Oligonucleotides with vaccine adjuvant and tumor therapeutic effects

Abstract

The present invention provides two single-stranded deoxyoligonucleotides, which can be used as adjuvants for microbial vaccines and tumor vaccines, exhibit anti-infection and anti-tumor effects, and can be used alone or in combination with other anti-tumor preparations to treat tumors.

Description

Technical field:

[0001] The present invention relates to two oligonucleotides and their uses as vaccine adjuvants and in treating tumors. Background technology:

[0002] Transforming growth factor beta 2 (TGF-β2) is a member of the TGF-β Cytokine Superfamily. The TGF-β superfamily has 40 cytokines, among which TGF-β1, TGF-β2 and TGF-β3 are named after TGF [Rider CC, et al. Molecules. 2017 Apr 29; 22(5).pii: E713; Travis MA, et al. Annu Rev Immunol. 2014; 32: 51-82], which are also commonly referred to as TGF-β. After translation and synthesis, TGF-β1, TGF-β2 and TGF-β3 are all precursor molecules, which are composed of a signal peptide, an N-terminal fragment (latency-associated peptide, LAP) and a C-terminal fragment (short C-terminal fragment). The released C-terminal fragment forms a dimer, which is the mature biologically active cytokine TGF-β. At the amino acid level, TGF-β1, TGF-β2 and TGF-β3 have 71-79% homology [Travis MA, et al. Annu Rev Immunol. 2014; 32: 51-82].

[0003] The TGF-β receptor (TβR) that can transduce TGF-β signals is a tetramer containing two TβRI and two TβRII molecules. TβRI and TβRII are serine / threonine kinases. Once the tetramer complex is formed, TβRII kinase is activated, recognizes specific serine residues of TβRI, and activates it [Ahmed S, et al. J Clin Med. 2017 Jan 5; 6(1). pii: E5]. The signal transduction activated by TGF-β2 depends on β-glycan, which is also called TGF-βRIII. β-glycan assists TGF-β2 to bind to TGF-βRII [Travis MA, et al. Annu Rev Immunol. 2014; 32: 51-82]. After activation, TGF-β receptor transduces signals through Smad proteins. There are three types of Smad proteins, receptor-regulated Smads (R-Smad), common mediator Smad (co-Smad) and inhibitory Smad (I-Smad). Smad2 and Smad3 are R-Smads, which are direct substrates of TβRI. TGF-β binds to and activates the TGF-β receptor. The intracellular end of the activated TGF-βRI recruits Smad2 and Smad3 and directly phosphorylates them. Phosphorylated Smad2 / 3 and Smad4 (co-Smad) form a trimer (Smad complex). The Smad complex translocates to the nucleus and regulates the expression of multiple genes. Activated I-Smad (Smad6 / 7) negatively regulates TGF-β signal transduction by promoting TβRI degradation, inhibiting Smad2 / Smad3 phosphorylation, and inhibiting Smad complex binding to chromatin [Ahmed S, et al. J Clin Med. 2017 Jan 5; 6(1). pii: E5; Travis MA, et al. Annu Rev Immunol. 2014; 32: 51-82]. TGF-β can transduce activation signals through the canonical pathway and the noncanonical pathway [Huang JJ, et al. Biochem Soc Trans. 2016 Oct 15; 44(5): 1441-1454].In humans and mice, TβRIII is mainly expressed in macrophages, neutrophils, megakaryocytes, dendritic cells and T cells [Ortega-Francisco S, et al. BiochemBiophys Res Commun. 2017 Dec 9; 494(1-2): 82-87], suggesting that these cells are also target cells of TGF-β2.

[0004] TGF-β has a negative regulatory effect on immune response, regulating a variety of adaptive immune response cells and innate immune response cells, including dendritic cells, T cells, B cells, NK cells, innate lymphoid cells and granulocytes [Kelly A, et al. Adv Immunol. 2017; 134: 137-233]. TGF-β2 downregulates the expression of major histocompatibility complex (MHC) molecules. Under in vitro culture conditions, TGF-β2 (1, 5, or 10 ng / ml) significantly downregulates MHC-I and MHC-II molecules on the surface of horse mesenchymal stem cells (MSCs), and can also partially antagonize the upregulation of IFN-γ on MSCs MHC-I and MHC-II molecules [Berglund AK, et al. Front Vet Sci. 2017 Jun 12; 4: 84]. In experimental allergic encephalomyelitis (EAE) mice, TGF-β2 significantly down-regulated the expression of MHC-II molecules by microglia [De Feo D, et al. J Clin Invest. 2017 Nov 1; 127(11): 3937-3953]. TGF-β2 can antagonize the induction of IFN-γ and TNF-d on the expression of MHC-II molecules by rat astrocytes [Schluesener HJ. J Neuroimmunol (1990) 27: 41-7]. TGF-β2 interferes with the function of antigen presenting cells. Bone marrow-derived dendritic cells (BMDCs) express TβR. Under in vitro culture conditions, TGF-β2 significantly inhibited CD40L-stimulated BMDCs to express and secrete IL-23 [De Feo D, et al. J Clin Invest. 2017 Nov 1; 127(11): 3937-3953]. This inhibition was almost completely eliminated by TGFBRI or TGFBRII tyrosine kinase inhibitor SB431542 [De Feo D, et al. J Clin Invest. 2017 Nov 1; 127(11): 3937-3953]. IL-23 is a mucosal immune inducer. In mice, whole influenza virus vaccine (WIV) (IL-23-WIV) supplemented with IL-23 significantly improved the efficacy of inducing virus-specific nasal IgA through mucosal immunization.High levels of anti-influenza virus IgA can be found in the serum and nasal lavage fluid of IL-23-WIV-immunized mice. This suggests that inhibition of TGF-β2 may play an adjuvant role by relieving the inhibition of IL-23 secretion. TGF-β2 inhibits the expression of cytokines. Under basal and inflammatory conditions, TGF-β2 inhibits the expression of IL8, CXCL5, CXCL1, chemokine (CC motif) ligand 20 (CCL20), IL1A, IL1B by keratinocytes [Li D, et al. J Clin Invest. 2015 Aug 3; 125(8): 3008-26]. In the immature small intestine, TGF-β2 attenuates the biological activity of LPS-induced IL-6, IL-1β and TNF-α [Nguyen DN, et al. Am J Physiol Gastrointest Liver Physiol. 2014 Oct 1; 307(7): G689-99]. TGF-β2 induces regulatory T cells (Treg). Antigen presenting cells (APCs) expressing TGF-β2 in mucosal and ocular tissues can induce Treg [Mir FA, et al. Immunology. 2015 Dec; 146(4): 547-56]. Anti-TβRIII blocking experiments have shown that TβRIII-mediated signals promote TGFβ-dependent CD4 + T cell differentiation [Ortega-Francisco S, et al. Biochem Biophys Res Commun. 2017 Dec 9; 494(1-2): 82-87].

[0005] TGF-β2 is a tumor-promoting factor. 95% of high-grade glioma cells express high levels of TGF-β2, and its expression level is positively correlated with the invasiveness of glioma cells [Zhang C, et al. J Exp Clin Cancer Res. 2017 Nov 16; 36(1): 162]. TGF-β2 is named as glioblastoma-derived T-cell suppressor factor. In glioma patients, TGF-β2 induces an immunosuppressive state and a loss of immune surveillance function. Glioma patients with high levels of TGF-β2 in the blood have accelerated disease progression and poor prognosis [Hau P, et al. Curr Pharm Biotechnol. 2011 Dec; 12(12): 2150-7]. The level of TGF-β2 in bladder cancer tissue is positively correlated with the degree of muscle infiltration of bladder cancer. TGF-β2 can be used as a biomarker for early diagnosis of tumors [Mahdavinezhad A, et al. Investig Clin Urol. 2017 Mar; 58(2): 140-145]. In oral cancer, TGF-β2 secreted by cancer-associated fibroblasts may maintain the malignant phenotype of oral cancer cells by weakening the adhesion between glial cells [Cirillo N, et al. Carcinogenesis. 2017 Jan; 38(1): 76-85]. TGF-β-2 induces epithelial-mesenchymal transition. Epithelial-mesenchymal transition (EMT) is a key process for tumor cell invasion [Cirillo N, et al. Carcinogenesis. 2017 Jan; 38(1): 76-85] and migration. Summary of the invention:

[0006] 1. The present invention provides two single-stranded deoxy oligonucleotides (oligonucleotides for short), the sequences of which are shown in the sequence table <400> 1 and <400> 2. They can reduce transforming growth factor beta 2 (TGF-β2) mRNA, inhibit the expression of TGF-β2 protein, and weaken and inhibit the immunosuppressive effect mediated by TGF-β2, thereby enhancing the individual's immune response to pathogenic microorganisms and tumor cells, showing anti-infection and anti-tumor effects.

[0007] 2. These two oligonucleotides can undergo various chemical modifications or be restructured.

[0008] 3. These two oligonucleotides can be used as adjuvants for pathogenic microorganism vaccines and tumor vaccines, and have anti-infection and anti-tumor effects.

[0009] 4. These two oligonucleotides can be used for tumor treatment.

[0010] 5. These two oligonucleotides can be used in combination with other adjuvants or innate immune enhancers to enhance an individual's protective immune response against pathogenic microorganisms and tumor cells.

[0011] 6. These two oligonucleotides can be used in combination with other anti-tumor agents and tumor therapeutic cells to treat tumors.

[0012] Terms used in the invention:

[0013] Unless otherwise specified, the terms in the present invention have the common meanings that can be understood by those skilled in the art. If there is a conflict in meaning, the interpretation, definition or description in the present invention shall prevail.

[0014] "Oligonucleotide": An oligonucleotide is a molecule composed of multiple nucleotides, and the number of nucleotides can be several or dozens. Nucleotide is the basic unit of nucleic acid and oligonucleotide. Nucleotide is composed of nucleoside and phosphate. Nucleoside is composed of pentose and base. Pentose includes ribose and deoxyribose. Pentose molecules and bases are linked to form nucleosides. Nucleosides are linked by phosphate groups to form nucleotides. Nucleotides are linked by phosphodiester bonds to form oligonucleotides. The bases that make up nucleosides include pyrimidine and purine. Pyrimidine includes thymine (abbreviated as T or t) and cytosine (abbreviated as C or c). Purine includes adenine (abbreviated as A or a) and guanine (abbreviated as G or g). The base in the oligonucleotide can be a rare base. Rare bases include, but are not limited to, 5-hydroxymethylcytosine, 7-methylguanine and 5-hydroxymethylcytosine. Oligonucleotide can be a molecule of single-strand, double-strand, circular or circular structure. In the present invention, oligonucleotide (Oligonucleotide, ODN) can be replaced by its English abbreviation ODN. The nucleotide sequence in the oligonucleotide constitutes its primary structure, and this order is also called nucleotide sequence. Nucleotide sequence can be represented by base sequence, therefore, the sequence of nucleotides is also called base sequence. The sequence of deoxyoligonucleotide can be represented by the English abbreviation of base, T or t represents thymine, C or c represents cytosine, A or a represents adenine, and G or g represents guanine.

[0015] "Oligonucleotides provided by the present invention" refers to the two oligonucleotides described in Example 1, which are named TIO1 and TIO3. TIO1 has the sequence shown in the sequence table <400> 1, TIO3 has the sequence shown in the sequence table <400> 2. The oligonucleotide provided by the present invention is a single-stranded deoxy oligonucleotide, referred to as oligonucleotide (ODN).

[0016] "Chemical modification": Compared with natural DNA, the oligonucleotides provided by the present invention can be chemically modified (chemical modification). Chemical modification of oligonucleotides is a phenomenon or method in which the covalent structure of the oligonucleotides is changed by introducing or removing any chemical gene. The chemical modification sites of oligonucleotides can occur at the phosphodiester bond, ribose and base. Chemical modification of oligonucleotides can occur at the 5' end or the 3' end, and can be performed during or after synthesis. The chemical modifications involved in the present invention include, but are not limited to, modifications to the oligonucleotide backbone, such as thio modification (the non-bridging oxygen atom of the phosphate in the phosphodiester bond between nucleotides is replaced by a sulfur atom) and substitution modification (including substitution of alkyl, aromatic or any other chemical groups). Chemical modification of oligonucleotides also includes base substitution and base modification, and the substituted base can be a rare base or a derivative of various bases. Chemical modification of oligonucleotides also includes connecting one or more nucleotides and / or any other chemical groups at its 5' end and / or 3' end.

[0017] "UTR": refers to the untranslated region (UTR) of an mRNA molecule, which is located at both ends of the coding region of the mRNA molecule. The one located at the 5' end is called the 5' UTR, and the one located at the 3' end is called the 3' UTR.

[0018] "TGF-β2 mediated immunosuppressive effect": The oligonucleotides provided by the present invention can inhibit or weaken the immunosuppressive effect mediated by TGF-β2. The immunosuppressive effect mediated by TGF-β2 refers to the inhibitory effect of TGF-β2 on the immune response induced by pathogenic microorganisms, and also refers to the inhibitory effect on the immune response of tumor cells. Reducing or interfering with TGF-β2 mRNA can inhibit the immunosuppressive effect mediated by TGF-β2; inhibiting or reducing the production of TGF-β2 protein can also inhibit the immunosuppressive effect mediated by TGF-β2. TGF-β2 is an immunosuppressive factor. Inhibiting the immunosuppressive effect mediated by TGF-β2 can enhance the individual's immune response to pathogenic microorganisms and produce an anti-infection effect. Inhibiting the immunosuppressive effect mediated by TGF-β2 can break the individual's immune tolerance to tumor cells and enhance the individual's anti-tumor ability. The anti-tumor effect can be produced by inhibiting the immunosuppressive effect mediated by TGF-β2 [Kim S, et al. Tumour Biol. 2016 Aug; 37(8): 11397-407].

[0019] "Reduce the level of TGF-β2 mRNA": The oligonucleotides provided by the present invention can reduce the level of TGF-β2 mRNA in immune cells. "Reduce" and "reduce" have the same meaning. Reducing the level of TGF-β2 mRNA can weaken and inhibit the immunosuppressive effect mediated by TGF-β2, thereby showing an enhancing effect on the immune response of pathogenic microorganisms and tumor cells. Preparations with reduced levels of TGF-β2 mRNA can become adjuvants for pathogenic microorganism vaccines or tumor vaccines, and can also be used alone for the treatment of tumors.

[0020] "Inhibit the expression of TGF-β2 protein": The oligonucleotides provided by the present invention can inhibit the expression of TGF-β2 protein. Inhibiting and reducing the production of TGF-β2 protein have the same meaning, and inhibiting the translation of TGF-β2 mRNA and inhibiting the expression of TGF-β2 protein also have the same meaning. The reduction of TGF-β2 protein can weaken and inhibit the immunosuppressive effect mediated by TGF-β2, thereby showing an enhancement effect on the immune response of pathogenic microorganisms and tumor cells. The preparation with the ability to inhibit the expression of TGF-β2 can be used as an adjuvant for pathogenic microorganism vaccines or tumor vaccines, and can also be used alone for the treatment of tumors.

