WO2017101744A1 - 具有抗原增效作用和肿瘤治疗作用的寡核苷酸 - Google Patents

具有抗原增效作用和肿瘤治疗作用的寡核苷酸 Download PDF

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WO2017101744A1
WO2017101744A1 PCT/CN2016/109451 CN2016109451W WO2017101744A1 WO 2017101744 A1 WO2017101744 A1 WO 2017101744A1 CN 2016109451 W CN2016109451 W CN 2016109451W WO 2017101744 A1 WO2017101744 A1 WO 2017101744A1
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tumor
cells
vaccine
antigen
stranded
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French (fr)
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王丽颖
于永利
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苏州派动生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the use of two oligonucleotides, as well as their synergistic antigens and in the treatment of tumors.
  • T Lymphocyte activation is a key condition that stimulates an individual's immune response to an antigen and an anti-tumor immune response.
  • T lymphocyte activation requires two activation signals.
  • the first activation signal comes from T lymphocytes through the T cell antigen receptor (T Cell antigen receptor (TCR) recognizes antigen presenting cells (antigen-presenting) Cell, APC) or antigenic peptide-MHC complex on the surface of target cells.
  • TCR T Cell antigen receptor
  • APC antigen presenting cells
  • the second activation signal is derived from T lymphocytes that recognize, bind to APC or B7 molecules on the surface of target cells via CD28 molecules (including CD80). And CD86).
  • the second activation signal is also called a costimulatory signal. Signals).
  • T lymphocytes that obtain the first activation signal are not fully activated and may even enter an immune tolerance state. Only after obtaining the first and second activation signals can the T lymphocytes be fully activated and function [ Sharma P. et al. Science. 2015 Apr 3; 348(6230): 56-61; Greenwald RJ, et al. Annu Rev Immunol.2005;23:515-48].
  • T lymphocytes also activate an inhibitory signaling pathway during activation, which activates the activation of T lymphocytes and inhibits their function [Sharma P. et al. Science. 2015 Apr 3;348(6230):56 -61; Greenwald RJ, et al. Annu Rev Immunol. 2005; 23: 515-48].
  • Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4, also known as CD152, is a protein that mediates this inhibitory signaling pathway. CTLA-4 is expressed on activated T lymphocytes (including CD4 + T) The surface of lymphocytes and CD8 + T lymphocytes compete with CD28 for binding to B7 molecules (CD80 and CD86) on the surface of APC or target cells.
  • CTLA-4 recognizes binding to B7 molecules. Inhibition signals (negative regulatory signals) are activated in T lymphocyte transduction (TL Walunas et al., Immunity 1, 405-413 (1994).
  • Interfering with, attenuating, and blocking the interaction of CTLA-4 and B7 molecules can enable T cells to continue to be fully activated, producing two potencies in the individual: 1. Enhancing the individual's immune response to the antigen; 2. Enhancing the tumor cells. Endogenous immune responses. Because of the first potency, interfering with, attenuating, and blocking the interaction of CTLA-4 and B7 molecules enhances the immunogenic potency of the antigen, so preparations that interfere with, attenuate, and block the interaction of CTLA-4 and B7 molecules can be used. It enhances the efficacy of the antigen and has the function of a vaccine adjuvant.
  • CTLA-4 An antibody is a preparation that interferes with, attenuates, and blocks the interaction between CTLA-4 and B7 molecules. It recognizes and binds to CTLA-4 and inhibits the transduction of T lymphocyte activation inhibition signals, thereby allowing T cells to be continuously and fully activated. . CTLA-4 antibody can enhance the immune response of individual cells to tumor cells and has become a drug for treating tumors.
  • CTLA-4 antibodies can treat a variety of tumors, including breast cancer [Hurwitz A. A., et Al. Proc Natl Acad Sci U S A.1998 Aug 18;95(17):10067-71] , melanoma [van Elsas A., Et al. J Exp Med. 1999 Aug 2;190(3):355-66] and prostate cancer [Waitz R, et al, Oncoimmunology.2012 Jul 1;1(4):544-546].
  • Clinical trials have shown that CTLA-4 antibody (Ipilimumab) has a therapeutic effect on tumors [ Leach DR, et al. Science.
  • CTLA-4 antibodies can enhance the immune potency of an individual in an individual.
  • the present invention provides two oligonucleotides having sequence numbers ⁇ 400> 1 and ⁇ 400> 2 The sequences shown, which inhibit CTLA-4 expression by interfering with CTLA-4 mRNA, which in turn Interfering with, attenuating, and blocking the interaction of CTLA-4 and B7 molecules inhibits the inhibition signal of T cell activation, and as a result, enhances the individual's immune response to the antigen and enhances the individual's anti-microbial and anti-tumor immune response.
  • oligonucleotides can be modified or modified by various chemicals.
  • These two oligonucleotides can be used as adjuvants for microbial antigens and tumor antigens, and have anti-infective and anti-tumor effects.
  • These two oligonucleotides can be used for tumor therapy.
  • oligonucleotides can be used in combination with other adjuvants to enhance an individual's immune response to microbial antigens and tumor antigens.
  • oligonucleotides can be used in combination with other anti-tumor agents and tumor-treated cells to treat tumors.
  • oligonucleotide is a molecule composed of a plurality of nucleotides, and the number of nucleotides thereof may be several or several. Nucleotides are the basic building blocks of nucleic acids and also oligonucleotides. Nucleotides consist of nucleosides and phosphoric acid. Nucleosides consist of pentose and base. Pentose sugars include ribose and deoxyribose. The pentose molecule is linked to the base to form a nucleoside. Nucleosides are linked by a phosphate group to form a nucleotide.
  • Nucleotides are linked by phosphodiester bonds to form oligonucleotides.
  • the bases constituting the nucleoside include pyrimidine and purine. Pyrimidines include thymine (abbreviated as T) Or t) and cytosine (cytosine, abbreviated as C Or c). ⁇ includes adenine (abbreviated as A or a) and guanine (abbreviated as G or g).
  • the base in the oligonucleotide can be a rare base. Rare bases include, but are not limited to, 5-hydroxymethylcytosine, 7-methylguanine, and 5-hydroxymethylcytosine.
  • the oligonucleotide may be a single stranded, double stranded, circular or molecularly circular structure.
  • an oligonucleotide (Oligonucleotide, ODN) can be replaced by its English abbreviation ODN.
  • ODN oligonucleotide
  • the nucleotide sequence in the oligonucleotide constitutes its primary structure, which is also referred to as the nucleotide sequence.
  • the nucleotide sequence can be represented by a base sequence, and therefore, the sequence of the nucleotide is also referred to as a base sequence.
  • the sequence of the deoxyoligonucleotide can be represented by the abbreviation of the base, T or t represents thymine, C Or c represents cytosine, A or a represents adenine, and G or g represents guanine.
  • the oligonucleotide provided by the present invention can be chemically modified compared to natural DNA (chemical Modification).
  • a chemical modification of an oligonucleotide is a phenomenon or method in which a covalent structure is altered by introducing or removing any chemical gene. Chemically modified sites of oligonucleotides can occur in phosphodiester bonds, ribose, and bases. Chemical modification of the oligonucleotide can occur at the 5' or 3' end and can be carried out either at the time of synthesis or after synthesis.
  • Chemical modifications involved in the present invention include, but are not limited to, modifications to the oligonucleotide backbone, such as thio modifications (the non-bridged oxygen atoms of the phosphoric acid in the internucleotide phosphate diester bond are replaced by sulfur atoms) and substitution modifications (including a substitution of an alkyl group, an aryl group or any other chemical group).
  • Chemical modifications of oligonucleotides also include base substitutions and base modifications, and alternative bases can 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 group at its 5' and/or 3' end.
  • 'UTR' refers to the untranslated region of the mRNA molecule (untranslated region, UTR), located at both ends of the coding region of the mRNA molecule polypeptide, is called the 5' UTR at the 5' end and the 3' UTR at the 3' end.
  • immune response immune Response
  • immune response is the individual's immune cells including B lymphocytes, T lymphocytes, NK cells, ⁇ T cells, NKT cells, dendritic cells, macrophages, and granulocytes, etc. against antigens or other stimuli [eg pathogen-associated model molecules (pathogen) Associated molecular pattern (PAMP) and damage associated molecular Pattern, DAMP) response.
  • PAMP pathogen-associated model molecules
  • DAMP damage associated molecular Pattern
  • the result of an immune response is the selective destruction or removal of invading pathogenic microorganisms or endogenous tumor cells.
  • the immune response includes an innate immune response and an adaptive immune response. Adaptive immune responses include cellular immune responses and humoral immune responses.
  • An immune response elicited by a vaccine made with a microbial antigen can give an immunized individual the ability to resist microbial infection.
  • An immune response elicited by a vaccine made using a tumor antigen can have a tumor therapeutic effect in the immunized individual. Promoting an immune response to an individual against a microorganism can have an anti-infective effect. Promoting an individual's immune response to tumor cells can have an anti-tumor effect.
  • lymphocyte refers to a single nucleated leukocyte that is present in blood, lymph, and lymphoid tissue without phagocytosis, including B lymphocytes (also known as B cells) and T lymphocytes (also known as T cells). . T cells can be divided into T cells (CD4 + T cells) that express CD4 molecules on the surface and T cells (CD8 + T cells) that express CD8 molecules on the surface.
  • CTLA-4 T lymphocyte-associated antigen 4
  • CTLA-4 antigen also known as CD152 or CTLA-4 antigen (CTLA- 4 antigen)
  • CTLA-4 binds to the B7 molecule on the surface of antigen presenting cells.
  • the B7 molecule is a ligand for CTLA-4 and a ligand for CD28 on the surface of T cells.
  • CTLA-4 Compared to CD28, CTLA-4 has a higher affinity for B7, which competes with CD28 for binding to B7, thereby attenuating the CD28-dependent costimulatory signal (the first signal for T cell activation). CTLA-4 also mediates direct inhibition of the MHC-TCR pathway signal (the second signal of T cell activation) [Nirschl CJ., et al. Clin Cancer Res. 2013 Sep 15; 19(18): 4917-24]. Activated T cells up-regulate the expression of CTLA-4, and CTLA-4 binds to B7 to inhibit the activation of T cells. In the tumor microenvironment, CTLA-4 mediated inhibition of T cell activation can impair the individual's anti-tumor immune response.
  • Blocking the function of CTLA-4 enhances the individual's anti-tumor activity and produces a tumor therapeutic effect [Leach DR., et al. Science. 1996 Mar 22; 271 (5256): 1734-6]. Blocking the function of CTLA-4 enhances an individual's immune response to an antigen or vaccine [van Elsas A., et al. J Exp Med. 1999 Aug 2;190(3):355-66].
  • T cell receptor T cell receptor
  • TCR T cell receptor
  • V variable
  • C constant
  • the V region is the domain in which the TCR recognizes an epitope.
  • TCR cannot directly recognize antigenic epitopes and can only recognize antigen-presenting cells or antigenic peptide-MHC (major histocompatibility complex) molecular complexes on the surface of target cells.
  • MHC molecule is the major histocompatibility complex (major histocompatibiltiy Complex, MHC) A protein molecule encoded by a gene.
  • MHC molecules include MHC class I molecules and MHC class II molecules.
  • CD28 is a protein expressed on the surface of T cells and recognizes the B7 molecule on the surface of antigen-presenting cells. After recognition of the B7 molecule, CD28 provides a costimulatory signal to T cells (co-stimulatory Signals).
  • B7 molecule is a protein molecule mainly expressed on the surface of antigen-presenting cells, especially dendritic cells, including B7.1 (CD80) and B7.2 (CD86), except for lymphoma cells, tumor cells do not express B7 molecules [Sharma, J.P. Allison. Science 348, 56, 2015].
  • Co-stimulatory molecule' costimulatory molecule
  • costimulatory molecule are protein molecules expressed on the surface of antigen presenting cells (APCs) or tumor cells, providing activation during the immune response A second activation signal for CD4+ T lymphocytes and CD8+ T lymphocytes.
  • T lymphocyte activation' Two signals are required for activation of T lymphocytes.
  • the first signal is obtained by its TCR recognition of an antigenic peptide-MHC molecular complex on the surface of APC or target cells (cells that can be killed by CD8 + T cells); a second signal, also known as a costimulatory signal, recognizes APC by its CD28 molecule or The B7 molecule on the surface of the target cell is obtained.
  • T cells initiate activation of signal transduction pathways, which in turn proliferate and differentiate. Only the first signal and no second activation signal can cause the T cells to enter a non-responding state.
  • a T lymphocyte reaction occurs after T lymphocyte activation.
  • T lymphocyte response and 'T lymphocyte activity' or 'function of T lymphocyte exercise' are interchangeable terms in the present invention.
  • T lymphocyte responses include T lymphocyte proliferation and/or differentiation into helper T lymphocytes (Th), cytotoxic T lymphocytes (Tc) or regulatory T lymphocytes (Treg), and also provided by Th to B lymphocytes Signals assist in the 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 a CD4 + T cell and Tc is a CD8 + T cell.
  • CD4 + T cells help B cells produce antibodies that help CD8 + T cells kill target cells such as virus-infected cells and tumor cells.
  • CD8 + T cells kill virus-infected and tumor cells. Promoting and maintaining T cell activation enhances the individual's immune response to microbial antigens and produces anti-infective effects, and also promotes the immune response of individual immune cells to tumor cells to produce tumor therapeutic effects.
  • An individual (subject or individual) in the present invention refers to a human and a non-human vertebrate.
  • target cell refers to a cell in which an individual can be attacked, killed or acted upon by an immune cell, and may be a tumor cell, a virus-infected cell, or a cell which is acted upon by the oligonucleotide provided by the present invention.
  • CTLA-4 Antibody' is an antibody that specifically binds to CTLA4, which blocks the inhibition signal of CTLA-4 transduction, enables T lymphocytes to be fully activated by tumor antigens, and prolongs the survival of tumor patients.
  • Ipilimumab Is a fully humanized IgG1 CTLA-4 monoclonal antibody [Lipson EJ., et al. Clin Cancer Res. 2011 Nov 15;17(22):6958-62], approved by the US FDA as a drug for the treatment of advanced melanoma in 2011 [Sharma P. et al. Science. 2015 Apr 3; 348 (6230): 56-61].
  • Tremelimumab is another humanized IgG2 CTLA-4 monoclonal antibody [Ribas A., et al. Oncologist. 2007 Jul;12(7):873-83]. In clinical trials, Tremelimumab is used to treat hepatocellular carcinoma [Sangro B., et Al. J Hepatol. 2013 Jul;59(1):81-8], gastric cancer and esophageal cancer [Ralph C. et al. Clin Cancer Res. 2010 Mar 1;16(5):1662-72].
  • the antibody of CTLA-4 belongs to an immunoassay molecule inhibitor.
  • Immunospotpoint molecules include, but are not limited to, CTLA-4 molecule, PD-1 molecule, PD-L1/2 molecule, lymphocyte-activation gene 3 (lymphocyte-activation) Gene 3, LAG-3), TIM-3 (T cell immunoglobulin and mucin domain-containing 3), TIGIT (T cell immunoreceptor with immunoglobulin and ITIM domains) and BTLA (B and T Lymphocyte attenuator).
  • Closed immune checkpoint refers to a method for inhibiting the function of an immune cardinal molecule to transmit an inhibitory signal in an immune cell to promote and maintain T cell activation. Blocking the immune card point enhances the individual's immune response to the microbial antigen and exhibits anti-infective effects, and also promotes the anti-tumor activity of the individual's immune cells to exhibit tumor therapeutic effects [ Melero I, et al. Nat Rev Cancer. 2015 Aug; 15(8): 457-72].
  • the 'tumor' in the present invention that is, a tumor defined by modern medicine, can be classified into a benign tumor and a malignant tumor.
  • Tumors and cancers are used interchangeably and have the same meaning.
  • Tumors include solid tumors, soft tissue sarcomas, and myeloid or lymphoid tumors.
  • the oligonucleotide provided by the invention can enhance the anti-tumor effect of an individual's immune response to a tumor antigen, and the tumors involved include, but are not limited to, esophageal cancer, gallbladder cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, adrenal gland Cortical, kidney, liver, lung, ovarian, cervical, uterine, vaginal, pancreatic, rectal, prostate, gastric, skin, melanoma Sarcoma, penile cancer, retinoblastoma, leukemia, lymphoma and myeloma.
  • the tumors involved include, but are not limited to, esophageal cancer, gallbladder cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, adrenal gland Cortical, kidney, liver, lung, ovarian, cervical, uterine, vaginal, pancreatic, rectal, prostate, gastric, skin, melanoma Sarcoma, penile cancer, reti
  • 'Treatment' The use of oligonucleotides provided by the present invention to prevent or delay the onset of symptoms and complications of diseases such as tumors. Treatment can also be preventive.
  • the treatment of tumors also refers to the individual's control of tumor progression, prolonging the survival of tumor patients, improving the quality of life, reducing symptoms, shrinking or even eliminating tumors, and curbing tumor metastasis.
  • 'tumor treatment' has the same meaning as 'antitumor effect' or 'treatment of tumor'.
  • Anti-tumor effects include treatment of tumors, as well as prevention of tumorigenesis, recurrence, and metastasis.
  • 'antigen' can be recognized by B cell receptors or T cell receptors to stimulate an individual's adaptive immune response (adaptive immune response) The substance or molecule of response.
  • the antigen may be from the outside of the individual, such as a microbial antigen; it may also be from the interior of the individual, such as a tumor antigen.
  • Microbial antigens are microbially recognized by B cell receptors or T cell receptors to stimulate an individual's adaptive immune response (adaptive) Ill The substance or molecule of response.
  • Vaccines prepared using microbial antigens when administered to an individual, can confer immunity to the pathogen, protecting them from re-exposure to the same or similar pathogens. Vaccines made with tumor antigens can stimulate their adaptive immune response to tumor cells and then produce tumor therapeutic effects.
  • the antigen can be extracted from microorganisms or tumor cells, or can be produced by recombinant DNA technology or by other methods.
  • Vaccine is a biological product for artificial active immunization made of an attenuated or killed pathogenic organism or a component thereof. Antigens and adjuvants are the main components of the vaccine. The purpose of vaccination is to protect an individual from immunity to the pathogen and then re-contact it with the corresponding pathogen.
  • the oligonucleotides provided by the present invention can be used in combination with human vaccines to enhance their immunological efficacy, including but not limited to vaccines that prevent the following infectious diseases [ Stanley A. Plotkin, Walter A. Orenstein, Paul A. Offit, Vaccines, Sixth Edition, An imprint of Elsevier Inc.
  • Vaccines contemplated by the present invention also include tumor vaccines.
  • Tumor antigens and adjuvants are the main components of tumor vaccines.
  • Tumor vaccines can treat or prevent tumors.
  • the 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 present invention can be combined with tumor vaccines to enhance their efficacy in preventing and/or treating tumors, and such vaccines include, but are not limited to, human papillomavirus vaccine for cervical cancer prevention, for use in Provenge vaccine for prostate cancer, vaccines made with various tumor antigens, vaccines made with various tumor cells, and vaccines made with tumor cell lysates.
  • the oligonucleotides provided by the present invention can be used in combination with animal vaccines to enhance their immunological efficacy. These vaccines include, but are not limited to, vaccines that prevent the following infectious diseases: swine foot and mouth disease, porcine blue ear disease (porcine reproductive and respiratory syndrome).
  • piglet pig pseudorabies, porcine circovirus infection, porcine parvovirus infection, Streptococcus suis, porcine transmissible gastroenteritis, swine asthma, Haemophilus parasuis infection, swine erysipelas, swine lung disease, pig Atrophic rhinitis, porcine transmissible gastroenteritis, swine JE, swine flu, swine brucellosis, piglet diarrhea, swine parainfluenza virus infection, swine flu, Mycoplasma hyopneumoniae infection, piglet edema disease, Salmonella choleraesuis, pig Epidemic diarrhea, swine plague; bovine carbuncle, bovine anthrax, bovine epidemic fever, bovine Pasteurellosis, bovine Brucella infection, bovine tetanus, Clostridium perfringens infection, Clostridium botulin
  • Tumor antigens can be used in combination with tumor antigens to promote an individual's immune response to tumor antigens.
