CN110229813B - Oligonucleotides with vaccine adjuvant and tumor therapeutic effects - Google Patents

Abstract

The invention provides two single-stranded deoxyoligonucleotides which can be used as an adjuvant of a microbial vaccine and a tumor vaccine, show anti-infection and anti-tumor effects, and can be used for treating tumors singly or in combination with other anti-tumor preparations.

Description

Oligonucleotides with vaccine adjuvant and tumor therapeutic effects

Technical field:

the present invention relates to two oligonucleotides and their use as vaccine adjuvants and in the treatment of tumors.

The background technology is as follows:

Transforming growth factor-beta 2 (transforming growth factor beta, TGF-beta 2) is a member of the transforming growth factor-beta cytokine superfamily (TGF-beta Cytokine Superfamily). There are 40 cytokines in the TGF-beta superfamily, among which TGF-beta 1, TGF-beta 2 and TGF-β3[Rider CC,et al.Molecules.2017 Apr 29;22(5).pii：E713;Travis MA,et al.Annu Rev Immunol.2014;32：51-82], are also commonly referred to as TGF-beta. The TGF-beta 1, TGF-beta 2 and TGF-beta 3 after translation synthesis are all precursor molecules, and consist of a signal peptide, an N-terminal fragment (LAP-associated peptide) 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-beta. TGF-beta 1, TGF-beta 2 and TGF-beta 3 share 71-79% homology at the amino acid level [ Travis MA, et al Annu Rev Immunol.2014;32:51-82].

TGF-beta receptors (TβR) capable of transducing TGF-beta signals are tetramers containing 2 TβRI and 2 TβRII molecules, the TβRI and TβRII being serine/threonine kinases. Once the tetrameric complex is formed, the TβRII kinase activates, recognizing the specific serine residue of TβRI, causing it to activate [ Ahmed S, et al J Clin Med.2017 Jan 5;6 (1). Pii: E5]. TGF- β2 activated signaling relies on β -glycans (β -glycan), also known as TGF- βRIII. Beta-glycans assist TGF-beta 2 in binding TGF-beta RII [ Travis MA, et al Annu Rev Immunol.2014;32:51-82]. TGF- β receptors transduce signals through Smad proteins upon activation. Smad proteins are of three classes, receptor-regulated Smads (R-Smads), common mediator Smad (co-Smad) and inhibitory Smads (I-Smad). Smad2 and Smad3 are R-Smads, direct substrates for TβRI. TGF-beta binding activates the TGF-beta receptor, and the intracellular end of activated TGF-beta RI recruits Smad2 and Smad3 and phosphorylates them directly. Phosphorylated Smad2/3 and Smad4 (co-Smad) form trimers (Smad complexes). Smad complexes translocate to the nucleus, regulating the expression of a variety of genes. Activated I-Smad (Smad 6/7) negatively regulates TGF- β signaling [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 by promoting T beta RI degradation, inhibiting Smad2/Smad3 phosphorylation, inhibiting Smad complex binding chromatin, and can transduce activation signals through both the classical (canonical pathway) and non-classical (noncanonical pathway) pathways [ Huang JJ, et al Biochem Soc Trans.2016 Oct 15;44 (5): 1441-1454]. In humans and mice, T.beta.RIII is expressed predominantly in macrophages, neutrophils, megakaryocytes, dendritic cells and T cells [ Ortega-Francisco S, et al biochem Biophys Res Commun.2017 Dec 9;494 (1-2): 82-87], suggesting that these cells are also target cells for TGF-. Beta.2.

TGF-beta has a negative regulatory effect on immune responses, modulating a variety of adaptive immune response cells and innate immune response cells, including dendritic cells, T cells, B cells, NK cells, innate immune lymphoid cells (innate lymphoid cells) and granulocytes [ Kelly A, et al Adv immunol.2017;134:137-233]. TGF-beta 2 down regulates expression of major histocompatibility complex (major histocompatibility complex, MHC) molecules. Under in vitro culture conditions, TGF-beta 2 (1, 5, or 10 ng/ml) significantly down-regulates MHC class I and MHC class II molecules on the surface of mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs) and also partially antagonizes the upregulation of MHC class I and MHC class II molecules by IFN-gamma [ Berglund AK, et al front Vet Sci.2017 Ju 12;4:84]. TGF-. Beta.2 significantly down-regulates microglial expression of MHC class II molecules in experimental allergic encephalitis (experimental allergic encephalomyelitis, EAE) mice [ De Feo D, et al J Clin invest.2017Nov 1;127 (11): 3937-3953]. TGF-. Beta.2 antagonizes IFN-. Gamma.and TNF-. D induction of MHC class II expression by rat astrocytes [ Schluesener HJ Neuroimmunol (1990) 27:41-7]. TGF-beta 2 interferes with the function of antigen presenting cells. Bone marrow derived dendritic cells (BMDCs) express T beta R. Under in vitro culture conditions, TGF-beta 2 significantly inhibits CD40L from stimulating BMDCs expression and secretion of IL-23[De Feo D,et al.J Clin Invest.2017Nov 1;127 (11): 3937-3953]. This inhibition was almost completely abolished 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 immunity inducer. In mice, the efficacy of IL-23-supplemented whole influenza virus vaccines (whole influenza virus vaccine, WIV) (IL-23-WIV) in inducing virus-specific nasal IgA via mucosal immunization was significantly improved. High levels of anti-influenza IgA can be present in serum and nasal washes of IL-23-WIV immunized mice. This suggests that inhibition of TGF-. Beta.2 may act as an adjuvant by relieving inhibition of IL-23 secretion. TGF-. Beta.2 inhibits cytokine expression. Under basal and inflammatory conditions, TGF- β2 inhibits keratinocyte expression IL8,CXCL5,CXCL1,chemokine(C-C motif)ligand 20(CCL20),IL1A,IL1B[Li D,et al.J Clin Invest.2015 Aug 3;125(8)：3008-26]. in the immature small intestine, TGF- β2 attenuates LPS-induced IL-6, IL-1β and TNF- α biological activities [ Nguyen DN, et al Am J Physiol Gastrointest Liver Physiol.2014Oct 1;307 (7): G689-99]. TGF- β2 induces suppressor T cells (regulatory T cell, treg). Antigen presenting cells (ANTIGEN PRESENTING CELLS, APCs) expressing TGF-beta 2 in mucosal and ocular tissues can induce Treg [ Mir FA, et al immunology 2015 Dec;146 (4): 547-56]. T-beta RIII mediated signaling was demonstrated to promote TGF-beta-dependent CD4 ⁺ T cell differentiation to Ortega-Francisco S, et al biochem Biophys Res Commun.2017 Dec 9;494 (1-2): 82-87.

TGF-. Beta.2 is a neoplastic factor. 95% of highly fractionated glioma cells express high levels of TGF-. Beta.2, the expression levels of which are positively correlated with the invasiveness of glioma cells [ Zhang C, et al J Exp CLIN CANCER Res.2017 Nov 16;36 (1): 162]. TGF-. Beta.2 was named glioma-derived T-cell inhibitor (glioblastoma-derived T-cell suppressor factor). In glioma patients, TGF-. Beta.2 induces immunosuppression and loss of immune monitoring function. Patients with glioma with high blood TGF-beta 2 levels have increased disease progression and poor prognosis [ Hau P, et al Curr Pharm Biotechnol.201110ec; 12 (12): 2150-7]. TGF- β2 levels in bladder cancer tissue are positively correlated with the extent of muscle infiltration of bladder cancer. TGF-. Beta.2 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 cancers, TGF-beta 2 secreted by Cancer-associated fibroblasts (Cancer-associated fibroblasts) may maintain the malignant phenotype of oral Cancer cells by attenuating the adhesion between glial cells [ Cirillo N, et al, carcinogenic.2017 Jan;38 (1): 76-85]. TGF-beta-2 induces epithelial mesenchymal transition. Epithelial cell 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.

The invention comprises the following steps:

1. The invention provides two single-stranded deoxyoligonucleotides (short oligonucleotides), the sequences of which are shown as a sequence table <400>1 and a sequence table <400> 2. They reduce the mRNA of transforming growth factor-beta 2 (transforming growth factor beta, TGF-beta 2), inhibit the expression of TGF-beta 2 protein and weaken and inhibit the TGF-beta 2 mediated immunosuppression, thereby enhancing the immune response of individuals to pathogenic microorganisms and tumor cells and exhibiting anti-infective and anti-tumor efficacy.

2. Both oligonucleotides may be subjected to various chemical modifications or altered.

3. The two oligonucleotides can be used as adjuvant of pathogenic microorganism vaccine and tumor vaccine, and have anti-infection and anti-tumor effects.

4. Both oligonucleotides can be used for tumor treatment.

5. These two oligonucleotides may be used in combination with other adjuvants or innate immunity enhancers to enhance the protective immune response of an individual against pathogenic microorganisms and tumor cells.

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

Terminology in the invention:

Unless specifically stated otherwise, terms used herein have the ordinary meaning as would be understood by one of ordinary skill in the art to which the present invention pertains. In the event of a conflict in sense, the present specification, including definitions, definitions or explanations, will control.

An "oligonucleotide" is a molecule composed of a plurality of nucleotides, the number of which may be several or several tens. Nucleotides (nucleotides) are the basic building blocks of nucleic acids, also oligonucleotides. The nucleotide consists of a nucleoside (nucleoside) and a phosphate. Nucleosides consist of pentoses (pentose) and bases (base). Pentoses include ribose and deoxyribose. Pentose molecules and bases are linked to form nucleosides (nucleoside). Nucleosides are linked by phosphate groups to form nucleotides. Nucleotides are linked by phosphodiester bonds to form oligonucleotides. Bases constituting nucleosides include pyrimidines and purines. Pyrimidine includes thymine (abbreviated as T or T) and cytosine (abbreviated as C or C). Purines include adenine (abbreviated as a or a) and guanine (abbreviated as G or G). The bases in the oligonucleotide may be rare bases. Rare bases include, but are not limited to, 5-hydroxymethylcytosine, 7-methylguanine and 5-hydroxymethylcytosine. The oligonucleotide may be single-stranded, double-stranded, circular, or a molecule having a circular structure. In the present invention, an Oligonucleotide (ODN) may be replaced with its english abbreviation ODN. The sequence of the nucleotide arrangement in an oligonucleotide constitutes its primary structure, which sequence is also called nucleotide sequence. The nucleotide sequence can be represented by a base sequence, and thus, the sequence of the nucleotide is also referred to as a base sequence. The sequence of the deoxyoligonucleotide may be represented by the base english Wen Suxie, T or T represents thymine, C or C represents cytosine, a or a represents adenine, and G or G represents guanine.

"The oligonucleotides provided by the invention" means two oligonucleotides as described in example 1, designated TIO1 and TIO3.TIO1 has a sequence shown in a sequence table <400>1, and TIO3 has a sequence shown in a sequence table <400> 2. The oligonucleotides provided by the invention are single-stranded deoxyoligonucleotides, which are called Oligonucleotides (ODNs) for short.

"Chemical modification" the oligonucleotides provided by the invention may be chemically modified compared to the natural DNA (chemical modification). Chemical modification of an oligonucleotide is a phenomenon or process whereby the covalent structure of the oligonucleotide is altered by the introduction or removal of any chemical moiety. Chemical modification sites of oligonucleotides can occur at phosphodiester linkages, ribose and bases. Chemical modification of the oligonucleotide may occur at the 5 'end or the 3' end, either during or after synthesis. Chemical modifications to which the present invention relates include, but are not limited to, modifications to the oligonucleotide backbone such as thio modifications (substitution of non-bridging oxygen atoms of phosphates in internucleotide phosphodiester linkages with sulfur atoms) and substitution modifications (including substitution of alkyl groups, aromatic groups, or any other chemical groups). Chemical modifications of oligonucleotides also include base substitutions and base modifications, the substituted bases may be rare bases or derivatives of various bases. Chemical modification of an oligonucleotide also includes attachment of one or more nucleotides and/or any other chemical groups at its 5 'and/or 3' end.

"UTR" means an untranslated region (untranslated region, UTR) of an mRNA molecule, located at both ends of the coding region of the mRNA molecule, at the 5 'end, known as the 5' UTR, and at the 3 'end, known as the 3' UTR.

The oligonucleotide provided by the invention can inhibit and weaken TGF-beta 2 mediated immunosuppression. TGF- β2 mediated immunosuppression refers to the inhibition of the immune response induced by pathogenic microorganisms by TGF- β2, and also refers to the inhibition of the immune response to tumor cells. Reducing and interfering TGF-beta 2mRNA can inhibit TGF-beta 2 mediated immunosuppression, and inhibiting and reducing TGF-beta 2 protein production can also inhibit TGF-beta 2 mediated immunosuppression. TGF-. Beta.2 is an immunosuppressive factor. Inhibition of TGF-beta 2 mediated immunosuppression can enhance an individual's immune response to pathogenic microorganisms, resulting in an anti-infective effect. Inhibiting TGF-beta 2 mediated immunosuppression can break the immune tolerance of an individual to tumor cells and enhance the anti-tumor capability of the individual. Attenuation of TGF-beta 2 mediated immunosuppression by inhibition of TGF-beta 2 mediated immunosuppression may result in an anti-tumor effect [ Kim S, et al Tumour biol.2016Aug;37 (8): 11397-407].

The oligonucleotides provided herein can reduce the level of TGF-beta 2mRNA in immune cells. "decrease" and "decrease" have the same meaning. Reducing the level of TGF-beta 2mRNA can attenuate and inhibit TGF-beta 2 mediated immunosuppression, thus exhibiting an enhancement of immune responses to pathogenic microorganisms and tumor cells. Formulations with reduced levels of TGF-beta 2mRNA can be used as adjuvants for pathogenic microorganism vaccines or tumor vaccines, and can be used alone for tumor treatment.