[0021] "Immune response": The oligonucleotides provided by the present invention can enhance the immune response to pathogenic microorganism antigens and tumor antigens. Immune response and immune reaction have the same meaning. Immune response is the response of an individual's immune cells, including B lymphocytes, T lymphocytes, NK cells, γδT cells, NKT cells, dendritic cells, macrophages and granulocytes, to antigens or other stimuli [such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)]. The result of the immune response is the selective destruction or removal of invading pathogenic microorganisms or endogenous tumor cells. The immune response includes innate immune response and adaptive immune response. The adaptive immune response includes cellular immune response and humoral immune response. The immune response stimulated by a vaccine made of pathogenic microorganism antigens can enable the immunized individual to acquire the ability to resist infection by pathogenic microorganisms. The immune response stimulated by a vaccine made of tumor antigens can have a tumor therapeutic effect in the immunized individual. Promoting an individual's immune response to pathogenic microorganisms can have an anti-infection effect. Promoting an individual's immune response to tumor cells can have an anti-tumor effect. Cytokines with immunosuppressive effects are produced during the immune response process. TGF-β2 is a cytokine with immunosuppressive effects. TGF-β2 can inhibit immune responses. Reducing the level of TGF-β2 mRNA and inhibiting the expression of TGF-β2 can promote immune responses to pathogenic microbial antigens or tumor antigens, and exhibit immunopreventive or immunotherapeutic effects. The oligonucleotides provided by the present invention can be used as adjuvants for microbial vaccines and tumor vaccines or used in the treatment of tumors by enhancing the immune response to microbial antigens and tumor antigens.

[0022] "Lymphocyte": Lymphocyte refers to mononuclear leukocytes without phagocytic ability that exist in blood, lymph and lymphatic tissue, including B lymphocytes (also called B cells) and T lymphocytes (also called T cells). T cells can be divided into T cells that express CD4 molecules on their surface (CD4 + T cells) and T cells expressing CD8 molecules on their surface (CD8 + T cells).

[0023] "CD4 + T cells": The oligonucleotides provided by the present invention can increase the number of specific CD4 T cells in the draining lymph nodes. + The number of T cells. CD4 +T cells are T cells that express CD4 molecules on their surface and are called helper T lymphocytes (Th). Th cells help B cells produce antibodies and also help CD8 + T cells kill virus-infected cells and tumor cells. Stimulate CD4 + Agents that proliferate (increase in cell number) T cells can enhance an individual's immune response to pathogenic microorganisms and tumor cells.

[0024] “CD19 + "Lymphocytes": The oligonucleotides provided by the present invention can increase the specific CD19 in the draining lymph nodes + The number of lymphocytes. CD19 + Lymphocytes are lymphocytes that express CD19 molecules on their surface, also known as CD19 + B lymphocytes or CD19 + Cells. Stimulation of CD19 + Lymphocyte proliferation (as evidenced by an increase in cell number) preparations can enhance an individual's humoral immune response to pathogenic microorganisms and are used as adjuvants for vaccines against pathogenic microorganisms.

[0025] "Antigen presenting cells" The oligonucleotides provided by the present invention can increase the number of antigen presenting cells (APC) in the draining lymph nodes. APC is a type of immune cell that can present peptides from microbial antigens or tumor antigens on its surface to activate antigen-specific T cells, including dendritic cells (DC), macrophages and B lymphocytes. CD11c is a surface marker of DC. Increasing the number of APCs in the draining lymph nodes promotes the activation of antigen-specific T cells, thereby enhancing the immune response to pathogenic microorganisms and tumor cells.

[0026] "CD86": The oligonucleotides provided by the present invention can upregulate and maintain the expression of CD86 by dendritic cells. CD86 is a co-stimulatory molecule expressed on the surface of antigen presenting cells (APC) or tumor cells. In the process of immune response, CD86 can initiate a second signal to activate T lymphocytes. T lymphocyte activation is a key condition for stimulating an individual's immune response to pathogenic microorganisms and tumor cells. T lymphocyte activation requires two activation signals. The first activation signal comes from T lymphocytes recognizing antigen peptide-MHC complexes on the surface of APC or target cells through T cell antigen receptors (TCR). The second activation signal comes from T lymphocytes recognizing and binding to co-stimulatory molecules including CD86 on the surface of APC or target cells through CD28 molecules. The second activation signal is also called co-stimulatory signals. T lymphocytes that only receive the first activation signal cannot be fully activated and may even enter a state of immune tolerance. Only after receiving the first and second activation signals at the same time, can T lymphocytes be fully activated and function [Sharma P. et al. Science. 2015 Apr 3; 348 (6230): 56-61; Greenwald RJ, et al. Annu Rev Immunol. 2005; 23: 515-48]. TGF-β2 can reduce the costimulatory molecules of APC, thereby weakening the second signal of T lymphocyte activation. The level of TGF-β2 mRNA and the inhibition of TGF-β2 expression can maintain or increase the level of CD86, enhance the second signal of T cell activation, and then promote the immune response to pathogenic microorganisms and tumor cells, and enhance the individual's anti-infection and anti-tumor capabilities.

[0027] "CD28": CD28 is a protein expressed on the surface of T cells that can recognize CD86 molecules on the surface of antigen-presenting cells. After recognizing CD86 molecules, CD28 provides co-stimulatory signals (also known as the second signal) to T cells.

[0028] "T cell receptor": T cell receptor (TCR) is an antigen receptor expressed on the surface of T cells and is a heterodimeric transmembrane protein. TCR consists of a variable (V) region and a constant (C) region. The V region is the domain that TCR recognizes antigen epitopes. TCR cannot directly recognize antigen epitopes, but can only recognize the antigen peptide-MHC (major histocompatibility complex) molecule complex on the surface of antigen presenting cells or target cells.

[0029] "MHC molecule": MHC molecules are protein molecules encoded by major histocompatibility complex (MHC) genes. MHC molecules include MHC class I molecules and MHC class II molecules.

[0030] "Activation of T lymphocytes": T lymphocyte activation requires two signals. The first signal is through its TCR recognition of APC or target cells (can be CD8 + The first signal is obtained by the antigen peptide-MHC molecule complex on the surface of the cell killed by the T cell; the second signal, also known as the co-stimulatory signal, is obtained by its CD28 molecule recognizing the B7 molecule on the surface of the APC or target cell. Under the action of the dual signals, the T cell starts the activation signal transduction pathway, and then proliferates, differentiates and performs its function. Only the first signal without the second activation signal can make the T cell enter a non-responsive state. T lymphocyte response occurs after T lymphocyte activation.

[0031] "T lymphocyte response": T lymphocyte response and "T lymphocyte activity" or "functions performed by T lymphocytes" are interchangeable terms in the present invention. T lymphocyte response includes T lymphocyte proliferation and / or differentiation into helper T lymphocytes (Th), cytotoxic T lymphocytes (Tc) or regulatory T lymphocytes (Treg), and also includes Th providing signals to B lymphocytes to assist them in producing antibodies, Tc killing target cells and releasing soluble factors such as cytokines to regulate the functions of other immune cells. Th is CD4 + T cells, Tc is CD8 + T cells. After activation, CD4 + T cells assist B cells in producing antibodies, assisting CD8 + T cells kill target cells such as virus-infected cells and tumor cells. After activation, CD8 + T cells can kill virus-infected cells and tumor cells. Promoting and maintaining T cell activation will enhance the individual's immune response to microbial antigens and produce anti-infection effects, and will also promote the immune response of individual immune cells to tumor cells to produce tumor therapeutic effects. Reducing the level of TGF-β2 mRNA and inhibiting the expression of TGF-β2 can enhance T lymphocyte response. The oligonucleotides provided by the present invention can be used as adjuvants for pathogenic microorganism vaccines and tumor vaccines or used in the treatment of tumors by enhancing the immune response to pathogenic microorganisms and tumor cells by enhancing the T lymphocyte response.

[0032] "Subject": In the present invention, subject or individual refers to humans and non-human vertebrates.

[0033] "Target cell": Target cell refers to a cell in an individual that can be attacked, killed or acted upon by immune cells, and may be a tumor cell, a virus-infected cell, or a cell acted upon by the oligonucleotide provided by the present invention.

[0034] "Tumor": "Tumor" in the present invention refers to the tumor defined by modern medicine, which can be divided into benign tumors and malignant tumors. Tumor and cancer can be used interchangeably and have the same meaning. Tumors include solid tumors, soft tissue sarcomas, and tumors of the medullary or lymphoid systems. The oligonucleotides provided by the present invention can enhance the individual's immune response to tumor antigens to produce anti-tumor effects, and the tumors involved include but are not limited to: esophageal cancer, gallbladder cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, adrenal cortical tumors, kidney cancer, liver cancer, lung cancer, ovarian cancer, cervical cancer, uterine cancer, vaginal cancer, pancreatic cancer, rectal cancer, prostate cancer, gastric cancer, skin cancer, melanoma, brain tumor, glioma, bone tumor, sarcoma, penile cancer, retinoblastoma, leukemia, lymphoma and myeloma.

[0035] "Treatment": includes the use of the oligonucleotides provided by the present invention to prevent or delay the onset of symptoms and complications of a disease (such as a tumor). Treatment can also be preventive. The treatment of a tumor also refers to controlling the progression of a tumor in an individual, prolonging the survival of a tumor patient, improving the quality of life, alleviating symptoms, shrinking or even eliminating a tumor, and curbing tumor metastasis. In the present invention, "tumor treatment" and "anti-tumor effect" or "tumor treatment" have the same meaning. Anti-tumor effect includes the treatment of a tumor, and also includes the prevention of tumor occurrence, recurrence and metastasis. The oligonucleotides provided by the present invention can be used for the treatment of a tumor.

[0036] "Antigen": The oligonucleotides provided by the present invention can enhance the individual's immune response to antigens. Antigens are substances or molecules that can be recognized by B cell receptors or T cell receptors to stimulate the individual's adaptive immune response. Antigens can come from the outside of the individual, such as microbial antigens; they can also come from the inside of the individual, such as tumor antigens. Microbial antigens are substances or molecules on microorganisms that can be recognized by B cell receptors or T cell receptors to stimulate the individual's adaptive immune response. Vaccines prepared using microbial antigens can enable individuals to gain immunity to the microorganism after being applied to individuals, so that they are protected when they are exposed to the same or similar pathogens again. Vaccines made using tumor antigens can stimulate their adaptive immune response to tumor cells after being applied to individuals, thereby producing a tumor therapeutic effect. Antigens can be extracted from microorganisms or tumor cells, or they can be produced using recombinant DNA technology or synthesized by other methods. Tumor cell lysates contain a variety of tumor antigens and can be used as antigens in tumor vaccines.

[0037] "Vaccine": A vaccine is a biological product used for artificial active immunization, made from attenuated or killed pathogenic organisms or their components as antigens. Antigens and adjuvants are the main components of vaccines. The purpose of vaccination is to enable individuals to gain immunity to pathogens and thus be protected when they are exposed to the corresponding pathogens again. The oligonucleotides provided by the present invention can be used in combination with human vaccines to enhance their immune efficacy. These vaccines include but are not limited to vaccines that can prevent the following infectious diseases [Stanley A. Plotkin, Walter A. Orenstein, Paul A. Offit, Vaccines, Sixth Edition, An imprint of Elsevier Inc. 2013, ISBN-13: 9781455700905]: diphtheria, tetanus, yellow fever, pertussis, Haemophilus influenzae b infection, poliomyelitis, measles, mumps, rubella, typhus, rabies, rotavirus infection, hepatitis A, hepatitis B, hepatitis E, influenza, tuberculosis, malaria, cholera, chickenpox, epidemic encephalitis B, forest encephalitis, cholera, Lyme disease, pneumococcal infection, meningococcal infection, typhoid, smallpox, anthrax, Ebola virus infection, green monkey virus (Marburg viruses, MARV) infection and Venezuelan equine encephalitis virus (VEEV) infection. The vaccines involved in the present invention also include tumor vaccines. Tumor antigens and adjuvants are the main components of tumor vaccines. Tumor vaccines can treat or prevent tumors. The antigens in tumor vaccines can be tumor antigens, cells presenting tumor antigens, cells expressing tumor antigens or tumor cell lysates. The oligonucleotides provided by the present invention can be used in combination with tumor vaccines to enhance their effectiveness in preventing and / or treating tumors. Such vaccines include but are not limited to: human papillomavirus vaccines for preventing cervical cancer, Provenge vaccines for treating prostate cancer, vaccines made with various tumor antigens, vaccines made with various tumor cells and vaccines made with tumor cell lysates.The oligonucleotides provided by the present invention can be used in combination with animal vaccines to enhance their immune efficacy. These vaccines include but are not limited to vaccines that can prevent the following infectious diseases: swine foot-and-mouth disease, porcine blue ear disease (porcine reproductive and respiratory syndrome), swine fever, swine pseudorabies, porcine circovirus infection, porcine parvovirus infection, swine streptococci, porcine transmissible gastroenteritis, swine asthma, parasuis infection, swine erysipelas, swine plague, porcine atrophic rhinitis, porcine transmissible gastroenteritis, swine Japanese encephalitis, swine influenza, swine brucellosis, piglet diarrhea, swine parainfluenza virus infection, swine influenza, swine pneumonia Mycoplasma infection, piglet edema, swine cholera salmonella, porcine epidemic diarrhea, swine plague; bovine emphysematous anthrax, bovine anthrax, bovine epidemic fever, bovine pasteurellosis, bovine brucellosis infection, bovine tetanus, bovine Clostridium perfringens infection, bovine Clostridium bovis infection, bovine foot-and-mouth disease, bovine paratyphoid, cowpox, bovine diarrhea; sheep rapid disease, sheep anthrax, sheep enterotoxemia, lamb dysentery, Sheep black disease (infectious necrotizing hepatitis), goat pox, sheep tetanus, sheep anthrax, lamb Escherichia coli disease, sheep abortion chlamydia infection, sheep mouth sores, sheep infectious pleuropneumonia, sheep brucellosis, sheep botulism Clostridium poisoning, sheep pox, sheep streptococcosis, sheep rabies, sheep foot-and-mouth disease, sheep mouth sores, sheep blue tongue; horse infectious anemia, horse influenza; pig, horse, cattle, sheep and dog encephalitis; chicken Newcastle disease, chicken infectious bursal disease, chicken Marek's disease, avian influenza, avian cholera, fowl pox, chicken infectious bronchitis, chicken viral hepatitis, chicken egg drop syndrome; duck influenza, duck plague Riemerella vaccine, duck plague, duck infectious serositis, duck Escherichia coli disease; gosling plague, goose paramyxovirus disease, goose influenza; rabies, dog fever, Vaccines for canine parvovirus disease, canine leptospirosis, canine infectious hepatitis, canine infectious bronchitis, canine parainfluenza disease, canine encephalitis and kennel cough. The oligonucleotides provided by the present invention can enhance the efficacy of vaccines.

