuguugaa ugucgugaag gaagcaguuc cucuggaagc 840

uucuugaaga caaacaacgu cuguagcgac ccuuugcagg cagcggaacc ccccaccugg 900

cgacaggugc cucugcggcc aaaagccacg uguauaagau acaccugcaa aggcggcaca 960

accccagugc cacguuguga guuggauagu uguggaaaga gucaaauggc ucuccucaag 1020

cguauucaac aaggggcuga aggaugccca gaagguaccc cauuguaugg gaucugaucu 1080

ggggccucgg ugcacaugcu uuacaugugu uuagucgagg uuaaaaaacg ucuaggcccc 1140

ccgaaccacg gggacguggu uuuccuuuga aaaacacgau gauaauaugg ccacaacguc 1200

gacgccacca uggcuccuag gagagccaga ggguguagga cacugggacu gccagcucug 1260

cugcugcugc ugcugcugag accuccagcu acaaggggaa ucaccugccc uccuccuaug 1320

agcguggagc acgccgacau uugggugaag agcuacagcc uguacagccg ggagcgcuac 1380

auuugcaaca gcggcuucaa gaggaaggcc ggaacaagcu cucucaccga gugcgugcug 1440

aacaaggcca ccaacguggc ccauuggaca accccuagcc ugaagugcau cagggaccca 1500

gcacuggugc accagagacc agcuccuccu agcacaguga ccacagccgg agugacaccu 1560

cagccagaaa gccugagccc uagcggaaaa gaaccagccg ccucuagccc cagcagcaau 1620

aauaccgccg ccacaacagc cgcuauugug ccaggaagcc agcugaugcc uagcaagagc 1680

ccuagcaccg gcacaacaga gaucagcagc cacgagagca gccacggaac accuagccag 1740

accacagcca agaauuggga gcugaccgcc agcgccagcc accagccucc aggaguguac 1800

ccucagggac acagcgauac caccguggcc aucucuacca gcacagugcu gcugugcgga 1860

cugucagcug ugucccugcu ggcuugcuac cugaagagca gacagacccc uccucuggcc 1920

agcguggaaa uggaggcuau ggaggcccug ccagugacuu ggggaaccuc uagcagagac 1980

gaggaccugg agaauugcag ccaccaccug uaggaauucg cuggagccuc gguagccguu 2040

ccuccugccc gcugggccuc ccaacgggcc cuccuccccu ccuugcaccg gcccuuccug 2100

gucuuuggcu ggagccucgg uagccguucc uccugcccgc ugggccuccc aacgggcccu 2160

ccuccccucc uugcaccggc ccuuccuggu cuuugaaaaa aaaaaaaaaa aaaaaaaaaa 2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2316

<210> 5

<211> 946

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gagacaagcu ugcaugccug caggucgacu cuagaggauc caccggucgc caccaugauc 60

gaaacauaca accaaacuuc uccccgaucu gcggccacug gacugcccau cagcaugaaa 120

auuuuuaugu auuuacuuac uguuuuucuu aucacccaga ugauuggguc agcacuuuuu 180

gcuguguauc uucauagaag guuggacaag auagaagaug aaaggaaucu ucaugaagau 240

uuuguauuca ugaaaacgau acagagaugc aacacaggag aaagauccuu auccuuacug 300

aacugugagg agauuaaaag ccaguuugaa ggcuuuguga aggauauaau guuaaacaaa 360

gaggagacga agaaagaaaa cagcuuugaa augcaaaaag gugaucagaa uccucaaauu 420

gcggcacaug ucauaaguga ggccagcagu aaaacaacau cuguguuaca gugggcugaa 480

aaaggauacu acaccaugag caacaacuug guaacccugg aaaaugggaa acagcugacc 540

guuaaaagac aaggacucua uuauaucuau gcccaaguca ccuucuguuc caaucgggaa 600

gcuucgaguc aagcuccauu uauagccagc cucugccuaa agucccccgg uagauucgag 660

agaaucuuac ucagagcugc aaauacccac aguuccgcca aaccuugcgg gcaacaaucc 720

auucacuugg gaggaguauu ugaauugcaa ccaggugcuu cgguguuugu caaugugacu 780

gauccaagcc aagugagcca uggcacuggc uucacguccu uuggcuuacu caaacucuga 840

agcggccgcg gauccccggg uaccgagcuc gaauucuuaa uuaaaaaaaa aaaaaaaaaa 900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 946