The oligonucleotide provided by the invention can inhibit the expression of TGF-beta 2 protein. Inhibiting and reducing the production of TGF-beta 2 protein has the same meaning, inhibiting the translation of TGF-beta 2mRNA and inhibiting the expression of TGF-beta 2 protein. The reduction of TGF-beta 2 protein can weaken and inhibit TGF-beta 2 mediated immunosuppression, thus showing enhancement of immune response to pathogenic microorganisms and tumor cells. Formulations with inhibition of TGF-beta 2 expression may be used as adjuvants for pathogenic microorganism vaccines or tumor vaccines, and may also be used alone for tumor treatment.

"Immune response" the oligonucleotides provided by the invention can be used to enhance immune responses to pathogenic microbial antigens and tumor antigens. Immune response (immune response) and immune response have the same meaning. The immune response is a response of an individual's immune cells including B lymphocytes, T gonocytes, NK cells, γδ T cells, NKT cells, dendritic cells, macrophages and granulocytes, etc., to an antigen or other stimulus such as a pathogen-associated model molecule (pathogen associated molecular pattern, PAMP) and a damage-associated model molecule (damage associated molecular pattern, DAMP). The result of the immune response is the selective destruction or elimination of invading pathogenic microorganisms or endogenous tumor cells. The immune response includes both an innate immune response and an adaptive immune response. Adaptive immune responses include cellular immune responses and humoral immune responses. The immune response elicited by vaccines made with antigens of pathogenic microorganisms can confer resistance to infection by pathogenic microorganisms to the individual being immunized. The immune response elicited by vaccines made with tumor antigens can exert a tumor therapeutic effect in the individual being immunized. An anti-infective effect may occur by promoting an immune response in an individual to a pathogenic microorganism. An anti-tumor effect can occur by promoting an individual's immune response to tumor cells. Cytokines with immunosuppressive effects are produced during the immune response. TGF-. Beta.2 is a cytokine with immunosuppressive effects. TGF-. Beta.2 can suppress immune responses. Reducing the level of TGF-beta 2mRNA and inhibiting the expression of TGF-beta 2 promotes the immune response to pathogenic microorganism antigens or tumor antigens, and exhibits immunoprophylaxis or immunotherapeutic effects. The oligonucleotides provided by the invention can be used as an adjuvant for microbial vaccines and tumor vaccines or used for the treatment of tumors by enhancing the immune response to microbial antigens and tumor antigens.

"Lymphocyte" means a mononuclear leukocyte without phagocytic capacity present in blood, lymph fluid and lymphoid tissue, and includes B lymphocyte (also called B cell) and T lymphocyte (also called T cell). T cells can be classified into T cells that surface express the CD4 molecule (CD 4 ⁺ T cells) and T cells that surface express the CD8 molecule (CD 8 ⁺ T cells).

"CD4 ⁺ T cells". The oligonucleotides provided herein can increase the number of specific CD4 ⁺ T cells in draining lymph nodes. CD4 ⁺ T cells are T cells that surface express CD4 molecules, and are helper T lymphocytes (Th). Th assist B cells in producing antibodies and also assist CD8 ⁺ T cells in killing virus-infected cells and tumor cells. Formulations that stimulate CD4 ⁺ T cell proliferation (manifested as an increase in cell number) can enhance an individual's immune response to pathogenic microorganisms and tumor cells.

"CD19 ⁺ lymphocytes" the oligonucleotides provided by the invention can increase the number of specific CD19 ⁺ lymphocytes in draining lymph nodes. CD19 ⁺ lymphocytes are lymphocytes that surface express CD19 molecules, also known as CD19 ⁺ B lymphocytes or CD19 ⁺ cells. Formulations that stimulate CD19 ⁺ lymphocyte proliferation (manifested as an increase in cell number) can enhance the humoral immune response of an individual to a pathogenic microorganism and are used as adjuvants for pathogenic microorganism vaccines.

"Antigen presenting cells" the oligonucleotides provided herein can increase the number of draining lymph node antigen presenting cells (ANTIGEN PRESENTING CELLS, APC). APCs are a class of immune cells that can present peptide fragments derived from microbial or tumor antigens on their surface to activate antigen-specific T cells, including dendritic cells (DENDRITIC CELL, DCs), macrophages, B lymphocytes, and the like. CD11c is a surface marker for DC. Increasing the number of draining lymph node APCs promotes activation of antigen-specific T cells, thereby enhancing immune responses to pathogenic microorganisms and tumor cells.

"CD86" the oligonucleotides provided by the invention can up-regulate and maintain expression of CD86 by dendritic cells. CD86 is a costimulatory molecule (costimulatory molecules) expressed on the surface of antigen presenting cells (ANTIGEN PRESENTING EELLS, APCs) or tumor cells. During the course of the immune response, CD86 may initiate a second signal that activates T lymphocytes. T lymphocyte activation is a key condition that triggers an individual's immune response to pathogenic microorganisms and tumor cells. T lymphocyte activation requires two activation signals to be obtained. The first activation signal is from the recognition of an antigen peptide-MHC complex on the surface of an APC or target cell by a T lymphocyte via a T cell antigen receptor (TCR). The second activation signal is from a costimulatory molecule, including CD86, that is recognized by T lymphocytes through CD28 molecules, binds to APC or the surface of target cells. The second activation signal is also referred to as a co-stimulus signal (costimulatory signals). T lymphocytes that acquire only the first activation signal are not sufficiently activated and may even enter an immune tolerance state. Only after the first and second activation signals are obtained simultaneously, the T lymphocytes are sufficiently activated to function [Sharma P.et al.Science.2015Apr 3;348(6230)：56-61;Greenwald RJ,et al.Annu Rev Immunol.2005;23：515-48]. TGF-β2 to reduce the co-stimulatory molecules of the APC, thereby attenuating the second signal of T lymphocyte activation. The level of TGF-beta 2mRNA and the inhibition of TGF-beta 2 expression can maintain or increase the level of CD86, enhance the second signal of T cell activation, further promote the immune response to pathogenic microorganisms and tumor cells, and enhance the anti-infection and anti-tumor capabilities of individuals.

"CD28 is a protein expressed on the surface of T cells and recognizes CD86 molecules on the surface of antigen presenting cells. After recognition of the CD86 molecule, CD28 provides a co-stimulatory signal (co-stimulatory signals), also known as a second signal, to the T cell.

"T Cell Receptor (TCR) is an antigen receptor expressed on the surface of T cells and is a heterodimeric transmembrane protein. The TCR consists of a variable (V) region and a constant (C) region. The V region is the domain of the TCR that recognizes an epitope. TCRs cannot directly recognize an epitope, but only antigen peptide-MHC (major histocompatibility complex) molecular complexes on the surface of antigen presenting cells or target cells.

"MHC molecules" are protein molecules encoded by the major histocompatibility complex (major histocompatibiltiy complex, MHC) genes. MHC molecules include MHC class I molecules and MHC class II molecules.

"Activation of T lymphocytes": activation of T lymphocytes requires two signals. The first signal is obtained by its TCR recognizing an antigen peptide-MHC molecule complex on the surface of an APC or target cell (a cell that can be killed by a CD8 ⁺ T cell), and the second signal, also called co-stimulatory signal, is obtained by its CD28 molecule recognizing a B7 molecule on the surface of an APC or target cell. Under the action of the double signals, the T cells start and activate signal transduction pathways, and then proliferate and differentiate to function. Only the first signal and no second activation signal may put the T cell into a non-responsive state. T lymphocyte responses occur following T lymphocyte activation.

"T lymphocyte reaction": T lymphocyte reaction (T lymphocyte response) and "T lymphocyte activity" (T lymphocyte activity) or "function performed by T lymphocytes" are interchangeable terms in the present invention. T lymphocyte responses include proliferation and/or differentiation of T lymphocytes into helper T lymphocytes (Th), cytotoxic T lymphocytes (Tc) or regulatory T lymphocytes (Treg), as well as providing signals from Th to B lymphocytes to assist in their production of antibodies, killing target cells by Tc, and releasing soluble factors such as cytokines to regulate the function of other immune cells. Th is CD4 ⁺ T cells and Tc is CD8 ⁺ T cells. After activation, CD4 ⁺ T cells assist B cells in producing antibodies, helping 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 can enhance the immune response of an individual to microbial antigens to produce anti-infection effect, and can also promote the immune response of the individual immune cells to tumor cells to produce tumor treatment effect. Reducing the level of TGF- β2mRNA and inhibiting the expression of TGF- β2 enhances T lymphocyte responses. The oligonucleotides provided by the invention can be used as an adjuvant for pathogenic microorganism vaccines and tumor vaccines or used for tumor treatment by enhancing T lymphocyte reaction and enhancing immune response to pathogenic microorganisms and tumor cells.

"Subject" in the context of the present invention refers to human and non-human vertebrates.

The term "target cell" (TARGET CELL) refers to a cell that an individual can be challenged, killed or affected by an immune cell, which can be a tumor cell, a virus-infected cell, or a cell that can be affected by an oligonucleotide provided by the present invention.

"Tumor" in the present invention, i.e., a tumor defined by modern medicine, can be classified into benign tumor and malignant tumor. Tumor (tumor) and cancer (cancer) can be used interchangeably and have the same meaning. Tumors include solid tumors, soft tissue sarcomas, and myeloid or lymphoid system tumors. The oligonucleotides provided herein can enhance an individual's immune response to a tumor antigen to produce an anti-tumor effect, and tumors of interest include, but are not limited to, esophageal cancer, gall bladder cancer, breast cancer, colon cancer, colorectal cancer, adrenocortical tumor, kidney cancer, liver cancer, lung cancer, ovarian cancer, cervical cancer, uterine cancer, vaginal cancer, pancreatic cancer, rectal cancer, prostate cancer, stomach cancer, skin cancer, melanoma, brain tumor, glioma, bone tumor, sarcoma, penile cancer, retinoblastoma, leukemia, lymphoma, and myeloma.

"Treatment" includes the use of the oligonucleotides provided herein to prevent or delay the appearance of symptoms and complications of a disease, such as a tumor. Treatment may also be prophylactic. Treatment of tumors also refers to controlling tumor progression in individuals, prolonging survival of tumor patients, improving quality of life, alleviating symptoms, shrinking or even eliminating tumors, and suppressing tumor metastasis. In the present invention, "tumor treatment" and "antitumor effect" or "treatment of tumor" have the same meaning. Antitumor effects include treatment of tumors, as well as prevention of tumorigenesis, recurrence, and metastasis. The oligonucleotides provided by the invention can be used for treating tumors.

"Antigen". The oligonucleotides provided herein can enhance an individual's immune response to an antigen. An antigen is a substance or molecule that is recognized by a B cell receptor or a T cell receptor to elicit an adaptive immune response (adaptive immune response) in an individual. The antigen may be derived from the outside of the individual, such as a microbial antigen, or may be derived from the inside of the individual, such as a tumor antigen. Microbial antigens are substances or molecules on a microorganism that are recognized by B cell receptors or T cell receptors to elicit an adaptive immune response (adaptive immune response) in an individual. Vaccines prepared using microbial antigens allow the vaccine to be used to obtain immunity to the microorganism after application to an individual, and to be protected from exposure to the same or a similar pathogen. The vaccine prepared from the tumor antigen can excite the adaptive immune response of the vaccine to tumor cells after being applied to an individual so as to generate a tumor treatment effect. The antigen may be extracted from a microorganism or tumor cell, or may be produced by recombinant DNA techniques or synthesized by other methods. Tumor cell lysates contain a variety of tumor antigens and can be used as antigens in tumor vaccines.

The vaccine vaccine is a biological product for artificial active immunization, which is made of attenuated or killed pathogenic organisms or their components as antigens. Antigens and adjuvants are the main components of the vaccine. The purpose of vaccination is to protect the individual from the pathogen and thus from re-exposure to the corresponding pathogen. The oligonucleotides provided by the invention can be used in combination with human vaccines to enhance their immune efficacy, including but not limited to vaccines [Stanley A.Plotkin,Walter A.Orenstein,Paul A.Offit, Vaccines,Sixth Edition,An imprint of Elsevier Inc.2013,ISBN-13：9781455700905]： for preventing infectious diseases such as 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, varicella, epidemic encephalitis B, forest encephalitis, cholera, influenza B virus infection, and vaccine, lyme disease, pneumococcal infection, meningococcal infection, typhoid, smallpox, anthrax, ebola virus infection, marburg virus (MARV) infection, and venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus, VEEV) infection. Vaccines to which the invention relates also include tumour vaccines. Tumor antigens and adjuvants are the main components of tumor vaccines. The tumor vaccine can treat or prevent tumor. The antigen in the tumor vaccine may be a tumor antigen, a cell presenting a tumor antigen, a cell expressing a tumor antigen, or a tumor cell lysate. The oligonucleotides provided by the invention can be used in combination with tumor vaccines to enhance the efficacy of preventing and/or treating tumors, such vaccines include, but are not limited to, human papilloma virus 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 oligonucleotide provided by the invention can be used in combination with animal vaccines to enhance the immune efficacy, wherein the vaccines comprise but are not limited to vaccines capable of preventing infectious diseases such as foot and mouth disease of pigs, blue ear disease of pigs (porcine reproductive and respiratory syndrome), swine fever, pseudorabies, porcine circovirus infection, porcine parvovirus infection, streptococcus suis, transmissible gastroenteritis of pigs, asthma of pigs, haemophilus parasuis infection, swine erysipelas, swine plague, atrophic rhinitis of pigs, transmissible gastroenteritis of pigs, epidemic encephalitis of pigs, swine brain, swine influenza, brucellosis of pigs, diarrhea of pigs, infection of parainfluenza virus of pigs, swine influenza, mycoplasma hyopneumoniae infection, edema disease of pigs, salmonella cholerae, and salmonella cholerae, Porcine epidemic diarrhea, swine plague, bovine air tumor, bovine anthrax, bovine epidemic fever, bovine pasteurellosis, bovine brucellosis infection, bovine tetanus, niu Weishi clostridium infection, beef toxin clostridium infection, bovine foot and mouth disease, bovine paratyphoid, vaccinia, bovine diarrhea, caprae seu ovis sudden carbuncle, caprae seu ovis enterotoxemia, lamb dysentery, caprae seu ovis (infectious necrotic hepatitis), capripomoea, caprine tetanus, caprine anthrax, lamb colibacillosis, chlamydia infection, caprine aphtha, capricosis, contagious pleuropneumonia, brucellosis, clostridium botulinum poisoning, capripox, capricosis, hoof and mouth disease, caprine and blue tongue disease, equine infectious anemia, Horse flu, swine, horse, cattle and sheep canine encephalitis, newcastle disease, infectious bursal disease, chicken Marek's disease, avian influenza, fowl cholera, chicken pox, infectious bronchitis, chicken viral hepatitis, chicken egg drop syndrome, duck flu, riemerella anatipestifer vaccine, duck plague, infectious serositis of the duck, duck colibacillosis, gosling plague, goose paramyxovirus disease, goose flu, rabies, canine warmth, canine parvovirus disease, canine leptospirosis, canine infectious hepatitis, canine infectious bronchitis, canine parainfluenza disease, canine encephalitis, and canine nest cough vaccine. The oligonucleotides provided herein can enhance the efficacy of a vaccine.