  • tumor antigen Tumor Antigen
  • TAA tumor associated antigen Antigen
  • Tumor antigens can elicit an anti-tumor immune response, and the immune response against tumor antigens produces 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, ⁇ -catenin-m, HSP70-2/m (heat shock-related 70 kDa Protein 2 mutated), KRAS, GM2 (ganglioside GM2), MUC1 (mucin-1), hepatitis B virus gene encoding antigen, hepatitis C virus gene-encoded antigen, human papillomavirus gene encoding antigen (e
  • Tumor antigens also include inactivation of whole tumor cells and tumor cell lysates.
  • the tumor antigen may be a neo-antigen expressed by a gene mutation, or may be a unique 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].
  • the oligonucleotides provided by the present invention can be used in combination with tumor vaccines to enhance the efficacy of their tumor treatment.
  • a tumor vaccine is a biological product that induces an individual to develop an anti-tumor adaptive immune response.
  • the tumor vaccine according to the present invention includes a vaccine prepared by using a tumor antigen, a tumor cell lysate, a tumor cell, and a cell presenting a tumor antigen in a certain dosage form.
  • Such vaccines include, but are not limited to, the following [ [Zielinski C, et al. Nat Rev Clin Oncol.
  • Tumor vaccines for prostate cancer including prostate acid phosphatase (for sipuleucel-T), (Provenge®) and PSA; tumor vaccines for breast cancer, including tumor antigens including HER2-derived polypeptide, MUC1 And WT1 (Wilms tumour protein) antigen; a tumor vaccine for treating lung cancer, the tumor antigens used include MUC1, melanoma-associated antigen-3 (MAGE-A3) polypeptide, telomerase reverse transcriptase and EGF; vaccine for the treatment of melanoma, including antigens included --catenin, gp100, MAGE-A3, MART1 (melanoma antigen recognized by T cells 1), NY-ESO-1 and Survivin; a tumor vaccine for treating a pancreatic cancer vaccine, comprising a tumor antigen comprising a telomerase peptide, an allogeneic tumor cell, and a mutant RAS synthetic peptide; a tumor vaccine for treating
  • 'Tumor cell lysate' tumor cell lysate can pass The broken tumor cells are obtained and contain various tumor antigens. Methods of disrupting tumor cells include, but are not limited to, repeated freeze-thaw, sonication, and mechanical disruption.
  • the oligonucleotides provided by the present invention can be combined with tumor cell lysates to promote an anti-tumor immune response in an individual.
  • Primary tumors, secondary or metastatic tumor cells can be used to prepare tumor cell lysates.
  • the tumor cell line can be obtained by culturing the primary tumor and the secondary or metastatic tumor cells in vitro. Cell line cells, which can also be used to prepare tumor cell lysates. Tumor cells used to prepare tumor cell lysates can be autologous, allogeneic or xenogeneic.
  • the oligonucleotides provided herein can be used in combination with one or more adjuvants to enhance the immunopotency of microbial antigens and tumor antigens.
  • An adjuvant is a substance that is used together with an antigen in a vaccine, and has the following activities: (1) reducing the number of times of vaccination of the vaccine; (2) prolonging the duration of immunization of the vaccine; and (3) promoting humoral immunity by stimulating an innate immune response. Responsive and cellular immune responses; (4) extended antigen-induced cross-protective immune response; (5) enhanced immune response to antigens in individuals with weak immune responses such as aging individuals or immunodeficient individuals; (6) reduced dose of antigen.
  • aluminum salt adjuvant (vaccine adjuvant with aluminum hydroxide or aluminum phosphate as the main component), AS04 adjuvant (aluminum phosphate adjuvant for adsorption of MPL (MPL is a chemically attenuated Gram-negative bacterium) Lipopolysaccharide), MF59 adjuvant (a water-in-oil emulsifier in which the oil phase is squalene), AS03 adjuvant (an oil-in-water adjuvant using squalene as the oil phase), AF03 Agent (an oil-in-water emulsifier using squalene as the oil phase), Montanide ISA 51 adjuvant (water-in-oil emulsifier with mineral oil as the oil phase), Freund's adjuvant, Freund's incomplete Agent, virosome adjuvant, N-oxidized polyethylene to dinitrogen ring derivative (polyoxidonium) adjuvant,
  • the oligonucleotides provided by the present invention can be associated with pathogen-associated molecular patterns (pathogen-associated molecular patterns, PAMPs and their analogues are used in combination to enhance the immune effects of microbial antigens and tumor antigens to exert anti-infective or anti-tumor effects.
  • PAMPs are various conserved components of microorganisms, such as bacterial and fungal cell wall components and viral nucleic acids.
  • Innate immune cells pass pattern recognition receptors Pattern-recognition receptors, PRRs) are activated by recognition of PAMPs.
  • 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 Receptor (CLRs) and AIM-2 like receptors; also includes intracellular nucleic acid receptors (intracellular) Sensors of nucleic acids), OAS protein and cGAS [ Iwasaki A et al. Nat Immunol. 2015 Apr;16(4):343-53].
  • the oligonucleotide provided by the present invention can be combined with one or more Toll-like receptors (Toll-like Receptors,TLRs)
  • TLRs Toll-like Receptors
  • the agonist is used in combination to enhance the immune effect of the microbial antigen and the tumor antigen, and exhibit anti-infective or anti-tumor activity.
  • TLR agonists that can be used in conjunction with the oligonucleotides provided herein include, but are not limited to, TLR7 and TLR8 agonists including Imiquimod and imidazoquinolines; TLR7 agonists including 852A; including VTX-2337 TLR8 agonist; including IMO-2055, CPG TLR9 agonist, 7909, MGN1703 and other CpG ODNs (including CpG deoxyoligonucleotides); including BCG TLR2/TLR4 agonist (BCG); includes OM-174, monophosphoryl lipid A, aminoalkyl glucosamine Phosphates and other lipids A (lipid A) TLR4 agonists including analogs; TLR9 agonists including viral nucleic acids and bacterial nucleic acids; TLR5 agonists including bacterial flagellin, TLR2/TLR6 agonists including zymosan; Glycosyl nucleoside nucleotide (poly TLR3 agonists
  • the oligonucleotides provided by the present invention can be associated with damage-related patterns (damage-associated Molecular patterns, DAMPs) To enhance the immune effect of microbial antigens and tumor antigens. DAMPs are cellular components released by damaged cells that stimulate the innate immune response. DAMPs can stimulate the body's innate immune response through PRR.
  • DAMPs damage-associated Molecular patterns
  • DAMPs include, but are not limited to, heat shock proteins, HMGB1 (high-mobility group box 1), hyaluronan Fragments, glycans, glycoconjugates, ATP, (Adenosine 5'-triphosphate), adenosine, uric acid, S100 Protein, heparin sulfate, Galectins, nuclear DNA, N-formylated peptides, Antimicrobial Peptides, mitochondrial DNA and calreticulin [ Krysko DV et al. Nat Rev Cancer. 2012 Dec;12(12):860-75;Pouwels SD et al. Mucosal Immunol. 2014 Mar;7(2):215-26] .
  • Cyclic dinucleotides [Cell, 2013 154(5), 962-970] can be derived from bacteria or can be synthesized in mammalian cells.
  • Bacterial CDNs include, but are not limited to, cyclic guanosine monophosphate (cyclic di-GMP, cdG), cyclic di-AMP (cdA) and cyclic adenosine monophosphate AMP-GMP, cAMP-GMP).
  • CDN Bacterial CDN is a class of PAMP that agonizes the innate immune response. CDN can also occur in mammalian cells, such as cyclic guanosine monophosphate (cyclic) Guanosine monophosphate-adenosine monophosphate, cGAMP) [Wu J et al. Science. 2013 Feb 15;339(6121):826-3].
  • cyclic guanosine monophosphate cyclic Guanosine monophosphate-adenosine monophosphate
  • cGAMP cyclic guanosine monophosphate
  • anti-tumor agent is Formulations for treating tumors after application to an individual, including but not limited to tumor vaccines, immunosuppressive receptors, costimulatory receptor activators, chemotherapeutic agents, radiotherapeutic agents, hormone inhibitors or hormones , cytokines, antibodies for tumor therapy, small molecule kinase inhibitors, PARP inhibitors, angiogenesis inhibitors, oncolytic viruses, and the like.
  • an immune checkpoint inhibitor is a substance that inhibits the function of an immune card molecule [Merero I et al. Nat Rev Cancer. 2015 Aug; 15(8):457-72]. Inhibition of the function of the immune-spot molecule allows the signal of activation of the immune cell to be highlighted, thereby allowing the immune cell to be continuously activated.
  • CD4 + T cells in a continuously activated state assist B cells in producing antibodies, and also assist CD8 + T cells in killing target cells such as virus-infected cells and tumor cells. In a continuous active state CD8 + T cells capable of killing tumor cells and virus-infected cells.
  • a preparation that maintains a T cell activation state such as an immunosuppressive inhibitor, enhances the immunological potency of an antigen or vaccine in an individual, and also has a tumor therapeutic effect in a cancer patient (individual).
  • the oligonucleotides provided by the present invention can be used in combination with an immunosuppression inhibitor to treat tumors and enhance the immune efficacy of an antigen or vaccine.
  • Immunosuppressive molecules inhibited by immunosuppression 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) [Pardoll DM. Nat Rev Cancer. 2012 Mar 22; 12(4): 252-64].
  • Antibodies that inhibit immune cardlet function are immunosuppressive inhibitors including, but not limited to, CTLA-4 antibodies, CD-1 antibodies, and CD-L antibodies.
  • PD-1 Programmed cell death Protein-1
  • CD279 is a membrane protein expressed on activated T cells, activated B cells, NK cells, and monocytes [Chen L. Nat Rev Immunol. 2004 May; 4(5): 336-47].
  • PD-1 induces depletion and incompetence of effector T cells.
  • PD-1 binds to the B7 family ligand PD-L1 on APC (programmed Death ligand-1, B7-H1) and PD-L2 (programmed death ligand-2, B7-DC) [ Ito A. Biomed Res Int. 2015; 2015: 605478] Initiation of transduction of immune cell activation inhibition signals.
  • Anti-PD-1 Antibodies are monoclonal antibodies to the PD1 molecule and have the function of inhibiting PD-1 mediated negative regulation of immune cells, and are immunosuppressive inhibitors.
  • two PD-1 Antibodies pembrolizumab and nivolumab are approved by the US FDA for use in cancer therapy.
  • Nivolumab Is a fully humanized IgG4 monoclonal antibody that binds to and inhibits the function of PD-1 [ Topalian SL et al.
  • Pidilizumab (CT-011) is a humanized IgG-1 ⁇ Monoclonal antibodies that bind to and inhibit the function of PD-1 are used to treat diffuse large B-cell lymphoma and follicular lymphoma.
  • antibodies to PD-1 include Pembrolizumab (MK-3475), an IgG-4 ⁇ monoclonal antibody that binds to and inhibits PD-1 function [Ito A et al. Biomed Res Int. 2015;2015:605478].
  • PD-L is a ligand for PD1 molecule, including PD-L1 (programmed death Ligand-1, B7-H1) and PD-L2 (programmed death Ligand-2).
  • PD-L1 is also called B7-H1.
  • PD-L2 is also called B7-DC.
  • PD-L1 and PD-L2 agonize the transduction of immune cell activation inhibition signals upon binding to PD-1 molecules.
  • PD-L1 and PD-L2 can be expressed on the surface of a variety of tumor cells, and the increased expression level may indicate poor prognosis in tumor patients. [ Ito A et al. Biomed Res Int. 2015; 2015: 605478].
  • Anti-PD-L antibody' an antibody that recognizes, binds to PD-L1 or PD-L2. After binding to PD-L, it can block PD-1 binding to the surface of immune cells, thereby blocking the transduction of immune cell activation inhibition signals, maintaining the activation state of immune cells, and thereby enhancing the immune response of the individual to antigen or tumor cells.
  • Anti-PD-L1 Antibodies have been shown to be useful in the treatment of tumors, including but not limited to BMS-936559, MPDL3280A, MEDI4736, and MSB0010718 [Ito A, et al. Biomed Res Int. 2015; 2015: 605478].
  • BMS-936559 is a fully humanized IgG4 anti-PD-L1 monoclonal antibody used to treat melanoma, non-small cell lung cancer, ovarian cancer and kidney cancer.
  • MPDL3280A is a humanized IgG-1 ⁇ anti-PD-L1 monoclonal antibody used to treat melanoma and bladder cancer.
  • MEDI4736 is a humanized IgG-1 ⁇ monoclonal antibody that prolongs 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].
  • 'PD-1-PD-L1/2 pathway' refers to the transduction of immune cell activation inhibition signal by PD-1 induced by PD-L1/2 expressed by tumor cells in immune cells, thereby inhibiting immune cell activation Way. Tumor cells can escape the immune cell attack by using the PD-1-PD-L1/2 pathway [Zou W., Chen L. Nature Reviews Immunology. 2008; 8(6): 467-477]. Block this pathway Enhance the anti-tumor activity of individual immune cells [ Topalian S. L., et al. Current Opinion in Immunology . 2012; 24 (2): 207-212].
  • the oligonucleotide provided by the present invention can be used in combination with a co-receptor activator to enhance an individual's immune response to a vaccine or antigen, and to enhance an individual's anti-tumor immune response.
  • Co-stimulatory receptors are receptors expressed on the surface of immune cells that mediate the transduction of immune cell activation signals upon activation by activators, thereby promoting an individual's immune response to an antigen or vaccine and enhancing the individual's anti-tumor immune activity.
  • a costimulatory receptor activator is a preparation that activates immune cells by binding to a costimulatory receptor.
  • Costimulatory receptor-activating monoclonal antibody is a costimulatory receptor activator that enhances the efficacy of an antigen or vaccine and enhances an individual's anti-tumor immune response.
  • Co-stimulatory receptors targeted by such activating monoclonal antibodies Receptors include, but are not limited to, CD137 (41BB), OX40, CD40, GITR, ICOS, and CD27 (Glucocorticoid-induced Tumour necrosis factor receptor family-related protein) [ Melero I et al. Nat Rev Cancer. 2015 Aug;15(8):457-72; Sanmamed MF et al. Semin Oncol.2015 Aug; 42(4): 640-55].
  • the oligonucleotides provided by the present invention can be used in combination with chemotherapeutic drugs to treat tumors.
  • Chemotherapy drugs It is a chemical that can treat tumors by inhibiting and killing tumor cells.
  • the chemical drugs referred to in the present invention include, but are not limited to, alkylating agents, antimetabolites, anti-microtubule preparations, topoisomerase inhibitors, and cytotoxic antibiotics.
  • Alkylating agents include nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatin and derivatives thereof, and non-classical alkylating agents.
  • Nitrogen mustard Mustards include mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide, and busulfan.
  • Nitrosoureas include N-Nitroso-N-methylurea, carmustine, lomustine, semustine, fotemustine, and streptozotocin.
  • Tetrazines include dacarbazine, mitozolomide and temozolomide.
  • Aziridines include thiotepa, mytomycin and diaziquone.
  • Cisplatin and its derivatives include cisplatin (cisplatin), carboplatin and oxaliplatin.
  • Non-classical alkylating agents include procarbazine And hexamethylmelamine.
  • Anti-metabolic classes include antifolates, fluorouracils, deoxynucleoside analogs, and steroids.
  • Antifolates include methotrexate and pemetrexed.
  • Fluorouracils include 5-fluorouracil.
  • Deoxynucleoside analogue drugs including cytarabine, gemcitabine (gemcitabine), decitabine, vidaza, fludarabine, nelarabine, cladribine, clofarabine and pentostatin.
  • Thiopurines include thioguanine and mercaptopurine.
  • Antimicrotubule preparations include vinca alkaloids, taxanes, and podotoxins.
  • Vinca alkaloids (vinca The alkaloids class includes vincristine, vinblastine, vinorelbine, vindesine, and vinflunine. Taxanes include paclitaxel and docetaxel.
  • the class of Podophyllotoxin includes etoposide and teniposide.
  • Topoisomerase inhibitor Inhibitors include topoisomerase I inhibitor and topoisomerase II Inhibitor. Topoisomerase I inhibitors include irinotecan and topotecan.
  • Topoisomerase II inhibitors include topoisomerase II Poisons and catalytic inhibitors. Topoisomerase II Poisons include etoposide, doxorubicin, mitoxantrone, and teniposide.
  • Catalytic inhibitors include Novobiocin, merbarone and aclarubicin.
  • Cytotoxic antibiotics include doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, mitoxantrone, gactinomycin, bleomycin (bleomycin), plicamycin and mitomycin.
  • a radiotherapy preparation is a substance that produces radiation.
  • a method of treating a tumor with a radiotherapy preparation is called chemotherapy.
  • the oligonucleotides provided by the present invention can be used in combination with radiotherapy to treat tumors.
  • the substance used for radiotherapy is a radioisotope that produces alpha, beta, and gamma rays.
  • Radiation for radiation therapy can also be produced by a machine, such as an x-ray treatment machine or an accelerator.
  • the oligonucleotides provided by the present invention can be used in combination with hormone inhibitors or hormones for tumor therapy to treat tumors, such inhibitors including, but not limited to, hormone synthesis inhibitors, hormone receptor antagonists And supplements with hormones.
  • Hormone synthesis inhibitor include aromatase inhibitors and gonadotropin-releasing hormone (GnRH) analogs.
  • GnRH analogs include Leuprolide and ghostre.
  • Hormone receptor antagonists include selective estrogen receptor modulators and androgen receptor antagonists.
  • Selective estrogen receptor modulator include Tamoxifen, Raloxifene, Toremifene and fulvestrant. Androgen receptor antagonists include Flutamide and Bicalutamide. Supplemental hormones are supplemental hormones (Hormone) Supplementation) A method of treating a tumor comprising a progestogen, an androgen, an estrogen, a progesterone-like drug, a testosterone-like drug, an estrogen agonist, and a somatostatin analog.
  • Progesterone-like drug Includes megestrol acetate and medroxyprogesterone acetate.
  • Testosterone-like drugs Fluoxymesterone.
  • Estrogen antagonists include diethylstilbestrol, Estrace and Polyestradiol phosphate.
  • Somatostatin analogs include Octreotide.
  • cytokine' The oligonucleotide provided by the invention can be combined with a cytokine to treat a tumor and enhance the immune effect of the vaccine.
  • cytokines include, but are not limited to, interleukin (IL)-2, thrombopoietin (IL-11), granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte colony-stimulating factor (GM-CSF) And interferon a.
  • the oligonucleotide provided by the present invention can be used for tumor therapeutic antibody (tumor therapeutic) Antibodies) for the treatment of tumors.
  • Antibodies for tumor therapy are antibodies that prolong their survival after application to a tumor subject, including but not limited to CD20, ErbB2, epidermal growth factor receptor, including CTLA-4 and PD-1.
  • Tumor Therapeutic Antibodies include, but are not limited to, Tositumomab (Bexxar), Rituximab (Rituxan) and Ofatumumab targeting CD20 (Arzerra; Genmab); Trastuzumab (Herceptin) targeting ErbB2; Panitumumab targeting epidermal growth factor receptor (Vectibix) and Cetuximab (Erbitux); humanized antibody targeting PD-1; humanized antibody targeting CTLA-4; Bevacizumab (Avastin) targeting vascular endothelial growth factor receptor; Brentuximab (vedotin) targeting CD30; Alemtuzumab targeting CD52 (Campath) and Gemtuzumab ozogamicin (Mylotarg; Wyeth) targeting CD33 [ Scott AM et al. Nat Rev Cancer. 2012 Mar 22;12(4):278-87].
  • oligonucleotide provided by the present invention can be combined with a small molecule kinase inhibitor (small-molecule Kinase Inhibitors are used in combination to treat tumors.