<210> 6

<211> 552

<212> RNA

<213> Encephalomyocarditis virus

<400> 6

acguuacugg ccgaagccgc uuggaauaag gccggugugc guuugucuau auguuauuuu 60

ccaccauauu gccgucuuuu ggcaauguga gggcccggaa accuggcccu gucuucuuga 120

cgagcauucc uaggggucuu uccccucucg ccaaaggaau gcaaggucug uugaaugucg 180

ugaaggaagc aguuccucug gaagcuucuu gaagacaaac aacgucugua gcgacccuuu 240

gcaggcagcg gaacccccca ccuggcgaca ggugccucug cggccaaaag ccacguguau 300

aagauacacc ugcaaaggcg gcacaacccc agugccacgu ugugaguugg auaguugugg 360

aaagagucaa auggcucucc ucaagcguau ucaacaaggg gcugaaggau gcccagaagg 420

uaccccauug uaugggaucu gaucuggggc cucggugcac augcuuuaca uguguuuagu 480

cgagguuaaa aaacgucuag gccccccgaa ccacggggac gugguuuucc uuugaaaaac 540

acgaugauaa ua 552

<210> 7

<211> 842

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gagaccggcc ucgagcagcu gaagcuuccu gcaggucgac ucuagagcca ccauggcgca 60

cgcugggaga acaggguacg auaaccggga gauagugaug aaguacaucc auuauaagcu 120

gucgcagagg ggcuacgagu gggaugcggg agaugugggc gccgcgcccc cgggggccgc 180

ccccgcaccg ggcaucuucu ccucccagcc cgggcacacg ccccauccag ccgcaucccg 240

ggacccgguc gccaggaccu cgccgcugca gaccccggcu gcccccggcg ccgccgcggg 300

gccugcgcuc agcccggugc caccuguggu ccaccugacc cuccgccagg ccggcgacga 360

cuucucccgc cgcuaccgcc gcgacuucgc cgagaugucc agccagcugc accugacgcc 420

cuucaccgcg cggggacgcu uugccacggu gguggaggag cucuucaggg acggggugaa 480

cugggggagg auuguggccu ucuuugaguu cggugggguc augugugugg agagcgucaa 540

ccgggagaug ucgccccugg uggacaacau cgcccugugg augacugagu accugaaccg 600

gcaccugcac accuggaucc aggauaacgg aggcugggau gccuuugugg aacuguacgg 660

ccccagcaug cggccucugu uugauuucuc cuggcugucu cugaagacuc ugcucaguuu 720

ggcccuggug ggagcuugca ucacccuggg ugccuaucug ggccacaagu gauuaauuaa 780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840

aa 842

Claims (1)

1. Use of an immunopotentiator composition for the preparation of a dendritic cell reagent for increasing the proportion of IFN-. Gamma.and/or TNF-. Alpha.positive cells in T lymphocytes, wherein said immunopotentiator composition comprises nucleic acid molecules encoding a Bcl2 protein, a CD40L protein, an IL15 receptor alpha protein and a survivin antigen, wherein the amino acid sequence of CD40L is represented by SEQ ID NO. 1, the amino acid sequence of IL15 is represented by SEQ ID NO. 2, the amino acid sequence of IL15 receptor alpha is represented by SEQ ID NO. 3, and the amino acid sequence of Bcl2 is represented by SEQ ID NO. 4.

Images