The oligonucleotide provided by the invention can be used as an adjuvant of tumor antigens to enhance the immune response to the tumor antigens. In the present invention, tumor antigen (Tumor antigen), tumor cell antigen (Tumor CELL ANTIGEN) and Tumor-associated antigen (Tumor associated antigen, TAA) can be used interchangeably and have the same meaning. Tumor antigens can elicit an anti-tumor immune response that can produce a tumor therapeutic effect. 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)、 hepatitis B virus gene-encoded antigen, hepatitis C virus gene-encoded antigen, human papilloma virus gene-encoded antigen (e.g., L1 protein, E6 protein, and E7 protein), and EB virus gene-encoded antigen (Epstein Barr Virus peptides)[Zielinski C,et al.Nat Rev Clin Oncol.2014Sep;11(9)：509-24;AdamsJL,et al.Nat Rev Drug Discov.2015Sep;14(9)：603-22]. tumor antigens, as well as whole tumor cells and tumor cell lysates that have been inactivated. The tumor antigen may be a neo-antigen expressed by a gene mutation, or may be a idiotype antigen of a B cell tumor and a heat shock protein tumor cell peptide complex extracted from a tumor cell [ Suot, R & Srivastava, P (1995) Science 269:1585-1588;Tamura,Y.et al (1997) Science 278:117-120].

"Tumor vaccine" the oligonucleotides provided by the invention can be used as adjuvants for tumor vaccines to enhance their efficacy in tumor treatment. Tumor vaccines refer to biological products that are capable of inducing an anti-tumor adaptive immune response in an individual. The tumor vaccine comprises a vaccine prepared from tumor antigen, tumor cell lysate, tumor cells and cells presenting the tumor antigen in a certain dosage form. Such vaccines include, but are not limited to, the tumor vaccine described below [ Zielinski C, et al Nat Rev Clin Oncol.2014 Sep;11 (9): 509-24] for the treatment of prostate cancer, using tumor antigens including prostatectomy phosphatase (for sipuleucel-T),And PSA, tumor vaccines for treating breast cancer, tumor antigens including HER 2-derived polypeptides, MUC1 and WT1 (Wilms tumour protein) antigens, tumor vaccines for treating lung cancer, tumor antigens including MUC1, melanoma-associated antigen-3 (MAGE-A3) polypeptides, telomerase REVERSE TRANSCRIPTASE and EGF, vaccines for treating melanoma, antigens including beta-catenin, gp100, MAGE-A3, MART1 (melanoma antigen recognized by T cells 1), NY-ESO-1 and survivin, tumor vaccines for treating pancreatic cancer vaccines, tumor antigens including telomerase peptides, allogeneic tumor cells and mutated RAS synthetic peptides, tumor vaccines for treating colorectal cancer, tumor antigens including carcinoembryonic antigen (carcinoembryonic antigen, CEA), MUC1 and whole tumor cells, tumor vaccines for treating renal cell carcinoma, tumor antigens including tumor cell RNA, blood tumor therapeutic vaccines, and tumor antigens including WT1, MAGE, MUC1, PRAME (preferentially expressed antigen of melanoma) and idiotype antigens. The oligonucleotides provided by the invention can enhance the immune response to tumor vaccines.

"Tumor cell lysate" the oligonucleotides provided by the invention can enhance the efficacy of the tumor cell lysate in inducing an anti-tumor immune response, and can be used as an adjuvant for tumor vaccines with tumor cell lysate as an antigen. Tumor cell lysates can be obtained by disrupting tumor cells and contain various tumor antigens. Methods of disrupting tumor cells include, but are not limited to, repeated freeze thawing, sonication, and mechanical disruption. Primary, secondary or metastatic tumor cells can be used to prepare tumor cell lysates. The primary tumor and secondary or metastatic tumor cells are cultured in vitro to obtain tumor cell line (tumor cell line) cells, which can also be used to prepare tumor cell lysates. The tumor cells used to prepare the tumor cell lysate may be from autologous, allogeneic or xenogeneic individuals.

"Adjuvant" the oligonucleotides provided herein may be used in combination with one or more adjuvants to enhance the immune efficacy of microbial antigens and tumor antigens. An adjuvant is a substance used in a vaccine together with an antigen, and has the following activities (1) to reduce the number of times of vaccination, (2) to prolong the immune duration of the vaccine, (3) to promote humoral and cellular immune responses by agonizing innate immune responses, (4) to expand antigen-induced cross-protective immune responses, (5) to enhance the immune response of individuals with weak immune responses such as aged individuals or immunodeficient individuals to the antigen, and (6) to reduce the amount of antigen used. Adjuvants [Stanley A.Plotkin,Walter A. Orenstein,Paul A.Offit,Vaccines,Sixth Edition,An imprint of Elsevier Inc.2013,ISBN-13：9781455700905] which may be used in combination with the oligonucleotides provided by the present invention include, but are not limited to, aluminum salt adjuvants (vaccine adjuvants comprising aluminum hydroxide or aluminum phosphate AS the major component), AS04 adjuvants [ aluminum phosphate adjuvants adsorbing MPL (chemically attenuated gram negative bacterial lipopolysaccharide), MF59 adjuvants (a water-in-oil type emulsifier, with squalene AS the oil phase), AS03 adjuvants (an oil-in-water type adjuvant, with squalene AS the oil phase), AF03 adjuvants (an oil-in-water type emulsifier, with squalene AS the oil phase), and, Montanide ISA 51 adjuvant (water-in-oil emulsifier with mineral oil as oil phase), freunds adjuvant, freunds incomplete adjuvant, virosome adjuvant (virosome adjuvant), polyethylene oxide-on-dinitrogen hexacyclic derivative (polyoxidonium) adjuvant, toll-like receptor agonist, pathogen-related pattern molecules and mimics thereof (analogues), lesion-related pattern molecules and mimics thereof (analogues), cyclic dinucleotides and mimics thereof (analogues), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-12, oily emulsifiers (AS 02, AS03, AF03, MF59 and Montanide ^TM ISA-51), white oil adjuvants, montanide ^TM ISA-206 adjuvants, QS21, polylactide co-glycolide, viral (Adenovirus, vaccinia, fowlpox) vector adjuvants, BCG (bacillus Calmette-Guerin), double-stranded RNA including poly (I: C) and its analogues (mimetics), lipid A analogues including MPL, flagellin, imidazoquinolines including Imiquimod and R848, cpG ODN, saponin including QS21, C-lectin ligand including TDB, CD1d ligand including alpha-galactosylceramide, AS01 (including MPL, QS21 and liposomes), cpG ODN, and combinations thereof, AS02 (containing MPL, QS21 and emulsifying agent), AS15 (containing MPL, QS21, cpG ODN and liposome), GLA-SE (containing GLA and emulsifying agent), IC31 (containing CpG ODN and cationic polypeptide), CAF01 (containing TDB, and cationic liposome) and ISCOMs (containing saponin and phospholipid) )[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].

The oligonucleotide provided by the invention can be combined with the innate immunity activator to play the role of anti-infection and anti-tumor. Innate immune activators include pathogen-associated model molecules and analogs thereof, toll-like receptor agonists and analogs thereof, lesion-associated model molecules and analogs thereof, and cyclic dinucleotides and analogs thereof. Analogs and mimetics have the same meaning.

The oligonucleotide provided by the invention can be combined with pathogen-related model molecules (pathogen-associated molecular patterns, PAMPs) and analogues thereof to enhance the immune efficacy of pathogenic microorganism antigens and tumor antigens to exert anti-infective and antitumor 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 (pattern-recognition receptors, PRRs). PRR includes 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, and also includes intracellular nuclear acid receptors (intracellular sensors of nucleic acids) for intracellular recognition of nucleic acids, OAS proteins and cGAS [ IWASAKI A ET al. Nat. Immunol.2015 Apr;16 (4): 343-53].

The oligonucleotide provided by the invention can be used in combination with one or more Toll-like receptors (TLRs) agonists to enhance the immune effect of pathogenic microorganism antigens and tumor antigens and exhibit anti-infective or anti-tumor activity. TLR agonists that can be used in combination with the oligonucleotides provided herein 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 (CPG-containing deoxyoligonucleotides), TLR2/TLR4 agonists including BCG, TLR4 agonists including OM-174, monophosphoryl lipid A, aminoalkyl glucosamine phosphates and other lipid a (lipid a) analogs, TLR9 agonists including viral nucleic acids, bacterial nucleic acids, TLR5 agonists including bacterial flagellin, TLR 2/6 agonists including yeast polysaccharides, TLR3 agonists including polyinosinic acid cytosine nucleotides (poly IC), viral double stranded RNAs or mimics thereof and TLR 7/8 single stranded RNA [ adjs ET AL NAT REV.603-2015 ] including viral RNA or mimics thereof.

The oligonucleotides provided herein can be used in combination with damage-related model molecules (damage-associated molecular patterns, DAMPs) and analogs thereof to enhance the immunopotency of microbial and tumor antigens. DAMPs are cellular components released by injured cells that elicit an innate immune response. DAMPs can stimulate the innate immune response of the body through PRRs. These DAMP 'S include, but are not limited to, heat shock protein, HMGB1 (high-mobility group box 1), hyaluronan fragment, glycans, glycoconjugates, ATP, (Adenosine' -triphosphate), adenylate, uric acid, S100 protein, hepatin sulfate, GALECTINS, nuclear DNA, N-formylated peptides, antimicrobial peptide, mitochondrial DNA, and calreticulin[KryskoDV et al.Nat Rev Cancer.2012 Dec;12(12)：860-75;Pouwels SD et al.Mucosal Immunol.2014 Mar;7(2)：215-26].

"Cyclic dinucleotides" the oligonucleotides provided by the invention can be used in combination with cyclic dinucleotides and mimics thereof to enhance the immune effect of microbial antigens and tumor antigens. The cyclic dinucleotides (Cyclic dinucleotides, CDNs) may be of bacterial origin or may be produced in mammalian cells. Bacteria CDNs include, but are not limited to, cyclic di-GMP (cdG), cyclic di-AMP (cdA), and cyclic di-guanylate (CYCLIC AMP-GMP, cAMP-GMP). Bacterial CDNs are a class of PAMPs that agonize the innate immune response. CDNs, such as cyclic guanylate-adenylate, may also occur in mammalian cells (cyclic guanosine monophosphate-adenosine monophosphate,cGAMP)[Wu J et al.Science.2013 Feb 15;339(6121)：826-3].

An "anti-tumor agent" is an agent that can treat tumors after application to an individual, and includes, but is not limited to, tumor vaccines, immunocapture inhibitors, co-stimulatory receptor activators, chemotherapeutic agents, radiotherapeutic agents, hormonal inhibitors or hormones, cytokines, antibodies for tumor treatment, small molecule kinase inhibitors, PARP inhibitors, angiogenesis inhibitors, oncolytic viruses, and the like.

An "immunocapture molecule" is a protein molecule expressed on immune cells that, upon recognition of its ligand, initiates an inhibitory signaling pathway and thus inhibits activation of immune cells. Immunocapture molecules include, but are not limited to, CTLA-4 molecules, PD-1 molecules, PD-L1/2 molecules, lymphocyte activation genes 3(lymphocyte-activation gene 3,LAG-3)、TIM-3(T cell immunoglobulin and mucin domain-containing 3)、TIGIT(T cell immunoreceptor with immunoglobul in and ITIM domains) and BTLA (B and T lymphocyte attenuator). Blocking the immune checkpoint (immune checkpoint blockade) refers to a method of inhibiting the function of immune checkpoint molecules in immune cells to transduce inhibition signals to promote and maintain T cell activation. Blocking the immunocapture enhances the immune response of the individual to the microbial antigen, shows anti-infective effect, can promote the anti-tumor activity of individual immune cells, and shows tumor treatment effect [ Melero I, et al Nat Rev cancer.2015 Aug;15 (8): 457 72].

The oligonucleotide provided by the invention can be used as an adjuvant of a microorganism vaccine or a tumor vaccine in combination with the immune checkpoint inhibitor, and can also be used for treating tumors in combination with the immune checkpoint inhibitor. The immune checkpoint inhibitor (immune checkpoint inhibitor) is a substance capable of inhibiting the function of immune checkpoint molecules [ Melero I et al.Nat Rev cancer.2015Aug;15 (8): 457-72]. Inhibiting the function of the immunocapture molecules highlights the signal of immune cell activation, thus placing the immune cells in a sustained activation state. CD4 ⁺ T cells in a sustained activation state will assist B cells in producing antibodies and also assist CD8 ⁺ T cells in killing target cells such as virus infected cells and tumor cells. CD8 ⁺ T cells in a sustained activated state can kill virus-infected cells and tumor cells. Thus, agents that maintain T cell activation, such as immunoblotter inhibitors, increase the immune efficacy of an antigen or vaccine in an individual, and also exert a tumor therapeutic effect in a cancer patient (individual). 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 inhibit immune checkpoint function are immune checkpoint inhibitors, including but not limited to CTLA-4 antibodies, CD-1 antibodies, and CD-L antibodies.