  • Small molecule kinase inhibitors are a class of small molecule compounds that can exert tumor therapeutic effects by inhibiting protein kinase activity, including but not limited to the following: Imatinib targeting Bcr-Abl; Afatinib targeting EGFR/ErbB2; targeting VEGFR1/ Axitinib of VEGFR2/VEGFR3/PDGFRB/c-KIT; Bosutinib targeting BcrAbl/SRC; Crizotinib targeting ALK/Met; Erlotinib targeting ErbB1; Fostamatinib targeting Syk; Gefitinib targeting EGFR; Targeting BTK Ibrutinib; Lapatinib targeting ErbB1/ErbB2; Lenvatinib targeting VEGFR2/VEGFR2; Nilotinib targeting Bcr-Abl; targeting VEGFR2/PDGFR/c-kit Pazopanib; Ruxolitinib targeting JAK; vemurafenib (
  • Small molecule protein kinase inhibitors also include all small molecule compounds that treat tumors by inhibiting the following protein kinases: Bcr-Ab, EGFR/ErbB2 VEGFR1/VEGFR2/VEGFR3/PDGFRB/c-KIT, BcrAbl/SRC, ALK/Met, ErbB1, Syk, EGFR, BTK, ErbB1/ErbB2, VEGFR2/VEGFR2, Bcr-Abl, VEGFR2/PDGFR/c-kit, JAK, BRAF and MEK [ Adams JL et Al Nat Rev Drug Discov. 2015 Sep;14(9):603-22].
  • Poly adenosine diphosphate ribose polymerase inhibitor' the oligonucleotide provided by the present invention and poly ADP-ribose polymerase inhibitor (poly ADP ribose Polymerase, PARP) is used in combination to treat tumors.
  • Poly ADP-ribose polymerase inhibitor abbreviated as PARP inhibitor, is a preparation that can treat tumors by inhibiting PARP activity.
  • PARP inhibitors include, but are not limited to, Iniparib for the treatment of breast cancer and lung squamous cell carcinoma; Talazoparib for the treatment of breast cancer (BMN-673); Olaparib for the treatment of breast, colon, ovarian and advanced prostate cancer; Rucaparib for the treatment of breast and ovarian cancer; Veliparib for the treatment of metastatic melanoma and breast cancer and CEP for the treatment of non-small cell lung cancer 9722 [Nature Reviews Clinical Oncology 12, 27-41, 2015].
  • 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: vascular endothelial growth factor including Avastin or bevacizumab Humanized monoclonal antibodies (VEGF); endostatin including Endo (ENDOSTAR) and anti-inhibition including pegaptinib The aptamer of vascular endothelial growth factor.
  • angiogenesis inhibitors include but not limited to: vascular endothelial growth factor including Avastin or bevacizumab Humanized monoclonal antibodies (VEGF); endostatin including Endo (ENDOSTAR) and anti-inhibition including pegaptinib The aptamer of vascular endothelial growth factor.
  • Oncolytic virus is a virus that can treat tumors by lysing tumor cells, including but not limited to Newcastle disease virus, herpes simplex virus, adenovirus, poxvirus, gram virus, respiratory enterovirus, measles virus, poliovirus, filter Bubble stomatitis virus, Seneca Valley Virus, parvovirus and retrovirus [ Kaufman HL et al. Nat Rev Drug Discov.2015 Sep 1;14(9):642-62] .
  • the oligonucleotides provided by the present invention can be used in combination with cells for treating tumors to treat tumors.
  • the cells for tumor treatment are cells that can exert an anti-tumor effect after application to an individual, including but not limited to tumor cells, dendritic cells, T lymphocytes, and NK cells.
  • Tumor cells can be used in combination with tumor cells to treat tumors.
  • Tumor cells can elicit an anti-tumor immune response in an individual and can be used as a tumor vaccine.
  • Such tumor cells include, but are not limited to, autologous tumor cells, allogeneic tumor cells, and tumor cell line cells, as well as transfected cytokines (such as GM-CSF and IL-2) encoding genes and/or costimulatory molecules (eg, B7).
  • cytokines such as GM-CSF and IL-2
  • B7 costimulatory molecules
  • the oligonucleotides provided by the present invention can be combined with autologous dendritic cells (Dendritic Cells, DC) are used in combination to treat tumors.
  • Dendritic cells that can present tumor antigens can be used to elicit an anti-tumor immune response when applied to an individual, and such cells can also be used as dendritic cell vaccines.
  • Dendritic cells for tumor therapy include, but are not limited to, dendritic cells loaded with a single tumor antigen (protein antigen or antigen peptide such as prostatic acid phosphatase), dendritic cells loaded with tumor cell lysate, dendrites loaded with tumor cell RNA Cells and dendritic cells loaded with autologous tumor cells eluting peptides [Nestle, F. et al. (1998) Nature Medicine 4: 328-332; Palucka K et al. Nat Rev Cancer. 2012 Mar 22;12(4):265-77]. Dendritic cells for tumor therapy also include gene transfection of dendritic cells.
  • a single tumor antigen protein antigen or antigen peptide such as prostatic acid phosphatase
  • dendritic cells loaded with tumor cell lysate dendrites loaded with tumor cell RNA Cells
  • dendritic cells loaded with autologous tumor cells eluting peptides [Nestle, F. et al. (1998)
  • Genes for transfecting dendritic cells include, but are not limited to, tumor antigen encoding genes, cytokines (such as IL-2, GM-CSF) encodes a gene and a costimulatory molecule encoding gene.
  • cytokines such as IL-2, GM-CSF
  • Dendritic cells for tumor therapy also include dendritic cells and tumor cell fusion cells (Kugler, A. et al. (2000) Nature Medicine 6: 332-336).
  • T cells for tumor therapy are autologous T cells used for tumor therapy, including tumor infiltrating T cells and genetically engineered T cells (Genetically Engineered T cells). Genetically engineered T cells assemble a chimeric antigen receptor that recognizes tumor antigens Receptor, CAR), T cells expressing this type of receptor are called CAR-T. Tumor-infiltrating T cells and genetically engineered T cells can be reinfused into tumor patients after expansion with IL-2 in vitro [ Kershaw MH et al. Nat Rev Cancer. 2013 Aug; 13(8): 525-41].
  • NK cells Natural killer cells for tumor therapy
  • NK cells can be isolated from the peripheral blood or umbilical cord blood of an individual, or can be induced from hematopoietic precursor cells, embryonic stem cells or pluripotent stem cells. Isolated or induced NK cells can be exported to individuals after amplification with IL-2 and IL-15 in vitro [Childs RW, et al. Nat Rev Drug Discov. 2015 Jul;14(7):487-98].
  • the oligonucleotides provided by the present invention can exert an anti-infective effect when applied alone or as a vaccine adjuvant. Anti-infective and therapeutic or prophylactic effects on microbial infections are interchangeable terms.
  • the anti-infective effect produced by the oligonucleotide provided by the present invention alone is particularly suitable for infections caused by microorganisms which have no effective vaccine, such as HIV and HCV; and also suitable for antigenic susceptible pathogens such as HIV, HCV influenza virus and malaria protozoan.
  • the oligonucleotides provided by the present invention can be used for the treatment of pathogenic viruses, pathogenic bacteria, pathogenic fungi, and pathogenic parasitic infections, It can be used as an adjuvant for pathogenic viruses, pathogenic bacteria, pathogenic fungi and pathogenic parasite vaccines.
  • pathogenic virus' the oligonucleotide provided by the present invention is used alone or as a vaccine adjuvant to exert an anti-pathogenic virus (pathogenic) The role of infection.
  • pathogenic viruses include hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, Flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory Syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, Parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum Virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.
  • herpes virus e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Ep
  • pathogenic bacteria the oligonucleotides provided by the present invention are used alone or as a vaccine adjuvant to exert anti-pathogenic bacteria (pathogenic Bacteria) the role of infection.
  • pathogenic bacteria include chlamydia, rickettsial bacteria, mycobacteria, Staphylococci, streptococci, pneumonococci, meningococci and conococci, Klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, Bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lymes Disease bacteria.
  • pathogenic fungi' the oligonucleotides provided by the present invention are used alone or as a vaccine adjuvant to exert anti-pathogenic fungi (pathogenic) Fungi) The role of infection.
  • pathogenic fungi include Candida, Cryptococcus neoformans, Aspergillus, Genus Mucorales, Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides Brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
  • 'Pathogenic parasites' The oligonucleotides provided by the present invention are used alone or as vaccine adjuvants to exert anti-pathogenic parasites (pathogenic The role of parasites). These pathogenic parasites include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma Gondi and Nippostrongylus brasiliensis.
  • the oligonucleotide drug provided by the present invention can be combined with a pharmaceutically acceptable carrier ( Pharmaceutical acceptable carrier )
  • the pharmaceutical composition may comprise an oligonucleotide provided by the present invention.
  • the dosage of the oligonucleotide in the composition is an effective dose (Effective Dosages) .
  • the composition can be administered in combination with an antigen, a vaccine, an adjuvant, and a preparation or cell having a tumor therapeutic effect.
  • the pharmaceutical compositions can be formulated into a dosage form including, but not limited to, solutions, emulsions, liposomes, and lyophilized powders and the like.
  • oligonucleotide drug provided by the present invention can be combined with a pharmaceutically acceptable carrier ( " pharmaceutically acceptable carrier ) constitutes a pharmaceutical composition.
  • pharmaceutically acceptable carrier Carrier means one or more solid or liquid fillers, diluents or encapsulating substances. Such vectors are suitable for applying the oligonucleotides provided herein to an individual.
  • the carrier can be organic, inorganic, natural or synthetic.
  • the pharmaceutically acceptable carrier can be pharmaceutically acceptable Acceptable solvents (aqueous and non-aqueous solutions), dispersants, suspensions, emulsifiers, powders , diluents, liposomes, antibacterial agents, antifungal agents, isotonic preparations, delayed absorption preparations, lyoprotectants, and other immunostimulating effects of nucleotide-enhancing vaccines or antigens suitable for use in the present invention to produce a therapeutic effect on tumors Preparation.
  • the aqueous solution includes, but is not limited to, water, physiological saline, PBS buffer, balanced salt solution, and glucose solution.
  • the solvent or dispersing agent may include water, ethanol, polyol (such as glycerin, propylene glycol, polyethylene glycol, etc., and also a mixture of these solvents or dispersing agents.
  • lecithin may be used in order to maintain the fluidity of the pharmaceutical composition.
  • a surfactant may be used in order to bring the pharmaceutical composition into a desired particle state.
  • a sugar, a polyol including mannitol and sorbitol, sodium chloride, or the like may be added to the pharmaceutical composition.
  • a sustained release agent such as stearate and gelatin may be added to the pharmaceutical composition.
  • Emulsifiers may include oil-in-water emulsifiers, water-in-oil emulsifiers or water-in-oil-in-water emulsifiers.
  • Pharmaceutically acceptable carriers also include pharmaceutically acceptable antioxidants ( Pharma-acceptable antioxidants ), these antioxidants include: water-soluble antioxidants such as ascorbic acid, Cystine hydrochloride, sodium bisulfate, sodium metabisulfite and sodium Sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT), lecithin, propyl Gallate and alpha-tocopherol; metal chelators such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.
  • antioxidants include: water-soluble antioxidants such as ascorbic acid, Cystine hydrochloride, sodium bisulfate, sodium metabisul
  • Effective Dosages of the oligonucleotides provided by the present invention include ' Effective doses that enhance antigen immunopotency 'and 'effective doses for tumor treatment'. Enhancing the effectiveness of antigen immunization Effective doses are doses of oligonucleotides that can significantly enhance the efficacy of antigen or vaccine immunization after administration to an individual. They also mean the optimal dose of oligonucleotides to prevent or treat infectious diseases or tumors after administration to an individual. .
  • a therapeutically effective dose of a tumor is a dose of an oligonucleotide that produces a therapeutic effect on a tumor after administration to an individual.
  • the amount of the dose is determined by criteria well known to those skilled in the art, with reference to other factors including, but not limited to, the size and health of the individual and the severity of the disease.
  • the oligonucleotide provided by the invention can be applied to an individual single or multiple times, and the dosage range of each time can be 1 m g to 1000 The range of mg. To achieve the desired effect, one skilled in the art can adjust the dosage of the oligonucleotide, which can range from 10 to 1000 times the aforementioned range.
  • the oligonucleotides provided by the present invention may be administered in dosage units when administered to an individual. .
  • Each unit contains a quantitative amount of a oligonucleotide that produces a prophylactic or therapeutic effect and the desired pharmaceutically acceptable composition.
  • the basis for the definition of a dosage unit is the characteristic activity characteristic of the oligonucleotide producing therapeutic effect and the sensitivity of the individual to the oligonucleotide upon treatment with the oligo-glycolic acid. If desired, oligonucleotides applied in dosage units can be applied twice, three times, four times, five times or more per day at regular intervals.
  • Oligonucleotides provided by the present invention may be in units of individual units Host body weight application, dose range is 0.0001 to 100 The interval between mg/kg and application may be once every two weeks or once a month or once every three to six months or other time interval suitable for the prophylactic or therapeutic effect.
  • the dose of the oligonucleotide may be 1-1000 Gg/ml. Effective dose includes therapeutically effective dose (therapeutically effective Dose) and prophylactically-effective dose.
  • the oligonucleotide provided by the present invention is The parenteral, topical or inhaled route of administration may be employed alone or in combination with other agents such as antigens, adjuvants and anti-tumor agents.
  • Parenteral routes of administration include intravenous, intraperitoneal, intrathecal, intramuscular, subcutaneous, intradermal, topical, paraneoplastic lymph nodes, direct injection of tumor tissue, and Intralymphatic injection.
  • Topical routes of administration include transdermal, oral, ocular, otic, and nasal. Inhalation can be through the nasal mucosa and lungs.
  • a pharmaceutical composition comprising an oligonucleotide provided by the present invention can be used by those skilled in the art.
  • the treatment device is applied to the individual.
  • Therapeutic devices include, but are not limited to, needle-free injection devices, implant devices, modules, implantable microinfusion pumps (implantable) Micro-infusion pump), infusion pump, and osmotic drug delivery system.
  • the oligonucleotides of the invention can be applied via a delivery vehicle.
  • Delivery vehicles include, but are not limited to, steroids (such as cholesterol), complexes, emulsions, immunostimulating complexes (ISCOMs), lipids (such as cationic lipids and anionic lipids), liposomes, bacterial carriers (such as Salmonella) , Escherichia coli, Mycobacterium bacillus, Lactobacillus, viral vectors (such as vaccinia, adenovirus, herpes simplex virus), virions, virus-like particles, microspheres, nucleic acid vaccines, polymer materials (such as carboxymethyl cellulose, chitosan) and cyclic polymers.
  • the delivery vehicle can also be a ligand for a specific receptor or a targeting molecule for the cell.
  • FIG. 1 Verification of PCV2b capsid protein expressed by Bac-to-Bac system
  • Figure-2 Effect of oligonucleotide (A1, A2) on CD4 + T cell activation induced by recombinant protein antigen re-stimulation
  • Figure 3 Effect of oligonucleotide (A1, A2) on the expression of CTLA-4 mRNA in mouse spleen cells induced by antigen re-stimulation
  • Figure 4 Effect of oligonucleotide (A1, A2) on the expression of CTLA-4 in CD4 + T cells induced by antigen re-stimulation
  • the 3'UTR sequence was designed with two single-stranded deoxyoligonucleotides, named A1 and A2, respectively, with sequence listings ⁇ 400> 1 and ⁇ 400>. 2 shows the sequence.
  • sequence of A1 is 5' ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1).
  • sequence of A2 is 5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2).
  • sequences of A1 and A2 are complementary to the sequence of the CTLA-4 mRNA 3'UTR.
  • A1 and A2 are synthesized by Takara Bio Co., Ltd., and their skeleton is fully thiolated.
  • A1 and A2 were dissolved in sterile PBS or other vehicle and stored without heat and frozen at -20 °C.
  • the endotoxin containing the A1 and A2 solutions was detected by the morphing cell lysis method.
  • A1 and A2 in solution The content is determined by spectrophotometer (260 nm wavelength) and can also be estimated by agarose gel (3%) electrophoresis (estimated according to known content of single-stranded deoxyoligonucleotide standards).
  • mice were immunized with a recombinant protein antigen vaccine, and the mouse spleen cells were subjected to an antigen recall reaction after immunization, and an antigen and an oligonucleotide (A1) or an oligonucleotide (A2) were added to the cell culture system.
  • A1 or A2 an antigen and an oligonucleotide
  • A2 an oligonucleotide
  • CD69 is a cell membrane glycoprotein molecule involved in the induction of T cell proliferation. At the surface of resting CD4 + T cells, the level of CD69 expression is very low.
  • CD69 expressed on the surface of CD4 + T cells After being subjected to TCR stimulation, CD69 expressed on the surface of CD4 + T cells rapidly up-regulated, and its expression peaked at 16-24 and then decreased.
  • the CD69 molecule is an early marker of CD4 + T cell activation.
  • CTLA-4 inhibits the expression of CD69 on the surface of CD4 + T cells [ Chambers CA. et al. Annu Rev Immunol. 2001; 19:565-94].
  • ICR mice (animal room of the Jilin University Medical Department), female, weighing 17-18 grams.
  • the PCV2b capsid protein (abbreviated as P protein) expressed by the Bac-to-Bac system can be assembled into a virus like particls (VLP).
  • VLP virus like particls
  • VLPs virus like particles
  • P protein was diluted to 60 ⁇ g/ml with PBS, this P protein solution and ISA 35
  • the emulsifier was mixed in a 1:1 (volume: volume) vaccine.
  • the vaccine was named P-ISA 35. 100 ⁇ l of P-ISA 35 contained 3 ⁇ g of P protein.
  • ICR mice were first immunized on day 0 by injecting 100 ⁇ l of P-ISA 35, the injection site is the right hind limb muscle, single injection. ICR mice were boosted on day 14 by injecting 100 ⁇ l of P-ISA 35 at the injection site for the right hind limb muscles, a single injection.
  • RPIM 1640 Medium and fetal bovine serum (FBS) were purchased from Gibco.
  • FBS fetal bovine serum
  • FITC-labeled anti-mouse CD4 antibody and PE-labeled anti-mouse CD69 antibody were purchased from BD.
  • P protein (referred to as P, see 2.1.1.2).
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • mice immunized with P-ISA 35 on day 48 after booster immunization with P-ISA 35 soaked in 75% ethanol for 2 min, aseptically spleen, and placed in 10 mL FBS 1640 medium containing 5 mL ice bath In the plate.
  • the spleen was ground into individual cells using ground glass.
  • the spleen cell single cell suspension was collected by filtration through a 300 mesh nylon mesh.
  • the red blood cells were removed by erythrocyte lysis solution ACK (0.155 mol/L NH4Cl, 0.01 mol/L KHCO3, 0.1 mol/L EDTA, pH 7.4).
  • the supernatant was discarded by centrifugation (1,200 rpm for 5 min) with 10% FBS 1640 (8 mL).
  • the spleen cell suspension of red blood cells was suspended with 10% FBS 1640.
  • the cells were counted and adjusted to a cell concentration of 5 ⁇ 10 6 /ml with 10% FBS 1640.
  • the cells were added to a 24-well culture plate at 1 ml per well, and the number of cells per well was 5 ⁇ 10 6 .
  • cells were harvested. The cells were lightly blown with a pipette, placed in a 1.5 ml EP tube, centrifuged at 2000 rpm for 5 min. The supernatant was discarded, the cells were blunted, and the cells were washed with 1 ml of PBS, centrifuged at 2000 rpm for 2 min. The cells were resuspended in 40 ⁇ l PBS and placed in two 1.5 EP tubes, 20 ⁇ l/tube. FITC-labeled anti-mouse CD4 fluorescent antibody, PE-labeled anti-mouse CD69 fluorescent antibody. Incubate on ice for 30 min in the dark.
  • the cells were washed with 1 ml of PBS, centrifuged at 2000 rpm for 2 min. It was resuspended in 200 ⁇ l of PBS, filtered through a 300-mesh filter, and detected by flow cytometry.
  • A1 or A2 treatment can significantly increase the CD69 expression of CD4 + T lymphocytes (Fig. 2), suggesting that A1 or A2 can promote the activation of CD4 + T lymphocytes by inhibiting the function of CTLA.