[0038] "Tumor antigen": The oligonucleotides provided by the present invention can be used as adjuvants for tumor antigens to enhance the immune response to tumor antigens. In the present invention, tumor antigen, tumor cell antigen and tumor associated antigen (TAA) can be used interchangeably and have the same meaning. Tumor antigens can stimulate anti-tumor immune responses, and immune responses against tumor antigens can produce tumor therapeutic effects. Tumor antigens include, but are not limited to, BAGE (B melanoma antigen), GAGE ​​(G antigen 12B / C / D / E), MAGE (melanoma antigen-encoding gene), NY-ESO-1; CEA (carcinoembryonic antigen), gp100 (glycoprotein 100), Melan-A (melanoma antigen recognized by T cells 1), PSA (prostate-specific antigen), tyrosinase, HER2 (human epidermal growth factor receptor 2, hTERT (telomerase transcriptase), p53, survivin, β-catenin-m, HSP70-2 / m (heat shock-related 70kDa protein 2mutated), KRAS, GM2 (ganglioside GM2), MUC1 (mucin-1), antigens encoded by hepatitis B virus genes, antigens encoded by hepatitis C virus genes, antigens encoded by human papillomavirus genes (such as L1 protein, E6 protein and E7 protein) and antigens encoded by Epstein Barr virus genes (Epstein Barr Virus peptides) [Zielinski C, et al. Nat Rev Clin Oncol. 2014 Sep; 11 (9): 509-24; Adams JL, et al. Nat Rev Drug Discov. 2015 Sep; 14 (9): 603-22]. Tumor antigens also include inactivated whole tumor cells and tumor cell lysates.Tumor antigens can be neo-antigens expressed due to gene mutations, or they can be complexes of idiotypic antigens of B cell tumors and heat shock protein tumor cell peptides extracted from tumor cells [Suot, R & Srivastava, P (1995) Science 269: 1585-1588; Tamura, Y. et al. (1997) Science 278: 117-120].

[0039] "Tumor vaccine" The oligonucleotides provided by the present invention can be used as adjuvants for tumor vaccines to enhance their efficacy in tumor treatment. Tumor vaccines refer to biological products that can induce individuals to produce anti-tumor adaptive immune responses. The tumor vaccines involved in the present invention include vaccines made in a certain dosage form using tumor antigens, tumor cell lysates, tumor cells and cells presenting tumor antigens. Such vaccines include but are not limited to the following [Zielinski C, et al. Nat Rev Clin Oncol. 2014 Sep; 11(9): 509-24]: Tumor vaccines for the treatment of prostate cancer, the tumor antigens used include prostatic acid phosphatase (used for sipuleucel-T), and PSA; tumor vaccines for the treatment of breast cancer, using tumor antigens including HER2-derived peptides, MUC1 and WT1 (Wilms tumour protein) antigens; tumor vaccines for the treatment of lung cancer, using tumor antigens including MUC1, melanoma-associated antigen-3 (MAGE-A3) peptide, telomerase reverse transcriptase and EGF; vaccines for the treatment of melanoma, using antigens including β-catenin, gp100, MAGE-A3, MART1 (melanoma antigen recognized by T cells 1), NY-ESO-1 and survivin; tumor vaccines for the treatment of pancreatic cancer, using tumor antigens including telomerase peptide, allogeneic tumor cells and mutant RAS synthetic peptides; tumor vaccines for the treatment of colorectal cancer, using tumor antigens including carcinoembryonic antigen (carcinoembryonic antigen The oligonucleotides provided by the present invention can enhance the immune response to tumor vaccines.

[0040] "Tumor cell lysate" The oligonucleotides provided by the present invention can enhance the efficacy of tumor cell lysate in inducing anti-tumor immune responses, and can be used as adjuvants for tumor vaccines with tumor cell lysate as antigen. Tumor cell lysate can be obtained by crushing tumor cells and contains various tumor antigens. Methods for crushing tumor cells include but are not limited to repeated freezing and thawing, ultrasonic treatment and mechanical crushing. Primary tumors, secondary or metastatic tumor cells can be used to prepare tumor cell lysates. Primary tumors and secondary or metastatic tumor cells are cultured in vitro to obtain tumor cell line cells, which can also be used to prepare tumor cell lysates. The tumor cells used to prepare tumor cell lysates can be from autologous, allogeneic or xenogeneic individuals.

[0041] "Adjuvant" The oligonucleotides provided by the present invention can be used in combination with one or more adjuvants to enhance the immune efficacy of microbial antigens and tumor antigens. Adjuvants are substances used together with antigens in vaccines, which have the following activities: (1) reduce the number of vaccinations; (2) prolong the duration of vaccine immunity; (3) promote humoral and cellular immune responses by stimulating innate immune responses; (4) extend the cross-protective immune response induced by antigens; (5) enhance the immune response of individuals with weak immune responses, such as elderly individuals or immunodeficient individuals to antigens; (6) reduce the amount of antigen used. Adjuvants that can be used in combination with the oligonucleotides provided by the present invention [Stanley A. Plotkin, Walter A. Orenstein, Paul A. Offit, Vaccines, Sixth Edition, An imprint of Elsevier Inc. 2013, ISBN-13: 9781455700905] include but are not limited to: aluminum salt adjuvants (vaccine adjuvants with aluminum hydroxide or aluminum phosphate as the main component), AS04 adjuvants [aluminum phosphate adjuvant adsorbed with MPL (chemically attenuated Gram-negative bacterial lipopolysaccharide)], MF59 adjuvants (an oil-in-water emulsifier with squalene as the oil phase), AS03 adjuvants (an oil-in-water adjuvant with squalene as the oil phase), AF03 adjuvants (an oil-in-water emulsifier with squalene as the oil phase), Montanide ISA 51 adjuvant (water-in-oil emulsion with mineral oil as the oil phase), Freund's adjuvant, Freund's incomplete adjuvant, virosome adjuvant, N-oxide polyethylene diazinon derivative (polyoxidonium) adjuvant, Toll-like receptor agonists, pathogen-related pattern molecules and their mimics (analogs), damage-related pattern molecules and their mimics (analogs), cyclic dinucleotides and their mimics (analogs), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-12, oily emulsions (AS02, AS03, AF03, MF59 and Montanide TM ISA-51), white oil adjuvant, Montanide TMISA-206 adjuvant, QS21, polylactide co-glycolide, virus (Adenovirus, vaccinia, fowlpox) carrier adjuvant, BCG (BCG, bacillus Calmette-Guérin), double-stranded RNA including poly(I:C) and its analogs (mimics), lipid A analogs including MPL, flagellin, Imidazoquinolines including Imiquimod and R848, CpG ODN, saponins including QS21, C-type lectin ligands including TDB, CD1d ligands including α-galactosylceramide, AS01 (containing MPL, QS21 and liposomes), AS02 (containing MPL, QS21 and emulsifiers), AS15 (containing MPL, QS21, CpG ODN and liposomes), GLA-SE (containing GLA and emulsifiers), IC31 (containing CpG ODN and cationic polypeptide), CAF01 (containing TDB and cationic liposomes) and ISCOMs (containing saponin and phospholipids) [Reed SG, et al. Nat Med. 2013 Dec; 19(12): 1597-608; Melero I, et al. Nat Rev Clin Oncol. 2014 Sep; 11(9): 509-24].

[0042] "Innate immune activator": The oligonucleotides provided by the present invention can be used in combination with innate immune activators to exert anti-infection and anti-tumor effects. Innate immune activators include pathogen-related model molecules and analogs thereof, Toll-like receptor agonists and analogs thereof, damage-related model molecules and analogs thereof, and cyclic dinucleotides and analogs thereof. Analogs and mimetics have the same meaning.

[0043] "Pathogen-associated molecular patterns": The oligonucleotides provided by the present invention can be used in combination with pathogen-associated molecular patterns (PAMPs) and their analogs to enhance the immune efficacy of pathogenic microbial antigens and tumor antigens to exert anti-infection and anti-tumor effects. PAMPs are various conserved components of microorganisms, including bacterial and fungal cell wall components and viral nucleic acids. Innate immune cells are activated by recognizing PAMPs through pattern recognition receptors (PRRs). PRRs include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (Nod)-, leucine-rich repeat-containing receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and AIM-2 like receptors; they also include intracellular sensors of nucleic acids, OAS proteins and cGAS [Iwasaki A et al. Nat Immunol. 2015 Apr; 16(4): 343-53].

[0044] "Toll-like receptor agonist": The oligonucleotides provided by the present invention can be used in combination with one or more Toll-like receptors (TLRs) agonists to enhance the immune effects of pathogenic microbial antigens and tumor antigens, and exhibit anti-infection or anti-tumor activity. TLR agonists that can be used in combination with the oligonucleotides provided by the present invention include, but are not limited to, TLR7 and TLR8 agonists including Imiquimod and imidazoquinolines; TLR7 agonists including 852A; TLR8 agonists including VTX-2337; TLR9 agonists including IMO-2055, CPG 7909, MGN1703 and other CpG ODN (containing CpG deoxyoligonucleotides); TLR2 / TLR4 agonists including Bacillus Calmette-Guérin (BCG); TLR4 agonists including OM-174, monophosphoryl lipid A, aminoalkyl glucosamine phosphates and other lipid A analogs; TLR9 agonists including viral nucleic acids and bacterial nucleic acids; TLR5 agonists including bacterial flagellin, TLR2 / TLR6 agonists including yeast polysaccharide; TLR3 agonists including polyinosinic acid cytosine nucleotides (poly IC), TLR3 agonists including viral double-stranded RNA or its mimics, and TLR7 / TLR8 agonists including viral single-stranded RNA or its mimics [Adams JL et al Nat Rev Drug Discov. 2015 Sep; 14(9): 603-22].

[0045] "Damage-associated molecular patterns": The oligonucleotides provided by the present invention can be used in combination with damage-associated molecular patterns (DAMPs) and their analogs to enhance the immune efficacy of microbial antigens and tumor antigens. DAMPs are self-cell components released by damaged cells that can stimulate the innate immune response. DAMPs can stimulate the body's innate immune response through PRR. These DAMPs include, but are not limited to, heat shock proteins, HMGB1 (high-mobility group box 1), hyaluronan fragments, glycans, glycoconjugates, ATP, (Adenosine 5'-triphosphate), adenylate, uric acid, S100 protein, heparin sulfate, Galectins, nuclear DNA, N-formylated peptides, antimicrobial peptides, mitochondrial DNA and calreticulin [Krysko DV et al. Nat Rev Cancer. 2012 Dec; 12(12): 860-75; Pouwels SD et al. Mucosal Immunol. 2014 Mar; 7(2): 215-26].

[0046] "Cyclic dinucleotides" The oligonucleotides provided by the present invention can be used in combination with cyclic dinucleotides and their mimetics to enhance the immune effects of microbial antigens and tumor antigens. Cyclic dinucleotides (CDNs) can be derived from bacteria or produced in mammalian cells. Bacterial CDNs include but are not limited to cyclic di-GMP (cdG), cyclic di-AMP (cdA) and cyclic AMP-GMP (cAMP-GMP). Bacterial CDNs are a type of PAMP that can stimulate innate immune responses. CDNs can also appear in mammalian cells, such as cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) [Wu J et al. Science. 2013 Feb 15; 339(6121): 826-3].

[0047] "Anti-tumor agents": Anti-tumor agents are agents that can treat tumors after being applied to individuals. These agents include but are not limited to tumor vaccines, immune checkpoint inhibitors, co-stimulatory receptor activators, chemotherapy drugs, radiotherapy agents, hormone inhibitors or hormones, cytokines, antibodies for tumor treatment, small molecule kinase inhibitors, PARP inhibitors, angiogenesis inhibitors and oncolytic viruses.

[0048] “Immune checkpoint molecules”: Immune checkpoint molecules are protein molecules expressed on immune cells. After recognizing their ligands, they initiate inhibitory signaling pathways and thus inhibit the activation of immune cells. Immune checkpoint molecules include but are not limited to CTLA-4 molecules, PD-1 molecules, PD-L1 / 2 molecules, lymphocyte-activation gene 3 (LAG-3), TIM-3 (T cell immunoglobulin and mucindomain-containing 3), TIGIT (T cell immunoreceptor with immunoglobul in andITIM domains) and BTLA (B and T lymphocyte attenuator). Immune checkpoint blockade refers to a method of inhibiting the function of immune checkpoint molecules in transmitting inhibitory signals to immune cells to promote and maintain T cell activation. Blocking immune checkpoints can enhance an individual's immune response to microbial antigens, exhibiting an anti-infection effect, and can also promote the anti-tumor activity of individual immune cells, exhibiting a tumor treatment effect [Melero I, et al. Nat Rev Cancer. 2015 Aug; 15(8): 457-72].

[0049] "Immune checkpoint inhibitors": The oligonucleotides provided by the present invention can be used in combination with immune checkpoint inhibitors as adjuvants for microbial vaccines or tumor vaccines, and can also be used in combination with immune checkpoint inhibitors to treat tumors. Immune checkpoint inhibitors are substances that can inhibit the function of immune checkpoint molecules [Melero I et al. Nat Rev Cancer. 2015Aug; 15(8): 457-72]. Inhibiting the function of immune checkpoint molecules allows the signal of immune cell activation to be highlighted, thereby keeping the immune cells in a continuously activated state. CD4 + T cells assist B cells in producing antibodies and also assist CD8 +T cells kill target cells such as virus-infected cells and tumor cells. + T cells can kill virus-infected cells and tumor cells. Therefore, preparations that maintain the activation state of T cells, such as immune checkpoint inhibitors, will increase the immune efficacy of antigens or vaccines in individuals, and will also have a tumor therapeutic effect in cancer patients (individuals). Immune checkpoint molecules inhibited by immune checkpoint inhibitors include but are not limited to CTLA4, PD1, LAG3 (Lymphocyte activation gene 3), 2B4 (CD244), BTLA (B and T lymphocyte attenuator), TIM3 (T cell membrane protein 3) and A2aR (adenosine A2a receptor) [Pardoli DM. Nat Rev Cancer. 2012 Mar 22; 12 (4): 252-64]. Antibodies that can inhibit the function of immune checkpoints belong to immune checkpoint inhibitors, including but not limited to CTLA-4 antibodies, CD-1 antibodies and CD-L antibodies.