The oligonucleotide provided by the invention can be used for treating tumors in combination with the immune checkpoint molecular antibody or used as an adjuvant of a microbial vaccine and a tumor vaccine. Immune checkpoint molecule antibodies include, but are not limited to, CTLA-4 antibodies, PD-1 molecule antibodies, and PD-L1/2 antibodies. The CTLA-4 antibody can specifically bind to CTLA4, can block the inhibition signal of CTLA-4 transduction, enables T lymphocytes to be fully activated by tumor antigens, and can prolong the survival time 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], approved by the FDA in the United states as a drug for the treatment of advanced melanoma [ Shalma P.et al science.2015 Apr 3;348 (6230): 56-61]. Tremelimumab is another humanized IgG2 CTLA-4 monoclonal antibody [ Ribas A., et al Oncolognoist.2007 Jul;12 (7): 873-83]. In clinical trials, tremelimumab was used to treat hepatocellular carcinoma [ Sangro B., et al J hepatol.2013 Jul;59 (1): 81-8], gastric and esophageal carcinoma [ Ralph C.et al Clin Cancer Res.2010 Mar 1;16 (5): 1662-72]. The antibody of PD-1 (Anti-PD-1 Antibodies) is a monoclonal antibody of PD1 molecules, has the function of inhibiting PD-1 mediated immune cell down regulation signal transduction and is an immune checkpoint inhibitor. 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 binds to and inhibits 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-1 kappa monoclonal antibody that binds to and inhibits the function of PD-1 and is used for the treatment of diffuse large B-cell lymphoma and follicular lymphoma. In addition, antibodies to PD-1 include Pembrolizumab (MK-3475), which is an IgG-4 kappa monoclonal antibody that binds to and inhibits PD-1 function [ Ito A et al biomed Res int.2015;2015:605478]. An anti-PD-L antibody is an antibody that recognizes, binds to PD-L1 or PD-L2. After the PD-L is combined, the PD-L can be blocked from combining with the PD-1 on the surface of immune cells, so that the transduction of immune cell activation inhibition signals is blocked, the activation state of the immune cells is maintained, and the immune response of an individual to an antigen or tumor cells is further enhanced. A variety of Anti-PD-L1 Antibodies (Anti-PD-L1 Antibodies) have been shown to have 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 for the treatment of melanoma, non-small cell lung cancer, ovarian cancer, and renal cancer. MPDL3280A is a humanized IgG-1 kappa anti-PD-L1 monoclonal antibody, and is used for treating melanoma and bladder cancer. MEDI4736 is a humanized IgG-1 kappa monoclonal antibody that can extend 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].

The oligonucleotide provided by the invention can be used in combination with a co-receptor activator to enhance the immune response of an individual to a vaccine or antigen and enhance the anti-tumor immune response of the individual. The co-stimulatory receptor is a receptor expressed on the surface of immune cells, which upon activation by an activator mediates transduction of immune cell activation signals, thus promoting an immune response of an individual to an antigen or vaccine and enhancing the anti-tumor immune activity of the individual. A co-stimulatory receptor activator is a preparation that activates immune cells upon binding to a co-stimulatory receptor. Co-stimulatory receptor activating monoclonal antibodies (Co-stimulatory receptor activating monoclonal antibody) are Co-stimulatory receptor activators that enhance the efficacy of an antigen or vaccine and enhance an anti-tumor immune response in an individual. Co-stimulatory receptors (Co-stimulatory receptor) targeted by such activating monoclonal antibodies include, but are not limited to, CD137 (41 BB), 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.2015Aug;42(4)：640-55].

"Chemotherapeutic agents" the oligonucleotides provided herein can be used in combination with chemotherapeutic agents to treat tumors. Chemotherapeutic agents are chemical agents that can treat tumors by inhibiting and killing tumor cells. The chemical agents referred to in the present invention include, but are not limited to, alkylating agents, antimetabolites, antimicrotubule 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 (nitrogen mustards) include mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide and busulfan. Nitrosoureas include N-Nitroso-N-methylurea, carmustine, lomustine, semustine, fotemustine and streptozotocin. Tetrazine (tetrazines) type drugs include dacarbazine, mitozolomide and temozolomide. Aziridine (aziridines) drugs include thiotepa, mytomycin and diaziquone. Cisplatin and its derivatives include cisplatin (cispratin), carboplatin (carboplatin) and oxaliplatin (oxaliplatin). Non-classical alkylating agents include procarbazine (procarbazine) and hexamethylmelamine. Antimetabolites include antifeedics, fluorouracils, deoxynucleoside analogues and mercaptopurine drugs. Antifolates include methotrexate (methotrexate) and pemetrexed. Fluorouracil drugs include 5-fluorouracil (5-fluorouracil). Deoxynucleoside analog drugs include cytarabine, gemcitabine (gemcitabine), decitabine, vidaza, fludarabine, nelarabine, cladribine, clofarabine, and pentastatin. Mercaptopurine (thiopurines) drugs include thioguanine and mercaptopurine. The microtubule-resisting preparation comprises vinca alkaloids, taxane and podophyllotoxin. Vinca alkaloids (vinca alkaloids) include vincristine (vincristine), vinblastine (vinblastine), vinorelbine (vinorelbine), vindesine and vinflunine. Taxanes (Taxanes) include paclitaxel (paclitaxel) and Docetaxel (Docetaxel). The class of podophyllotoxins (Podophyllotoxin) includes etoposide (etoposide) and teniposide. Topoisomerase inhibitors (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 (epirubicin), idarubicin, pirarubicin, aclarubicin, mitoxantrone, gactinomycin, bleomycin (bleomycin), plicamycin, and mitomycin.

The oligonucleotide provided by the invention can be used for treating tumors in combination with radiotherapy. Radiotherapeutic agents are radiation-producing substances. The treatment of tumors with radiotherapeutic agents is known as chemotherapy. The substances used for radiotherapy are radioisotopes that produce alpha, beta, gamma rays. Radiation for radiotherapy may also be generated by machines, including x-ray therapy machines or accelerators.

The oligonucleotide provided by the invention can be combined with the hormone inhibitor or hormone for treating tumors. Hormone inhibitors include, but are not limited to, inhibitors of hormone synthesis, hormone receptor antagonists and complementing hormones. Inhibitors of hormone synthesis include aromatase inhibitors and gonadotrophin releasing hormone (GnRH) analogues. 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. Supplemental hormones are methods of treating tumors using supplemental hormones (Hormone supplementation) including progestins, androgens, estrogens, progesterone-like drugs, testosterone-like drugs, estrogen agonists, and somatostatin analogs. Progesterone-like drugs include megestrol acetate and medroxyprogesterone acetate. Testosterone-like drugs include Fluoxymesterone. Estrogen antagonists include diethylstilbestrol, estrace and Polyestradiol phosphate. The somatostatin analog includes Octraotide.

The oligonucleotide provided by the invention can be used for treating tumors by combining with cytokines, so that the immune effect of the vaccine is enhanced. Such 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 alpha.

"Antibody for tumor treatment" the oligonucleotide provided by the present invention can be used in combination with an antibody (tumor therapeutic antibodies) for tumor treatment to treat tumors. Tumor therapeutic antibodies are antibodies that increase the survival of an individual suffering from a tumor after administration, and these antibodies target molecules including, but 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. Tumor therapeutic antibodies of interest 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 EGF receptor, humanized antibody targeting PD-1, humanized antibody targeting CTLA-4, bevacizumab (Avastin) targeting VEGF receptor, brentuximab (vedotin) targeting CD30, alemtuzumab (Campath) targeting CD52 and Gemtuzumab ozogamicin (Mylotarg; wyeth) [ Scott AM et al Nat Rev cancer cell 2012 Mar22; 12 (4): 278-87].

The oligonucleotide provided by the invention can be combined with a small molecule kinase inhibitor (small-molecule kinase inhibitors) to treat tumors. Small molecule kinase inhibitors are a class of small molecule compounds that can exert a tumor therapeutic effect by inhibiting protein kinase activity, including, but not limited to, 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, gefitinib targeting EGFR, 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 ：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 discovery.2015 Sep;14 (9): 603-22] that treat tumors by inhibiting the protein kinases described below.

The oligonucleotide provided by the invention can be combined with the poly (A-ribose) diphosphate polymerase inhibitor (poly ADP ribose polymerase, PARP) to treat tumors or be used as an adjuvant for microbial vaccines and tumor vaccines. A poly (adenosine diphosphate) ribose polymerase inhibitor, PARP inhibitor for short, is a formulation that can treat tumors by inhibiting PARP activity. PARP inhibitors include, but are not limited to Iniparib for the treatment of breast and lung squamous cell carcinomas, talazoparib for the treatment of breast cancer (BMN-673), olaparib for the treatment of breast, colon, ovarian and advanced prostate cancers, rucaparib for the treatment of breast and ovarian cancers, veliparib for the treatment of metastatic melanoma and breast cancers and CEP 9722[Nature Reviews Clinical Oncology 12,27-41,2015 for the treatment of non-small cell lung cancers.

The oligonucleotide provided by the invention can be combined with the angiogenesis inhibitor 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 monoclonals antibodies to endothelial growth factor (VEGF) including Avastin or bevacizumab, vascular endothelial inhibins including Endolichos (ENDOSTAR) and aptamer (aptamer) to vascular endothelial growth factor including pegaptinib.

The oligonucleotide provided by the invention can be used for treating tumors by combining with oncolytic viruses. 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, kessa virus, respiratory enterovirus, measles virus, polio virus, follicular stomatitis virus, senican valley virus, parvovirus, and retrovirus [ Kaufman HL et al Nat Rev Drug discovery 1;14 (9): 642-62].

The oligonucleotide provided by the invention can be combined with the cell for tumor treatment to treat tumors. Tumor therapeutic cells are cells that exert an anti-tumor effect after application to an individual, and include, but are not limited to, dendritic cells, T lymphocytes, and NK cells.

The oligonucleotide provided by the invention can be combined with autologous Dendritic Cells (DC) to treat tumors. DCs are immune cells expressing CD11 c. The DC presenting tumor antigen can trigger anti-tumor immune response after being applied to individual, and the cell can be used as DC vaccine. Tumor therapeutic DCs include, but are not limited to, DCs loaded with a single tumor antigen (protein antigen or antigenic peptide, such as prostaacid phosphatase), DCs loaded with tumor cell lysates, DCs loaded with tumor cell RNAs, and DC[Nestle,F.et al.(1998) Nature Medicine 4：328-332;Palucka K et al.Nat Rev Cancer.2012 Mar 22;12(4)：265-77]. tumor therapeutic DCs loaded with autologous tumor cell eluting peptides, as well as gene transfected DCs. Genes for transfection of DCs include, but are not limited to, tumor antigen encoding genes, cytokine (e.g., IL-2, gm-CSF) encoding genes, and costimulatory molecule encoding genes. DCs for tumor therapy also include DCs and tumor cell fusion cells [ Kugler, A.et al (2000) Nature Medicine 6:332-336].

The oligonucleotide provided by the invention can be combined with T cells for tumor treatment to treat tumors. T cells for tumor therapy include tumor infiltrating T cells and genetically engineered T cells (GENETICALLY ENGINEERED T CELLS). Genetically engineered T cells may be assembled with chimeric antigen receptors (CHIMERIC ANTIGEN receptors, CARs) that recognize tumor antigens, and T cells expressing such receptors are referred to as CAR-T. Tumor-infiltrating T cells and CAR-T can be reinfused into tumor patients after in vitro IL-2 expansion [ KERSHAW MH ET al Nat Rev cancer.2013 Aug;13 (8): 525-41].

The oligonucleotide provided by the invention can be combined with natural killer cells [ Natural Killer (NK) cells ] for tumor treatment to treat tumors. Natural Killer (NK) cells for tumor therapy may be isolated from peripheral blood or umbilical cord blood of an individual, or may be induced to be produced from hematopoietic precursor cells, embryonic stem cells or pluripotent stem cells. The NK cells isolated or induced can be reinfused to individuals after in vitro amplification with IL-2 and IL-15 [ Childs RW, et al Nat Rev Drug discovery.2015 Jul;14 (7): 487-98].

The oligonucleotide provided by the invention can play an anti-infective role when being singly used or used as a vaccine adjuvant. Anti-infective refers to the treatment and prevention of diseases caused by pathogenic microorganisms. The oligonucleotides provided by the invention have anti-infective effect on pathogenic microorganisms.

"Pathogenic microorganism". The oligonucleotide provided by the present invention can exert an effect of resisting pathogenic microorganism infection, alone or as a vaccine adjuvant. Pathogenic microorganisms include pathogenic viruses and pathogenic bacteria.

The oligonucleotide provided by the invention can be used singly or as a vaccine adjuvant to play a role in resisting pathogenic virus (pathogenic viruses) infection. 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 (HSV-II), cytomegalovirus (CMV), EB virus, adenovirus, influenza virus, arbovirus, epstein-Barr virus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, circovirus, foot-and-mouth disease virus, human T-cell tropic virus (HTLV), dengue virus, papilloma virus, molluscum contagiosum virus, polio virus, rabies virus, polyoma virus and arbovirus encephalitis virus.

"Pathogenic bacteria". The oligonucleotides provided by the invention can be used alone or as vaccine adjuvants to act against pathogenic bacterial (pathogenic bacteria) infections. These pathogenic bacteria include Chlamydia, rickettsia, mycobacterium, staphylococcus, streptococcus, pneumococcus, meningococcus, klebsiella, serratia, pseudomonas, diphtheria, salmonella, vibrio cholerae, tetanus, botulinum, bacillus anthracis, plague and Lyme disease bacteria.