  • A1 with this activity or A2 can be used in individuals to enhance their immune response to microbial antigens (vaccines), tumor antigens (vaccines), and can also be used in individuals to enhance their anti-tumor response.
  • the I protein was immunized as described in Example 2 using the P protein as described in Example 2.
  • the mouse spleen cells were subjected to a recall reaction using the P protein, and the oligonucleotide A1 was added to the cell culture system. Or oligonucleotide A2.
  • PCR polymerase chain reaction
  • P protein (referred to as P, see 2.1.1.2).
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • RPIM 1640 medium and fetal bovine serum (FBS) were purchased from Gibco.
  • TRIzol reagent was purchased from Invitrogen, USA. EasyScript First-Strand cDNA Synthesis SuperMix, Top Green qPCR SuperMix (TransStartTM) The kit was purchased from Transgen Corporation;
  • GAPDH-specific primers for amplification of GAPDH-specific cDNA, GAPDH is a housekeeping gene
  • the sequence of the upstream primer is: 5'-ATCACCATCTTCCAGGAGCGA-3'
  • the sequence of the downstream primer is 5'-TCTCGTGGTTCACACCCATCA-3'.
  • CTLA4 specific primer for amplification of CTLA4 specificity cDNA
  • sequence of the upstream primer is: 5'-CCCAGTCTTCTCTGAAGCCATA-3'
  • sequence of the downstream primer is: 5'-TCTCTGTGAATGTCGTGGCA-3'.
  • GAPDH-specific primers and CTLA4-specific primers are all produced by Bao Bioengineering (Dalian) Co., Ltd. (Takara) Bio) synthesis.
  • 24-well culture plate plate, ground glass plate, 300 mesh filter, small forceps, cell counting plate, dropper, and sampler.
  • CO 2 cell culture incubator (SANYO Corporation, Japan), cell culture inverted microscope (Olympus, Japan), centrifuge (Biofuge Fresco, Germany), and real-time PCR instrument (Applied Biosystems, USA: model: ABI Prism 7300).
  • the spleen cell suspension of red blood cells was suspended with 10% FBS 1640.
  • the cells were counted and adjusted to a cell concentration of 5 ⁇ 10 6 /ml with 10% FBS 1640.
  • the cells were added to 24-well culture plate, each well of 1ml, the number of cells per well of 5 ⁇ 10 6 th.
  • After 24 hours of culture, cells were harvested. Total RNA was extracted by the Trizol method.
  • GAPDH-specific cDNA and CTLA4-specific cDNA were amplified by qPCR using GAPDH-specific primers and CTLA4-specific primers using EasyScript First-Strand cDNA Synthesis SuperMix and Top Green qPCR SuperMix (TransStartTM) kits.
  • a real-time PCR instrument (ABI Prism 7300) was used for quantitative PCR analysis.
  • A1 and A2 inhibit P protein re-stimulation-induced mouse immune cell CTLA-4 mRNA expression ( Figure-3) and exercise its function. This shows that A1 and A2 can inhibit CTLA-4 The expression and inhibition of mRNA exerts its function, thereby promoting and maintaining the activation of T cells. This result indicates that the mouse immune cell CTLA-4 has an inhibitory effect on P protein re-stimulation induction.
  • the expression of mRNA and the function of A1 or A2 can be used by individuals to enhance their immune response to microbial antigens (vaccines), tumor antigens (vaccines), and can also be used in individuals to enhance their anti-tumor response.
  • the I protein was immunized as described in Example 2 using the P protein as described in Example 2.
  • the mouse spleen cells were subjected to a recall reaction using the P protein, and the oligonucleotide A1 was added to the cell culture system. Or oligonucleotide A2.
  • cells were harvested and stained with fluorescently labeled CTLA-4 antibody for flow cytometry.
  • CTLA-4 is a membrane signaling molecule that negatively regulates T cells. After T cell receptor activation (identification of antigenic peptide activation), T cells rapidly express CTLA-4 [Sakaguchi S, et al. Science. 2011 Apr 29; 332 (6029): 542-3].
  • CD4 + and CD8 + T cells express CTLA-4.
  • CD4 + T cells express higher levels of CTLA-4 than CD8 + T cells. Inhibition of CTLA-4 expression promotes and maintains T cell activation and proliferation. In this case, CD4 + T cells CD8 + T cell activation more pronounced than proliferation [Chan DV, et al Genes Immun 2014 Jan; 15 (1):.. 25-32].
  • P protein (referred to as P, see 2.1.1.2).
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • RPIM 1640 medium and fetal bovine serum (FBS) were purchased from Gibco.
  • FITC-labeled anti-mouse CD4 antibody and PE-labeled anti-murine CTLA4 antibody were purchased from BD.
  • the cells were lightly blown with a pipette, placed in a 1.5 ml EP tube, centrifuged at 2000 rpm for 5 min. The supernatant was discarded, the cells were blunted, and the cells were washed with 1 ml of PBS, centrifuged at 2000 rpm for 2 min. The cells were resuspended in 40 ⁇ l PBS and placed in two 1.5 EP tubes, 20 ⁇ l/tube. FITC-labeled anti-mouse CD4 fluorescent antibody was added, and PE-labeled anti-mouse CTLA4 fluorescent antibody was added, and incubated on ice for 30 min in the dark.
  • the cells were washed with 1 ml of PBS, centrifuged at 2000 rpm for 2 min. It was resuspended in 200 ⁇ l of PBS, filtered through a 300-mesh filter, and detected by flow cytometry.
  • A1 inhibits the expression of CTLA4 in mouse CD4 + T cells induced by P protein re-stimulation (Fig. 4). This indicates that A1 can inhibit the expression of CTLA-4, thus promoting and maintaining the activation of T cells; A1 with this activity can be used in individuals to enhance their immune response to microbial antigens (vaccines) and tumor antigens (vaccines). It can also be used in individuals to enhance its anti-tumor response.
  • vaccines microbial antigens
  • vaccines tumor antigens
  • Example 5 Effects of oligonucleotides (A1 and A2) on recombinant protein antigen re-stimulation of mouse antigen-presenting cells CD80 and CD86:
  • Antigen-activated T cells express CTLA-4, which binds to and internalizes CD80 or CD86 molecules on the surface of antigen-presenting cells, rendering antigen-presenting cells lack these two costimulatory molecules. Antigen-presenting cells lacking costimulatory molecules cannot provide a second activation signal to T cells by binding to CD28, thus limiting T cell activation [ Sakaguchi S et al. Science. 2011 Apr 29; 332 (6029): 542-3].
  • CTLA-4 expressed by regulatory T cells (Treg) also binds to and internalizes CD80 or CD86 molecules on the surface of antigen-presenting cells, thus making T cells immune-tolerant [ Sansom DM.Science.2015 Jul 24; 349 (6246): 377-8].
  • a decrease in CD80 or CD86 on the surface of antigen-presenting cells by A1 and A2 is a response to inhibition of CTLA-4 expression.
  • ICR mice (animal room of the Jilin University Medical Department), female, weighing 17-18 grams.
  • PCV2b capsid protein (abbreviated as D protein) expressed in E. coli, this protein can be assembled into virus-like particles (virus like Particls, VLP).
  • D protein (D) was shown to have a molecular weight of 28 on SDS-PAGE. The KD zone was observed under electron microscopy (40,000 magnification), and the D protein formed a typical VLP of approximately 17 nM, see Figure-5.
  • D protein was diluted to 100 ⁇ g/ml with PBS, this D protein solution and ISA 35
  • the emulsifier was mixed in a ratio of 1:1 (volume: volume) to make a vaccine, which was named D-ISA 35.
  • 100 ⁇ l of D-ISA 35 contained 5 ⁇ g of D protein.
  • ICR mice were first immunized on day 0 by injecting 100 ⁇ l D-ISA 35, the injection site is the right hind limb muscle, single injection. ICR mice were boosted on day 14 by primary immunization of antigens and methods.
  • RPIM 1640 Medium and fetal bovine serum (FBS) were purchased from Gibco.
  • FBS fetal bovine serum
  • FITC-labeled anti-mouse CD4 antibody, PE-labeled anti-mouse CD80 antibody and PE-labeled anti-mouse 86 antibody were purchased from BD.
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • the cells were counted and adjusted to a cell concentration of 5 ⁇ 10 6 /ml with 10% FBS 1640.
  • the cells were added to a 24-well culture plate at 1 ml per well. Add D protein (final concentration 10 ⁇ g/ml), or add D protein (final concentration 10 ⁇ g/ml) and A1 (final concentration 5 ⁇ g/ml), or add D protein (final concentration 5 ⁇ g/ml) and A2 (final concentration 5 ⁇ g/ Ml). After 24 hours of culture, cells were harvested.
  • the cells were lightly blown with a pipette, placed in a 1.5 ml EP tube, centrifuged at 2000 rpm for 5 min. The supernatant was discarded, the cells were blunted, and the cells were washed with 1 ml of PBS, centrifuged at 2000 rpm for 2 min. The cells were resuspended in 40 ⁇ l PBS and placed in two 1.5 EP tubes, 20 ⁇ l/tube. FITC-labeled anti-murine CD11c antibody, PE-labeled anti-mouse CD80 antibody or PE-labeled anti-mouse CD86 antibody. Incubate on ice for 30 min in the dark.
  • the cells were washed with 1 ml of PBS, centrifuged at 2000 rpm for 2 min. It was resuspended in 200 ⁇ l of PBS, filtered through a 300-mesh filter, and detected by flow cytometry.
  • A2 treatment can make CD11c cells CD80 ( Figure-6) and CD86 ( Figure-7) Increased.
  • A1 or A2 can increase the number of costimulatory molecules on the surface of the antigen presenting cells by inhibiting CTLA, thereby promoting the activation of T lymphocytes by the microbial antigen and tumor antigen by providing a second activation signal.
  • A1 or A2 having this activity can be used in an individual to enhance its immune response to a microbial antigen (vaccine), a tumor antigen (vaccine), and can also be used in an individual to enhance its antitumor response.
  • ICR mice (animal room of the Jilin University Medical Department), female, weighing 17-18 grams.
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • D-ISA 35 Adjusts concentration of D protein to 600 ⁇ g/ml with D protein assembly solution, and this D protein solution and ISA 35
  • the emulsifier was mixed in a ratio of 1:1 (volume: volume) to make a vaccine, which was named D-ISA 35.
  • 100 ⁇ l of D-ISA 35 contained 30 ⁇ g of D protein.
  • the D protein in A2 was 30 ⁇ g, and both A1 and A2 were 5 ⁇ g.
  • the serum of the mice to be immunized was collected one day before the initial immunization. Blood was collected from the tail vein of the mouse, and the blood collection amount was ⁇ 100 ⁇ l. The collected whole blood (in a 1.5 ml EP tube) was allowed to stand at room temperature for 30 minutes. 11000rpm Centrifuge for 15 minutes, collect the serum, dispense, and store at -20 °C.
  • ICR mice were first immunized on day 0 by injecting 100 ⁇ l D-ISA 35 or D-ISA 35 A1 or D-ISA 35 A2, the injection site is the right hind limb muscle, single injection. ICR mice were boosted on day 14 in the same manner as the first immunization.
  • the immunized mouse serum was collected 7 days and 14 days after the second immunization. Methods such as pre-immune blood collection (6.1.3).
  • the circovirus (PCV2b)-specific antibody in mouse serum was detected by ELISA.
  • Enzyme label (combined microplate), 0.5 ml EP tube, 1.5 ml EP tube, sampler head, pipette, multichannel pipette, plate (diameter 9 cm), graduated glass bottle.
  • the enzyme label was coated in the coating solution with inactivated PCV2b, 100 ⁇ l/well, overnight at 4 °C. Dry liquid, use blocking solution at 37 ° C Blocked for 2 hours, 200 ⁇ l/well. Add serum to be tested, 37 ° C, 1 hour. The solution was dried, and the washing solution (300 ⁇ l/well) was added, and the mixture was dried at room temperature for 3 min, and the washing was repeated twice. HRP-labeled secondary antibody (goat anti-mouse) IgG (diluted 1:5000 with washing solution) was added. 100 Ll/well, 37 ° C for 1 hour.
  • the solution was dried, washed with a washing solution, 300 ⁇ l/well, room temperature for 3 min, and the solution was dried and washed twice.
  • Add substrate solution 100 Ll / hole, room temperature protected from light (wrapped tin foil) color for 15min.
  • Stop solution diluted sulfuric acid 2 mmol/L, 50 ⁇ l/well. Detected with a microplate reader (wavelength 549 nm).
  • A1 or A2 enhances the immune potency of the recombinant protein vaccine (circle virus vaccine) ( Figure-8). This means A1 or A2 can be used in an individual to enhance its immune response to a microbial antigen (vaccine) to become an adjuvant for a novel microbial antigen or microbial vaccine.
  • vaccine microbial antigen
  • mice Female, weighing 17-18 g, were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.
  • Hepatitis B surface antigen (HBsAg) vaccine (Watson Biotechnology Co., Ltd.) formulated with aluminum adjuvant, referred to as HBsAg vaccine or HBsAg.
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1 )]
  • oligonucleotide (A2) [5'tttcatggaa aatattgagt taaaa 3' (sequence list ⁇ 400> 2 ],
  • HBsAg-A1 and HBsAg-A2 were added to the HBsAg vaccine to form an oligonucleotide-containing HBsAg vaccine, which was named HBsAg-A1 and HBsAg-A2, respectively. Every 100 ⁇ l The HBsAg in HBsAg-A1 and HBsAg-A2 was 1 ⁇ g, and both A1 and A2 were 5 ⁇ g.
  • the serum of the mice to be immunized was collected one day before the initial immunization.
  • the method is as described in 6.1.3 of Example 6.
  • Balb/c mice Primary immunization of Balb/c mice on day 0 by injection of 100 ⁇ l HBsAg vaccine or HBsAg-A1 or HBsAg-A2.
  • the injection site is the right hind limb muscle, a single injection.
  • Balb/c mice were boosted on day 14 in the same manner as the first immunization.
  • the immunized mouse serum was collected 7 days and 14 days after the second immunization.
  • the method is as described in 6.1.3 of Example 6.
  • Hepatitis B surface antigen (HBsAg)-specific antibodies in mouse serum were detected by ELISA.
  • HBsAg hepatitis B surface antigen
  • the enzyme label was coated with HBsAg in the coating solution, 100 ⁇ l/well (0.1 ⁇ g of HBsAg per well), and overnight at 4 °C.
  • the remaining operation steps are the same as in the sixth embodiment.
  • A1 or A2 enhances the immune efficacy of the recombinant protein vaccine (hepatitis B virus vaccine) ( Figure-9). This means, A1 or A2 can be used in an individual to enhance its immune response to a microbial antigen (vaccine) to become an adjuvant for a novel microbial antigen or microbial vaccine.
  • a microbial antigen vaccine
  • mice weighing 18-22 grams, were obtained from the Animal Department of the Jilin University School of Medicine.
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • A1 or A2 was added to the rabies vaccine to form an oligonucleotide-containing rabies vaccine, which was named rabies vaccine-A1 and rabies vaccine-A2, respectively.
  • Each 500 ⁇ l of the oligonucleotide-containing vaccine contained 10 ⁇ g of A1 and A2.
  • mice to be immunized were collected two days before the initial immunization as described in 6.1.3 of Example 6.
  • mice were intraperitoneally injected with 0.5 ml of vaccine (rabies vaccine or rabies vaccine-A1 or rabies vaccine-A2) on days 0, 3, 7, 14 and 28, with 8 mice in each group, half male and half female.
  • vaccine rabies vaccine or rabies vaccine-A1 or rabies vaccine-A2
  • mice The sera of the immunized mice were collected on the 35th day as described in 6.1.3 of Example 6.
  • the rabies virus neutralizing antibody in mouse serum was detected by the rapid rabies vaccine fluorescent focus inhibition assay (RFFIT).
  • RFFIT rapid rabies vaccine fluorescent focus inhibition assay
  • A2 enhances the immune efficacy of the inactivated virus vaccine (rabies vaccine) ( Figure-10). This shows that A1 or A2 can be used in an individual to enhance its immune response to a microbial antigen (vaccine) to become an adjuvant for a novel microbial antigen or microbial vaccine.
  • vaccine microbial antigen
  • mice Female, weighing about 18 grams.
  • H5 vaccine Avian influenza virus H5 subtype bivalent inactivated vaccine (H5N1, Re-1 strain + Re-4 strain) (Harbin Vico Biotechnology Development Company), this vaccine is referred to as H5 vaccine.
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • H5 vaccine-A1 and H5 vaccine-A2 were added to the H5 vaccine to form an oligonucleotide H5-containing vaccine, which was named H5 vaccine-A1 and H5 vaccine-A2, respectively. Every 100 ⁇ l Both A1 and A2 in the H5 vaccine-A1 and H5 vaccine-A2 were 10 ⁇ g.
  • Influenza virus-specific antibodies in mouse serum were detected by ELISA.
  • the coating solution Na2CO3 1.59) was used as the coating antigen (see below) with the antigen extracted from the avian influenza virus H5 subtype bivalent inactivated vaccine.
  • g NaHCO3 2.93 g, pH 9.6, dilute to 1 L) 1:2 dilution, coated with 100 ⁇ l / well ELISA plate, sealed, 4 ° C overnight; wash the plate with washing solution (PBS containing 0.05% Tween-20), 300 ⁇ l / well, wash 3 times; add blocking solution (PBS containing 5% FBS), 200 ⁇ l / well, 37 ° C 2 hours; use
  • the mouse serum was diluted with PBS (1:400 dilution), the diluted serum was added to the microtiter plate, 100 ⁇ l/well was added, and the mixture was allowed to stand at 37 ° C for 1 hour; washed with washing solution 3 times, and then labeled with horseradish peroxidase.
  • Mouse secondary antibody (diluted 1:1000 with blocking solution), 100 ⁇ l /well, place at 37 ° C for 1 hour; wash 3 times with washing solution, add ready-to-use substrate solution (10 mL of citric acid 0.01M, Na2HPO4 0.02 M, ultrapure water 9 mL, 30% H2O2 15 ⁇ l, OPD 4 mg), 100 m L/well, color development at room temperature for 20 minutes; add stop solution (20% sulfuric acid), 50 ⁇ l/well; measure the OD value of each well at A492.
  • the coated antigen was extracted from the avian influenza virus H5 subtype bivalent inactivated vaccine.
  • This vaccine uses a water-in-oil adjuvant that can be broken and stratified by freezing and high-speed centrifugation.
  • the vaccine was frozen and thawed at -70 ° C / room temperature 2 times, 11000 After centrifugation at r/m for 20 minutes, the lower aqueous phase was separated and centrifuged at 11,000 r/m at room temperature to reveal clear stratification.
  • the upper layer was white chylomicron (oil phase) and the lower layer was clear and transparent (aqueous phase). As detected by the Bradford method, the upper layer contained no protein and the lower layer contained protein.
  • the lower aqueous phase is the coating solution containing the H5 influenza virus antigen.
  • A1 or A2 enhances the immune efficacy of the viral vaccine (influenza virus vaccine) ( Figure-11). This means, A1 or A2 can be used in an individual to enhance its immune response to a microbial antigen (vaccine) to become an adjuvant for a novel microbial antigen or microbial vaccine.
  • vaccine microbial antigen
  • Example 10 Synergistic effect of oligonucleotides (A1, A2) on glioma cell lysate vaccine:
  • C57BL/6 mice were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.
  • GL261 cells are C57BL/6 mouse-derived glioma cells from the Institute of Military Research, Beijing Military Research Institute
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • GL261 cells were cultured in RPIM 1640 medium (Gibco) containing 10% (v/v) fetal bovine serum, 100 IU penicillin/ml and 100 IU of streptomycin/ml at 37 ° C, 5% CO 2 . . 1 ml of GL261 cells (1 ⁇ 10 7 /ml) in good growth state were inoculated into the peritoneal cavity of healthy C57BL/6 mice. After the mice were bulged in the abdomen, the mice were sacrificed and immersed in 75% ethanol for 2-3 minutes for disinfection. The mouse peritoneal cavity was opened in a clean bench, and solid GL261 cells were formed (Fig. 12). The GL261 cell solid tumor tissue was removed aseptically.