[0050] "Immune checkpoint molecule antibody": The oligonucleotides provided by the present invention can be used in combination with immune checkpoint molecule antibodies to treat tumors or as adjuvants for microbial vaccines and tumor vaccines. Immune checkpoint molecule antibodies include but are not limited to CTLA-4 antibodies, PD-1 molecule antibodies and PD-L1 / 2 antibodies. CTLA-4 antibodies are antibodies that can specifically bind to CTLA4, block the inhibitory signals transduced by CTLA-4, enable T lymphocytes to be fully activated by tumor antigens, and prolong the survival of tumor patients. Ipilimumab is a fully humanized IgG1 CTLA-4 monoclonal antibody [Lipson EJ., et al. Clin Cancer Res. 2011 Nov 15; 17(22): 6958-62], and was approved by the US FDA in 2011 as a drug for the treatment of advanced melanoma [Sharma P. et al. Science. 2015 Apr 3; 348(6230): 56-61]. Tremelimumab is another humanized IgG2 CTLA-4 monoclonal antibody [Ribas A., et al. Oncologist. 2007 Jul; 12(7): 873-83]. In clinical trials, tremelimumab has been used to treat hepatocellular carcinoma [Sangro B., et al. J Hepatol. 2013 Jul; 59(1): 81-8], gastric cancer and esophageal cancer [Ralph C. et al. Clin Cancer Res. 2010 Mar 1; 16(5): 1662-72]. Antibodies to PD-1 (Anti-PD-1 Antibodies) are monoclonal antibodies to PD-1 molecules that have the function of inhibiting PD-1-mediated negative regulatory signal transduction of immune cells and are immune checkpoint inhibitors. In 2014, two PD-1 antibodies (pembrolizumab and nivolumab) were approved by the US FDA for tumor treatment. Nivolumab is a fully humanized IgG4 monoclonal antibody that can bind to and inhibit the function of PD-1 [Topalian SL et al. Curr Opin Immunol. 2012Apr; 24(2): 207-12] and is used to treat melanoma, non-small cell lung cancer, ovarian cancer and renal cancer [Ito A et al. Biomed Res Int. 2015; 2015: 605478]. Pidilizumab (CT-011) is a humanized IgG-1κ monoclonal antibody that can bind to and inhibit the function of PD-1 and is used to treat diffuse large B-cell lymphoma and follicular lymphoma.In addition, PD-1 antibodies also include Pembrolizumab (MK-3475), which is an IgG-4κ monoclonal antibody that can bind to and inhibit the function of PD-1 [Ito A et al. Biomed Res Int. 2015; 2015: 605478]. Anti-PD-L antibodies are antibodies that recognize and bind to PD-L1 or PD-L2. After binding to PD-L, it can block its binding to PD-1 on the surface of immune cells, thereby blocking the transduction of immune cell activation inhibitory signals, maintaining the activation state of immune cells, and thus enhancing the individual's immune response to antigens or tumor cells. A variety of anti-PD-L1 antibodies (Anti-PD-L1 Antibodies) have shown tumor therapeutic effects, including but not limited to BMS-936559, MPDL3280A, MEDI4736 and MSB0010718 [Ito A, et al. Biomed Res Int. 2015; 2015: 605478]. BMS-936559 is a fully humanized IgG4 anti-PD-L1 monoclonal antibody used to treat melanoma, non-small cell lung cancer, ovarian cancer and renal cancer. MPDL3280A is a humanized IgG-1κ anti-PD-L1 monoclonal antibody used to treat melanoma and bladder cancer. MEDI4736 is a humanized IgG-1κ monoclonal antibody that can prolong the survival of tumor-bearing individuals. MSB0010718 is a humanized IgG1 PD-L1 monoclonal antibody [Ito A, et al. Biomed Res Int. 2015; 2015: 605478].

[0051] "Co-stimulatory receptor activator": The oligonucleotides provided by the present invention can be used in combination with co-receptor activators to enhance an individual's immune response to vaccines or antigens, and enhance an individual's anti-tumor immune response. Co-stimulatory receptors are receptors expressed on the surface of immune cells, which mediate the transduction of immune cell activation signals after being activated by an activator, thereby promoting an individual's immune response to antigens or vaccines and enhancing an individual's anti-tumor immune activity. Co-stimulatory receptor activators are preparations that activate immune cells by binding to co-stimulatory receptors. Co-stimulatory receptor activating monoclonal antibodies are co-stimulatory receptor activators that can enhance the efficacy of antigens or vaccines and enhance an individual's anti-tumor immune response. The co-stimulatory receptors targeted by this type of activating monoclonal antibodies include but are not limited to CD137 (41BB), OX40, CD40, GITR, ICOS and CD27 (Glucocorticoid-induced tumour necrosis factor receptor family-related protein) [Melero I et al. Nat Rev Cancer. 2015 Aug; 15(8): 457-72; Sanmamed MF et al. Semin Oncol. 2015 Aug; 42(4): 640-55].

[0052] "Chemotherapeutic drugs" The oligonucleotides provided by the present invention can be used in combination with chemotherapeutic drugs to treat tumors. Chemotherapeutic drugs are chemical drugs that can treat tumors by inhibiting and killing tumor cells. The chemical drugs referred to in the present invention include, but are not limited to, alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors and cytotoxic antibiotics. Alkylating agents include nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatin and its derivatives, and non-classical alkylating agents. Nitrogen mustards include mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide and busulfan. Nitrosoureas include N-Nitroso-N-methylurea, carmustine, lomustine, semustine, fotemustine and streptozotocin. Tetrazines include dacarbazine, mitozolomide and temozolomide. Aziridines include thiotepa, mytomycin, and diaziquone. Cisplatin and its derivatives include cisplatin, carboplatin, and oxaliplatin. Nonclassical alkylating agents include procarbazine and hexamethylmelamine. Antimetabolites include antifolates, fluorouracils, deoxynucleoside analogs, and thiopurines. Antifolates include methotrexate and pemetrexed. Fluorouracils include 5-fluorouracil. Deoxynucleoside analogs include cytarabine, gemcitabine, decitabine, vidaza, fludarabine, nelarabine, cladribine, clofarabine, and pentostatin. Thiopurines include thioguanine and mercaptopurine. Antimicrotubule agents include vinca alkaloids, taxanes, and podophyllotoxins. Vinca alkaloids include vincristine, vinblastine, vinorelbine, vindesine, and vinflunine.Taxanes include paclitaxel and docetaxel. Podophyllotoxins include etoposide and teniposide. Topoisomerase inhibitors include topoisomerase I inhibitors and topoisomerase II inhibitors. Topoisomerase I inhibitors include irinotecan and topotecan. Topoisomerase II inhibitors include topoisomerase II poisons and catalytic inhibitors. Topoisomerase II poisons include etoposide, doxorubicin, mitoxantrone, and teniposide. Catalytic inhibitors include novobiocin, merbarone, and aclarubicin. Cytotoxic antibiotics include doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, mitoxantrone, gactinomycin, bleomycin, plicamycin, and mitomycin.

[0053] "Radiotherapeutic agents": The oligonucleotides provided herein can be used in combination with radiotherapy to treat tumors. Radiotherapy agents are substances that produce radiation. The use of radiotherapy agents to treat tumors is called chemotherapy. Substances used in radiotherapy are radioactive isotopes that produce alpha, beta, or gamma rays. Radiation used in radiotherapy can also be produced by machines, such as x-ray therapy machines or accelerators.

[0054] "Hormone inhibitors or hormones": The oligonucleotides provided by the present invention can be used in combination with hormone inhibitors or hormones for tumor treatment to treat tumors. Hormone inhibitors include, but are not limited to, hormone synthesis inhibitors, hormone receptor antagonists and supplementary hormones. Hormone synthesis inhibitors include aromatase inhibitors and gonadotropin-releasing hormone (GnRH) analogs. Aromatase inhibitors include Letrozole, anastrozole and Aminoglutethimide. GnRH analogs include Leuprolide and gosereli. Hormone receptor antagonists include selective estrogen receptor modulators and androgen receptor antagonists. Selective estrogen receptor modulators include Tamoxifen, Raloxifene, Toremifene and fulvestrant. Androgen receptor antagonists include Flutamide and bicalutamide. Supplementary hormones are a method of treating tumors using hormone supplementation, and the hormones used include progesterone, androgen, estrogen, progesterone-like drugs, testosterone-like drugs, estrogen agonists and somatostatin analogs. Progesterone-like drugs include megestrol acetate and medroxyprogesteroneacetate. Testosterone-like drugs include Fluoxymesterone. Estrogen antagonists include diethylstilbestrol, Estrace, and Polyestradiol phosphate. Somatostatin analogs include Octraotide.

[0055] "Cytokines": The oligonucleotides provided by the present invention can be used in combination with cytokines to treat tumors and enhance the immune effect of vaccines. These cytokines include, but are not limited to, interleukin (IL)-2, granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF) and interferon α.

[0056] "Tumor therapeutic antibodies" The oligonucleotides provided by the present invention can be used in combination with tumor therapeutic antibodies to treat tumors. Tumor therapeutic antibodies are antibodies that can prolong the survival of individuals with tumors after application. The molecules targeted by these antibodies include but are not limited to CD20, ErbB2, epidermal growth factor receptor, immune checkpoint molecules including CTLA-4 and PD-1, vascular endothelial growth factor receptor, CD30, CD52 and CD33. The tumor therapeutic antibodies involved include, but are not limited to, Tositumomab (Bexxar), Rituximab (Rituxan) and Ofatumumab (Arzerra; Genmab) targeting CD20; Trastuzumab (Herceptin) targeting ErbB2; Panitumumab (Vectibix) and Cetuximab (Erbitux) targeting epidermal growth factor receptor; humanized antibody targeting PD-1; humanized antibody targeting CTLA-4; Bevacizumab (Avastin) targeting vascular endothelial growth factor receptor; Brentuximab (vedotin) targeting CD30; Alemtuzumab (Campath) targeting CD52 and Gemtuzumab ozogamicin (Mylotarg; Wyeth) targeting CD33 [Scott AM et al. Nat Rev Cancer. 2012 Mar 22; 12(4): 278-87].

[0057] "Small-molecule kinase inhibitors": The oligonucleotides provided by the present invention can be used in combination with small-molecule kinase inhibitors to treat tumors. Small-molecule kinase inhibitors are a class of small-molecule compounds that can treat tumors by inhibiting the activity of protein kinases, including but not limited to the following: Imatinib targeting Bcr-Abl; Afatinib targeting EGFR / ErbB2; Axitinib targeting VEGFR1 / VEGFR2 / VEGFR3 / PDGFRB / c-KIT; Bosutinib targeting BcrAbl / SRC; Crizotinib targeting ALK / Met; Erlotinib targeting ErbB1; Fostamatinib targeting Syk; Ge fitinib; ibrutinib targeting BTK; lapatinib targeting ErbB1 / ErbB2; lenvatinib targeting VEGFR2 / VEGFR2; nilotinib targeting Bcr-Abl; pazopanib targeting VEGFR2 / PDGFR / c-kit; ruxolitinib targeting JAK; vemurafenib (Zelboraf) and dabrafenib (Tafinlar) targeting mutant BRAF and trametinib (Mekinist) targeting MEK. Small molecule protein kinase inhibitors also include all small molecule compounds that treat tumors by inhibiting the following protein kinases: Bcr-Ab, EGFR / ErbB2, VEGFR1 / VEGFR2 / VEGFR3 / PDGFRB / c-KIT, BcrAbl / SRC, ALK / Met, ErbB1, Syk, EGFR, BTK, ErbB1 / ErbB2, VEGFR2 / VEGFR2, Bcr-Abl, VEGFR2 / PDGFR / c-kit, JAK, BRAF and MEK [Adams JL et al Nat Rev Drug Discov. 2015 Sep; 14(9): 603-22].

[0058] "Poly ADP ribose polymerase inhibitor": The oligonucleotides provided by the present invention can be used in combination with poly ADP ribose polymerase inhibitors (PARP) to treat tumors or be used as adjuvants for microbial vaccines and tumor vaccines. Poly ADP ribose polymerase inhibitors, referred to as PARP inhibitors, are preparations that can treat tumors by inhibiting PARP activity. PARP inhibitors include, but are not limited to: Iniparib for the treatment of breast cancer and squamous cell lung carcinoma; Talazoparib (BMN-673) for the treatment of breast cancer; Olaparib for the treatment of breast cancer, colon cancer, ovarian cancer and advanced prostate cancer; Rucaparib for the treatment of breast and ovarian cancer; Veliparib for the treatment of metastatic melanoma and breast cancer and CEP 9722 for the treatment of non-small cell lung cancer [Nature Reviews Clinical Oncology 12, 27-41, 2015].

[0059] "Angiogenesis inhibitors": The oligonucleotides provided by the present invention can be used in combination with angiogenesis inhibitors to treat tumors. Angiogenesis inhibitors are agents that can treat tumors by inhibiting angiogenesis [Albini A et al. Nat Rev Clin Oncol. 2012 Sep; 9(9): 498-509], including but not limited to: humanized monoclonal antibodies to vascular endothelial growth factor (VEGF), including Avastin (Avastin or bevacizumab); endostatins including ENDOSTAR and anti-vascular endothelial growth factor aptamers including pegaptinib.

[0060] "Oncolytic virus": The oligonucleotides provided by the present invention can be used in combination with oncolytic viruses to treat tumors. Oncolytic viruses are viruses that can treat tumors by lysing tumor cells, including but not limited to Newcastle disease virus, herpes simplex virus, adenovirus, poxvirus, california virus, respiratory enterovirus, measles virus, poliovirus, follicular stomatitis virus, Seneca Valley virus, parvovirus and retrovirus [Kaufman HL et al. Nat Rev Drug Discov. 2015 Sep 1; 14(9): 642-62].

[0061] "Tumor therapeutic cells": The oligonucleotides provided by the present invention can be used in combination with tumor therapeutic cells to treat tumors. Tumor therapeutic cells are cells that can exert anti-tumor effects after being applied to an individual, and these cells include but are not limited to dendritic cells, T lymphocytes and NK cells.

[0062] "Dendritic cells": The oligonucleotides provided by the present invention can be used in combination with autologous dendritic cells (DC) to treat tumors. DC is an immune cell that expresses CD11c. DC that can present tumor antigens can stimulate anti-tumor immune responses when applied to individuals, and such cells can be used as DC vaccines. DC for tumor treatment include, but are not limited to, DC loaded with a single tumor antigen (protein antigen or antigen peptide, such as prostatic acid phosphatase), DC loaded with tumor cell lysate, DC loaded with tumor cell RNA, and DC loaded with autologous tumor cell eluted peptides [Nestle, F. et al. (1998) Nature Medicine 4: 328-332; Palucka K et al. Nat Rev Cancer. 2012 Mar 22; 12 (4): 265-77]. DC for tumor treatment also includes gene-transfected DC. Genes used to transfect DC include, but are not limited to, genes encoding tumor antigens, genes encoding cytokines (such as IL-2, GM-CSF), and genes encoding co-stimulatory molecules. DCs for tumor therapy also include cells fused with DCs and tumor cells [Kugler, A. et al. (2000) Nature Medicine 6: 332-336].

[0063] "T cells for tumor treatment": The oligonucleotides provided by the present invention can be used in combination with T cells for tumor treatment to treat tumors. T cells for tumor treatment include tumor-infiltrating T cells and genetically engineered T cells. Genetically engineered T cells can be assembled with chimeric antigen receptors (CAR) that recognize tumor antigens, and T cells expressing such receptors are called CAR-T. Tumor-infiltrating T cells and CAR-T can be amplified in vitro with IL-2 and then infused back into the tumor patient [Kershaw MH et al. Nat Rev Cancer. 2013 Aug; 13(8): 525-41].