The oligonucleotide medicine provided by the invention can be combined with a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) to form a medicine composition (pharmaceutical compositions). The oligonucleotide in the composition is administered in an effective amount (EFFECTIVE DOSAGES). The composition can be combined with antigen, vaccine, adjuvant, and preparation or cell with tumor therapeutic effect. The pharmaceutical composition may be formulated into dosage forms including, but not limited to, solutions, emulsions, liposomes, and lyophilized powders.

The oligonucleotide medicine provided by the invention can form a medicine composition (pharmaceutical compositions) with a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier). Pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) refers to one or more solid or liquid fillers, diluents or encapsulating substances. Such vectors are suitable for use with the oligonucleotides provided herein in an individual. The support may be organic, inorganic, natural or synthetic. Pharmaceutically acceptable carriers can be pharmaceutically acceptable solvents (aqueous and non-aqueous), dispersing agents, suspending agents, emulsifying agents, powders, diluents, liposomes, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, lyoprotectants and other agents suitable for the immune efficacy of the oligonucleotide vaccine or antigen provided herein, which produce a therapeutic effect on tumors. Aqueous solutions include, but are not limited to, water, physiological saline, PBS buffer, balanced salt solution, and dextrose solution. Solvents or dispersants may include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), as well as mixtures of such solvents or dispersants. Lipids including lecithin may be used in order to maintain fluidity of the pharmaceutical composition. Surfactants may be used in order to provide the pharmaceutical composition in the desired particulate state. For proper osmotic pressure, sugar, polyols including mannitol and sorbitol, sodium chloride, and the like may be added to the pharmaceutical composition. In order to prolong the action time, sustained release agents such as stearate and gelatin can be added to the pharmaceutical composition. The emulsifier may include an oil-in-water emulsifier, a water-in-oil emulsifier or a water-in-oil-in-water emulsifier. Pharmaceutically acceptable carriers also include pharmaceutically acceptable antioxidants (pharmaceutically-acceptable antioxidants) including water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like, oil soluble antioxidants such as ascorbyl palmitate, butylhydroxyanisole, butylhydroxytoluene, lecithin, propyl gallate, and alpha-wheat germ phenol, and the like, metal chelators such as citric acid, EDTA, sorbitol, tartaric acid, and phosphoric acid.

Effective dosages of the oligonucleotides (EFFECTIVE DOSAGES) provided herein include "effective dosages for enhancing the immune efficacy of an antigen" and "effective dosages for treating tumors". An effective dose for enhancing the immune efficacy of an antigen is an amount of an oligonucleotide that, upon administration to an individual, significantly enhances the immune efficacy of the antigen or vaccine, and also an amount of an oligonucleotide that, upon administration to an individual, results in the desired prevention or treatment of an infectious disease or tumor. A tumor-treating effective dose is a dose of an oligonucleotide that produces a tumor-treating effect upon administration to an individual. The amount of the dosage will depend on criteria which will be appreciated by those skilled in the art, and reference will be made to other factors including, but not limited to, the size and health of the individual and the severity of the disease. The oligonucleotides provided by the invention can be applied to an individual in a single or multiple times, and each dosage can be in the range of 1 mug to 1000 mg. The dosage of the oligonucleotide can be adjusted by those skilled in the art to achieve the desired effect, and can be in the range of 10 to 1000 times the aforementioned range. The oligonucleotides provided herein may be in dosage units for use in an individual. Each unit contains a quantity of the oligonucleotide that produces a prophylactic or therapeutic effect and a desired pharmaceutically acceptable composition. The basis for the definition of the dosage unit is the characteristic activity profile of the oligonucleotide that produces a therapeutic effect and the sensitivity of the individual to the oligonucleotide when receiving the oligonucleotide therapy. The oligonucleotides applied in dosage units may be applied 2,3, 4, 5 or more times per day at intervals, if desired. The oligonucleotides provided by the invention can be applied in individual body weight (host body weight) at a dosage ranging from 0.0001 to 100mg/kg at intervals of once every two weeks or once a month or once every 3 to 6 months or other time intervals suitable for producing a prophylactic or therapeutic effect. The dose of the oligonucleotide may be 1-1000. Mu.g/ml when used in combination with an antigen or vaccine. Effective dosages include both therapeutically effective dosages (therapeutically effective dose) and prophylactically effective dosages (prophylactically-EFFECTIVE DOSE).

The oligonucleotide provided by the present invention may be administered parenterally, topically or by inhalation, alone or in combination with other agents such as antigens, adjuvants, innate immune activators and anti-tumor agents. Parenteral routes of administration include intravenous, intraperitoneal, intrathecal, intramuscular, subcutaneous, intradermal, topical, paraneoplastic lymph node, direct injection of neoplastic tissue and intralymph node injection. Routes of administration for external use include transdermal, oral, ocular, aural and nasal. Inhalation may be via the nasal mucosa and lungs.

"Therapeutic device" pharmaceutical compositions comprising the oligonucleotides provided herein may be applied to an individual using therapeutic devices known to those skilled in the art. Treatment devices include, but are not limited to, needleless injection devices, implant devices, chamber devices (modules), implantable micro-infusion pumps (implantable micro-infusion pumps), infusion pumps, and osmotic drug delivery systems (osmotic drug DELIVERY SYSTEM).

"Delivery vehicle" the oligonucleotides of the invention may be applied via a delivery vehicle. Delivery vehicles include, but are not limited to, steroids (e.g., cholesterol), complexes, emulsions, immunostimulatory complexes (ISCOMs), lipids (e.g., cationic lipids and anionic lipids), liposomes, bacterial vectors (e.g., salmonella, escherichia coli, shigella mycobacteria, lactobacillus), viral vectors (e.g., vaccinia, adenovirus, herpes simplex virus), virosomes, virus-like particles, microspheres, nucleic acid vaccines, polymeric materials (e.g., carboxymethyl cellulose, chitosan), and cyclic polymers. The delivery vehicle may also be a ligand for a specific receptor or a targeting molecule for a cell.

Description of the drawings:

FIG. 1 synergistic effect of TIO1 and TIO3 on recombinant protein vaccine (circovirus vaccine)

In FIG. 1, CP represents CP-ISA35, TIO1, TIO2 and TIO3 represent CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3, respectively, ODN represents a single stranded deoxyoligonucleotide, ns represents no statistical significance, the level of antibody is represented by absorbance value (A492 OD value), and each of the circular symbol, square symbol, right triangle symbol and inverted triangle symbol represents a mouse, respectively.

FIG. 2 inhibition of TIO1 and TIO3 on expression of TGF beta-2 by immune recall responsive mouse immune cells

In FIG. 2, CP represents CP-ISA35, TIO1 or TIO3 represents CP-ISA35+TIO1 or CP-ISA35+TIO3, respectively, ODN represents single-stranded deoxyoligonucleotides, TGF-beta 2relative expression represents the percentage of membrane-bound TGF-beta 2 positive cells in draining lymph node cells, and each of the circular, square, and regular triangle symbols represents a mouse, respectively.

FIG. 3 reduction of IL-4 induced immune cell TGF-2 mRNA levels by TIO1 and TIO3

In FIG. 3, ODN represents single stranded deoxyoligonucleotides, the level of TGF-2 mRNA is indicated by relative expression, and each of the circular symbol, square symbol, right triangle symbol, inverted triangle symbol, and diamond symbol represents one sample.

FIG. 4 TIO1 and TIO3 effect on LPS-induced reduction of immune cell TGF-2 mRNA level

In FIG. 4, ODN represents single stranded deoxyoligonucleotides, the level of TGF-2 mRNA is indicated by relative expression, and each of the circular symbol, square symbol, inverted triangle symbol, diamond symbol, and regular triangle symbol represents one sample.

FIG. 5 increasing effect of TIO1 and TIO3 on CD86 expressing dendritic cells in lymph node cells of immunocompetent mice

In FIG. 5, CP stands for CP-ISA35, TIO1, TIO2 or TIO3 for CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3, respectively, ODN stands for single stranded deoxyoligonucleotides, and each of the circular, square, and regular triangle symbols stands for a mouse, respectively.

FIG. 6 increasing effect of TIO1 and TIO3 on CD4 expressing T lymphocytes in lymph node cells of immunocompetent mice

In FIG. 6, CP represents CP-ISA35, TIO1 or TIO3 represents CP-ISA35+TIO1 or CP-ISA35+TIO3, respectively, ODN represents single-stranded deoxyoligonucleotides, each circle symbol, square symbol, right triangle symbol represents a mouse, respectively, and CD4 ⁺% represents the number of CD4 ⁺ cells in the draining lymph node cells analyzed.

FIG. 7 increasing effect of TIO1 and TIO3 on CD19 expressing B lymphocytes in lymph node cells of immunocompetent mice

In FIG. 7, CP represents CP-ISA35, TIO1, TIO2 or TIO3 represents CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3, respectively, ODN represents single stranded deoxyoligonucleotides, each circle symbol, square symbol, right triangle and inverted triangle symbol represents a mouse, respectively, and CD19 ⁺ cell% represents the number of CD19 ⁺ cells in the draining lymph node cells analyzed.

FIG. 8 synergistic effect of TIO1 and TIO3 on hepatitis B Virus vaccine

In FIG. 8, HBsAg represents an HBsAg vaccine, TIO1, TIO2, TIO3 represent the addition of TIO1, TIO2 or TIO3, respectively, to the HBsAg vaccine, ODN represents a single-stranded deoxyoligonucleotide, each of the circle symbol, square symbol, right triangle and inverted triangle symbol represents a mouse, respectively, and OD Value (A492 nm) represents the level of HBsAg-specific antibody in serum to be tested.

FIG. 9 synergistic effect of TIO1 or TIO3 on influenza Virus vaccine

In FIG. 9, FM1 represents an inactivated vaccine of FM1 influenza virus, TIO1, TIO2 and TIO3 represent respectively that TIO1 or TIO3 is added into FM1, ODN represents a single-stranded deoxyoligonucleotide, each of a circular symbol, a square symbol and a regular triangle symbol represents respectively one mouse, and OD Value (A492 nm) represents the level of an influenza virus specific antibody in serum to be tested.

FIG. 10 synergistic effect of TIO1 or TIO3 on rabies virus vaccine

In FIG. 10, TIO1, TIO2, TIO3 represent the addition of TIO1, TIO2 or TIO3, respectively, to a rabies vaccine, ODN represents a single stranded deoxyoligonucleotide, and each of the circle, square, right triangle and inverted triangle symbols represent a mouse, respectively. Efficacy of the vaccine is expressed as IU/ml.

FIG. 11 synergistic effect of TIO3 on glioma cell lysate vaccine

In FIG. 11, GTL represents glioma cell lysate vaccine, GTL+TIO3 represents glioma cell lysate vaccine containing TIO3, days after inoculation of glioma cells, and Percent survivin represents the Percent of surviving mice.

FIG. 12 therapeutic effect of TIO3 on gastric cancer

In FIG. 12, PBS represents PBS injection, PBS+TIO3 represents PBS-dissolved TIO3 injection, days after inoculation with gastric cancer cells, and Percent survivin represents the percentage of surviving mice.

The specific implementation method comprises the following steps:

Example 1 design and Synthesis of oligonucleotides (TIO 1, TIO 3)

Three single stranded deoxyoligonucleotides were designed based on the sequence of the 3' UTR of human and mouse transforming growth factor beta 2 (TGF-. Beta.2) mRNA, designated as TIO1, TIO2 and TIO3, respectively, TIO1 having the sequence shown as sequence table <400>1 and TIO3 having the sequence shown as sequence table <400> 2.

The TIO1 sequence is 5'-tggcaaagtatttggtctcca-3' (sequence Listing <400> 1).

The TIO3 sequence is 5'-ttaccactagagcaccaca-3' (sequence Listing <400> 2).

The sequence of TIO2 is 5'-tccttaagccatccatgagttt-3'

The sequences of TIO1, TIO2 and TIO3 are complementary to the sequence of the TGF-. Beta.2mRNA 3' UTR.

TIO1, TIO2 and TIO3 were synthesized by Takara Bio, inc. (Dalian Co., ltd.) and the backbones thereof were modified with total thio.

In application, TIO1 or TIO3 is dissolved with sterile PBS or other vehicle that has been heat-treated. Frozen at-20 ℃.

Endotoxin in the solution containing TIO1, TIO2 and TIO3 was detected by limulus amoebocyte lysis method.

The contents of TIO1, TIO2 and TIO3 in the solution were determined using a spectrophotometer (260 nm wavelength) and also estimated using agarose gel (3%) electrophoresis (estimated from single stranded deoxyoligonucleotide standards of known content).

Example 2 potentiation of the immunopotency of TIO1 and TIO3 against recombinant protein vaccines (circovirus vaccines):

2.1 materials

2.1.1 Mice

ICR mice (animal room, university of Jilin medical department), females, weigh 17-18 grams.

2.1.2 Antigens

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

2.1.3 Adjuvants

ISA 35 emulsifier (Seppic)

2.1.4 Oligonucleotides

TIO1 (having the sequence shown in sequence Listing <400> 1), TIO2 (having the sequence shown in example 1) and TIO3 (having the sequence shown in sequence Listing <400> 2), as described in example 1.

2.2 Preparation of vaccine

The CPs were dissolved in PBS and mixed with an ISA35 emulsifier in a 1:1 (volume: volume) ratio to prepare a vaccine designated CP-ISA 35. 100. Mu.l of CP-ISA35 contained 10. Mu.g of CP. CP-ISA35 containing TIO1, TIO2 and TIO3 was prepared separately, and the doses of TIO1, TIO2 and TIO3 were 10. Mu.g/100. Mu.l. CP-ISA35 containing TIO1, TIO2 and TIO3 is named CP-ISA35+ TIO1, CP-ISA35+ TIO2 or CP-ISA35+ TIO3, respectively.

2.3 Immunization of mice

Mice were immunized initially on day 0. Mu.l of CP-ISA35, CP-ISA35+ TIO1, CP-ISA35+ TIO2 or CP-ISA35+ TIO3 were injected separately, the injection site was the right hindlimb muscle, single point injection. Mice were boosted on day 14 in the same manner as the primary immunization.