  • GTL vaccine The protein concentration of GTL was adjusted to 4 mg/ml with PBS. This GTL and ISA 35 The emulsifier was mixed in a 1:1 (volume: volume) vaccine, and the vaccine was named GTL vaccine. A G1 vaccine containing A1 or A2 formulated with A1 or A2 is added to the GTL vaccine. These two vaccines are called GTL-A1 vaccine and GTL-21-A2 vaccine, wherein the concentrations of A1 and A2 were both 10 ⁇ g/100 ⁇ l.
  • GTL vaccine or GTL-A1 vaccine or GTL-21-A2 vaccine were injected into the left and right axilla of the forelimb of mouse mice.
  • GTL vaccine or GTL-A1 vaccine or GTL-21-A2 vaccine were injected into the left and right axilla of the forelimb of mouse mice.
  • mice Lifetime (days) A1+GL261 vaccine 8 81 ⁇ 13 GL261 vaccine 8 34 ⁇ 6 A2+GL261 vaccine 8 78 ⁇ 9
  • A1 or A2 enhanced the immunogenicity of glioma tumor cell lysates (tumor antigen vaccine) (Table-1), which was prolonged survival of tumor-bearing mice (p ⁇ 0.05). This means, A1 or A2 can be used in an individual to enhance its immune response to tumor antigens (vaccines) and become an adjuvant to a novel tumor vaccine.
  • Example 11 synergistic effect of oligonucleotides (A1, A2) on lung cancer cell lysate vaccine:
  • Lewis lung carcinoma (LLC) cells (derived from ATCC, USA). Incubate at 37 ° C, 5% CO 2 .
  • the medium used was 10% (v/v) inactivated fetal bovine serum, 100 IU penicillin/ml and 100 IU streptomycin/ml of IMDM medium.
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • LLC cell lysates were prepared, identified, and preserved as described in Example 10. Each 100 ⁇ l of LTL was made up of 2 x 10 6 LLC cells.
  • the GTL and ISA 35 will be prepared
  • the emulsifier was mixed in a ratio of 1:1 (volume: volume) to make a vaccine, which was named LTL vaccine.
  • A1 or A2-containing LTL vaccine formulated with A1 or A2 is added to the LTL vaccine.
  • These two vaccines are called The LTL-A1 vaccine and the LTL-A2 vaccine, wherein the concentrations of A1 and A2 were both 10 ⁇ g/100 ⁇ l.
  • mice at 6 weeks of age on day 0 and day 14 were injected subcutaneously with 100 ⁇ l on the right side.
  • mice were injected subcutaneously with 100 ⁇ l of LLC cell suspension on the left side of the left side.
  • the LLC cell suspension was prepared by inoculating in vitro cultured LLC cells (1 ⁇ 10 7 /100 ⁇ l) into the C57BL/6J male mice and subcutaneously growing into solid tumors. The mice were sacrificed and the tumor tissues were removed aseptically and digested with 0.25% trypsin-0.04% EDTA for 30 min to prepare a single cell suspension. The cell concentration was adjusted to 1 ⁇ 10 6 / 100 ⁇ l per PBS. The mice were sacrificed on the 18th day after the tumor was inoculated, and the tumor pieces were dissected and weighed.
  • A1 or A2 enhanced the immunological efficacy of lung cancer tumor cell lysates (tumor antigen vaccine) (Table-2), which reduced tumors (p ⁇ 0.05). This means, A1 or A2 can be used in an individual to enhance its immune response to tumor antigens (vaccines) and become an adjuvant to a novel tumor vaccine.
  • B16 cells are melanoma cells derived from C57BL/6 mice (derived from ATCC, USA). Incubate at 37 ° C, 5% CO 2 .
  • the medium used was RPMI 1640 medium (GIBCO) containing 10% fetal bovine serum, 100 IU penicillin/ml and 100 IU of streptomycin/ml.
  • Oligonucleotide (A1) [5'ttctttgggc tgtgccattc cctaa 3' (sequence list ⁇ 400>1)] and oligonucleotide (A2) [5'tttcatggaa aatattgagt taaa 3' (sequence list ⁇ 400> 2 ], as described in Example 1.
  • the B16 cells cultured in vitro were inoculated subcutaneously on the dorsal side of the left hind limb of the mice on day 0, and the injection volume was 200 ⁇ l, which contained 1 ⁇ 10 6 B16 cells.
  • Mice were injected with A1 on the 10th day after inoculation of B16 cells, and then injected once every two days for a total of 6 injections (Fig. 13).
  • the injection site was subcutaneously inoculated into the left posterior limb of the tumor lymphatic drainage area. Each injection volume was 100 ⁇ l, which contained 25 ⁇ g of A1. Control mice were injected with PBS in the same procedure. The survival time of the mice was observed and recorded.
  • A1 alone can exert an anti-melanoma effect in mice (Fig. 13), prolonging the survival of tumor-bearing mice.
  • A1 or A2 can be used in individuals to treat tumors including melanoma and become a novel anti-tumor preparation.

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Abstract

提供两种单链脱氧寡核苷酸，它们具有抗原增效作用和肿瘤治疗作用。

Description

具有抗原增效作用和肿瘤治疗作用的寡核苷酸 技术领域
本发明涉及两种寡核苷酸以及其增效抗原和治疗肿瘤的用途。
背景技术
T 淋巴细胞激活是激发个体对抗原发生高效免疫应答和抗肿瘤免疫应答的关键条件。T淋巴细胞激活需要获得两个激活信号。第一激活信号来自T淋巴细胞通过T细胞抗原受体（T cell antigen receptor，TCR）识别抗原提呈细胞（antigen-presenting cell，APC)或靶细胞表面的抗原肽-MHC复合物。第二激活信号来自T淋巴细胞通过CD28分子识别、结合APC或靶细胞表面的B7分子 (包括CD80 和CD86)。第二激活信号也称为共刺激信号（costimulatory signals）。仅获得第一激活信号的T淋巴细胞不能充分激活，甚至会进入免疫耐受状态。只有获得第一和第二激活信号后，T淋巴细胞才能被充分激活而行使功能 [ Sharma P. et al. Science. 2015 Apr 3;348(6230):56-61 ；Greenwald RJ, et al. Annu Rev Immunol.2005;23:515-48 ] 。
T 淋巴细胞在激活过程中也激活抑制信号通路，该抑制信号通路的激活可抑制T淋巴细胞的激活因而抑制其行使功能 [ Sharma P. et al. Science. 2015 Apr 3;348(6230):56-61 ；Greenwald RJ, et al. Annu Rev Immunol.2005;23:515-48 ] 。细胞毒性T淋巴细胞抗原4（Cytotoxic T-Lymphocyte Antigen 4，CTLA-4, 也称CD152，是一种介导该抑制信号通路的蛋白质。CTLA-4表达在激活的T淋巴细胞（包括CD4+T淋巴细胞和CD8+T淋巴细胞）的表面，能和CD28竞争结合APC或靶细胞表面的B7分子（CD80和CD86）。和转导激活信号的CD28不同，CTLA-4在识别结合B7分子后，在T淋巴细胞转导激活抑制信号（负调节信号）(T. L. Walunas et al., Immunity 1, 405-413 (1994)。
干扰、减弱、封闭CTLA-4和B7分子的相互作用可使T细胞处于持续充分的激活状态，会在个体产生两种效能：1、增强个体对抗原的免疫应答；2、增强对肿瘤细胞的内源性免疫应答（endogenous immune responses ）。因为具有第一种效能，干扰、减弱、封闭CTLA-4和 B7分子的相互作用能增强抗原的免疫效力，因此具有干扰、减弱、封闭CTLA-4和B7分子的相互作用的制剂可被用来增强抗原的效力而具有疫苗佐剂的功能。因为具有第二种效能，干扰、减弱、封闭CTLA-4和B7分子的相互作用能增强个体抗肿瘤细胞免疫反应，因此具有干扰、减弱、封闭CTLA-4和B7分子相互作用的制剂可成为一种治疗肿瘤的药物 [Sharma P. et al. Science. 2015 Apr 3;348(6230):56-61; R. J. Greenwald RJ, et al. Annu Rev Immunol.2005;23:515-48] 。
CTLA-4 抗体是可以干扰、减弱、封闭CTLA-4和B7分子的相互作用的制剂，它可以识别、结合CTLA-4而抑制T淋巴细胞激活抑制信号的转导，进而使T细胞处于持续充分的激活状态。CTLA-4抗体能增强个体对肿瘤细胞的免疫应答，已成为治疗肿瘤的药物。
临床前实验证明，用CTLA-4 抗体可治疗多种肿瘤，包括乳腺癌 [Hurwitz A. A., et al. Proc Natl Acad Sci U S A.1998 Aug 18;95(17):10067-71] 、 黑色素瘤 [van Elsas A., et al. J Exp Med. 1999 Aug 2;190(3):355-66] 和 前列腺癌 [Waitz R, et al, Oncoimmunology.2012 Jul 1;1(4):544-546] 。临床实验表明，CTLA-4 抗体(Ipilimumab)对肿瘤有治疗作用 [ Leach DR, et al. Science.1996 Mar 22;271(5256):1734-6] ，涉及的肿瘤包括黑色素瘤 [Hodi FS. et al., N Engl J Med. 2010 Aug 19;363(8):711-23] 、 肾细胞癌 [Yang JC et al., J Immunother. 2007 Nov-Dec;30(8):825-30] 、 前列腺癌 [ van den Eertwegh AJ. et al., Lancet Oncol.2012 May;13(5):509-17] 、 膀胱癌 [Carthon B.C. et al., Clin Cancer Res.2010 May 15;16(10):2861-71] 、 卵巢癌 [Hodi FS., et al., Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):3005-10] 和 非何杰金淋巴瘤 [Ansell SM., et al. Clin Cancer Res. 2009 Oct 15 ; 15 ( 20 ):6446-53] 。2011年，一种抗CTLA-4抗体(Ipilimumab)被美国食品药品管理局（US Food and Drug Administration，FDA)批准治疗黑色素瘤 [ Sharma P, et al. Nat Rev Cancer. 2011 Oct 24;11(11):805-12 ；Pardoll DM, Nat Rev Cancer. 2012 Mar 22;12(4):252-64]。
临床前实验表明，CTLA-4 抗体可以在个体增强抗原的免疫效力, 涉及的抗原有经照射处理的表达粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor，GM-CSF)的乳腺癌细胞 [ Hurwitz AA, et al. Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):10067-71] 、 经照射处理的表达GM-CSF的黑色素瘤细胞 [ van Elsas A, et al. J Exp Med.1999 Aug 2;190(3):355-66] 、 转染GM-CSF基因的同种异体前列腺癌细胞 [ van den Eertwegh AJ, et al., Lancet Oncol. 2012 May;13(5):509-17] 和 黑色素瘤特异性gp100多肽抗原 [Hodi, F.S. et al. N Engl J Med. 2010 Aug 19;363(8):711-23]。
发明内容:
1 、本发明提供了两种寡核苷酸，具有 如序列表 <400> 1 和 <400> 2 所示的序列 , 它们可通过干扰CTLA-4 mRNA而抑制 CTLA-4 的表达，进而 干扰、减弱、封闭CTLA-4和B7分子的相互作用，使T细胞激活的抑制信号受到抑制，结果是增强个体对抗原的免疫应答、增强个体的抗微生物和抗肿瘤免疫反应。
2 、这两种寡核苷酸可以经过各种化学修饰或改构。
3 、这两种寡核苷酸可被用作微生物抗原和肿瘤抗原的佐剂，具有抗感染和抗肿瘤作用。
4 、这两种寡核苷酸可被用于肿瘤治疗。
5 、这两种寡核苷酸可以和其它的佐剂联合应用来增强个体对微生物抗原和肿瘤抗原的免疫应答。
6 、这两种寡核苷酸可以和其它抗肿瘤制剂和肿瘤治疗细胞联合应用来治疗肿瘤。
发明中的术语：
除非特别强调，本发明中的术语具有可以被本发明所属领域内技术人员所理解的通常意义。若出现含义上的冲突，应遵从本发明中的解释、界定或说明。
'寡核苷酸'：寡核苷酸是由多个核苷酸组成的分子，其核苷酸的数量可以是几个或几十个。核苷酸（nucleotide）是核酸、也是寡核苷酸的基本组成单位。核苷酸由核苷（nucleoside）和磷酸组成。核苷由戊糖（pentose）和碱基（base）组成。戊糖包括核糖和脱氧核糖。戊糖分子和碱基连接形成核苷（nucleoside）。核苷由磷酸基团连接形成核苷酸。核苷酸通过磷酸二酯键连接形成寡核苷酸。组成核苷的碱基包括嘧啶和嘌呤。嘧啶包括胸腺嘧啶（thymine，缩写为T 或t）和胞嘧啶（cytosine，缩写为C 或c）。嘌呤包括腺嘌呤（adenine，缩写为A或a）和鸟嘌呤（guanine，缩写为G或g）。寡核苷酸中的碱基可以是稀有碱基。稀有碱基包括但不限于5-羟甲基胞嘧啶、7-甲基鸟嘌呤和5-羟甲基胞嘧啶。寡核苷酸可以是单链、双链、环形或具有环形结构的分子。在本发明中，寡核苷酸(Oligonucleotide, ODN)可以用它的英文缩写ODN来代替。寡核苷酸中的核苷酸排列序顺序构成其一级结构，这种顺序也称核苷酸序列。核苷酸序列可用碱基顺序代表，因此，核苷酸的序列也称为碱基序列。脱氧寡核苷酸的序列可用碱基的英文缩写表示，T或t代表胸腺嘧啶，C 或c代表胞嘧啶，A或a代表腺嘌呤，G或g代表鸟嘌呤。
'化学修饰'：与自然的DNA相比，本发明提供的寡核苷酸可被化学修饰（chemical modification）。寡核苷酸的化学修饰是通过引入或除去任何化学基因而使其共价结构发生改变的现象或方法。寡核苷酸的化学修饰部位可发生在磷酸二酯键、核糖和碱基。寡核苷酸的化学修饰可发生在5'端或3'端，可在合成时或合成后进行。本发明涉及的化学修饰包括但不限于对寡核苷酸骨架的修饰，如硫代修饰（核苷酸间磷酸二酯键中磷酸的非桥性氧原子被硫原子取代）和取代修饰（包括烷基、芳香基或其它任何化学基团的取代）。寡核苷酸的化学修饰还包括碱基替代和碱基修饰，替代的碱基可以是稀有碱基或各种碱基的衍生物。寡核苷酸的化学修饰还包括在其5'端和/或3'端连接一或多个核苷酸和/或其它任何化学基团。
'UTR': 是指mRNA分子的非翻译区（untranslated region, UTR），位于mRNA分子多肽编码区的两端，位于5'端的被称为5'端UTR，位于3'端的被称为3'端UTR。
'免疫应答'：免疫应答（immune response）和免疫反应具有相似的意义。免疫应答是个体的免疫细胞包括 B淋巴细胞、T淋巴细胞、NK细胞、γδT细胞、NKT细胞、树突细胞、巨噬细胞和粒细胞等对抗原或其它刺激[如病原体相关的模式分子(pathogen associated molecular pattern, PAMP)和损伤相关的模式分子(damage associated molecular pattern, DAMP)做出的反应。免疫应答的结果是选择性破坏或清除入侵的病原微生物或内生的肿瘤细胞。免疫应答包括固有免疫应答和适应性免疫应答。适应性免疫应答括细胞免疫应答和体液免疫应答。由采用微生物抗原制成的疫苗所激发的免疫应答可以使被免疫的个体获得抵抗微生物感染的能力。由采用肿瘤抗原制成的疫苗所激发的免疫应答可在被免疫的个体发生肿瘤治疗作用。促进对个体对微生物的免疫应答可发生抗感染作用。促进个体对肿瘤细胞的免疫应答可发生抗肿瘤作用。