[0064] "Natural killer cells for tumor treatment": The oligonucleotides provided by the present invention can be used in combination with natural killer (NK) cells for tumor treatment to treat tumors. Natural killer (NK) cells for tumor treatment can be isolated from the peripheral blood or umbilical cord blood of an individual, or induced from hematopoietic cell precursor cells, embryonic stem cells or pluripotent stem cells. The isolated or induced NK cells can be amplified in vitro with IL-2 and IL-15 and then returned to the individual [Childs RW, et al. Nat Rev Drug Discov. 2015 Jul; 14 (7): 487-98].

[0065] "Anti-infection": The oligonucleotides provided by the present invention can exert anti-infection effects when used alone or as vaccine adjuvants. Anti-infection refers to the treatment and prevention of diseases caused by pathogenic microorganisms. The oligonucleotides provided by the present invention have anti-infection effects on pathogenic microorganisms.

[0066] "Pathogenic microorganisms": The oligonucleotides provided by the present invention can be used alone or as vaccine adjuvants to fight against infection by pathogenic microorganisms. Pathogenic microorganisms include pathogenic viruses and pathogenic bacteria.

[0067] "Pathogenic viruses": The oligonucleotides provided by the present invention can be used alone or as a vaccine adjuvant to play an anti-pathogenic virus (pathogenic viruses) infection effect. These pathogenic viruses include hepatitis A virus, hepatitis B virus, hepatitis C virus, varicella-zoster virus (VZV), herpes simplex virus type I (HSV-1), herpes simplex virus type II (HSV-II), cytomegalovirus (CMV), Epstein-Barr virus, adenovirus, influenza virus, arbovirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, circovirus, foot-and-mouth disease virus, hand-foot-and-mouth disease virus, human T-lymphotropic virus (HTLV), dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, polyomavirus and arboviral encephalitis virus.

[0068] "Pathogenic bacteria": The oligonucleotides provided by the present invention can be used alone or as a vaccine adjuvant to exert an effect against pathogenic bacteria infection. These pathogenic bacteria include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus, Klebsiella, Serratia, Pseudomonas, Diphtheria, Salmonella, Vibrio cholerae, Tetanus, Clostridium botulinum, Bacillus anthracis, Yersinia pestis and Lyme disease bacteria.

[0069] "Pharmaceutical composition": The oligonucleotide drug provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition. The dosage of the oligonucleotide in the composition is an effective dose. The composition can be combined with an antigen, a vaccine, an adjuvant, and a preparation or cell with a tumor therapeutic effect. The pharmaceutical composition can be prepared into a certain dosage form, which includes but is not limited to a solution, an emulsion, a liposome, and a lyophilized powder.

[0070] "Pharmaceutically acceptable carrier": The oligonucleotide drugs provided by the present invention can be combined with pharmaceutically acceptable carriers to form pharmaceutical compositions. Pharmaceutically acceptable carriers refer to one or more solid or liquid fillers, diluents or encapsulating materials. Such carriers are suitable for applying the oligonucleotides provided by the present invention to individuals. The carrier can be organic, inorganic, natural or synthetic. Pharmaceutically acceptable carriers can be pharmaceutically acceptable solvents (aqueous solutions and non-aqueous solutions), dispersants, suspending agents, emulsifiers, powders, diluents, liposomes, antibacterial agents, antifungal agents, isotonic preparations, delayed absorption preparations, lyophilization protectants and other preparations suitable for the immunological efficacy of the oligonucleotide vaccine or antigen provided by the present invention, and the production of therapeutic tumor effects. Aqueous solutions include but are not limited to water, physiological saline, PBS buffer, balanced salt solution and glucose solution. Solvents or dispersants may include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and also mixtures composed of these solvents or dispersants. In order to maintain the fluidity of the pharmaceutical composition, lipids including lecithin can be used. In order to make the pharmaceutical composition in an ideal granular state, a surfactant can be used. In order to have a suitable osmotic pressure, sugars, polyols including mannitol and sorbitol, and sodium chloride can be added to the pharmaceutical composition. In order to prolong the duration of action, sustained-release agents such as stearates and gelatin can be added to the pharmaceutical composition. Emulsifiers can include water-in-oil emulsifiers, oil-in-water emulsifiers, or water-in-oil-in-water emulsifiers. Pharmaceutically acceptable carriers also include pharmaceutically acceptable antioxidants, which include: water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate and alpha wheat germ phenol; metal chelators such as citric acid, EDTA, sorbitol, tartaric acid and phosphoric acid.

[0071] "Effective dose": The effective doses of oligonucleotides provided by the present invention include "effective doses for enhancing antigen immune efficacy" and "effective doses for tumor treatment". The effective dose for enhancing antigen immune efficacy is the dose of oligonucleotides that can significantly enhance the immune efficacy of antigens or vaccines after being applied to an individual, and also refers to the dose of oligonucleotides that can produce an ideal prevention or treatment of infectious diseases or tumors after being administered to an individual. The effective dose for tumor treatment is the dose of oligonucleotides that can produce tumor treatment effects after being applied to an individual. The amount of the dose is determined by the standards that should be known to those skilled in the art, and other factors should also be referred to, including but not limited to the size and health of the individual and the severity of the disease. The oligonucleotides provided by the present invention can be applied to an individual once or multiple times, and the dosage range of each time can be in the range of 1μg to 1000mg. In order to achieve the ideal effect, those skilled in the art can adjust the dosage of this oligonucleotide, and the dosage range can be 10 times to 1000 times the aforementioned range. When applied to an individual, the oligonucleotides provided by the present invention can be used in dosage units. Each unit contains a quantitative oligonucleotide that can produce a preventive or therapeutic effect and a desired pharmaceutically acceptable composition. The basis for defining the dosage unit is the unique active characteristics of the oligonucleotide producing the therapeutic effect and the sensitivity of the individual to the oligonucleotide when receiving the oligonucleotide treatment. If necessary, the oligonucleotide applied in the dosage unit can be applied 2 times, 3 times, 4 times, 5 times or more at certain time intervals per day. The oligonucleotide provided by the present invention can be applied according to the host body weight, the dosage range is 0.0001 to 100 mg / kg, and the application interval can be once every two weeks or once a month or once every 3 to 6 months or other time intervals suitable for producing preventive and therapeutic effects. When used in combination with an antigen or vaccine, the dosage of the oligonucleotide can be 1-1000 μg / ml. The effective dose includes a therapeutically effective dose and a prophylactically effective dose.

[0072] "Route of administration": The oligonucleotides provided by the present invention can be administered parenterally, externally or by inhalation when used alone or in combination with other agents such as antigens, adjuvants, innate immune activators and anti-tumor agents. Parenteral administration routes include intravenous, peritoneal, intrathecal, intramuscular, subcutaneous, intradermal, topical, paratumoral lymph nodes, direct injection into tumor tissue and intralymph node injection. External administration routes include through the skin, mouth, eyes, ears and nose. Inhalation can be through the nasal mucosa and lungs.

[0073] "Therapeutic device": A pharmaceutical composition containing an oligonucleotide provided by the present invention can be applied to an individual using a therapeutic device known to those skilled in the art. Therapeutic devices include, but are not limited to, needle-free injection devices, implantation devices, modules, implantable micro-infusion pumps, infusion pumps, and osmotic drug delivery systems.

[0074] "Delivery vector": The oligonucleotides of the present invention can be applied via a delivery vector. Delivery vectors include, but are not limited to, steroids (such as cholesterol), complexes, emulsions, immunostimulatory complexes (ISCOMs), lipids (such as cationic lipids and anionic lipids), liposomes, bacterial vectors (such as Salmonella, Escherichia coli, Mycobacterium Shigella, Lactobacillus), viral vectors (such as vaccinia, adenovirus, herpes simplex virus), virosomes, virus-like particles, microspheres, nucleic acid vaccines, polymer materials (such as carboxymethyl cellulose, chitosan) and cyclic polymers. The delivery vector can also be a ligand for a specific receptor or a targeting molecule for a cell. Description of the drawings:

[0075] Figure 1 The synergistic effect of TIO1 and TIO3 on recombinant protein vaccine (circovirus vaccine)

[0076] exist Figure 1 In the figure, CP stands for CP-ISA35, TIO1, TIO2 and TIO3 stand for CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3, respectively, ODN stands for single-stranded deoxy oligonucleotide, ns stands for no statistical significance, the antibody level is expressed by the absorbance value (A492 OD value), and each circle, square, righteous triangle and inverted triangle stand for one mouse.

[0077] Figure 2 Inhibitory effects of TIO1 and TIO3 on TGFβ-2 expression in immune cells of mice induced by immune recall response

[0078] exist Figure 2In the figure, CP represents CP-ISA35, TIO1 or TIO3 represents CP-ISA35+TIO1 or CP-ISA35+TIO3, respectively, ODN represents single-stranded deoxyoligonucleotide, TGF-β2relative expression represents the percentage of membrane-bound TGF-β2-positive cells in the draining lymph node cells, and each circle symbol, square symbol, and equilateral triangle symbol represents a mouse.

[0079] Figure 3 TIO1 and TIO3 reduce the level of TGFβ-2 mRNA in immune cells induced by IL-4

[0080] exist Figure 3 In the figure, ODN represents single-stranded deoxyoligonucleotide, the level of TGFβ-2 mRNA is represented by relative expression, and each circle symbol, square symbol, upright triangle symbol, inverted triangle symbol and diamond symbol represents a sample respectively.

[0081] Figure 4 TIO1 and TIO3 reduce the level of TGFβ-2 mRNA in immune cells induced by LPS

[0082] exist Figure 4 In the figure, ODN represents single-stranded deoxyoligonucleotide, the level of TGFβ-2 mRNA is represented by relative expression, and each circle symbol, square symbol, inverted triangle symbol, diamond symbol and upright triangle symbol represents a sample respectively.

[0083] Figure 5 Effects of TIO1 and TIO3 on the increase of dendritic cells expressing CD86 in lymph node cells of mice induced by immune recall response

[0084] exist Figure 5 In the figure, CP stands for CP-ISA35, TIO1, TIO2 or TIO3 stands for CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3, respectively, ODN stands for single-stranded deoxyoligonucleotide, and each circle symbol, square symbol and equilateral triangle symbol represents a mouse.

[0085] Figure 6 Effects of TIO1 and TIO3 on the increase of CD4-expressing T lymphocytes in lymph node cells of mice induced by immune recall response

[0086] exist Figure 6In the table, CP represents CP-ISA35, TIO1 or TIO3 represents CP-ISA35+TIO1 or CP-ISA35+TIO3, respectively, ODN represents single-stranded deoxyoligonucleotide, and each circle, square, and triangle symbol represents a mouse, CD4 + % represents CD4 + The number of cells among the draining lymph node cells analyzed.

[0087] Figure 7 Effects of TIO1 and TIO3 on the increase of B lymphocytes expressing CD19 in lymph node cells of mice induced by immune recall response

[0088] exist Figure 7 In the table, CP represents CP-ISA35, TIO1, TIO2 or TIO3 represent CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3, respectively, ODN represents single-stranded deoxyoligonucleotide, each circle symbol, square symbol, equilateral triangle symbol and inverted triangle symbol represent one mouse, CD19 + cell% represents CD19 + The number of cells among the draining lymph node cells analyzed.

[0089] Figure 8 TIO1 and TIO3 enhance the efficacy of hepatitis B virus vaccine

[0090] exist Figure 8 In the figure, HBsAg represents HBsAg vaccine; TIO1, TIO2, TIO3 represent the addition of TIO1, TIO2 or TIO3 to HBsAg vaccine, respectively; ODN represents single-stranded deoxyoligonucleotide; each circle symbol, square symbol, up-right triangle symbol and inverted triangle symbol represents a mouse, respectively; OD Value (A492nm) represents the level of HBsAg-specific antibodies in the serum to be tested.

[0091] Fig. 9 TIO1 or TIO3 enhances the efficacy of influenza virus vaccines

[0092] exist Fig. 9 In the figure, FM1 represents FM1 influenza virus inactivated vaccine; TIO1, TIO2, TIO3 represent TIO1 or TIO3 added to FM1, respectively; ODN represents single-stranded deoxyoligonucleotide; each circle symbol, square symbol, and triangle symbol represents a mouse, respectively; OD Value (A492nm) represents the level of influenza virus-specific antibodies in the serum to be tested.

[0093] Fig.10The synergistic effect of TIO1 or TIO3 on rabies virus vaccine

[0094] exist Fig.10 In the figure, TIO1, TIO2, and TIO3 represent the addition of TIO1, TIO2, or TIO3 to the rabies vaccine, respectively; ODN represents single-stranded deoxyoligonucleotide; each circle symbol, square symbol, up-right triangle symbol, and inverted triangle symbol represent a mouse, respectively. The potency of the vaccine is expressed in IU / ml.

[0095] Figure-11 The synergistic effect of TIO3 on glioma cell lysate vaccine

[0096] exist Fig.11 In the figure, GTL, stands for glioma cell lysate vaccine; GTL+TIO3, stands for glioma cell lysate vaccine containing TIO3; days, the number of days after glioma cell inoculation; Percent survival, stands for the percentage of surviving mice.

[0097] Figure-12 The therapeutic effect of TIO3 on gastric cancer

[0098] exist Fig.12 In the figure, PBS represents the injection of PBS; PBS+TIO3 represents the injection of TIO3 dissolved in PBS; days represents the number of days after inoculation of gastric cancer cells; Percent survival represents the percentage of surviving mice. Specific implementation method:

[0099] Example 1 Design and synthesis of oligonucleotides (TIO1, TIO3)

[0100] Three single-stranded deoxy oligonucleotides were designed based on the sequences of human and mouse transforming growth factor β2 (TGF-β2) mRNA 3'UTR. The three oligonucleotides were named TIO1, TIO2 and TIO3. TIO1 has the following sequence: <400> 1, TIO3 has the sequence shown in the sequence table <400> The sequence shown in 2.

[0101] The sequence of TIO1 is 5'-tggcaaagtatttggtctcca-3' (Sequence Listing <400> 1).

[0102] The sequence of TIO3 is 5′-ttaccactagagcaccaca-3′ (Sequence Listing <400> 2).

[0103] The sequence of TIO2 is 5'-tccttaagccatccatgagttt-3'

[0104] The sequences of TIO1, TIO2 and TIO3 are complementary to the sequence of TGF-β2 mRNA 3'UTR.

[0105] TIO1, TIO2 and TIO3 were synthesized by Takara Bio (Dalian) Co., Ltd. (Takara Bio), and their backbones were fully thiolated.

[0106] When used, dissolve TIO1 or TIO3 in sterile PBS or other solvents treated with pyrogen-free water. Store frozen at -20℃.

[0107] The endotoxin in the solution containing TIO1, TIO2 and TIO3 was detected by the horseshoe crab amebocyte lysis method.

[0108] The contents of TIO1, TIO2 and TIO3 in the solution were determined by a spectrophotometer (260 nm wavelength). The contents were also estimated by agarose gel (3%) electrophoresis (based on single-stranded deoxyoligonucleotide standards with known contents).