2.4. Post-immunization blood collection

Immunized mice serum was collected on day 28 post boost. The tail vein is used for blood sampling, the blood sampling amount is more than or equal to 100 mu l. The collected whole blood (in 1.5mlEP tube) was left at room temperature for 30 minutes. Centrifugation at 11000rpm for 15 minutes, serum collection, split charging and storage at-20 ℃.

2.5. Mouse serum specific antibody detection

ELISA was used to detect antibodies specific for circovirus (PCV 2 b) in mouse serum.

2.5.1 Materials

2.5.1.1 Inactivated circovirus (PCV 2 b) (TCID 50/ml=7.0, tianjin rap biotechnology Co., ltd.).

2.5.1.2 Test Equipment

ELISA strips (combined ELISA plates), 0.5mlEP tube, 1.5 tube mlEP tube, a sample applicator head, a pipette, a multichannel pipette, a plate (diameter 9 cm), a graduated glass bottle.

2.5.1.3 Reagent

Sodium carbonate (national pharmaceutical group chemical reagent Co., ltd.), naCl (national pharmaceutical group chemical reagent Co., ltd.), KCl (Beijing chemical Co., ltd.), na ₂HPO₄·12H₂ O (national pharmaceutical group chemical reagent Co., ltd.), KH ₂PO₄ (Tianjin GYO), tween 20 (Tianjin GYO), skimmed milk powder (Biotopped), citric acid (national pharmaceutical group 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).

2.5.1.4 Liquid

Coating (25% glutaraldehyde PBS), PBS (7.3 Mol/L NaCl,3mmol/L KCl,10mmol/L Na ₂HPO₄·12H₂ O and 17.6mmol/L KH ₂ PO4 in water), washing (0.05% Tween 20 in PBS), blocking (5% skim milk powder in washing), 0.1Mol/L citric acid in water, 0.2Mol/L Na ₂HPO₄.12H₂ O in water, 10 Xmethyl ethyl acetate [0.1Mol/L citric acid in water (volume): 0.2Mol/L Na ₂HPO₄.12H₂ O in water (volume) =94.5:100 ], substrate buffer [ ethyl methyl (ethyl methyl) with OPD (1 μg/ml) and 0.045% H2O2 ] and stop (20% concentrated sulfuric acid).

2.5.2 Experiments

The ELISA strips were coated with inactivated PCV2b in a coating solution at 100 μl/well overnight at 4 ℃. The liquid was spun off and blocked with blocking liquid at 37℃for 2 hours, 200. Mu.l/well. Serum to be tested (1:100 dilution) was added, 37℃for 1 hour. Spin-drying the liquid, adding the washing liquid (300 μl/hole), standing for 3min at room temperature, spin-drying the liquid, and repeating the washing twice. HRP-labeled secondary antibody (goat anti-mouse) IgG (1:5000 dilution with wash solution) was added. 100 μl/well, 37 ℃ for 1 hour. Spin-drying the liquid, washing with the washing liquid, 300 μl/well, and repeating the washing twice at room temperature for 3 min. The substrate solution was added thereto at 100. Mu.l/well, and the reaction was carried out at room temperature in the dark (tin coated paper) for 15 minutes. Stop solution (2 mmol/L of dilute sulfuric acid, 50. Mu.l/well. Detection was performed by an enzyme-labeled instrument (wavelength 492 nm).

2.5.3 Results

TIO1 and TIO3 enhance the immunopotency of recombinant protein vaccines (circovirus vaccines) (FIG. 1). The results demonstrate that TIO1 and TIO3 can be used in individuals to enhance their immune response to antigens (vaccines) of pathogenic microorganisms, including circovirus, and as adjuvants for vaccines of pathogenic microorganisms.

EXAMPLE 3 inhibition of TIO1 and TIO2 on the expression of TGF beta-2 in immune recall responsive mouse immune cells

3.1 Materials

3.1.1 Antigens

CP protein (CP for short), as described in example 2, 2.1.2.

3.1.2 Oligonucleotides

TIO1 (having a sequence as shown in sequence Listing <400> 1), T1O3 (having a sequence as shown in sequence Listing <400> 2), as described in example 1.

3.1.3 Medium

RPMI1640 medium (Gibco Co.). Complete RPMI1640 medium contained 10% Fetal Bovine Serum (FBS) (Gibco, inc.), 2mM L-glutamine, 100U/ml penicillin, 100. Mu.g/ml streptomycin. Serum-free RPMI1640 medium contained no fetal bovine serum.

3.1.4 Fluorescent antibodies

FITC-labeled anti-mouse TGF-beta 2 antibody (BD Co.).

3.1.5 Mice

ICR mice (animal room, university of Jilin medical department), females, weigh 17-18 grams.

3.1.6 Vaccine

CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3, formulated as described in example 2.2.

3.2. Main experimental equipment

24-Hole culture plate, ground glass sheet, 300-mesh filter screen, small tweezers, cell counting plate, dropper and sample feeder. CO2 cell culture incubator (SANYO Co., japan), cell culture inverted microscope (Olympus Co., japan), centrifuge (Biofuge Fresco, germany), flow cytometer as Accuri C6 flow cytometer (BD Bioscience), flow cytometer detection result analysis software as NovoExpress software (ACEA Biosciences).

3.3 Experiment

Mice were immunized initially on day 0. 100 μl of CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 were injected separately, the injection site was right hindlimb muscle, single point injection. Mice were boosted on day 14 in the same manner as the primary immunization. Recall response (recall) was induced 120 days after boost by injecting 100 μl CP-ISA35, CP-ISA35+ TIO1 or CP-ISA35+ TIO3, respectively, into each group of mice, the injection site being the right hind limb muscle, single point injection. Mice were sacrificed after 6 hours.

The sacrificed mice were immersed in 70% ethanol and removed rapidly. The skin and other tissues were removed using sterile surgical equipment, draining lymph nodes were removed under sterile conditions and placed in 0.5ml sterilized 4 ℃ precooled PBS. The lymph nodes were minced on ice, ground with a coarse glass plate, and the filter screen was used to obtain a single cell suspension. Cells were suspended in 4℃pre-chilled PBS or Fluorescence Activated Cell Separation (FACS) buffer (PBS containing 2% fetal bovine serum, 5mM EDTA and 1mM sodium azide).

Cells were washed by centrifugation (277 g, 5min, 4 ℃) with 5ml of 4℃pre-chilled PBS or FACS buffer. Cells were suspended with 4℃pre-chilled PBS or FACS buffer (200-1,000. Mu.l). Counting the number of cells and regulating the concentration of cells. Viability of cells was measured using the phenol blue exclusion assay.

1-10X10 ⁶ lymph node cells were transferred into a 5ml round bottom polystyrene tube. Cells were washed by centrifugation (277 g, 5min, 4 ℃) with FACS buffer. The supernatant was discarded and the cells were suspended in 50-100. Mu.l FACS buffer containing FITC-labeled anti-mouse TGF-beta 2 antibody. The content of each fluorescent antibody is 0.5-1 μg, and the mixture is applied on ice for 30-45min. The sample was protected from light. Cells were washed by centrifugation (277 g, 5min, 4 ℃) with FACS buffer. Cells were suspended in 50-100 μl FACS buffer for flow analysis.

3.4 Results

TIO1 and TIO3 significantly reduced TGF-2 protein expression by lymph node cells of immunocompetent (recall) mice (FIG. 2), as demonstrated by a significant reduction in membrane-bound TGF-2 protein [ Yang ZZ, et al PLoS one.2013;8 (3): e59456Miller MM, et al Virol J.2014 Jan 18;11:7 ]. This suggests that TIO1 and TIO3 inhibit translation of TGF-2 mRNA, and that TIO1 and TIO3 inhibit and attenuate TGF-2 mediated immunosuppression by reducing expression of TGF-2 by immune cells, thereby enhancing immune responses of individuals to pathogenic microorganisms and tumor cells, resulting in anti-infective and anti-tumor effects.

EXAMPLE 4 reducing Effect of TIO1 and TIO2 on the level of immunocyte TGF beta-2 mRNA

4.1 Materials

4.1.1 Cells

RAW 264.7 cells, mouse macrophage lineage cells [ Cell 1978 Sep,15 (1) 261-7], were obtained from ATCC.

4.1.2 Medium

RPMI1640 medium (Gibco Co.). Complete RPMI1640 medium contained 10% Fetal Bovine Serum (FBS) (Gibco, inc.), 2mM L-glutamine, 100U/ml penicillin, 100. Mu.g/ml streptomycin. Serum-free RPMI1640 medium contained no fetal bovine serum.

4.1.3 TGF-beta 2 inducers

Recombinant mouse IL-4 (IL-4), purchased from PeproTech. IL-4 stimulates RAW 264.7 cells to produce large amounts of TGF-beta 2[cell Biol Int.2017 Sep;41 (9): 960-968 under in vitro culture conditions.

LPS (lipopolysaccharide), 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-beta 2[PLoS One.2015 Dec 14;10 (12): e0144954].

4.1.4 Oligonucleotides

TIO1 (having the sequence shown in sequence Listing <400> 1), TIO2 (having the sequence shown in example 1) and TIO3 (having the sequence shown in sequence Listing <400> 2) are as described in example 1.

4.1.5 PCR reagent

TRIzol reagent was purchased from Invitrogen, inc. of America. EASYSCRIPT FIRST-STRAND CDNA SYNTHESIS Supermix, top GREEN QPCR SuperMix (TransStartTM) kit was purchased from full gold (Transgen);

GAPDH specific primers (for amplification of GAPDH specific cDNA, GAPDH is housekeeping gene), the sequence of the upstream primer is 5'-ATCACCATCTTCCAGGAGCGA-3' and the sequence of the downstream primer is 5'-TCTCGTGGTTCACACCCATCA-3'.

TGF-beta 2 specific primers (for amplification of TGF-beta 2 specific cDNA), the sequence of the upstream primer is 5'-ttgtgaaaaccagagcggagg-3' and the sequence of the downstream primer is 5'-agaggtgccatcaatacctgc-3'.

GAPDH-specific primers and TGF-. Beta.2-specific primers were synthesized by Takara Bio Inc. (Dai Co.).

4.2. Main experimental equipment and instrument

24-Hole culture plate, ground glass sheet, 300-mesh filter screen, small tweezers, cell counting plate, dropper and sample feeder. CO ₂ cell culture incubator (SANYO Co., japan), cell culture inverted microscope (Olympus Co., japan), centrifuge (Biofuge Fresco, germany), fluorescent quantitative PCR instrument (U.S. Applied Biosystems, model: ABI Prism 7300).

4.3 Experiment

RAW 264.7 cells were cultured using complete 1640 medium at 37℃under 5% CO ₂. 5X 10 ⁶ RAW 264.7 cells/ml was added to 24-well plates, 1ml of medium per well. IL-4 (25 ng/ml) was added and cells were harvested after 48 hours of incubation with TIO1, TIO2 or TIO3 (final concentration 10. Mu.g/ml). Total RNA was extracted by Trizol method. GAPDH specific cDNA and TGF-beta 2mRNA specific cDNA were amplified by qPCR using EASYSCRIPT FIRST-STRAND CDNA SYNTHESIS Supermix, top GREEN QPCR SuperMix (TransStartTM) kit with GAPDH specific primers and TGF-beta 2 specific primers, respectively. Fluorescence quantitative analysis was performed according to the kit instructions.

1Ml of medium containing RAW 264.7 cells was added to a 24-well plate with a cell count of 5X 10 ⁶ per well. LPS (2. Mu.g/mL) was added and incubated for 2 hours, and TIO1, TIO2 or TIO3 (final concentration 10. Mu.g/mL) was added. After 48 hours of culture, the cells were harvested. Total RNA was extracted by Trizol method. GAPDH specific cDNA and TGF-beta 2mRNA specific cDNA were amplified by qPCR using EASYSCRIPT FIRST-STRAND CDNA SYNTHESIS Supermix, top GREEN QPCR SuperMix (TransStartTM) kit with GAPDH specific primers and TGF-beta 2 specific primers, respectively. Fluorescence quantitative analysis was performed according to the kit instructions.

4.4. Results

TIO1 or TIO3 significantly reduced the TGF- β2mRNA expressed by IL-4 stimulated mouse macrophages (FIG. 3), as well as significantly reduced the level of TGF- β2mRNA expressed by LPS stimulated mouse macrophages (FIG. 4). These results demonstrate that either TIO1 or TIO3 can significantly reduce the level of TGF- β2mRNA in immune cells, including megaphaga cells, thereby reducing the synthetic release of TGF- β2, and thereby reducing or inhibiting TGF- β2 mediated immunosuppression. The results also demonstrate that TIO1 or TIO3 can enhance the immune response to microbial antigens (vaccines) or tumor antigens (vaccines) by reducing TGF-beta 2mRNA of immune cells, and thus can be used as an adjuvant to pathogenic microbial vaccines and tumor vaccines to produce anti-infective and anti-tumor effects, and also can be used in tumor therapy.

Example 5 effect of TIO1 and TIO3 on induction of immune recall response mouse antigen presenting cells express CD 86:

5.1 materials

ICR mice (animal room, university of Jilin medical department), females, weigh 17-18 grams.

5.1.1.2 Antigens

Recombinant circovirus 2b Capsid Protein (CP) was used as antigen [ vaccine.2016 Dec 7;34 (50): 6358-6366].

5.1.1.3 Adjuvant

ISA 35 emulsifier (Seppic)

5.1.1.4 Oligonucleotides

TIO1 (having the sequence shown in sequence Listing <400> 1), TIO2 (having the sequence shown in example 1) and TIO3 (having the sequence shown in sequence Listing <400> 2), as described in example 1.

5.1.1.5 Fluorescent antibodies

FITC-labeled anti-murine CD11c antibody and PE-labeled anti-murine CD86 antibody were both purchased from BD company.