'淋巴细胞'：淋巴细胞（lymphocyte）指在血液、淋巴液和淋巴组织中存在的没有吞噬能力的单个核白细胞，包括B淋巴细胞（也称B细胞）和T淋巴细胞（也称T细胞）。T细胞可被分为表面表达CD4 分子的T细胞（CD4+ T细胞）和为表面表达CD8分子的T细胞（CD8+ T细胞）。
' CTLA4 ': CTLA-4 (T lymphocyte-associated antigen 4) [ Rudd C. E., et al. Immunol Rev. 2009 May; 229(1):12-26 ] ，也称为CD152或CTLA-4抗原（ CTLA-4 antigen ） ，是一种可诱导的膜蛋白，表达在激活的 CD4+ T细胞和CD8+ T细胞的表面。CTLA-4 结合在抗原提呈细胞表面的B7分子。B7分子即是CTLA-4的配体，也是T细胞表面CD28的配体。同CD28相比，CTLA-4对B7的亲和力更高，它可和CD28竞争结合B7，因而减弱CD28依赖性共刺激信号（T细胞激活的第一信号）。CTLA-4也介导对 MHC-TCR通路信号（T细胞激活的第二信号）的直接抑制[Nirschl CJ., et al. Clin Cancer Res.2013 Sep 15;19(18):4917-24 ] 。 活化T细胞可上调表达 CTLA-4，CTLA-4与B7结合后可传导抑制信号抑制T细胞激活。 在肿瘤微环境中，CTLA-4介导的T细胞激活抑制可削弱个体的抗肿瘤免疫反应。阻断CTLA-4的功能可增强个体的抗肿瘤活性，产生肿瘤治疗作用 [ Leach DR., et al. Science.1996 Mar 22;271(5256):1734-6 ] 。 阻断CTLA-4的功能可增强个体对抗原或疫苗的免疫应答[van Elsas A., et al. J Exp Med. 1999 Aug 2;190(3):355-66 ] 。
'T细胞受体'：T细胞受体（T cell receptor, TCR）是表达在T细胞表面的抗原受体，为异二聚体跨膜蛋白。TCR由可变（V）区和恒定（C）区组成。V区是TCR识别抗原表位的结构域。TCR不能直接识别抗原表位，只能识别抗原递呈细胞或靶细胞表面的抗原肽-MHC（主要组织相容复合体）分子复合物。
'MHC分子'：MHC分子是主要组织相容复合体（major histocompatibiltiy complex，MHC）基因编码的蛋白类分子。MHC分子包括MHC I类分子和MHC II类分子。
'CD28'：CD28是表达在T细胞表面的蛋白，可识别抗原呈递细胞表面的B7分子。在识别B7分子后，CD28向T细胞提供共刺激信号（co-stimulatory signals）。
'B7分子'：B7分子是主要表达在抗原提呈细胞特别是树突细胞表面的蛋白分子，包括B7.1 (CD80)和B7.2 (CD86)两种，除淋巴瘤细胞外，肿瘤细胞多不表达 B7分子[Sharma, J.P. Allison. Science 348, 56，2015]。
'共刺激分子'：共刺激分子（co-stimulaory molecules）是表达在抗原提呈细胞（antigen presenting cells，APC）或肿瘤细胞表面的蛋白类分子，在免疫应答的过程中提供激活 CD4+T淋巴细胞和CD8+ T淋巴细胞的第二激活信号。
'T淋巴细胞的激活'：T淋巴细胞的激活需要来两个信号。第一信号通过其TCR识别APC或靶细胞（可被CD8+T细胞杀伤的细胞）表面的抗原肽-MHC分子复合物获得；第二信号，也称共刺激信号，通过其CD28分子识别APC或靶细胞表面的B7分子获得。在双信号的作用下，T细胞启动激活信号转导通路，进而增殖、分化行使功能。仅有第一信号而无第二激活信号可使T细胞进入不应答状态。T淋巴细胞激活后发生T淋巴细胞反应。
' T 淋巴细胞反应'：T淋巴细胞反应 （T lymphocyte response）和'T淋巴细胞活性'（T lymphocyte activity）或'T淋巴细胞行使的功能'在本发明中是可以互换的术语。T淋巴细胞反应包括T淋巴细胞增生和/或分化成辅助性T淋巴细胞（Th）、细胞毒性T淋巴细胞（Tc）或调节性T淋巴细胞（Treg），也包括由Th向B淋巴细胞提供信号辅助其产生抗体、由Tc杀伤靶细胞和释放可溶性因子如细胞因子来调节其它免疫细胞的功能。Th是CD4+ T细胞，Tc是CD8+ T细胞。在激活后，CD4+T细胞辅助B细胞产生抗体，辅助CD8+T细胞杀伤靶细胞如病毒感染细胞和肿瘤细胞。在激活后，CD8+T细胞可杀伤病毒感染细胞和肿瘤细胞。促进、维持T细胞激活会增强个体对微生物抗原的免疫应答而产生抗感染作用，也会促进个体免疫细胞对肿瘤细胞的免疫应答产生肿瘤治疗作用。
'个体'：在本发明中的个体（subject 或individual）指人和非人类的脊椎动物。
'靶细胞'：靶细胞（Target cell）指个体可被免疫细胞攻击、杀伤或作用的细胞，可以是肿瘤细胞、病毒感染细胞，也可以指被本发明提供的寡核苷酸作用的细胞。
'CTLA-4 抗体'：是能特异结合CTLA4的抗体，可以封闭CTLA-4转导的抑制信号，使T淋巴细胞被肿瘤抗原充分激活，能延长肿瘤患者的生存期。Ipilimumab 是一种完全人源化的IgG1 CTLA-4单克隆抗体[Lipson EJ., et al. Clin Cancer Res. 2011 Nov 15;17(22):6958-62]，在2011被美国FDA批准为治疗晚期黑色素瘤的药物[Sharma P. et al. Science. 2015 Apr 3;348(6230):56-61]。Tremelimumab 是另一种人源化的IgG2 CTLA-4单克隆抗体[Ribas A., et al. Oncologist. 2007 Jul;12(7):873-83]。在临床试验中，Tremelimumab被用于治疗肝细胞癌[Sangro B.,et al. J Hepatol. 2013 Jul;59(1):81-8]、胃癌和食管癌[Ralph C. et al. Clin Cancer Res. 2010 Mar 1;16(5):1662-72]。CTLA-4的抗体属免疫卡点分子抑制物。
'免疫卡点分子': 免疫卡点分子(immune checkpoint molecules) 是表达在免疫细胞上的蛋白分子，在识别其配体后启动抑制性信号通路因而抑制免疫细胞的激活。免疫卡点分子包括但不限于CTLA-4分子、PD-1分子、PD-L1/2分子、淋巴细胞激活基因3（lymphocyte-activation gene 3，LAG-3)、TIM-3 (T cell immunoglobulin and mucin domain-containing 3)、TIGIT (T cell immunoreceptor with immunoglobulin and ITIM domains)和BTLA（B and T lymphocyte attenuator)。封闭免疫卡点（immune checkpoint blockade）是指抑制免疫卡点分子在免疫细胞转递抑制信号的功能而促进、维持T细胞激活的方法。封闭免疫卡点增强个体对微生物抗原的免疫应答表现抗感染作用，也可促进个体免疫细胞的抗肿瘤活性表现肿瘤治疗作用[ Melero I, et al.Nat Rev Cancer.2015 Aug;15(8):457-72]。
'肿瘤'：本发明中的'肿瘤'即现代医学定义的肿瘤，可以分为良性肿瘤和恶性肿瘤。肿瘤（tumor）和癌症（cancer）可以互换使用，具有相同的含义。肿瘤包括实体瘤、软组织肉瘤和髓样或淋巴样系统肿瘤。本发明提供的寡核苷酸可增强个体对肿瘤抗原的免疫应答产生抗肿瘤作用，涉及的肿瘤包括但不限于：食道癌、胆囊癌、膀胱癌、乳腺癌、结肠癌、结直肠癌、肾上腺皮质肿瘤、肾癌、肝癌、肺癌、卵巢癌、子宫颈癌、子宫癌、阴道癌、胰腺癌、直肠癌、前列腺癌、胃癌、皮肤癌、黑色素瘤、脑肿瘤、胶质瘤、骨肿瘤、肉瘤、阴茎癌、视网膜母细胞瘤、白血病、淋巴瘤和骨髓瘤。
'治疗'：包括应用本发明提供的寡核苷酸来阻止或延缓疾病（如肿瘤）的症状和并发症的出现。治疗也可以是预防性。对肿瘤的治疗也指在个体控制肿瘤的进展，延长肿瘤患者的生存期，改善生活质量，减轻症状，使肿瘤缩小甚至消除，使肿瘤转移受到遏制。在本发明中'肿瘤治疗'和'抗肿瘤作用'或'治疗肿瘤'有相同的含义。抗肿瘤作用包括对肿瘤的治疗，也包括对肿瘤发生、复发和转移的预防。
'抗原'：是能被B细胞受体或T细胞受体识别而激发个体适应性免疫应答（adaptive immune response）的物质或分子。抗原可以来自个体的外部，如微生物抗原；也可来自个体的内部，如肿瘤抗原。微生物抗原是微生物上可被B细胞受体或T细胞受体识别而激发个体适应性免疫应答（adaptive immune response）的物质或分子。采用微生物抗原制备成的疫苗在给个体应用后能使其获得对该病原体的免疫力，使其在再次接触到同样或相似病原体时受到保护。采用肿瘤抗原制成的疫苗在给个体应用后能激发其对肿瘤细胞的适应性免疫应答进而产生肿瘤治疗作用。抗原可以从微生物或肿瘤细胞提取、也可以采用重组DNA技术生产或其它方法合成。
' 疫苗' ： 疫苗 （vaccine）是用减毒或杀死的病原生物或其组份为抗原制成的用于人工主动免疫的生物制品。抗原和佐剂是疫苗的主要成分。接种疫苗的目的是使个体获得对病原体的免疫力进而使其再接触到相应的病原体时受到保护。本发明提供的寡核苷酸可以和人用疫苗联合应用增强其免疫效力，这些疫苗包括但不限于可预防下述传染性疾病的疫苗[ Stanley A. Plotkin, Walter A. Orenstein, Paul A. Offit , Vaccines, Sixth Edition, An imprint of Elsevier Inc. 2013, ISBN-13: 9781455700905] ： 白喉、破伤风、黄热病、百日咳、流感嗜血杆菌b感染、脊髓灰质炎、麻疹、腮腺炎、风疹、斑疹伤寒、狂犬病、轮状病毒感染、甲型肝炎、乙型肝炎、戊型肝炎、流感、结核、疟疾、霍乱、 水痘、 流行性乙型脑炎、森林脑炎、霍乱、莱姆病、肺炎球菌感染、脑膜炎球菌感染、伤寒、天花、 炭疽、埃博拉病毒感染、绿猴病毒（ Marburg viruses, MARV) 感染和委内瑞拉马脑炎病毒（Venezuelan equine encephalitis virus，VEEV)感染。本发明涉及的疫苗也包括肿瘤疫苗。肿瘤抗原和佐剂是肿瘤疫苗的主要成分。肿瘤疫苗可治疗或预防肿瘤。肿瘤疫苗中的抗原可以是肿瘤抗原、提呈肿瘤抗原的细胞、表达肿瘤抗原的细胞或肿瘤细胞裂解物。 本发明提供的寡核苷酸可以和肿瘤疫苗联合应用增强其预防和/或治疗肿瘤的效力，这类疫苗包括但不限于：子宫颈癌预防的 人乳头瘤病毒疫苗、用于 治疗前列腺癌的Provenge疫苗、用各种肿瘤抗原制成的疫苗、用各种肿瘤细胞制成的疫苗和用肿瘤细胞裂解物制成的疫苗。本发明提供的寡核苷酸可以和动物用疫苗联合应用增强其免疫效力，这些疫苗包括但不限于可预防下述传染性疾病的疫苗：猪口蹄疫、猪蓝耳病（猪繁殖与呼吸综合征）、猪瘟、猪伪狂犬病、猪圆环病毒感染、猪细小病毒感染、猪链球菌、猪传染性胃肠炎、猪气喘病、副猪嗜血杆菌感染、猪丹毒、猪肺疫、猪萎缩性鼻炎、猪传染性胃肠炎、猪乙脑、猪流感、猪布鲁氏病、仔猪腹泻、猪副流感病毒感染、猪流感、猪肺炎支原体感染、仔猪水肿病、猪霍乱沙门氏菌、猪流行性腹泻、猪肺疫；牛气肿疽、牛炭疽、牛流行热、牛巴氏杆菌病、牛布鲁菌感染、牛破伤风、牛魏氏梭菌感染、牛肉毒梭菌感染、牛口蹄疫、牛副伤寒、牛痘、牛腹泻；羊快疫、羊猝疽、羊肠毒血症、羔羊痢疾、羊黑疫（传染性坏死性肝炎）、山羊痘病、羊破伤风、羊炭疽病、羔羊大肠杆菌病、羊流产衣原体感染、羊口疮、羊传染性胸膜肺炎、羊布氏杆菌病、羊肉毒梭菌中毒症、羊痘、羊链球苗病、羊狂犬病、羊口蹄疫、羊口疮病、羊蓝舌病；马传染性贫血、马流感；猪马牛羊犬脑炎；鸡新城疫、鸡传染性法氏囊炎、鸡马立克病、禽流感、禽霍乱病、鸡痘、鸡染性支气管炎、鸡病毒性肝炎、鸡减蛋综合征；鸭流感、鸭疫里默氏杆菌苗、鸭瘟、鸭传染性浆膜炎、鸭大肠杆菌病；小鹅瘟、鹅副黏病毒病、鹅流感；狂犬病、犬温热、犬细小病毒病、犬钩端螺旋体病、犬传染性肝炎、犬传染性支气管炎、犬副流感病、犬脑炎和犬窝咳疫苗。
'肿瘤抗原'：本发明提供的寡核苷酸可以和肿瘤抗原联合应用促进个体对肿瘤抗原的免疫应答。在本发明中，肿瘤抗原（Tumor antigen），肿瘤细胞抗原（tumor cell antigen）和肿瘤相关抗原（tumor associated antigen，TAA）可以交换应用，有相同的含义。肿瘤抗原能激发抗肿瘤免疫应答，针对肿瘤抗原的免疫应答产生肿瘤治疗作用。瘤抗原包括但不限于：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)、酪氨酸酶、HER2（human epidermal growth factor receptor 2, hTERT （telomerase transcriptase）、p53、survivin、β-catenin-m、HSP70-2/m (heat shock-related 70 kDa protein 2 mutated)、KRAS、GM2 (ganglioside GM2)、MUC1 (mucin-1)、乙型肝炎病毒基因编码抗原、丙型肝炎病毒基因编码的抗原、人乳头瘤病毒基因编码抗原（如L1蛋白、E6蛋白和E7 蛋白）和EB病毒基因编码的抗原（Epstein Barr Virus peptides）[Zielinski C, et al. Nat Rev Clin Oncol. 2014 Sep;11(9):509-24; Adams JL, et al. Nat Rev Drug Discov. 2015 Sep;14(9):603-22]。肿瘤抗原还包括经灭活处理全肿瘤细胞和肿瘤细胞裂解物。肿瘤抗原可以是因基因突变而表达的新抗原（neo-antigen），也可以是B细胞肿瘤的独特型抗原和自肿瘤细胞提取的热休克蛋白肿瘤细胞肽复合物[Suot, R & Srivastava, P (1995) Science 269: 1585-1588; Tamura, Y. et al. (1997) Science 278: 117-120]。
'肿瘤疫苗'本发明提供的寡核苷酸可以和肿瘤疫苗联合应用增强其肿瘤治疗的效力。肿瘤疫苗是指能诱生个体产生抗肿瘤适应性免疫应答的生物制品。本发明涉及的肿瘤疫苗包括采用肿瘤抗原、肿瘤细胞裂解物、肿瘤细胞和提呈肿瘤抗原的细胞以一定的剂型制成的疫苗。这类疫苗包括但不限于下述[ [Zielinski C, et al. Nat Rev Clin Oncol. 2014 Sep;11(9):509-24 ]: 治疗前列腺癌的肿瘤疫苗，采用的肿瘤抗原包括前列腺酸性磷酸酶（用于sipuleucel-T）、(Provenge®)和PSA；治疗乳腺癌的的肿瘤疫苗，采用的肿瘤抗原包括HER2来源的多肽、MUC1和WT1 (Wilms tumour protein)抗原；治疗肺癌的肿瘤疫苗，采用的肿瘤抗原包括MUC1、melanoma-associated antigen-3 (MAGE-A3) 多肽、telomerase reverse transcriptase和EGF；治疗黑色素瘤的疫苗，采用的抗原包括 β-catenin、gp100、MAGE-A3、MART1（melanoma antigen recognized by T cells 1）、NY-ESO-1和 survivin；治疗胰腺癌疫苗的肿瘤疫苗，采用的肿瘤抗原包括端粒酶肽、同种异体肿瘤细胞和突变RAS合成肽；治疗结直肠癌的的肿瘤疫苗，采用的肿瘤抗原包括癌胚抗原（carcinoembryonic antigen，CEA)、MUC1和全肿瘤细胞；治疗肾细胞癌的肿瘤疫苗，采用的肿瘤抗原包括肿瘤细胞RNA；血液肿瘤治疗用疫苗，采用的肿瘤抗原包括 WT1、MAGE、MUC1、PRAME（preferentially expressed antigen of melanoma )和独特型抗原。
'肿瘤细胞裂解物'肿瘤细胞裂解物可通过 破碎肿瘤细胞获得，含各种肿瘤抗原。破碎肿瘤细胞的方法包括但不限于反复冻融、超声处理和机械破碎。 本发明提供的寡核苷酸可以和肿瘤细胞裂解物联合应用促进个体的抗肿瘤免疫反应。原发肿瘤、继发或转移肿瘤细胞都可被用来制备肿瘤细胞裂解物。将原发肿瘤和继发或转移肿瘤细胞在体外培养，可获得肿瘤细胞系(tumor cell line)细胞，这些肿瘤细胞系细胞也可以被用来制备肿瘤细胞裂解物。用于制备肿瘤细胞裂解物的肿瘤细胞可以是自体、同种异体的或异种的。
'佐剂' 本发明提供的寡核苷酸可以和一种或多种佐剂联合使用来增强微生物抗原和肿瘤抗原的免疫效力。佐剂 （adjuvant）是在疫苗中和抗原一起应用的物质，具有下述活性（1）减少疫苗的接种次数；（2）延长疫苗的免疫持续期；（3）通过激动固有免疫应答促进体液免疫应答和细胞免疫应答 ；（4）扩展抗原诱导的交叉保护免疫应答；（5）增强弱免疫应答个体如老龄个体或免疫缺陷个体对抗原的免疫应答；（6）减少抗原的用量。可以和 本发明提供的寡核苷酸联合应用的佐剂 [Stanley A. Plotkin, Walter A. Orenstein, Paul A. Offit , Vaccines, Sixth Edition, An imprint of Elsevier Inc. 2013, ISBN-13: 9781455700905] 包括但不限于：铝盐佐剂（ 由氢氧化铝或磷酸铝为主要成分的疫苗佐剂）、 AS04 佐剂 （吸附MPL的磷酸铝佐剂（MPL是经化学减毒的革兰氏阴性菌脂多糖）、 MF59 佐剂 （ 一种油包水型乳化剂，其中的油相是角鲨烯）、 AS03 佐剂 （ 一种采用角鲨烯为油相的水包油佐剂）、 AF03 佐剂 （ 一种采用角鲨烯为油相的水包油型乳化剂）、 Montanide ISA 51 佐剂 （ 以矿物油为油相的油包水乳化剂）、弗氏佐剂、弗氏不完全佐剂 、病毒小体佐剂（virosome adjuvant）、N-氧化聚乙烯对二氮己环衍生物（polyoxidonium）佐剂、Toll样受体激动剂、病原体相关模式分子及其模拟物、损伤相关模式分子及其模拟物、环二核苷酸及其模拟物、GM-CSF（granulocyte-macrophage colony-stimulating factor）、IL-12、油性乳化剂（AS02, AS03，AF03, MF59和Montanide™ ISA-51）、白油佐剂、Montanide™ ISA-206佐剂、QS21、polylactide co-glycolide、病毒（Adenovirus, vaccinia, fowlpox）载体佐剂、卡介苗（BCG, bacillus Calmette-Guérin）、包括poly(I:C)在内的双链RNA及其类似物、包括MPL在内的类脂A类似物、鞭毛蛋白、包括Imiquimod和 R848在内的Imidazoquinolines、CpG ODN、包括QS21在
Figure 5167
的皂素、包括TDB在内的C-type lectin ligands、包括α- galactosylceramide在内的CD1d 配体、AS01 (MPL,QS21, liposomes)、AS02 (MPL,QS21, 乳化剂)、AS15 (MPL, QS21, CpG ODN, 脂质体)、GLA-SE (GLA, emulsion)、IC31 (CpG ODN, 阳离子多肽)、CAF01 (TDB, 阳离子脂质体)和ISCOMs (皂素，磷脂) [ 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]。
'病原体相关的模式分子'：本本发明提供的寡核苷酸可以和病原体相关的模式分子（pathogen-associated molecular patterns, PAMPs)及其类似物联合应用来增强微生物抗原和肿瘤抗原的免疫效果来发挥抗感染或抗肿瘤作用。PAMPs是微生物的各种保守成分，如细菌和真菌细胞壁成分和病毒核酸。固有免疫细胞通过模式识别受体（ pattern-recognition receptors, PRRs) 识别PAMPs而被激活。PRR包括 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) 和AIM-2 like receptors；也包括细胞内识别核酸的细胞内核酸感受器（intracellular sensors of nucleic acids）、OAS 蛋白和cGAS [ Iwasaki A et al. Nat Immunol. 2015 Apr;16(4):343-53] 。