[0109] Example 2 The enhancing effect of TIO1 and TIO3 on the immune efficacy of recombinant protein vaccine (circovirus vaccine):

[0110] 2.1 Materials

[0111] 2.1.1 Mice

[0112] ICR mice (Animal Room, School of Medicine, Jilin University), female, weighing 17-18 g.

[0113] 2.1.2 Antigen

[0114] Recombinant circovirus 2b capsid protein (CP protein or CP) was used as the antigen [Vaccine. 2016 Dec 7; 34(50): 6358-6366].

[0115] 2.1.3 Adjuvants

[0116] ISA 35 emulsifier (Seppic)

[0117] 2.1.4 Oligonucleotides

[0118] TIO1 (with sequence listing <400> 1), TIO2 (sequence as shown in Example 1) and TIO3 (sequence as shown in the sequence table <400> 2), as described in Example 1.

[0119] 2.2 Vaccine preparation

[0120] CP was dissolved in PBS and mixed with ISA35 emulsifier at a ratio of 1:1 (volume:volume) to prepare a vaccine, which was named CP-ISA35. 100μl CP-ISA35 contained 10μg CP. CP-ISA35 containing TIO1, TIO2 and TIO3 were prepared separately, and the dose of TIO1, TIO2 and TIO3 was 10μg / 100μl. CP-ISA35 containing TIO1, TIO2 and TIO3 was named CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3.

[0121] 2.3 Immunization of mice

[0122] The mice were given a primary immunization on day 0. 100 μl of CP-ISA35, CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3 were injected into the right hind limb muscle at a single point. The mice were given a booster immunization on day 14, using the same method as the primary immunization.

[0123] 2.4. Blood collection after immunization

[0124] Collect serum from immune mice on day 28 after booster immunization. Collect blood from tail vein, with a blood volume of ≥100 μl. Place the collected whole blood (in 1.5 ml EP tube) at room temperature for 30 minutes. Centrifuge at 11000 rpm for 15 minutes, collect serum, aliquot, and store at -20°C.

[0125] 2.5. Mouse serum specific antibody detection

[0126] The ELISA method was used to detect PCV2b-specific antibodies in mouse serum.

[0127] 2.5.1 Materials

[0128] 2.5.1.1 Inactivated circovirus (PCV2b) (Tianjin Ruipu Biotechnology Co., Ltd., TCID50 / ml=7.0).

[0129] 2.5.1.2 Test equipment

[0130] ELISA strips (combined ELISA plates), 0.5ml EP tubes, 1.5ml EP tubes, pipette tips, pipettes, multichannel pipettes, petri dishes (9cm in diameter), graduated glass bottles.

[0131] 2.5.1.3 Reagents

[0132] Sodium carbonate (Sinopharm Chemical Reagent Co., Ltd.), NaCl (Sinopharm Chemical Reagent Co., Ltd.), KCl (Beijing Chemical Plant), Na2HPO4·12H2O (Sinopharm Chemical Reagent Co., Ltd.), KH2PO4 (Tianjin Yongda Chemical Reagent Development Center), Tween 20 (Tianjin Guangfu Fine Chemical Research Institute), skimmed milk powder (Biotopped), citric acid (Sinopharm Chemical Reagent Co., Ltd.), OPD (Shanghai Sanpu Chemical Co., Ltd.), 30% H2O2 (Beijing Chemical Plant), concentrated sulfuric acid (Beijing Chemical Plant) and glutaraldehyde (Tianjin Fuchen Chemical Reagent Plant).

[0133] 2.5.1.4 Liquids

[0134] Coating solution (25% glutaraldehyde in PBS), PBS (7.3mol / L NaCl, 3mmol / L KCl, 10mmol / L Na2HPO4·12H2O and 17.6mmol / L KH2PO4 aqueous solution), washing solution (0.05% Tween 20 in PBS), blocking solution (5% skimmed milk powder in washing solution), 0.1Mol / L citric acid aqueous solution, 0.2Mol / L Na2HPO4.12H2O aqueous solution, 10× methyl and ethyl solution [0.1Mol / L citric acid aqueous solution (volume): 0.2Mol / L Na2HPO4.12H2O aqueous solution (volume) = 94.5:100], substrate buffer [methyl and ethyl solution containing OPD (1μg / ml) and 0.045% H2O2] and stopping solution (20% concentrated sulfuric acid).

[0135] 2.5.2 Experiment

[0136] Coat the enzyme label strip with inactivated PCV2b in the coating solution, 100μl / well, overnight at 4℃. Shake off the liquid, block with blocking solution at 37℃ for 2 hours, 200μl / well. Add the test serum (1:100 dilution), 37℃, 1 hour. Shake off the liquid, add washing solution (300μl / well), room temperature for 3min, shake off the liquid, and repeat washing twice. Add HRP-labeled secondary antibody (sheep anti-mouse) IgG (1:5000 dilution with washing solution). 100μl / well, 37℃ 1 hour. Shake off the liquid, wash with washing solution, 300μl / well, room temperature for 3min, shake off the liquid, and repeat washing twice. Add substrate solution, 100μl / well, color at room temperature in the dark (wrapped with tin foil) for 15min. Add stop solution (dilute sulfuric acid 2mmol / L, 50μl / well. Detect with an enzyme marker (wavelength 492nm).

[0137] 2.5.3 Results

[0138] TIO1 and TIO3 can enhance the immune efficacy of recombinant protein vaccines (circovirus vaccines) (Figure 1). This result shows that TIO1 and TIO3 can be used in individuals to enhance their immune response to pathogenic microorganism antigens (vaccines) including circoviruses and can be used as adjuvants for pathogenic microorganism vaccines.

[0139] Example 3 Inhibitory effects of TIO1 and TIO2 on TGFβ-2 expression in immune cells of mice induced by immune recall response

[0140] 3.1 Materials

[0141] 3.1.1 Antigen

[0142] CP protein (abbreviated as CP), as described in 2.1.2 of Example 2.

[0143] 3.1.2 Oligonucleotides

[0144] TIO1 (with sequence listing <400> 1), T1O3 (having the sequence shown in the sequence table <400> 2), as described in Example 1.

[0145] 3.1.3 Culture medium

[0146] RPMI1640 medium (Gibco). Complete RPMI1640 medium contains 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin. Serum-free RPMI1640 medium does not contain fetal bovine serum.

[0147] 3.1.4 Fluorescent Antibodies

[0148] FITC-labeled anti-mouse TGF-β2 antibody (BD Biosciences).

[0149] 3.1.5 Mice

[0150] ICR mice (Animal Room, School of Medicine, Jilin University), female, weighing 17-18 g.

[0151] 3.1.6 Vaccines

[0152] CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 were prepared as described in 2.2 of Example 2.

[0153] 3.2. Main experimental equipment

[0154] 24-well culture plates, flat dishes, frosted glass slides, 300-mesh filters, small tweezers, cell counting plates, droppers, and pipettes. CO2 cell culture incubator (SANYO, Japan), cell culture inverted microscope (Olympus, Japan), centrifuge (Biofuge Fresco, Germany), flow cytometer Accuri C6 flow cytometer (BD Bioscience), and flow cytometer result analysis software NovoExpress software (ACEA Biosciences).

[0155] 3.3 Experiments

[0156] The mice were given a primary immunization on day 0. 100 μl of CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 were injected into the right hind limb muscle at a single point. The mice were given a booster immunization on day 14, using the same method as the primary immunization. Recall immunization was performed 120 days after the booster immunization, by injecting 100 μl of CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 into each group of mice at a single point in the right hind limb muscle. The mice were sacrificed 6 hours later.

[0157] Immerse the sacrificed mouse in 70% ethanol and remove it quickly. Use sterile surgical instruments to discard the skin and other tissues, and take the draining lymph nodes under sterile conditions, and place them in 0.5 ml of sterilized 4°C pre-cooled PBS. Place on ice and perform the following operations: cut the lymph nodes, grind them with a rough glass slide, and filter to obtain a single cell suspension. Suspend the cells in 4°C pre-cooled PBS or fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% fetal bovine serum, 5mM EDTA and 1mM sodium azide).

[0158] Wash the cells by centrifugation (277g, 5 minutes, 4°C) with 5 ml of 4°C pre-cooled PBS or FACS buffer. Resuspend the cells in 4°C pre-cooled PBS or FACS buffer (200-1,000 μl). Record the cell number and adjust the cell concentration. Detect the cell viability by trypan blue exclusion test.

[0159] 1-10X10 6Transfer lymph node cells to 5 ml round-bottom polystyrene tubes. Wash cells by centrifugation with FACS buffer (277g, 5 minutes, 4°C). Discard supernatant and suspend cells in 50-100 μl FACS buffer containing FITC-labeled anti-mouse TGF-β2 antibody. The content of each fluorescent antibody is 0.5-1 μg, and incubate on ice for 30-45 minutes. Protect samples from light. Wash cells by centrifugation with FACS buffer (277g, 5 minutes, 4°C). Suspend cells in 50-100 μl FACS buffer for flow cytometry analysis.

[0160] 3.4 Results

[0161] TIO1 and TIO3 can significantly reduce the expression of TGFβ-2 protein in lymph node cells of mice induced with immune recall response (Figure 2), which is manifested by a significant reduction in membrane-bound TGFβ-2 protein [Yang ZZ, et al PLoS One. 2013; 8(3): e59456 Miller MM, et al. Virol J. 2014 Jan 18; 11: 7.]. This indicates that TIO1 and TIO3 can inhibit the translation of TGFβ-2 mRNA; TIO1 and TIO3 can inhibit and weaken TGFβ-2-mediated immunosuppression by reducing the expression of TGFβ-2 in immune cells, thereby enhancing the individual's immune response to pathogenic microorganisms and tumor cells, and producing anti-infection and anti-tumor effects.

[0162] Example 4 The effect of TIO1 and TIO2 on reducing the level of TGFβ-2 mRNA in immune cells

[0163] 4.1 Materials

[0164] 4.1.1 Cells

[0165] RAW 264.7 cells: mouse macrophage cell line cells [Cell 1978 Sep, 15(1) 261-7], obtained from ATCC.

[0166] 4.1.2 Culture medium

[0167] RPMI1640 medium (Gibco). Complete RPMI1640 medium contains 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin. Serum-free RPMI1640 medium does not contain fetal bovine serum.

[0168] 4.1.3 TGF-β2 inducers

[0169] Recombinant mouse IL-4 (IL-4) was purchased from PeproTech. Under in vitro culture conditions, IL-4 stimulated RAW 264.7 cells to produce a large amount of TGF-β2 [cell Biol Int. 2017 Sep; 41(9): 960-968].

[0170] LPS (lipopolysaccharide) was extracted from E. coli serotype O111: B4 (Sigma, St. Louis, MI, USA). Under in vitro culture conditions, LPS stimulated RAW 264.7 cells to express TGF-β2 [PLoS One. 2015 Dec14; 10(12): e0144954].

[0171] 4.1.4 Oligonucleotides

[0172] TIO1 (with sequence listing <400> 1), TIO2 (sequence as shown in Example 1) and TIO3 (sequence as shown in the sequence table <400> 2) as described in Example 1.

[0173] 4.1.5 PCR reagents

[0174] TRIzol reagent was purchased from Invitrogen, USA. EasyScript First-Strand cDNASynthesis SuperMix and Top Green qPCR SuperMix (TransStartTM) kits were purchased from Transgen;

[0175] GAPDH specific primers (used to amplify GAPDH specific cDNA, GAPDH is a housekeeping gene), the sequence of the upstream primer is: 5'-ATCACCATCTTCCAGGAGCGA-3', and the sequence of the downstream primer is 5'-TCTCGTGGTTCACACCCATCA-3'.

[0176] TGF-β2 specific primers (used to amplify TGF-β2 specific cDNA), the sequence of the upstream primer is: 5'-ttgtgaaaaccagagcggagg-3' and the sequence of the downstream primer is: 5'-agaggtgccatcaatacctgc-3'.

[0177] GAPDH-specific primers and TGF-β2-specific primers were synthesized by Takara Biotechnology (Dalian) Co., Ltd. (TakaraBio).

[0178] 4.2. Main experimental equipment and instruments

[0179] 24-well culture plates, flat dishes, frosted glass slides, 300-mesh filters, small tweezers, cell counting plates, droppers, pipettes, CO2 cell culture incubators (SANYO, Japan), cell culture inverted microscopes (Olympus, Japan), centrifuges (Biofuge Fresco, Germany), and fluorescence quantitative PCR instruments (Applied Biosystems, USA, model: ABI Prism 7300).

[0180] 4.3 Experiments

[0181] RAW 264.7 cells were cultured in complete 1640 medium at 37°C and 5% CO2. 5×10 6 RAW264.7 cells / ml were added to a 24-well culture plate, with 1 ml of culture medium per well. IL-4 (25 ng / ml) was added, followed by TIO1, TIO2 or TIO3 (final concentration 10 μg / ml). After culturing for 48 hours, the cells were harvested. Total RNA was extracted using the Trizol method. EasyScript First-Strand cDNA Synthesis SuperMix and Top Green qPCR SuperMix (TransStartTM) kits were used to amplify GAPDH-specific cDNA and TGF-β2 mRNA-specific cDNA using qPCR with GAPDH-specific primers and TGF-β2-specific primers, respectively. Fluorescence quantitative analysis was performed according to the kit instructions.

[0182] Add 1 ml of culture medium containing RAW 264.7 cells to a 24-well culture plate, with 5 × 10 cells per well. 6 . Add LPS (2μg / mL), culture for 2 hours, and add TIO1, TIO2 or TIO3 (final concentration 10μg / ml). After 48 hours of culture, the cells were harvested. Total RNA was extracted using the Trizol method. EasyScript First-Strand cDNA Synthesis SuperMix and Top Green qPCR SuperMix (TransStartTM) kits were used to amplify GAPDH-specific cDNA and TGF-β2mRNA-specific cDNA by qPCR using GAPDH-specific primers and TGF-β2-specific primers, respectively. Fluorescence quantitative analysis was performed according to the kit instructions.

[0183] Results

[0184] TIO1 or TIO3 significantly reduced the TGF-β2 mRNA expressed by IL-4-stimulated mouse macrophages (Figure 3), and also significantly reduced the level of TGF-β2 mRNA expressed by LPS-stimulated mouse macrophages (Figure 4). These results indicate that TIO1 or TIO3 can significantly reduce the level of TGF-β2 mRNA in immune cells including macrophages, thereby reducing the synthesis and release of TGF-β2, and then weakening or inhibiting TGF-β2-mediated immunosuppression. The results also indicate that TIO1 or TIO3 can enhance the immune response to microbial antigens (vaccines) or tumor antigens (vaccines) by reducing TGF-β2 mRNA in immune cells, and thus can be used as adjuvants for pathogenic microbial vaccines and tumor vaccines to produce anti-infection and anti-tumor effects, and can also be used for tumor treatment.

[0185] Example 5 Effects of TIO1 and TIO3 on the Expression of CD86 by Antigen Presenting Cells in Mice Inducing Immune Recall Responses:

[0186] 5.1 Materials

[0187] ICR mice (Animal Room, School of Medicine, Jilin University), female, weighing 17-18 g.