5.1.1.6 Medium

RPMI1640 medium (Gibco Co.). Complete RPMI1640 medium contained 10% Fetal Bovine Serum (FBS) (Gibco, inc.), 2mM L-glutamine, 100U/ml penicillin, 100. Mu.g/ml streptomycin. Serum-free RPMI1640 medium contained no fetal bovine serum.

5.2 Main experimental apparatus

Plate, ground glass sheet, 300 mesh filter screen, small tweezers, cell counting plate, dropper, and sample applicator. CO2 cell culture incubator (SANYO Co., japan), cell culture inverted microscope (Olympus Co., japan), centrifuge (Biofuge Fresco, germany), flow cytometer as Accuri C6 flow cytometer (BD Bioscience), flow cytometer detection result analysis software as NovoExpress software (ACEA Biosciences).

5.3 Experiment

5.3.1 Formulation of vaccine

The CPs were dissolved in PBS and mixed with an ISA35 emulsifier in a 1:1 (volume: volume) ratio to prepare a vaccine designated CP-ISA 35. 100. Mu.l of CP-ISA35 contained 10. Mu.g of CP. CP-ISA35 containing TIO1, TIO2 and TIO3 was prepared separately, and the doses of TIO1, TIO2 and TIO3 were 10. Mu.g/100. Mu.l. The vaccine containing TIO1, TIO2 and TIO3 is named CP-ISA35+TIO1, CP-ISA35+TIO2 and CP-ISA35+TIO3, respectively.

5.3.2 Immunized mice

Mice were immunized initially on day 0. Mu.l of CP-ISA35, CP-ISA35+ TIO1, CP-ISA35+ TIO2 or CP-ISA35+ TIO3 were injected separately, the injection site was the right hindlimb muscle, single point injection. Mice were boosted on day 14 in the same manner as the primary immunization. The immunization was performed 120 days after boost by injecting 100. Mu.l of CP-ISA35, CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3, respectively, into each group of mice, the injection site being the right hind limb muscle, single point injection.

5.3.3. Isolation of draining mouse lymph node cells

Mice were sacrificed 6 hours after induction of immune recall response and mice were isolated draining lymph node cells (as described in example 3,3.3).

5.3.4. Fluorescent antibody staining and flow analysis of draining lymph node cells

1X10 ⁶ lymph node cells were transferred into a 5ml round bottom polystyrene tube. Cells were washed by centrifugation (277 g, 5min, 4 ℃) with FACS buffer. The supernatant was discarded and the cells were suspended in 50-100. Mu.l FACS buffer containing FITC-labeled anti-murine CD11c antibody and PE-labeled anti-murine CD86 antibody. The content of each fluorescent antibody is 0.5-1 μg, and the mixture is applied on ice for 30-45min. The sample was protected from light. And (5) performing flow analysis.

5.4 Results

TIO1 or TIO3 significantly increased CD11c positive cells (dendritic cells) expressing CD86 in mouse lymph node cells (FIG. 5). This result demonstrates that the use of TIO1 or TIO3 can significantly increase the co-stimulatory molecules on the surface of the antigen presenting cell by reducing the expression of TGF- β2, thereby allowing the antigen presenting cell to provide more secondary activation signals to the T cell to promote an adaptive immune response to microbial and tumor antigens. TIO1 or TIO3 having this activity may be used in individuals to enhance their immune response to microbial antigens (vaccines), tumor antigens (vaccines) and may also be used in individuals to enhance their anti-tumor response.

Example 6 potentiation of TIO1 and TIO3 on draining lymph node CD4+ T lymphocytes in mice inducing immune recall responses

6.1 Materials

6.1.1 Mice

ICR mice (animal room, university of Jilin medical department), females, weigh 17-18 grams.

6.1.2 Antigens

Recombinant circovirus 2b Capsid Protein (CP) was used as antigen [ vaccine.2016 Dec 7;34 (50): 6358-6366].

6.1.3 Adjuvants

ISA 35 emulsifier (Seppic)

6.1.4 Oligonucleotides

TIO1 (having the sequence shown in sequence Listing <400> 1) and TIO3 (having the sequence shown in sequence Listing <400> 2) are as described in example 1.

6.1.5 Fluorescent antibodies

FITC-labeled anti-murine CD4 antibodies were purchased from BD company.

6.1.6 Medium

RPMI1640 medium (Gibco Co.). Complete RPMI1640 medium contained 10% Fetal Bovine Serum (FBS) (Gibco, inc.), 2mM L-glutamine, 100U/ml penicillin, 100. Mu.g/ml streptomycin. Serum-free RPMI1640 medium contained no fetal bovine serum.

6.2 Main experimental equipment

Plate, ground glass sheet, 300 mesh filter screen, small tweezers, cell counting plate, dropper, and sample applicator. CO2 cell culture incubator (SANYO Co., japan), cell culture inverted microscope (Olympus Co., japan), centrifuge (Biofuge Fresco, germany), flow cytometer as Accuri C6 flow cytometer (BD Bioscience), flow cytometer detection result analysis software as NovoExpress software (ACEA Biosciences).

6.3 Experiment

6.3.1 Preparation of vaccine

The CPs were dissolved in PBS and mixed with an ISA35 emulsifier in a 1:1 (volume: volume) ratio to prepare a vaccine designated CP-ISA 35. 100. Mu.l of CP-ISA35 contained 10. Mu.g of CP. CP-ISA35 containing TIO1, TIO2 and TIO3 was prepared separately, and the doses of TIO1, TIO2 and TIO3 were 10. Mu.g/100. Mu.l. The vaccine containing TIO1, TIO2 and TIO3 is named CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3, respectively.

6.3.2 Immunized mice

Mice were immunized initially on day 0. 100 μl of CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 were injected separately, the injection site was right hindlimb muscle, single point injection. Mice were boosted on day 14 in the same manner as the primary immunization. The immunization was performed 120 days after boost by injecting 100. Mu.l of CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3, respectively, into each group of mice, the injection site being the right hind limb muscle, single point injection.

6.3.3. Isolation of draining mouse lymph node cells

Mice were sacrificed 6 hours after immunization of the recall response, and mice were isolated to drain lymph node cells (as described in example 3,3.3).

6.3.4. Fluorescent antibody staining and flow analysis

1X10 ⁶ lymph node cells were transferred into a 5ml round bottom polystyrene tube. Cells were washed by centrifugation (277 g, 5min, 4 ℃) with FACS buffer. The supernatant was discarded and the cells were suspended in 50-100. Mu.l FACS buffer containing FITC-labeled anti-murine CD 4. The content of fluorescent antibody is 0.5-1 μg, and the fluorescent antibody acts on ice for 30-45min. The sample was protected from light. And (5) performing flow analysis.

6.4 Results

TIO1 or TIO3 significantly increased CD4 expressing lymphocytes (CD 4 ⁺ cells) in mouse lymph node cells (FIG. 6). This result demonstrates that the use of TIO1 or TIO3 can significantly increase antigen-specific helper T cells by reducing TGF-beta 2 expression, thereby promoting an adaptive immune response to microbial and tumor antigens. The TIO1 or TIO3 having this activity can be used in individuals to enhance their immune response to microbial antigens (vaccines), tumor antigens (vaccines) and also in individuals to enhance their anti-tumor response.

Example 7 increasing Effect of TIO1 and TIO3 on CD19 ⁺ B lymphocytes in draining lymph nodes of immunocompetent mice

7.1 Materials

ICR mice (animal room, university of Jilin medical department), females, weigh 17-18 grams.

7.1.2 Antigens

Recombinant circovirus 2b Capsid Protein (CP) was used as antigen [ vaccine.2016 Dec 7;34 (50): 6358-6366].

7.1.3 Adjuvants

ISA 35 emulsifier (Seppic)

7.1.4 Oligonucleotides

TIO1 (having the sequence shown in sequence Listing <400> 1), TIO2 (having the sequence shown in example 1) and TIO3 (having the sequence shown in sequence Listing <400> 2) are as described in example 1.

7.1.5 Fluorescent antibodies

FITC-labeled anti-murine CD19 antibody, available from BD company.

7.1.6 Medium

RPMI1640 medium (Gibco Co.). Complete RPMI1640 medium contained 10% Fetal Bovine Serum (FBS) (Gibco, inc.), 2mM L-glutamine, 100U/ml penicillin, 100. Mu.g/ml streptomycin. Serum-free RPMI1640 medium contained no fetal bovine serum.

7.2 Main experimental apparatus

Plate, ground glass sheet, 300 mesh filter screen, small tweezers, cell counting plate, dropper, and sample applicator. CO2 cell culture incubator (SANYO Co., japan), cell culture inverted microscope (Olympus Co., japan), centrifuge (Biofuge Fresco, germany), flow cytometer as Accuri C6 flow cytometer (BD Bioscience), flow cytometer detection result analysis software as NovoExpress software (ACEA Biosciences).

7.3 Experiment

7.3.1 Preparation of vaccine

The CPs were dissolved in PBS and mixed with an ISA35 emulsifier in a 1:1 (volume: volume) ratio to prepare a vaccine designated CP-ISA 35. 100. Mu.l of CP-ISA35 contained 10. Mu.g of CP. CP-ISA35 containing TIO1, TIO2 and TIO3 was prepared separately, and the doses of TIO1, TIO2 and TIO3 were 10. Mu.g/100. Mu.l. The vaccine containing TIO1, TIO2 and TIO3 is named CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3, respectively.

7.3.2 Immunized mice

Mice were immunized initially on day 0. Mu.l of CP-ISA35, CP-ISA35+ TIO1, CP-ISA35+ TIO2 or CP-ISA35+ TIO3 were injected separately, the injection site was the right hindlimb muscle, single point injection. Mice were boosted on day 14 in the same manner as the primary immunization. The immunization was performed 120 days after boost by injecting 100. Mu.l of CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3, respectively, into each group of mice, the injection site being the right hind limb muscle, single point injection.

7.3.3. Isolation of draining mouse lymph node cells

Mice were sacrificed 6 hours after immunization of the recall response and the mice were isolated to drain lymph node cells (as described in the experimental 3,3.3).

7.3.4. Fluorescent antibody staining and flow analysis

1X10 ⁶ lymph node cells were transferred into a 5ml round bottom polystyrene tube. Cells were washed by centrifugation (277 g, 5min, 4 ℃) with FACS buffer. The supernatant was discarded and the cells were suspended in 50-100. Mu.l FACS buffer containing FITC-labeled anti-murine CD19 antibody. The content of each fluorescent antibody is 0.5-1 μg, and the mixture is applied on ice for 30-45min. The sample was protected from light. And (5) performing flow analysis.

7.4 Results

TIO1 or TIO3 significantly increased the expression of CD19 cells (B lymphocytes) in mouse lymph node cells (FIG. 7). CD19 is a surface marker for B lymphocytes, and B lymphocytes with increased specificity due to recall reactions rapidly differentiate into plasma cells that produce specific antibodies. This result demonstrates that the use of TIO1 or TIO3 increases the number of antigen-specific B lymphocytes by reducing the expression of TGF-beta 2. TIO1 or TIO3 having this activity may be used in individuals to enhance their long lasting humoral immune response to pathogenic microbial antigens (vaccines).

Example 8 potentiation of the immunopotency of TIO1 and TIO3 to hepatitis B vaccine:

8.1 materials

8.1.1. A mouse

Balb/c mice, females, weighing 17-18g, were purchased from Beijing Veitz laboratory animal Co.

8.1.2 Vaccine

Hepatitis B surface antigen (HBsAg) vaccine (Watson Biotechnology Co., ltd.) prepared by aluminum adjuvant, HBsAg vaccine or HBsAg for short.

8.1.3 Oligonucleotides

Oligonucleotides

TIO1 (having the sequence shown in sequence Listing <400> 1), TIO2 (having the sequence shown in example 1) and TIO3 (having the sequence shown in sequence Listing <400> 2) are as described in example 1.

8.1.4 Preparation of oligonucleotide-containing vaccine

TIO1, TIO2 or TIO3 is added into the HBsAg vaccine to prepare the oligonucleotide-containing HBsAg vaccine, which is named as HBsAg-TIO1, HBsAg-TIO2 and HBsAg-TIO3 respectively. 1. Mu.g HBsAg was included in each 100. Mu.l of vaccine. In HBsAg-TIO1, HBsAg-TIO2 and HBsAg-TIO3, the content of the oligonucleotide was 10. Mu.g.

8.1.5

Recombinant hepatitis B surface antigen (HBsAg) (Watson Biotechnology Co., ltd.), abbreviated as HBsAg.

8.2. Immunization of mice

Balb/c mice were immunized initially on day 0 by injection with 100. Mu.l of HBsAg vaccine, HBsAg-TIO1, HBsAg-TIO2 or HBsAg-TIO3. The injection site is the right hind limb muscle, single point injection. Balb/c mice were boosted on day 14 in the same manner as primary immunization.

8.3. Post-immunization blood collection

Immunized mice serum was collected on day 28 post boost. The tail vein is used for blood sampling, the blood sampling amount is more than or equal to 100 mu l. The collected whole blood (in 1.5mlEP tube) was left at room temperature for 30 minutes. Centrifugation at 11000rpm for 15 minutes, serum collection, split charging and storage at-20 ℃. .

8.4. Immune mouse serum specific antibody detection

ELISA was used to detect antibodies specific for hepatitis B surface antigen (HBsAg) in mouse serum. The ELISA strips were coated with HBsAg in a coating solution at 100. Mu.l/well (0.1. Mu.g HBsAg per well) overnight at 4 ℃. The dilution of the serum to be tested was 1:6400.

The remaining lower procedure for serum was as described in example 2, 2.5.

8.5. Results

TIO1 or TIO3 can enhance the immunopotency of recombinant protein vaccines (hepatitis B virus vaccines) (FIG. 8). This suggests that TIO1 or TIO3 may be used in individuals to enhance their immune response to pathogenic microbial antigens (vaccines) and become an adjuvant to novel microbial antigens or microbial vaccines.

EXAMPLE 9 synergistic effects of TIO1 or TIO3 on influenza Virus vaccines

9.1. Material

9.1.1 Mice

ICR mice (university of gilin laboratory animal center). Female, weighing about 18 g.