'Toll样受体激动剂'：本发明提供的寡核苷酸可以和一种或多种Toll 样受体（Toll-like receptors，TLRs） 激动剂联合应用来增强微生物抗原和肿瘤抗原的免疫效果，表现抗感染或抗肿瘤活性。能和本发明提供的寡核苷酸联合应用的TLR激动剂包括但不限于：包括Imiquimod和imidazoquinolines在内的TLR7和TLR8激动剂；包括852A在内的TLR7激动剂；包括VTX-2337在内的TLR8激动剂；包括IMO-2055、CPG 7909、MGN1703和其它CpG ODN（含CpG脱氧寡核苷酸）在内的TLR9激动剂；包括卡介苗 (BCG)在内的TLR2/TLR4激动剂；包括OM‑174、monophosphoryl lipid A、aminoalkyl glucosamine phosphates和其它类脂A（lipid A）类似物在内的TLR4激动剂；包括病毒核酸、细菌核酸在内的TLR9激动剂；包括细菌鞭毛蛋白在内的TLR5激动剂、包括酵母多糖在内的TLR2/TLR6激动剂；包括聚肌苷酸胞嘧啶核苷酸（poly IC）、病毒双链RNA或其模拟物在内的TLR3激动剂和包括病毒单链RNA或其模拟物的TLR7/TLR8激动剂[ Adams JL et al Nat Rev Drug Discov. 2015 Sep;14(9):603-22]。
'损伤相关的模式分子'：本发明提供的寡核苷酸可以和损伤相关的模式分子（damage-associated molecular patterns, DAMPs) 来增强微生物抗原和肿瘤抗原的免疫效果。DAMPs是由损伤细胞释放的、能激发固有免疫应答自身细胞成分。DAMPs能通过PRR激发机体的固有免疫应答。这些DAMP包括但不限于：热休克蛋白、 HMGB1(high-mobility group box 1) 、hyaluronan 片段、glycans、glycoconjugates、ATP，（Adenosine 5'-triphosphate）、腺苷酸、 尿酸、S100 蛋白、heparin sulfate、Galectins、细胞核DNA、N-formylated peptides、Antimicrobial peptides、线粒体DNA和calreticulin [ Krysko DV et al. Nat Rev Cancer. 2012 Dec;12(12):860-75；Pouwels SD et al. Mucosal Immunol. 2014 Mar;7(2):215-26] 。
'环二核苷酸' 本本发明提供的寡核苷酸可以和环二核苷酸及其模拟物联合应用来增强微生物抗原和肿瘤抗原的免疫效果。环二核苷酸（Cyclic dinucleotides，CDNs) [Cell,2013 154(5),962-970]可来源于细菌，也可在哺乳动物细胞内合成。细菌CDNs包括但不限于环二鸟苷酸（cyclic di-GMP，cdG）、环二腺苷酸（cyclic di-AMP，cdA)和环腺苷酸-鸟苷酸（cyclic AMP-GMP，cAMP-GMP)。细菌CDN是一类PAMP，可激动固有免疫应答。在哺乳动物细胞中也可出现CDN，如环鸟苷酸-腺苷酸（cyclic guanosine monophosphate-adenosine monophosphate，cGAMP) [Wu J et al. Science. 2013 Feb 15;339(6121):826-3]。
'抗肿瘤制剂'：抗肿瘤制剂（ anti-tumor agent ）是 应用于个体后可治疗肿瘤的制剂，这些制剂包括但不限于肿瘤疫苗、免疫卡点抑制物、共刺激受体激活剂、化疗药物、放疗制剂、 激素抑制物或激素 、细胞因子、肿瘤治疗用抗体、小分子激酶抑制剂、PARP抑制物、血管生成抑制剂和溶瘤病毒等。
'免疫卡点抑制物'：免疫卡点抑制物（immune checkpoint inhibitor）是能抑制免疫卡点分子功能的物质 [ Melero I et al. Nat Rev Cancer. 2015Aug;15(8):457-72] 。抑制免疫卡点分子的功能使免疫细胞激活的信号得以突显，因而使免疫细胞处于持续的激活状态。处于持续激活状态的 CD4+T 细胞 会 辅助B细胞产生抗体，也辅助CD8+T细胞杀伤靶细胞如病毒感染细胞和肿瘤细胞。 处于持续激活状态的 CD8+T 细胞能杀伤病毒感染细胞和肿瘤细胞。因此，维持T细胞激活状态的制剂如免疫加点抑制物会提高抗原或疫苗在个体的免疫效力，也会在癌症患者（个体）发生肿瘤治疗作用。 本发明提供的寡核苷酸可以和免疫卡点抑制剂联合应用治疗肿瘤和提高抗原或疫苗的免疫效力。免疫卡点抑制剂抑制的免疫卡点分子包括但不限于CTLA4、PD1、LAG3（Lymphocyte activation gene 3)、2B4 (CD244)、BTLA（B and T lymphocyte attenuator)、TIM3（T cell membrane protein 3)和A2aR（adenosine A2a receptor) [ Pardoll DM. Nat Rev Cancer. 2012 Mar 22;12(4):252-64] 。能抑制免疫卡点功能的抗体属免疫卡点抑制物，包括但不限于CTLA-4抗体、CD-1抗体和CD-L抗体。
'PD-1'：PD-1（Programmed cell death protein-1），也称CD279, 是表达在激活的T细胞、激活的B细胞、NK细胞和单核细胞上的膜蛋白[ Chen L. Nat Rev Immunol. 2004 May;4(5):336-47]。PD-1可诱导效应性T细胞耗竭和无能。PD-1结合APC上的B7家族配体PD-L1(programmed death ligand-1, B7-H1) 和PD-L2 (programmed death ligand-2, B7-DC) [ Ito A. Biomed Res Int.2015;2015:605478]启动免疫细胞激活抑制信号的转导。
'PD1 抗体'：PD-1的抗体（Anti-PD-1 Antibodies）是PD1分子的单克隆抗体，具有抑制PD-1介导的免疫细胞负调节信号转导的功能，为免疫卡点抑制物。2014年，两种PD-1 抗体(pembrolizumab和nivolumab)被美国FDA批准用于肿瘤治疗。Nivolumab 是全人源化IgG4单克隆抗体，可结合并抑制PD-1的功能 [ Topalian SL et al. Curr Opin Immunol.2012Apr;24(2):207-12]，被用于治疗黑色素瘤、非小细胞性肺癌、卵巢癌和肾癌 [ Ito A et al. Biomed Res Int. 2015;2015:605478]。Pidilizumab (CT-011)是人源化IgG-1κ 单克隆抗体，可结合并抑制PD-1的功能，被用于治疗弥散性大B细胞淋巴瘤和滤泡淋巴瘤。此外，PD-1的抗体还包括Pembrolizumab (MK-3475)，它是可结合并抑制PD-1功能的IgG-4κ单克隆抗体[ Ito A et al. Biomed Res Int. 2015;2015:605478]。
' PD-L1' ：PD-L是PD1分子的配体，包括PD-L1 (programmed death ligand-1, B7-H1) 和PD-L2 (programmed death ligand-2)。PD-L1也称B7-H1。PD-L2也称B7-DC。PD-L1和PD-L2在结合PD-1分子后可激动免疫细胞激活抑制信号的转导。PD-L1和PD-L2可表达在多种肿瘤细胞的表面，其表达水平的升高可提示肿瘤患者的不良预后 [ Ito A et al. Biomed Res Int. 2015;2015:605478] 。
'抗-PD-L抗体'：是识别、结合PD-L1或PD-L2的抗体。在结合PD-L后可阻断其结合免疫细胞表面的PD-1，因而阻断免疫细胞激活抑制信号的转导，维持免疫细胞的激活状态，进而增强个体对抗原或肿瘤细胞的免疫应答。多种抗-PD-L1抗体（Anti-PD-L1 Antibodies）表现了肿瘤治疗作用，这些抗体包括但不限于BMS-936559、MPDL3280A、MEDI4736和MSB0010718 [Ito A, et al.Biomed Res Int. 2015;2015:605478]。BMS-936559 是全人源化IgG4抗PD-L1单克隆抗体，被用于治疗黑色素瘤、非小细胞肺癌、卵巢癌和肾癌。MPDL3280A是人源化IgG-1 κ 抗PD-L1单克隆抗体，被用于治疗黑色素瘤和膀胱癌。MEDI4736是人源化 IgG-1 κ 单克隆抗体，可延长荷瘤个体的生存期。MSB0010718是人源化IgG1 PD-L1单克隆抗体 [Ito A, et al.Biomed Res Int. 2015;2015:605478]。
'PD-1-PD-L1/2途径'：是指肿瘤细胞通过其表达的PD-L1/2作用于免疫细胞表达的PD-1诱导免疫细胞激活抑制信号的转导，因而抑制免疫细胞激活的途径。肿瘤细胞可利用PD-1-PD-L1/2途径逃逸免疫细胞的攻击 [Zou W., Chen L.Nature Reviews Immunology.2008;8(6):467-477]。阻断该途径可 增强个体免疫细胞的抗肿瘤活性 [ Topalian S. L.,et al. Current Opinion in Immunology . 2012; 24 (2):207-212 ] 。
'共刺激受体激活剂'：本发明提供的寡核苷酸可以和共受体激活剂联合应用增强个体对疫苗或抗原的免疫应答，增强个体的抗肿瘤免疫反应。共刺激受体是表达在免疫细胞表面的受体,在受到激活剂激活后介导免疫细胞激活信号的转导，因而促进个体对抗原或疫苗的免疫反应、增强个体的抗肿瘤免疫活性。共刺激受体激活剂是通过结合共刺激受体后激活免疫细胞的制剂。共刺激受体激活性单克隆抗体（Co-stimulatory receptor activating monoclonal antibody）是共刺激受体激活剂，可增强抗原或疫苗的效力、增强个体的抗肿瘤免疫应答。这类激活性单克隆抗体靶向的共刺激受体（Co-stimulatory receptor）包括但不限于CD137(41BB)、OX40、CD40、GITR、ICOS和CD27 (Glucocorticoid-induced tumour necrosis factor receptor family-related protein)[ Melero I et al. Nat Rev Cancer. 2015 Aug;15(8):457-72; Sanmamed MF et al. Semin Oncol.2015 Aug;42(4):640-55]。
' 化疗药物' 本发明提供的寡核苷酸可以和化疗药物联合应用治疗肿瘤。化疗药物 是可以通过抑制、杀伤肿瘤细胞而治疗肿瘤的化学药物。本发明所指的化学药物包括但不限于烷化剂类、抗代谢类、抗微管制剂类、拓扑异构酶抑制剂类和细胞毒性抗生素类药物。烷化剂类药物包括氮芥类、亚硝基脲类、四嗪类、氮杂环丙烷类、顺铂及其衍生物类和非经典烷化剂类。氮芥（nitrogen mustards）类药物包括mechlorethamine、环磷酰胺、chlorambucil、ifosfamide和busulfan。亚硝基脲类包括N-Nitroso-N-methylurea、carmustine、lomustine、semustine、fotemustine和streptozotocin。四嗪（tetrazines）类药物包括氮烯唑胺(dacarbazine)、mitozolomide和temozolomide。氮杂环丙烷（aziridines）类药物包括thiotepa、mytomycin和diaziquone。顺铂及其衍生物类包括顺铂 (cisplatin)、 卡铂 (carboplatin)和奥沙利铂(oxaliplatin)。非经典烷化剂类包括甲基苄肼(procarbazine) 和hexamethylmelamine。抗代谢类包括抗叶酸类、氟尿嘧啶类、脱氧核苷类似物和巯嘌呤类药物。抗叶酸类包括甲氨喋呤（methotrexate）和pemetrexed。氟尿嘧啶类药物包括5-氟脲嘧啶(5-fluorouracil)。脱氧核苷类似物药物包括cytarabine、吉西他滨 (gemcitabine)、decitabine、vidaza、fludarabine、nelarabine、cladribine、clofarabine和pentostatin。巯嘌呤类（thiopurines）药物包括thioguanine和mercaptopurine。抗微管制剂药物包括长春花生物碱类、紫杉烷类和足叶草毒素类。长春花生物碱（vinca alkaloids）类包括长春新碱(vincristine)、长春碱(vinblastine)、长春瑞宾(vinorelbine)、vindesine和vinflunine。紫杉烷类（Taxanes）包括紫杉醇(paclitaxel)和多烯紫杉醇(Docetaxel)。足叶草毒素（Podophyllotoxin）类包括依托泊苷(etoposide)和teniposide。拓扑异构酶抑制剂（topoisomerase inhibitors）包括topoisomerase I 抑制剂和topoisomerase II 抑制剂。拓扑异构酶I抑制剂包括irinotecan和topotecan。拓扑异构酶II抑制剂包括 topoisomerase II poisons和催化抑制物。topoisomerase II poisons包括etoposide、doxorubicin、mitoxantrone和teniposide。催化抑制物包括 novobiocin、merbarone和aclarubicin。细胞毒性抗生素包括阿霉素、daunorubicin、表柔比星(epirubicin)、idarubicin、pirarubicin、aclarubicin、mitoxantrone、gactinomycin、博来霉素 (bleomycin)、plicamycin和mitomycin。
' 放疗制剂 '： 放疗制剂是可以产生射线的物质。采用放疗制剂治疗肿瘤的方法被称为化疗。 本发明提供的寡核苷酸可以和放疗联合应用治疗肿瘤。用于放疗的物质是产生α、β、γ射线的放射性同位素。用于放疗的射线也可由机器产生，这类机器包括x射线治疗机或加速器。
'激素抑制物或激素'：本发明提供的寡核苷酸可以和用于肿瘤治疗的激素抑制物或激素联合应用治疗肿瘤，这类抑制物包括但不限于激素合成抑制剂、激素受体拮抗剂和补充用激素。激素合成抑制剂 包 括芳香化酶 抑制剂 和促性腺激素释放激素( gonadotropin-releasing hormone,GnRH ) 类似物 。 芳香化酶 抑制剂 包括 Letrozole ，anastrozole和Aminoglutethimide。GnRH类似物包括Leuprolide和gosereli。 激素受体拮抗剂包括选择性雌激素受体调节剂 和雄激素受体拮抗剂。 选择性雌激素受体调节剂 包括Tamoxifen、Raloxifene、Toremifene和fulvestrant。雄激素受体拮抗剂包括 Flutamide和 bicalutamide。 补充用激素是采用补充激素（Hormone supplementation）治疗肿瘤的方法，采用的激素包括孕激素、雄激素、雌激素、孕酮样药物、睾丸酮样药物、雌激素激动剂和生长激素抑制素类似物。孕酮样药物 包括megestrol acetate 和medroxyprogesterone acetate。 睾丸酮样药物 包括 Fluoxymesterone。雌激素拮抗剂包括diethylstilbestrol，Estrace和 Polyestradiol phosphate。 生长激素抑制素类似物包括 Octreotide 。
'细胞因子'： 本发明提供的寡核苷酸可和细胞因子联合应用治疗肿瘤、增强疫苗的免疫效果。这些细胞因子包括但不限于：白细胞介素（IL）-2、血小板生成素(IL-11)、粒细胞集落刺激因子（G-CSF）、粒细胞-单核细胞集落刺激因子（GM-CSF）和干扰素 a 。
'肿瘤治疗用抗体' 本发明提供的寡核苷酸可和肿瘤治疗用抗体（tumor therapeutic antibodies）联合应用治疗肿瘤。肿瘤治疗用抗体是在给患肿瘤个体应用后能延长其生存期的抗体，这些抗体靶向的分子包括但不限于CD20、ErbB2、表皮细胞生长因子受体、包括CTLA-4和PD-1在内的免疫卡点分子、血管内皮细胞生长因子受体、CD30、CD52和CD33。所涉及到的肿瘤治疗抗体这些抗体包括但不限于靶向CD20的Tositumomab（Bexxar）、Rituximab（Rituxan）和Ofatumumab (Arzerra; Genmab)；靶向ErbB2的 Trastuzumab （Herceptin）；靶向表皮细胞生长因子受体的 Panitumumab （Vectibix）和Cetuximab （Erbitux）；靶向PD-1的人源化抗体；靶向CTLA-4的人源化抗体；靶向血管内皮细胞生长因子受体的Bevacizumab（Avastin）；靶向CD30的Brentuximab（vedotin）；靶向CD52的Alemtuzumab （Campath）和靶向CD33的Gemtuzumab ozogamicin (Mylotarg; Wyeth) [ Scott AM et al. Nat Rev Cancer. 2012 Mar 22;12(4):278-87]。
'小分子激酶抑制物'：本发明提供的寡核苷酸可和小分子激酶抑制物（small-molecule kinase inhibitors）联合应用治疗肿瘤。小分子激酶抑制物为一类能通过抑制蛋白激酶活性发挥肿瘤治疗作用的小分子化合物，包括但不限于下述：靶向Bcr-Abl的Imatinib；靶向EGFR/ErbB2的Afatinib；靶向VEGFR1/VEGFR2/VEGFR3/PDGFRB/c-KIT的Axitinib；靶向BcrAbl/SRC的Bosutinib；靶向ALK/Met的Crizotinib；靶向ErbB1的Erlotinib；靶向Syk的Fostamatinib；靶向EGFR的Gefitinib；靶向BTK的Ibrutinib； 靶向ErbB1/ErbB2的Lapatinib；靶向VEGFR2/VEGFR2的Lenvatinib；靶向Bcr-Abl的Nilotinib；靶向VEGFR2/PDGFR/c-kit 的Pazopanib；靶向JAK的Ruxolitinib；靶向突变BRAF的 vemurafenib (Zelboraf)和dabrafenib (Tafinlar)和靶向MEK的trametinib（Mekinist）。小分子蛋白激酶抑制物还包括所有的通过抑制下述蛋白激酶来治疗肿瘤的小分子化合物：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和MEK [ Adams JL et al Nat Rev Drug Discov. 2015 Sep;14(9):603-22]。
'多聚腺苷二磷酸核糖聚合酶抑制物'：本发明提供的寡核苷酸可和多聚腺苷二磷酸核糖聚合酶抑制物（poly ADP ribose polymerase，PARP)联合应用治疗肿瘤。多聚腺苷二磷酸核糖聚合酶抑制物，简称PARP抑制物，是可通过抑制PARP活性治疗肿瘤的制剂。PARP抑制物包括但不限于：治疗乳腺癌和肺鳞状细胞癌的Iniparib；治疗乳腺癌的Talazoparib (BMN-673)；治疗乳腺癌、结肠癌、卵巢癌和晚期前列腺癌的Olaparib；治疗乳腺和卵巢癌的Rucaparib；治疗转移性黑色素瘤和乳腺癌的Veliparib和治疗非小细胞肺癌的CEP 9722 [Nature Reviews Clinical Oncology 12, 27-41，2015]。
'血管生成抑制剂'： 本发明提供的寡核苷酸可和 血管生成抑制剂联合应用治疗肿瘤。血管生成抑制剂是可以通过抑制血管生成治疗肿瘤的制剂 [ Albini A et al. Nat Rev Clin Oncol. 2012 Sep;9(9):498-509] ， 包括但不限于 ： 包括阿瓦斯丁 （Avastin 或 bevacizumab）在内 血管内皮生长因子 （VEGF） 的人源化单克隆抗体 ； 包括恩度 (ENDOSTAR) 在内的 血管内皮抑制素和包括 pegaptinib 在内的抗 血管内皮细胞生长因子的核酸适体 （aptamer） 。
'溶瘤病毒'： 本发明提供的寡核苷酸可和 溶瘤病毒联合应用治疗肿瘤。溶瘤病毒是可以通过裂解肿瘤细胞治疗肿瘤的病毒，包括但不限于新城疫病毒、单纯疱疹病毒、腺病毒、痘病毒、克萨病毒、呼吸道肠道病毒、麻疹病毒，脊髓灰质炎病毒、滤泡性口炎病毒、塞尼卡谷 病毒、 细小病毒和逆转录病毒[ Kaufman HL et al. Nat Rev Drug Discov.2015 Sep 1;14(9):642-62] 。
'肿瘤治疗用细胞'：本发明提供的寡核苷酸可以和肿瘤治疗用细胞联合应用治疗肿瘤。肿瘤治疗用细胞是应用于个体后能行使抗肿瘤效应的细胞，这些细胞包括但不限于肿瘤细胞、树突细胞、T淋巴细胞和NK细胞。
'肿瘤细胞'：本发明提供的寡核苷酸可以和肿瘤细胞联合应用治疗肿瘤。肿瘤细胞在个体能激发抗肿瘤免疫应答，可被用作肿瘤疫苗。这类肿瘤细胞包括但不限于自体肿瘤细胞、同种异体肿瘤细胞和肿瘤细胞系细胞，也包括转染细胞因子（如GM-CSF和IL-2）编码基因和/或共刺激分子（如B7）编码基因的肿瘤细胞 ( Hurwitz, A. et al. (1998) Proc. Natl. Acad. Sci U.S.A. 1998; 95:10067-10071)。