[0188] 5.1.1.2 Antigens

[0189] Recombinant circovirus 2b capsid protein (CP) was used as the antigen [Vaccine. 2016 Dec 7; 34(50): 6358-6366].

[0190] 5.1.1.3 Adjuvants

[0191] ISA 35 emulsifier (Seppic)

[0192] 5.1.1.4 Oligonucleotides

[0193] TIO1 (with sequence listing <400> 1), TIO2 (sequence as shown in Example 1) and TIO3 (sequence as shown in the sequence table <400> 2), as described in Example 1.

[0194] 5.1.1.5 Fluorescent Antibodies

[0195] FITC-labeled anti-mouse CD11c antibody and PE-labeled anti-mouse CD86 antibody were purchased from BD Biosciences.

[0196] 5.1.1.6 Culture medium

[0197] RPMI1640 medium (Gibco). Complete RPMI1640 medium contains 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin. Serum-free RPMI1640 medium does not contain fetal bovine serum.

[0198] 5.2 Main experimental equipment

[0199] Flat dishes, frosted glass slides, 300-mesh filters, small tweezers, cell counting plates, droppers, and pipettes. CO2 cell culture incubator (SANYO, Japan), cell culture inverted microscope (Olympus, Japan), centrifuge (BiofugeFresco, Germany), flow cytometer Accuri C6 flow cytometer (BD Bioscience), and flow cytometer result analysis software NovoExpress software (ACEA Biosciences).

[0200] 5.3 Experiments

[0201] 5.3.1 Vaccine preparation

[0202] CP was dissolved in PBS and mixed with ISA35 emulsifier at a ratio of 1:1 (volume:volume) to prepare a vaccine, which was named CP-ISA35. 100μl CP-ISA35 contained 10μg CP. CP-ISA35 containing TIO1, TIO2 and TIO3 were prepared separately, and the dose of TIO1, TIO2 and TIO3 was 10μg / 100μl. The vaccines containing TIO1, TIO2 and TIO3 were named CP-ISA35+TIO1, CP-ISA35+TIO2 and CP-ISA35+TIO3, respectively.

[0203] 5.3.2 Immunization of mice

[0204] The mice were given a primary immunization on day 0. 100 μl of CP-ISA35, CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3 were injected into the right hind limb muscle at a single point. The mice were given a booster immunization on day 14, using the same method as the primary immunization. Recall immunization was induced 120 days after the booster immunization, by injecting 100 μl of CP-ISA35, CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3 into each group of mice, respectively, at a single point in the right hind limb muscle.

[0205] 5.3.3. Isolation of draining mouse lymph node cells

[0206] The mice were sacrificed 6 hours after immunization to induce immune recall response, and the mouse draining lymph node cells were isolated (as described in Example 3, 3.3).

[0207] 5.3.4. Fluorescent antibody staining and flow cytometry analysis of draining lymph node cells

[0208] 1x10 6 Transfer lymph node cells to 5 ml round-bottom polystyrene tubes. Wash cells by centrifugation with FACS buffer (277 g, 5 minutes, 4°C). Discard supernatant and suspend cells in 50-100 μl FACS buffer containing FITC-labeled anti-mouse CD11c antibody and PE-labeled anti-mouse CD86 antibody. The content of each fluorescent antibody is 0.5-1 μg, and the reaction is carried out on ice for 30-45 minutes. Protect samples from light. Perform flow cytometry analysis.

[0209] 5.4 Results

[0210] TIO1 or TIO3 can significantly increase the number of CD11c-positive cells (dendritic cells) expressing CD86 in mouse lymph node cells (Figure 5). This result shows that the application of TIO1 or TIO3 can significantly increase the co-stimulatory molecules on the surface of antigen-presenting cells by reducing the expression of TGF-β2, thereby enabling antigen-presenting cells to provide more second activation signals to T cells to promote adaptive immune responses to microbial antigens and tumor antigens. TIO1 or TIO3 with this activity can be used in individuals to enhance their immune responses to microbial antigens (vaccines) and tumor antigens (vaccines), and can also be used in individuals to enhance their anti-tumor responses.

[0211] Example 6 Effects of TIO1 and TIO3 on the increase of CD4+ T lymphocytes in draining lymph nodes of mice induced by immune recall response

[0212] 6.1 Materials

[0213] 6.1.1 Mice

[0214] ICR mice (Animal Room, School of Medicine, Jilin University), female, weighing 17-18 g.

[0215] 6.1.2 Antigens

[0216] Recombinant circovirus 2b capsid protein (CP) was used as the antigen [Vaccine. 2016 Dec 7; 34(50): 6358-6366].

[0217] 6.1.3 Adjuvants

[0218] ISA 35 emulsifier (Seppic)

[0219] 6.1.4 Oligonucleotides

[0220] TIO1 (with sequence listing <400> 1) and TIO3 (having the sequence shown in the sequence list <400> 2) as described in Example 1.

[0221] 6.1.5 Fluorescent Antibodies

[0222] FITC-labeled anti-mouse CD4 antibody was purchased from BD Biosciences.

[0223] 6.1.6 Culture medium

[0224] RPMI1640 medium (Gibco). Complete RPMI1640 medium contains 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin. Serum-free RPMI1640 medium does not contain fetal bovine serum.

[0225] 6.2 Main experimental equipment

[0226] Flat dishes, frosted glass slides, 300-mesh filters, small tweezers, cell counting plates, droppers, and pipettes. CO2 cell culture incubator (SANYO, Japan), cell culture inverted microscope (Olympus, Japan), centrifuge (BiofugeFresco, Germany), flow cytometer Accuri C6 flow cytometer (BD Bioscience), and flow cytometer result analysis software NovoExpress software (ACEA Biosciences).

[0227] 6.3 Experiments

[0228] 6.3.1 Vaccine preparation

[0229] CP was dissolved in PBS and mixed with ISA35 emulsifier at a ratio of 1:1 (volume:volume) to prepare a vaccine, which was named CP-ISA35. 100μl CP-ISA35 contained 10μg CP. CP-ISA35 containing TIO1, TIO2 and TIO3 was prepared, and the dose of TIO1, TIO2 and TIO3 was 10μg / 100μl. The vaccines containing TIO1, TIO2 and TIO3 were named CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3.

[0230] 6.3.2 Immunization of mice

[0231] The mice were given a primary immunization on day 0. 100 μl of CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 were injected into the right hind limb muscle at a single point. The mice were given a booster immunization on day 14, using the same method as the primary immunization. 120 days after the booster immunization, an immune recall response (Fecall) immunization was performed by injecting 100 μl of CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 into each group of mice at a single point in the right hind limb muscle.

[0232] 6.3.3. Isolation of draining mouse lymph node cells

[0233] The mice were sacrificed 6 hours after the induction of recall response immunization, and the mouse draining lymph node cells were isolated (as described in Example 3, 3.3).

[0234] 6.3.4. Fluorescent antibody staining and flow cytometry analysis

[0235] 1x10 6 Transfer lymph node cells to a 5 ml round-bottom polystyrene tube. Wash cells by centrifugation with FACS buffer (277 g, 5 min, 4°C). Discard supernatant and suspend cells in 50-100 μl FACS buffer containing FITC-labeled anti-mouse CD4. The content of fluorescent antibody is 0.5-1 μg, and the reaction is carried out on ice for 30-45 min. Protect samples from light. Perform flow cytometry analysis.

[0236] 6.4 Results

[0237] TIO1 or TIO3 can induce CD4-expressing lymphocytes (CD4 + The results showed that the application of TIO1 or TIO3 can significantly increase the number of antigen-specific helper T cells by reducing the expression of TGF-β2, thereby promoting the adaptive immune response to microbial antigens and tumor antigens. TIO1 or TIO3 with this activity can be used in individuals to enhance their immune response to microbial antigens (vaccines) and tumor antigens (vaccines), and can also be used in individuals to enhance their anti-tumor response.

[0238] Example 7 Effects of TIO1 and TIO3 on CD19 in draining lymph nodes of mice induced by immune recall response + Increased B lymphocytes

[0239] 7.1 Materials

[0240] ICR mice (Animal Room, School of Medicine, Jilin University), female, weighing 17-18 g.

[0241] 7.1.2 Antigens

[0242] Recombinant circovirus 2b capsid protein (CP) was used as the antigen [Vaccine. 2016 Dec 7; 34(50): 6358-6366].

[0243] 7.1.3 Adjuvants

[0244] ISA 35 emulsifier (Seppic)

[0245] 7.1.4 Oligonucleotides

[0246] TIO1 (with sequence listing <400> 1), TIO2 (sequence as shown in Example 1) and TIO3 (sequence as shown in the sequence table <400> 2) as described in Example 1.

[0247] 7.1.5 Fluorescent Antibodies

[0248] FITC-labeled anti-mouse CD19 antibody was purchased from BD Biosciences.

[0249] 7.1.6 Culture medium

[0250] RPMI1640 medium (Gibco). Complete RPMI1640 medium contains 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin. Serum-free RPMI1640 medium does not contain fetal bovine serum.

[0251] 7.2 Main experimental equipment

[0252] Flat dishes, frosted glass slides, 300-mesh filters, small tweezers, cell counting plates, droppers, and pipettes. CO2 cell culture incubator (SANYO, Japan), cell culture inverted microscope (Olympus, Japan), centrifuge (BiofugeFresco, Germany), flow cytometer Accuri C6 flow cytometer (BD Bioscience), and flow cytometer result analysis software NovoExpress software (ACEA Biosciences).

[0253] 7.3 Experiments

[0254] 7.3.1 Vaccine preparation

[0255] CP was dissolved in PBS and mixed with ISA35 emulsifier at a ratio of 1:1 (volume:volume) to prepare a vaccine, which was named CP-ISA35. 100μl CP-ISA35 contained 10μg CP. CP-ISA35 containing TIO1, TIO2 and TIO3 was prepared, and the dose of TIO1, TIO2 and TIO3 was 10μg / 100μl. The vaccines containing TIO1, TIO2 and TIO3 were named CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3.

[0256] 7.3.2 Immunization of mice

[0257] The mice were given a primary immunization on day 0. 100 μl of CP-ISA35, CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3 were injected into the right hind limb muscle at a single point. The mice were given a booster immunization on day 14, using the same method as the primary immunization. Recall immunization was performed 120 days after the booster immunization, by injecting 100 μl of CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 into each group of mice, using a single injection into the right hind limb muscle.

[0258] 7.3.3. Isolation of Draining Mouse Lymph Node Cells

[0259] The mice were sacrificed 6 hours after the induction of recall response immunization, and the mouse draining lymph node cells were isolated (as described in Example 3, Experiment 3.3).

[0260] 7.3.4. Fluorescent antibody staining and flow cytometry analysis

[0261] 1x10 6 Transfer lymph node cells to 5 ml round-bottom polystyrene tubes. Wash cells by centrifugation with FACS buffer (277 g, 5 minutes, 4°C). Discard supernatant and suspend cells in 50-100 μl FACS buffer containing FITC-labeled anti-mouse CD19 antibody. The content of each fluorescent antibody is 0.5-1 μg, and the reaction is carried out on ice for 30-45 minutes. Protect samples from light. Perform flow cytometry analysis.

[0262] 7.4 Results

[0263] TIO1 or TIO3 can significantly increase the number of cells (B lymphocytes) expressing CD19 in mouse lymph node cells (Figure 7). CD19 is a surface marker of B lymphocytes. B lymphocytes that specifically increase due to recall reactions will rapidly differentiate into plasma cells that produce specific antibodies. This result shows that the application of TIO1 or TIO3 can increase the number of antigen-specific B lymphocytes by reducing the expression of TGF-β2. TIO1 or TIO3 with this activity can be used in individuals to enhance their long-lasting humoral immune response to pathogenic microbial antigens (vaccines).

[0264] Example 8 The enhancing effect of TIO1 and TIO3 on the immune efficacy of hepatitis B vaccine:

[0265] 8.1 Materials

[0266] 8.1.1. Mice

[0267] Balb / c mice, female, weighing 17-18 g, were purchased from Beijing Weitonglihua Experimental Animal Co., Ltd.

[0268] 8.1.2 Vaccines

[0269] Hepatitis B surface antigen (HBsAg) vaccine (Watson Biotech Co., Ltd.) formulated with aluminum adjuvant, referred to as HBsAg vaccine or HBsAg.

[0270] 8.1.3 Oligonucleotides

[0271] Oligonucleotides

[0272] TIO1 (with sequence listing <400> 1), TIO2 (sequence as shown in Example 1) and TIO3 (sequence as shown in the sequence table <400> 2) as described in Example 1.

[0273] 8.1.4 Preparation of Oligonucleotide-Containing Vaccines

[0274] TIO1, TIO2 or TIO3 are added to HBsAg vaccine to prepare oligonucleotide HBsAg vaccine, which are named HBsAg-TIO1, HBsAg-TIO2 and HBsAg-TIO3 respectively. Each 100μl vaccine contains 1μg HBsAg. In HBsAg-TIO1, HBsAg-TIO2 and HBsAg-TIO3, the content of oligonucleotide is 10μg. 8.1.5

[0276] Recombinant hepatitis B surface antigen (HBsAg) (Watson Biotechnology Co., Ltd.), referred to as HBsAg.

[0277] 8.2. Immunization of mice

[0278] Balb / c mice were given a primary immunization on day 0 by injecting 100 μl of HBsAg vaccine, HBsAg-TIO1, HBsAg-TIO2 or HBsAg-TIO3. The injection site was the right hind limb muscle, with a single injection. Balb / c mice were given a booster immunization on day 14, using the same method as the primary immunization.

[0279] 8.3. Blood collection after immunization

[0280] Collect serum from immune mice on the 28th day after booster immunization. Collect blood from tail vein, the blood volume ≥ 100μl. Place the collected whole blood (in 1.5ml EP tube) at room temperature for 30 minutes. Centrifuge at 11000rpm for 15 minutes, collect serum, aliquot, and store at -20℃.

[0281] 8.4. Detection of specific antibodies in sera of immunized mice

[0282] ELISA method was used to detect the specific antibody of hepatitis B surface antigen (HBsAg) in mouse serum. HBsAg was coated on the enzyme label strip in the coating solution, 100 μl / well (0.1 μg HBsAg per well), and incubated at 4°C overnight. The dilution of the serum to be tested was 1:6400.

[0283] The remaining serum operation steps are the same as those described in 2.5 of Example 2.

[0284] Results

[0285] TIO1 or TIO3 can enhance the immune efficacy of recombinant protein vaccines (hepatitis B virus vaccines) (Figure 8). This shows that TIO1 or TIO3 can be used in individuals to enhance their immune response to pathogenic microbial antigens (vaccines) and become a new adjuvant for microbial antigens or microbial vaccines.

[0286] Example 9 The synergistic effect of TIO1 or TIO3 on influenza virus vaccine

[0287] 9.1. Materials

[0288] 9.1.1 Mice

[0289] ICR mice (Jilin University Experimental Animal Center). Female, weighing about 18 grams.