9.1.2 Oligonucleotides

TIO1 (having a sequence as shown in sequence Listing <400> 1) and TIO3 (having a sequence as shown in sequence Listing <400> 2), as described in example 1.

9.1.3 Vaccine

FM1 (mouse adapted influenza virus from the biological system of the institute of Japan) was amplified with 11 day old chick embryos and allantoic fluid was taken. The hemagglutination inhibition assay (hemagglutination, HA) was used to determine the hemagglutination unit (HA unit, HAU) of FM1 of allantoic fluid. FM1 was inactivated with 10% V/V formalin to prepare whole inactivated virus (whole-INACTIVATED VIRUSES, WIV) [ Vaccine 2016 Jan 20;34 (4) 495-502]. An ISA 35 emulsifier (Seppic) is adopted to prepare FM1 vaccine (FM 1 for short). Inactivated FM1 containing 700HAU per 100 μl FM1 vaccine. TIO1 or TIO3 is added into FM1 vaccine to prepare FM1 vaccine containing oligonucleotides named FM1-TIO1 and FM1-TIO3, respectively, wherein the oligonucleotide content is 10 mug.

9.2. Immunization and blood collection

Two immunizations were performed, and one immunization was boosted 22 days after the primary immunization. The vaccine was injected in a volume of 100 μl using hind leg intramuscular injection. Blood was collected via the tail vein at day 28 post boost, 50 μl/mouse. The collected blood was left at 37℃for 30 minutes, and after 3 hours at 4℃the serum was sufficiently separated, centrifuged at 4000r/m for 10 minutes, and the serum was aspirated and diluted 1:400 for ELISA detection.

9.3. Immune mouse serum specific antibody detection

ELISA method is used to detect influenza virus specific antibodies in mouse serum. The antigen of FM1 total inactivated virus is used as coating antigen, the coating liquid (Na 2CO3 1.59g,NaHCO3 2.93g,pH 9.6, constant volume to 1L) is used for 1:2 dilution, 100 mu L/hole is used for coating the ELISA plate, sealing is carried out, 4 ℃ is carried out overnight, the ELISA plate is washed by washing liquid (PBS containing 0.05% Tween-20), 300 mu L/hole is washed 3 times, sealing liquid (PBS containing 5% FBS) is added, 200 mu L/hole is carried out for 2 hours at 37 ℃, the mouse serum is diluted by PBS (1:4000 dilution), the diluted serum is added to the ELISA plate, 100 mu L/Kong Jiaru and 37 ℃ are placed for 1 hour, washing liquid is then added for 3 times, horseradish peroxidase is added for marking goat anti-mouse secondary antibody (diluted by sealing liquid 1:1000), 100 mu L/hole is placed for 1 hour, washing liquid is used for 3 times, ready-to-use substrate liquid (10 mL, 0.01M,Na2HPO4 0.02M is carried out by citric acid, 9mL,30% H2 mu L, OPD 4 mg) is added, 100 mu L/hole is carried out for 20 minutes, and the light-shielding value is measured for 20 mu L/hole at room temperature, and the light shielding value is 20% of the ultra pure water is added for 20 minutes.

9.4. Results

TIO1 or TIO3 may enhance the immunopotency of a viral vaccine (influenza vaccine) (FIG. 9). This suggests that TIO1 or TIO3 may be used in individuals to enhance their immune response to antigens (vaccines) of pathogenic microorganisms, including influenza viruses, as an adjuvant to novel microbial vaccines.

EXAMPLE 10 synergistic Effect of TIO1 or TIO3 on rabies vaccine

10.1 Materials

10.1.1 Mice

ICR/c mice weighing 18-22 g were from the university of Jilin department of medicine animal room.

10.1.2 Vaccine

Rabies vaccine (vinca biologicals institute).

10.1.3 Oligonucleotides

TIO1 (having the sequence shown in sequence Listing <400> 1), TIO2 (having the sequence shown in example 1) and TIO3 (having the sequence shown in sequence Listing <400> 2), as described in example 1.

10.1.4 Vaccine

TIO1, TIO2 or TIO3 is added into the rabies vaccine to prepare the rabies vaccine containing oligonucleotides, which are named as rabies vaccine-TIO 1, rabies vaccine-TIO 2 or rabies vaccine-TIO 3 respectively. The content of oligonucleotides in each 500. Mu.l of the oligonucleotide-containing rabies vaccine was 10. Mu.g.

10.2 Blood sampling before immunization

Mice to be immunized were collected two days prior to primary immunization. Blood was collected via the tail vein, 50. Mu.l/mouse. The collected blood was left at 37℃for 30 minutes, and after 3 hours at 4℃the serum was sufficiently separated, centrifuged at 4000r/m for 10 minutes, and the serum was aspirated and stored at-20 ℃.

10.3. Immunized mice

The mice were intraperitoneally injected with 0.5ml of rabies vaccine-TIO 1, rabies vaccine-TIO 2 or rabies vaccine-TIO 3 on days 0, 3, 7, 14, 28. Each group of mice has a male half and a female half.

10.4 Blood sampling

Immunized mice serum was collected on day 35, as described in 10.2.

10.5 Detection of neutralizing antibodies to serum rabies virus

The rapid rabies vaccine fluorescence focus inhibition assay (RFFIT) was used to examine the rabies virus neutralizing antibodies in mouse serum, and the results were expressed in International units per milliliter (IU/ml) [ Virol sin.2012 Jun;27 (3): 187-93].

10.6. Results

TIO1 or TIO3 enhanced the immunopotency of the inactivated virus vaccine (rabies vaccine) (FIG. 10). This suggests that TIO1 or TIO3 may enhance the immunity of an individual against rabies virus infection and may be used as an adjuvant for pathogenic microbial vaccines including rabies vaccines.

EXAMPLE 11 potentiating effect of TIO3 on glioma cell lysate vaccine

11.1 Materials

11.1.1 Mice

C57BL/6 mice, purchased from Peking Violet laboratory animals Co.

11.1.2GL261 cells

GL261 cells are glioma cells (ATCC) of C57BL/6 mouse origin.

11.1.3 Oligonucleotides

TIO3 (having a sequence as shown in sequence Listing <400> 2), as described in example 1.

11.1.4 Medium

RPMI1640 medium (Gibco Co., complete RPMI1640 medium contains 10% Fetal Bovine Serum (FBS) (Gibco Co., ltd.), 2mM L-glutamine, 100U/ml penicillin, 100. Mu.g/ml streptomycin serum-free RPMI1640 medium contains no fetal bovine serum

Preparation of GL261 cell lysate

GL261 cells were cultured using complete RPMI1640 medium at 37℃under 5% CO ₂. 1ml of GL261 cells (1X 10 ⁷/ml) in good growth state were inoculated into the peritoneal cavity of healthy C57BL/6 mice. After the abdomen of the mice swells, the mice are sacrificed and soaked in 75% ethanol for 2-3min for disinfection. The mice were opened to the abdominal cavity in a super clean bench and the GL261 cell solid tumor formed was seen. And taking out GL261 cell solid tumor according to aseptic operation. Cutting the tumor tissue into small pieces in a plate by using surgical scissors, and repeatedly flushing the tumor tissue and residual blood by using normal saline. Repeatedly grinding the cleaned tissue blocks in a tissue grinder, and collecting grinding liquid. The grinding fluid is placed in liquid nitrogen and is repeatedly frozen and thawed for 5 times at room temperature. After centrifugation at 2000rmp for 10min, the supernatant was collected. The supernatant was GL261 cell glioma tumor cell lysate (GTL). The protein content of the GTL was determined by Bradford method. The GTL was further analyzed by SDS-PAGE. The GTL is stored in a refrigerator at-80 ℃.

11.3 GTL vaccine and preparation of GTL vaccine containing oligonucleotide

The protein concentration of GTL was adjusted to 4mg/ml with PBS. The GTL and emulsifier are mixed 1:1 (volume: volume) to make a vaccine, which is designated as a GTL vaccine. TIO-3-containing GTL vaccine formulated with TIO-3, which is referred to as GTL-TIO-3 vaccine, was added to the GTL vaccine, wherein the concentration of TIO-3 was 10. Mu.g/100. Mu.l.

11.4 Immunized mice

100 Μl of GTL vaccine or GTL-TIO-3 vaccine was injected subcutaneously in the neck of mice on day 0 and day 9.

11.5 Inoculation of glioma cells

On day 14, mice were injected intracranially with 1X 10 ⁴ GL261 cells in a volume of 2. Mu.l to develop gliomas as described in literature (Prins RM, et al cancer Res.2003 Dec 1;63 (23): 8487-91). The survival of the mice was observed and recorded from the time of tumor inoculation, and after death of the mice, cadaveric lysis was performed to confirm that glioma had occurred intracranially in the mice.

11.6 Results

The TIO3 significantly enhanced the immunopotency of glioma cell lysates (tumor antigen vaccine) (fig. 11), resulting in a significant prolongation of the survival of tumor-bearing mice. The results indicate that TIO3 enhances an individual's immune response against gliomas and can be used as an adjuvant to tumor vaccines, including glioma vaccines.

Example 12, potentiation of lung cancer cell lysate vaccine by TIO1 or TIO 3:

12.1 materials

12.1.1 Mice

C57BL/6 male mice (Beijing Vitrendy laboratory animal technologies Co., ltd.) 6 weeks old.

12.1.2 Cells

Lewis lung carcinoma (Lewis lung carcinoma, LLC) cells (ATCC) originated in C57BL/6 mice. The cells were cultured using complete RPMI1640 medium (as described in 11.1.4) at 37 ℃ with 5% co ₂.

12.1.3 Oligonucleotides

TIO1 (having a sequence as shown in sequence Listing <400> 1) and TIO3 (having a sequence as shown in sequence Listing <400> 2), as described in example 1.

Preparation of Lewis lung cancer cell (LLC) lysate

LLC cells (1X 10 ⁶/0.1 ml) subcultured in vitro were inoculated into the back of C57BL/6J male mice and grown subcutaneously as solid tumors. The tumor was removed from the sacrificial mice and LLC lysate (LTL) was prepared, identified and stored as described in example 11.2. Mu.l of LTL was prepared per 1X 10 ⁶ LLC cells.

12.3 LTL vaccine and preparation of LTL vaccine containing oligonucleotide

The LTL and the emulsifier were mixed 1:1 (volume: volume) to prepare a vaccine, which was designated as LTL vaccine. To the LTL vaccine, a TIO1 or TIO 3-containing LTL vaccine prepared by TIO1 or TIO3 was added, and these two vaccines were called LTL-TIO1 vaccine and LTL-TIO3 vaccine, respectively, wherein the concentration of TIO1 or TIO3 was 10. Mu.g/100. Mu.l.

12.4 Immunization of mice

100 Μl of LTL vaccine, LTL-TIO1 vaccine or LTL-TIO3 vaccine was injected subcutaneously into the right back of 6 week old C57BL/6J male mice on day 0 and day 14. Each group had 10 mice.

12.5 Inoculation of Lewis lung carcinoma (Lewis lung cancer, LLC) cells

On day 21, mice were injected subcutaneously with 100 μl of LLC cell suspension on the left dorsal side. LLC cell suspensions were prepared by inoculating in vitro subcultured LLC cells (1X 10 ⁷/100 μl) into the back of C57BL/6J male mice to subcutaneously grow solid tumors. The mice were sacrificed, tumor tissues were removed by aseptic manipulation, and digested with 0.25% trypsin-0.04% edta for 30min to prepare single cell suspensions. The cell concentration was adjusted to 1X 10 ⁶/100. Mu.l in PBS. Mice were sacrificed on day 18 post tumor inoculation, tumor masses were dissected out and weighed.

12.6 Results (Table-1)

Table-1, TIO1 or TIO3 potentiating effect on lung cancer cell lysate vaccines

The results indicate that TIO-1 or TIO-3 can enhance the immunopotency of lung cancer cell lysates (Table-1), shrinking tumors (p < 0.05). This suggests that TIO-1 or TIO-3 may enhance an individual's immune response against lung cancer and may be used as an adjuvant to tumor vaccines, including lung cancer vaccines.

Example 13 therapeutic effect of TIO3 on gastric cancer:

13.1 materials

13.1.1 Mice

Balb/c female mice (Beijing Vitrenlhua laboratory animal technologies Co., ltd.) 18-22g.

13.1.2 Stomach cancer cells

The MFC cells (mouse GASTRIC CANCER CELL) are Balb/c mouse-derived gastric cancer cells (from ATCC in the United states). The cells were cultured in complete RPMI1640 medium (as described in 11.1.4) at 37 ℃ with 5% co ₂.

13.1.3 Oligonucleotides

TIO3 (having a sequence as shown in sequence Listing <400> 2), as described in example 1.

13.2 Major laboratory apparatus and instruments

100Ml cell culture flask, 1ml syringe, cell counting plate. CO ₂ cell culture incubator (SANYO Co., japan), cell culture inverted microscope (Olympus Co., japan), centrifuge (Biofuge Fresco, germany).

13.3 Experiment

The MFC subcutaneous engrafted tumor model cells cultured in vitro were inoculated subcutaneously on the dorsal side of the left hind limb of mice on day 0 in an injection volume of 200 μl containing 1X10 ⁶ MFC cells. Mice were injected with TIO3 (in PBS) starting on day 10 after inoculation with MFC cells, then re-injected every two days for 6 total injections. The injection site is the left hind limb inoculated tumor lymph node drainage area subcutaneous. Each injection had a volume of 100. Mu.l and contained 25. Mu.g of TIO3. Control mice were injected with PBS according to the same procedure. The survival time of the mice was observed and recorded.

13.4 Results

The use of TIO3 alone significantly prolonged the survival of tumor-bearing mice (FIG. 12). This suggests that TIO3 may enhance an individual's anti-gastric cancer cellular immune response and may be used to treat tumors including gastric cancer.

Claims (5)

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 of 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.