[0290] 9.1.2 Oligonucleotides

[0291] TIO1 (with sequence listing <400> 1) and TIO3 (having the sequence shown in the sequence list <400> 2), as described in Example 1.

[0292] 9.1.3 Vaccines

[0293] FM1 (mouse-adapted influenza virus, from the Department of Biology, Massachusetts Institute of Technology) was amplified using 11-day-old chicken embryos, and allantoic fluid was obtained. The hemagglutination inhibition test (HA) was used to determine the hemagglutination unit (HA unit, HAU) of FM1 in the allantoic fluid. FM1 was inactivated with 10% V / V formalin to prepare whole-inactivated viruses (WIV) [Vaccine 2016 Jan 20; 34(4)495-502]. ISA 35 emulsifier (Seppic) was used to prepare FM1 vaccine (abbreviated as FM1). Each 100μl FM1 vaccine contained 700HAU of inactivated FM1. TIO1 or TIO3 was added to the FM1 vaccine to prepare an oligonucleotide-containing FM1 vaccine, which was named FM1-TIO1 and FM1-TIO3, respectively, and the oligonucleotide content was 10μg.

[0294] 9.2. Immunization and blood collection

[0295] Immunization was performed twice, and a booster immunization was performed 22 days after the initial immunization. The hind leg muscle injection was used, and the volume of the vaccine injected was 100 μl. Blood was collected from the tail vein on the 28th day after the booster immunization, 50 μl / mouse. The collected blood was placed in a 37°C environment for 30 minutes, and then placed at 4°C for 3 hours to allow the serum to be fully separated. It was centrifuged at 4000r / m for 10 minutes, and the serum was aspirated and diluted 1:400 times for ELISA detection.

[0296] 9.3. Detection of specific antibodies in sera of immunized mice

[0297] The ELISA method was used to detect influenza virus-specific antibodies in mouse serum. The antigen of the FM1 inactivated virus was used as the coating antigen, and the coating solution (Na2CO3 1.59g, NaHCO3 2.93g, pH 9.6, fixed to 1L) was diluted 1:2, and the ELISA plate was coated with 100μl / well, sealed, and incubated at 4℃ overnight; the ELISA plate was washed with washing solution (PBS containing 0.05% Tween-20), 300μl / well, washed 3 times; the blocking solution (PBS containing 5% FBS) was added, 200μl / well, and incubated at 37℃ 2 hours; dilute mouse serum with PBS (1:4000 dilution), add the diluted serum to the ELISA plate, 100 μl / well, and place at 37°C for 1 hour; wash 3 times with washing solution, then add horseradish peroxidase-labeled goat anti-mouse secondary antibody (diluted 1:1000 with blocking solution), 100 μl / well, and place at 37°C for 1 hour; wash 3 times with washing solution, add ready-to-use substrate solution (0.01M citric acid, 0.02M Na2HPO4, 9mL ultrapure water, 15μl 30% H2O2, 4mg OPD in 10 mL), 100 μL / well, and develop color at room temperature in the dark for 20 minutes; add stop solution (20% sulfuric acid), 50 μl / well; measure the OD value of each well on A492.

[0298] Results

[0299] TIO1 or TIO3 can enhance the immune efficacy of virus vaccines (influenza virus vaccines) (Figure 9). This shows that TIO1 or TIO3 can be used in individuals to enhance their immune response to pathogenic microbial antigens (vaccines) including influenza viruses and become a new type of microbial vaccine adjuvant.

[0300] Example 10 The synergistic effect of TIO1 or TIO3 on rabies vaccine

[0301] 10.1 Materials

[0302] 10.1.1 Mice

[0303] ICR / c mice, weighing 18-22 g, were obtained from the Animal Laboratory of the Medical College of Jilin University.

[0304] 10.1.2 Vaccines

[0305] Rabies vaccine (Changchun Institute of Biological Products).

[0306] 10.1.3 Oligonucleotides

[0307] TIO1 (with sequence listing <400> 1), TIO2 (sequence as shown in Example 1) and TIO3 (sequence as shown in the sequence table <400> 2), as described in Example 1.

[0308] 10.1.4 Vaccines

[0309] TIO1, TIO2 or TIO3 are added to the rabies vaccine to prepare the oligonucleotide-containing rabies vaccine, which are named rabies vaccine-TIO1, rabies vaccine-TIO2 or rabies vaccine-TIO3 respectively. The content of oligonucleotide in each 500 μl of the oligonucleotide-containing rabies vaccine is 10 μg.

[0310] 10.2 Blood collection before immunization

[0311] Two days before the first immunization, serum was collected from the mice to be immunized. Blood was collected from the tail vein, 50 μl / mouse. The collected blood was placed in a 37°C environment for 30 minutes, and then placed at 4°C for 3 hours to allow the serum to be fully separated. The blood was centrifuged at 4000 r / m for 10 minutes, and the serum was aspirated and stored at -20°C.

[0312] 10.3. Immunization of mice

[0313] The mice were intraperitoneally injected with 0.5 ml of rabies vaccine, rabies vaccine-TIO1, rabies vaccine-TIO2 or rabies vaccine-TIO3 on days 0, 3, 7, 14 and 28. Each group of mice consisted of half male and half female.

[0314] 10.4 Blood Collection

[0315] On day 35, serum from immunized mice was collected using the method described in 10.2.

[0316] 10.5 Serum rabies virus neutralizing antibody detection

[0317] The rapid rabies vaccine fluorescent focus inhibition test (RFFIT) method was used to detect rabies virus neutralizing antibodies in mouse serum, and the results were expressed in international units / ml (IU / ml) [Virol Sin. 2012 Jun; 27(3): 187-93].

[0318] 10.6. Results

[0319] TIO1 or TIO3 can enhance the immune efficacy of inactivated virus vaccine (rabies vaccine) (Figure 10). This shows that TIO1 or TIO3 can enhance an individual's immunity against rabies virus infection and can be used as an adjuvant for pathogenic microorganism vaccines including rabies vaccines.

[0320] Example 11. The synergistic effect of TIO3 on glioma cell lysate vaccine

[0321] 11.1 Materials

[0322] 11.1.1 Mice

[0323] C57BL / 6 mice were purchased from Beijing Weitonglihua Experimental Animal Co., Ltd.

[0324] 11.1.2 GL261 cells

[0325] GL261 cells are glioma cells derived from C57BL / 6 mice (ATCC).

[0326] 11.1.3 Oligonucleotides

[0327] TIO3 (with sequence listing <400> 2), as described in Example 1.

[0328] 11.1.4 Culture medium

[0329] RPMI1640 medium (Gibco. Complete RPMI1640 medium contains 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin. Serum-free RPMI1640 medium does not contain fetal bovine serum

[0330] Preparation of GL261 cell lysate

[0331] GL261 cells were cultured in complete RPMI1640 medium at 37°C and 5% CO2. 1 ml of GL261 cells (1×10 7 / ml) was inoculated into the abdominal cavity of healthy C57BL / 6 mice. After the abdomen of the mouse was swollen, the mouse was killed and immersed in 75% ethanol for 2-3 minutes for disinfection. The abdominal cavity of the mouse was opened in the clean bench, and the GL261 cell solid tumor formed was visible. The GL261 cell solid tumor was removed according to sterile operation. It was cut into small pieces in a flat dish with surgical scissors, and repeatedly rinsed with physiological saline to remove tumor tissue and residual blood. The washed tissue blocks were repeatedly ground in a tissue grinder to collect the grinding liquid. The grinding liquid was repeatedly frozen and thawed in liquid nitrogen and room temperature for 5 times. After centrifugation at 2000rmp for 10 minutes, the supernatant was collected. This supernatant is the GL261 cell glioma tumor cell lysate (GTL). The protein content of GTL was determined by Bradford method. GTL was further analyzed by SDS-PAGE. GTL was stored in a -80℃ refrigerator.

[0332] 11.3 Preparation of GTL vaccines and oligonucleotide-containing GTL vaccines

[0333] The protein concentration of GTL was adjusted to 4 mg / ml with PBS. The GTL and emulsifier were mixed at 1:1 (volume:volume) to prepare a vaccine, which was named GTL vaccine. The GTL vaccine containing TIO-3 was prepared by adding TIO-3 to the GTL vaccine, which was called GTL-TIO-3 vaccine, wherein the concentration of TIO-3 was 10 μg / 100 μl.

[0334] 11.4 Immunization of mice

[0335] On days 0 and 9, mice were injected subcutaneously with 100 μl of GTL vaccine or GTL-TIO-3 vaccine in the neck.

[0336] 11.5 Inoculation of glioma cells

[0337] On day 14, 1 × 10 4 GL261 cells were injected into the brain of mice with a volume of 2 μl to induce glioma in mice, as described in the literature (Prins RM, et al. Cancer Res. 2003 Dec 1; 63(23): 8487-91). The survival of mice was observed and recorded from the time of tumor inoculation, and autopsies were performed after the death of mice to confirm that glioma had occurred in the brain of mice.

[0338] 11.6 Results

[0339] TIO3 can significantly enhance the immune efficacy of glioma cell lysate (tumor antigen vaccine) ( Fig.11 ), significantly prolonging the survival of tumor-bearing mice. This result suggests that TIO3 can enhance an individual's anti-glioma immune response and can be used as an adjuvant for tumor vaccines, including glioma vaccines.

[0340] Example 12, the synergistic effect of TIO1 or TIO3 on lung cancer cell lysate vaccine:

[0341] 12.1 Materials

[0342] 12.1.1 Mice

[0343] C57BL / 6 male mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.), 6 weeks old.

[0344] 12.1.2 Cells

[0345] Lewis lung carcinoma (LLC) cells (ATCC) were derived from C57BL / 6 mice and cultured in complete RPMI1640 medium (as described in 11.1.4) at 37°C and 5% CO2.

[0346] 12.1.3 Oligonucleotides

[0347] TIO1 (with sequence listing <400> 1) and TIO3 (having the sequence shown in the sequence list <400> 2), as described in Example 1.

[0348] 12.2. Preparation of Lewis lung cancer cell (LLC) lysate

[0349] LLC cells (1×10 6 / 0.1ml) was inoculated into the back of C57BL / 6J male mice subcutaneously to form solid tumors. The mice were sacrificed, the tumors were removed, and LLC lysates (LTL) were prepared, identified, and stored according to the method described in 11.2 of Example 11. 6 LLC cells were used to prepare 100 μl of LTL.

[0350] 12.3 Preparation of LTL vaccines and oligonucleotide-containing LTL vaccines

[0351] The prepared LTL and emulsifier were mixed at a ratio of 1:1 (volume:volume) to prepare a vaccine, which was named LTL vaccine. TIO1 or TIO3 was added to the LTL vaccine to prepare an LTL vaccine containing TIO1 or TIO3, which were respectively named LTL-TIO1 vaccine and LTL-TIO3 vaccine, wherein the concentration of TIO1 or TIO3 was 10 μg / 100 μl.

[0352] 12.4 Immunization of mice

[0353] 6-week-old C57BL / 6J male mice were injected subcutaneously on the right back with 100 μl of LTL vaccine, LTL-TIO1 vaccine or LTL-TIO3 vaccine on day 0 and day 14. There were 10 mice in each group.

[0354] 12.5 Inoculation of Lewis lung cancer (LLC) cells

[0355] On the 21st day, 100 μl LLC cell suspension was injected subcutaneously on the left back of the mice. LLC cell suspension was prepared by: LLC cells (1×10 7 / 100μl) was inoculated into the back of C57BL / 6J male mice to form solid tumors. The mice were sacrificed, and the tumor tissue was removed according to the aseptic operation method. It was digested with 0.25% trypsin-0.04% EDTA for 30 minutes to prepare a single cell suspension. The cell concentration was adjusted to 1×10 6 / 100 μl. On the 18th day after tumor inoculation, the mice were sacrificed, the tumor masses were dissected out, and their weights were measured.

[0356] 12.6 Results (Table 1)

[0357] Table-1. The synergistic effect of TIO1 or TIO3 on lung cancer cell lysate vaccine

[0358]

[0359] The results showed that TIO-1 or TIO-3 could enhance the immune efficacy of lung cancer cell lysates (Table 1) and shrink the tumor (p < 0.05). This indicates that TIO-1 or TIO-3 can enhance the individual's anti-lung cancer immune response and be used as an adjuvant for tumor vaccines including lung cancer vaccines.

[0360] Example 13 The therapeutic effect of TIO3 on gastric cancer:

[0361] 13.1 Materials

[0362] 13.1.1 Mice

[0363] Balb / c female mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.), 18-22 g.

[0364] 13.1.2 Gastric Cancer Cells

[0365] MFC cells (mouse gastric cancer cells) are gastric cancer cells derived from Balb / c mice (from ATCC, USA). The cells were cultured in complete RPMI1640 medium (as described in 11.1.4) at 37°C and 5% CO2.

[0366] 13.1.3 Oligonucleotides

[0367] TIO3 (with sequence listing <400> 2), as described in Example 1.

[0368] 13.2 Main experimental equipment and instruments

[0369] 100ml cell culture flask, 1ml syringe, cell counting plate, CO2 cell culture incubator (SANYO, Japan), cell culture inverted microscope (Olympus, Japan), centrifuge (Biofuge Fresco, Germany).

[0370] 13.3 Experiments

[0371] The in vitro cultured MFC subcutaneous transplant tumor model cells were inoculated subcutaneously on the dorsal left hind limb of mice on day 0. The injection volume was 200 μl, containing 1×10 6MFC cells. On the 10th day after inoculation of MFC cells, mice were injected with TIO3 (dissolved in PBS), and then injected once every two days, for a total of 6 injections. The injection site was subcutaneous in the left hind limb, where the tumor lymph node was drained. The injection volume for each injection was 100 μl, containing 25 μg of TIO3. The control group mice were injected with PBS according to the same procedure. The survival time of the mice was observed and recorded.

[0372] 13.4 Results

[0373] The use of TIO3 alone can significantly prolong the survival of tumor-bearing mice (Figure 12). This shows that TIO3 can enhance the individual's anti-gastric cancer cell immune response and can be used to treat tumors including gastric cancer.

Claims

1. Use of a single-stranded deoxyoligonucleotide having a sequence as shown in SEQ ID NO: 2 in the preparation of a drug for enhancing the immune efficacy of influenza virus vaccines.

2. Use of a single-stranded deoxyoligonucleotide having a sequence as shown in SEQ ID NO: 2 in the preparation of a drug for enhancing the immune efficacy of circovirus vaccines.

3. Use of the single-stranded deoxyoligonucleotide with the sequence shown in SEQ ID NO: 2 in the preparation of a drug for enhancing the immune efficacy of hepatitis B virus vaccine.

4. Use of the single-stranded deoxyoligonucleotide with the sequence shown in SEQ ID NO: 2 in the preparation of a drug for enhancing the immune efficacy of rabies virus vaccine.

5. The use according to claims 1-4, wherein the medicine further comprises a pharmaceutically acceptable carrier.

Images