'树突细胞'：本发明提供的寡核苷酸可以和自体树突细胞（Dendritic cells，DC)联合应用来治疗肿瘤。可提呈肿瘤抗原的树突细胞被应用于个体后可激发抗肿瘤免疫应答，这种细胞也可被用作树突细胞疫苗。肿瘤治疗用树突细胞包括但不限于装载单一的肿瘤抗原（蛋白抗原或抗原肽，如前列腺酸性磷酸酶）的树突细胞、装载肿瘤细胞裂解物的树突细胞、装载肿瘤细胞RNA的树突细胞和装载自体肿瘤细胞洗脱肽的树突细胞[Nestle, F. et al. (1998) Nature Medicine 4: 328-332；Palucka K et al. Nat Rev Cancer. 2012 Mar 22;12(4):265-77]。肿瘤治疗用树突细胞也包括基因转染树突细胞。用于转染树突细胞的基因包括但不限于肿瘤抗原编码基因、细胞因子（如IL-2, GM-CSF）编码基因和共刺激分子编码基因。肿瘤治疗用树突细胞也包括树突细胞和肿瘤细胞融合细胞(Kugler, A. et al.(2000) Nature Medicine 6:332-336)。
'肿瘤治疗用T细胞'：本发明提供的寡核苷酸可以和肿瘤治疗用T细胞联合应用治疗肿瘤。肿瘤治疗用T细胞是被用于肿瘤治疗的自体T细胞，包括肿瘤浸润T细胞和经遗传改造的T细胞（Genetically engineered T cells）。经遗传改造的T细胞组装有可识别肿瘤抗原的嵌合抗原受体（chimeric antigen receptor，CAR)，表达该类受体的T细胞被称为CAR-T。肿瘤浸润的T细胞和经遗传改造的T细胞可在体外用IL-2扩增后被回输到肿瘤患者体内[ Kershaw MH et al. Nat Rev Cancer. 2013 Aug;13(8):525-41]。
'肿瘤治疗用天然杀伤细胞'：本发明提供的寡核苷酸可以和肿瘤治疗用天然杀伤细胞[Natural killer (NK) cells]联合应用治疗肿瘤。肿瘤治疗用天然杀伤细胞也称肿瘤治疗用NK细胞。这种NK细胞可以从个体的外周血或脐带血分离，也可从造血细胞前体细胞、胚胎干细胞或多能干细胞诱导生成。分离或诱导生成的NK细胞可在体外用IL-2和IL-15扩增后回输给个体[Childs RW, et al. Nat Rev Drug Discov.2015 Jul;14(7):487-98]。
'抗感染'：本发明提供的寡核苷酸单独应用或作为疫苗佐剂应用可发挥抗感染作用。抗感染和对微生物感染产生治疗或预防作用是可以互换的术语。单独应用本发明提供的寡核苷酸产生的抗感染作用尤其适合于尚无有效疫苗的微生物引起的感染，如HIV和HCV；也适合抗原性易发生变化的病原体如HIV、HCV流感病毒和疟原虫。因为具有增强T细胞介导的免疫应答的功效，本发明提供的寡核苷酸可被用于致病性病毒、致病性细菌、致病性真菌和致病性寄生虫感染的治疗，也可被用作致病性病毒、致病性细菌、致病性真菌和致病性寄生虫疫苗的佐剂。
'致病性病毒'：本发明提供的寡核苷酸单独应用或作为疫苗佐剂应用发挥抗致病性病毒（pathogenic viruses）感染的作用。这些致病性病毒包括 hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus 和arboviral encephalitis virus。
'致病性细菌'：本发明提供的寡核苷酸单独应用或作为疫苗佐剂应用发挥抗致病性细菌（pathogenic bacteria）感染的作用。这些致病性细菌包括chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis和Lymes disease bacteria。
'致病性真菌'：本发明提供的寡核苷酸单独应用或作为疫苗佐剂应用发挥抗致病性真菌（pathogenic fungi）感染的作用。这些致病性真菌包括Candida, Cryptococcus neoformans, Aspergillus, Genus Mucorales, Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis和Histoplasma capsulatum。
'致病寄生虫'：本发明提供的寡核苷酸单独应用或作为疫苗佐剂应用发挥抗致病寄生虫（pathogenic parasites）的作用。这些致病性寄生虫（pathogenic parasites）包括Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi和Nippostrongylus brasiliensis。
' 药物组合物 '：本发明提供的寡核苷酸 药物可与药物学上可接受的载体 （ pharmaceutically acceptable carrier ） 组成药物组合物 （ pharmaceutical compositions ），该药物组合物 可以包含本发明所提供的 寡核苷酸。该组合物中的寡核苷酸的使用剂量为有效剂量（ Effective Dosages ） 。该组合物可和抗原、疫苗、佐剂和具有肿瘤治疗作用的制剂或细胞联合应。该药物组合物可被制成一定的剂型，这些剂型包括但不限于溶液、乳化液、脂质体和冻干粉等。
'药物学可接受的载体'： 本发明提供的寡核苷酸 药物可与药物学上可接受的载体 （ pharmaceutically acceptable carrier ） 组成药物组合物 （ pharmaceutical compositions ）。 药物学可接受的载体（pharmaceutically acceptable carrier）是指一种或多种固体或液体的填充剂、稀释剂或包封物质。这类载体适合将本发明提供的寡核苷酸应用于个体。该载体可以是有机的、无机的，天然的或合成的。药物学可接受的载体可以是药学 可接受的溶剂（水溶液和非水溶液）、分散剂、悬浊剂、乳化剂、粉剂 、稀释剂、脂质体、抗菌剂、抗真菌剂、等渗制剂、延缓吸收制剂、冻干保护剂和其他的适合本发明中的核苷酸增强疫苗或抗原的免疫效力，产生治疗肿瘤作用的制剂。水溶液包括单不限于水、生理盐水、PBS缓冲液、平衡盐溶液和葡萄糖溶液。溶剂或分散剂可包括水、乙醇、多元醇（如甘油、丙二醇、聚乙二醇等，也包括由这些溶剂或分散剂组成的混合物。为了维持药物组合物的流动性可应用包括卵磷脂在内的脂类。 为了使药物组合物处于理想的颗粒状态可以应用表面活性剂。为了有合适的渗透压，可在药物组合物中添加糖、 包括甘露醇和山梨醇在内的多元醇和氯化钠等。 为了延长作用时间，可在药物组合物中加入缓释剂如硬脂酸盐和明胶。 乳化剂可包括水包油乳化剂、油包水乳化剂或水包油包水乳化剂。药物学可接受的载体也包括药学可接受的抗氧化剂（ pharmaceutically-acceptable antioxidants ） ，这些抗氧化剂包括：水溶性抗氧化剂，如 ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite 和 sodium sulfite等；油溶性 抗氧化剂，如 ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate和alpha-tocopherol等；金属螯合剂，如枸橼酸、ethylenediamine tetraacetic acid (EDTA)、sorbitol、tartaric acid和phosphoric acid。
' 有效剂量'：本发明所提供的寡核苷酸的有效剂量（ Effective Dosages ）包括 ' 增强抗原免疫效力有效剂量 ' 和 '肿瘤治疗有效剂量'。 增强抗原免疫效力有效剂量是给个体应用后能显著增强抗原或疫苗免疫效力的寡核苷酸剂量，也指在给予个体后可产生理想的预防或治疗传染性疾病或肿瘤的 寡核苷酸 剂量。 肿瘤治疗有效剂量是给个体应用后能产生肿瘤治疗作用的寡核苷酸的剂量。 剂量的多少决定于本领域技术熟练人员应知的标准，还要参考其他因素，这些因素包括并不限于个体的大小和健康情况和疾病的严重程度。本发明提供的寡核苷酸可单次或多次应用于个体，每次的剂量范围可在 1 m g 到1000 mg的范围。为了达到理想的效果，本领域技术熟练人员可以对此寡核苷酸的剂量作调整，其剂量范围可以是前述范围的10倍到1000倍。在给个体应用时，本发明提供的寡核苷酸可采用剂量单位 。每一单位包含定量的可产生预防或治疗作用寡核苷酸和所需的药学可接受的组合物。 剂量单位界定的依据是寡核苷酸产生治疗作用的特有活性特征和个体在接受该寡合甘酸治疗时对该寡核苷酸的敏感性。如果需要，以剂量单位应用的寡核苷酸每天可以按一定的时间间隔应用2次、3次、4次、5次或多次。本发明提供的寡核苷酸可以按个体单位体 重（host body weight）应用，剂量范围是0.0001到100 mg/kg,应用的间隔可以是每两周一次或每月一次或每3到6个月一次或其它适合产生预防、治疗作用的时间间隔。在和抗原或疫苗联合应用时，该寡核苷酸的剂量可以是1-1000 μg/ml。有效剂量包括治疗有效剂量（therapeutically effective dose）和预防有效剂量（prophylactically-effective dose）。
'给药途径'：本发明提供的 寡核苷酸在 单独应用或和其它制剂如抗原、佐剂和抗肿瘤制剂联合应用时可采用肠外、外用或吸入的给药途径。肠外给药途径包括经静脉、腹膜、鞘内、肌肉、皮下、皮内、局部、瘤旁淋巴结、肿瘤组织直接注射和 淋巴结内注射。 外用给药途径包括经皮肤、口、眼、耳和鼻。吸入可经鼻粘膜和肺。
' 治疗装置 '含本发明提供的寡核苷酸的药物组合物可用 本领域技术熟练人员应知的 治疗装置应用于个体。治疗装置包括但不限于无针注射装置、植入装置、仓室装置（modules）、植入性微输液泵（implantable micro-infusion pump）、输液泵和渗透药物递送系统（osmotic drug delivery system）。
'递送载体'：本发明中的寡核苷酸可经递送载体应用。递送载体包括但不限于：类固醇（如胆固醇）、络和物、乳化物、免疫刺激复合物（ISCOMs）、脂质（如阳离子脂质和阴离子脂质）、脂质体、细菌载体(如沙门氏菌、大肠杆菌、分枝杆菌志贺(氏)杆菌、乳酸杆菌)、病毒载体（如牛痘苗、腺病毒、单纯疱疹病毒）、病毒小体、病毒样颗粒、微球、核酸疫苗、高分子材料（如羧甲基纤维素、脱乙酰壳多糖）和环状多聚物。递送载体也可以是特异性受体的配体或细胞的靶向分子。
附图说明
图-1：采用Bac-to-Bac系统表达的PCV2b衣壳蛋白的检定
图-2：寡核苷酸（A1，A2）对重组蛋白抗原再刺激诱导的 CD4+T细胞激活的影响
图-3 寡核苷酸（A1，A2）对抗原再刺激诱导的小鼠脾细胞CTLA-4mRNA表达的影响
图4：寡核苷酸（A1，A2）对抗原再刺激诱导的CD4+T细胞 CTLA-4 表达的影响
图5：采用大肠杆菌表达的PCV2b衣壳蛋白的检定
图-6 寡核苷酸（A1和A2）对重组蛋白抗原再激活小鼠抗原提呈细胞CD80 的影响
图-7 寡核苷酸（A1和A2）对重组蛋白抗原再激活小鼠抗原提呈细胞 CD86的影响
图-8 寡核苷酸（A1和A2）对圆环病毒疫苗免疫效力的增强作用
图-9 寡核苷酸（A1和A2）对乙型肝炎疫苗免疫效力的增强作用
图-10 寡核苷酸（A1和A2）对狂犬疫苗免疫效力的增强作用
图-11寡核苷酸（A1和A2）对流感病毒疫苗免疫效力的增强作用
图-12 GL261细胞胶质瘤肿瘤细胞裂解物的检定
图-13 寡核苷酸（A1，A2）对黑色素瘤的治疗作用
 本发明的实施方式
实施例1 寡核苷酸（A1，A2）的设计及合成 ：
根据人和小鼠CTLA-4 mRNA 3'UTR的序列，设计了两种单链脱氧寡核苷酸，这两种寡核苷酸分别被命名为A1和A2, 具有 如序列表 <400> 1 和 <400> 2 所示的序列 。
A1 的序列是5' ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 ) 。
A2 的序列是5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ) 。
A1 和A2的序列和CTLA-4 mRNA 3'UTR的序列互补。
A1 和A2由宝生物工程（大连）有限公司（Takara Bio）合成，其骨架为全硫代修饰。
在应用时，A1和A2用无菌PBS或其它溶媒溶解，并经无热源处理， -20℃冻存。
含A1和A2溶液中的内毒素应用鲎变形细胞溶解法检测。
溶液中的A1和A2 的含量采用分光光度计（260nm波长）确定，也可采用琼脂糖凝胶（3%）电泳估算（根据已知含量单链脱氧寡核苷酸标准品估算）。
实施例2 寡核苷酸（A1，A2）对重组蛋白抗原再刺激导的 CD4+T细胞激活的影响：
采用重组蛋白抗原疫苗免疫小鼠，在免疫后取小鼠脾细胞做抗原回忆反应（recall）实验,在细胞培养体系中加入抗原和寡核苷酸（A1）或寡核苷酸（A2）。培养24小时后，用流式细胞仪检测CD4+ T细胞表面CD69分子的表达来判定A1或A2对CTLA-4功能的抑制作用。CD69是一种细胞膜糖蛋白分子，参与T细胞增生的诱导。在静息的CD4+ T细胞表面，CD69表达的水平很低。在受到抗原激活（TCR stimulation）后，表达在CD4+T细胞表面的CD69迅速上调，其表达在16-24达到高峰，然后下降。CD69分子是CD4+T细胞早期激活的标志物（early markers）。在CD4+T细胞激活的早期激活，CTLA-4抑制CD4+T细胞表面CD69的表达 [ Chambers CA. et al. Annu Rev Immunol.2001;19:565-94]。
2.1 免疫小鼠
2.1.1 材料
2.1.1.1 小鼠
ICR 小鼠（吉林大学医学部动物室），雌性，体重为17-18克。
2.1.1.2 抗原
采用Bac-to-Bac系统表达的PCV2b衣壳蛋白（简称P蛋白），此蛋白能组装成类病毒颗粒（virus like particls, VLP）。
经SDS-PAGE检测 ，P蛋白呈现为分子量为28 KD的区带。在电镜下（4万倍放大），P蛋白已组装成大小约为17 nM 的类病毒颗粒（virus like particles, VLPs，见图-1。
2.1.1.3 佐剂
ISA 35 乳化剂（Seppic）
2.1.2 疫苗的配制
将P蛋白用PBS稀释为60μg/ml,将此 P蛋白溶液和ISA 35 乳化剂按1:1(体积：体积)混合制成疫苗，此疫苗被命名为P-ISA 35。100μl P-ISA 35中含3μg P蛋白。
2.1.3. 小鼠免疫
于第0天对ICR小鼠进行初次免疫，方法是注射100μl P-ISA 35，注射部位是右后肢肌肉，单点注射。于第14天对ICR小鼠进行加强免疫，方法是注射100μl P-ISA 35，注射部位是右后肢肌肉，单点注射。
2.2. 寡核苷酸（A1，A2）对P蛋白再激活诱导的P-ISA 35免疫小鼠CD4+T细胞激活的影响
2.2.1 试剂
RPIM 1640 培养基和胎牛血清（FBS）购自Gibco公司。FITC标记的抗鼠CD4抗体和PE标记的抗鼠CD69抗体购自BD 公司。
P 蛋白（简称P，见2.1.1.2）。
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
2.2.2. 器材和仪器
24 培养板孔板、平皿、毛玻璃片、300目滤网、小镊子、细胞计数板、滴管、加样器。CO2细胞培养孵箱（日本SANYO公司）、细胞培养倒置显微镜（日本Olympus公司）、离心机（德国Biofuge Fresco）、ACEA NovoCyte流式细胞仪（美国艾森公司）。
2.2.3. 实验
在用P-ISA 35加强免疫后第48天处死P-ISA 35免疫小鼠，于75%乙醇内浸泡 2 min，无菌取脾，将脾放入含有5 mL 冰浴10％FBS 1640 培养基的平皿中。用毛玻璃片将脾研磨成单个细胞。用300 目尼龙网过滤，收取脾细胞单细胞悬液。用红细胞裂解液ACK（0.155 mol/L NH4Cl, 0.01 mol/L KHCO3, 0.1 mol/L EDTA, pH 7.4）去除其中的红细胞。用10％FBS 1640（8 mL），离心洗涤（1,200 rpm 5 min），弃上清。用10％FBS 1640悬起去红细胞的脾细胞悬液。计数细胞，用10％FBS 1640调细胞浓度为5×106个/ml。将细胞加入24孔培养板，每孔1ml，则每孔细胞个数为5×106个。加入P蛋白(终浓度10μg/ml)，或加入P蛋白(终浓度10μg/ml)和A1(终浓度5μg/ml)，或加入P蛋白(终浓度10μg/ml)和A2(终浓度5μg/ml)。培养24 小时后，收取细胞。用加样器轻吹细胞，置于1.5ml EP管中，2000rpm，离心5min。弃上清，将细胞弹匀，加入1ml PBS清洗细胞，2000rpm，离心2min。用40μl PBS重悬细胞，分别置于2个1.5 EP管中，20μl/管。加FITC标记的抗鼠CD4荧光抗体，PE标记的抗鼠CD69 荧光抗体。冰上，避光孵育30min。加入1ml PBS清洗细胞，2000rpm，离心2min。用200μl PBS重悬，用300目滤网过滤后，用流式细胞仪检测。
2.2.4. 结果
A1 或A2处理可使CD4+T淋巴细胞的CD69分子表达明显提高（图-2），提示A1或A2可通过抑制CTLA的功能维持促进CD4+T淋巴细胞的激活，具有这一活性的A1或A2可被用于个体来增强其对微生物抗原（疫苗）、肿瘤抗原（疫苗）的免疫应答，也可被用于个体来增强其抗肿瘤反应。
实施例3 寡核苷酸（A1，A2）对抗原再刺激诱导的小鼠脾细胞CTLA-4mRNA表达的抑制作用：
采用如实施例2所述P蛋白按实施例2所述免疫ICR小鼠，在免疫后取小鼠脾细胞用P蛋白做回忆反应（recall）实验,在细胞培养体系中加入寡核苷酸A1或寡核苷酸A2。培养24小时后，收取细胞，提取mRNA,做逆转录和聚合酶链式反应（PCR），检测CTLA-4 mRNA的表达。静息的Ｔ细胞几乎不表达CTLA-4 mRNA，在受到激活后T细胞表达的CTLA-4 mRNA水平迅速升高[Brunet JF,et al.Nature.1987 Jul 16-22;328(6127):267-70]，导致T细胞表面出现更多的CTLA。
3.1 材料
P 蛋白（简称P，见2.1.1.2）。
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
RPIM 1640 培养基和胎牛血清（FBS）购自Gibco公司。
TRIzol 试剂购自美国Invitrogen公司。EasyScript First-Strand cDNA Synthesis SuperMix、Top Green qPCR SuperMix (TransStartTM) 试剂盒购自全式金（Transgen）公司；
GAPDH 特异性引物（用于扩增GAPDH 特异性 cDNA, GAPDH为管家基因），上游引物的序列是：5'-ATCACCATCTTCCAGGAGCGA-3'，下游引物的序列是5'-TCTCGTGGTTCACACCCATCA-3'。
CTLA4 特异性引物（用于扩增CTLA4特异性 cDNA）,上游引物的序列为：5'-CCCAGTCTTCTCTGAAGCCATA-3'，下游引物的序列为：5'-TCTCTGTGAATGTCGTGGCA-3'。
GAPDH 特异性引物和CTLA4特异性引物均由宝生物工程（大连）有限公司（Takara Bio）合成。
3.2 小鼠
为P-ISA 35免疫ICR小鼠（见2.1.3）。
3.3. 主要实验器材和仪器
24 孔培养板、平皿、毛玻璃片、300目滤网、小镊子、细胞计数板、滴管、加样器。CO2细胞培养孵箱（日本SANYO公司）、细胞培养倒置显微镜（日本Olympus公司）、离心机（德国Biofuge Fresco）、荧光定量PCR仪（美国Applied Biosystems，型号：ABI Prism 7300）。
3.4 实验
牺牲P-ISA 35加强免疫ICR小鼠，于75%乙醇内浸泡 2 min，无菌取脾，将脾放入含有5 mL 冰浴10％FBS 1640 培养基的平皿中。用毛玻璃片将脾研磨成单个细胞。用300 目尼龙网过滤，收取脾细胞单细胞悬液。用红细胞裂解液ACK（0.155 mol/L NH4Cl, 0.01 mol/L KHCO3, 0.1 mol/L EDTA, pH 7.4）去除其中的红细胞。用10％FBS 1640（8 mL），离心洗涤（1,200 rpm 5 min），弃上清。用10％FBS 1640悬起去红细胞的脾细胞悬液。计数细胞，用10％FBS 1640调细胞浓度为5×106个/ml。将细胞加入24孔培养板，每孔1ml，则每孔细胞个数为5×106个。加入P蛋白(终浓度10μg/ml)，或加入P蛋白(终浓度10μg/ml)和A1(终浓度10μg/ml)，或加入P蛋白(终浓度10μg/ml)和A2(终浓度10μg/ml)。培养24 小时后，收取细胞。用Trizol法提取总RNA。采用EasyScript First-Strand cDNA Synthesis SuperMix、Top Green qPCR SuperMix (TransStartTM) 试剂盒，以GAPDH 特异性引物和CTLA4特异性引物做qPCR分别扩增GAPDH 特异性 cDNA 和 CTLA4特异性 cDNA。上荧光定量PCR仪（ABI Prism 7300）做荧光定量PCR分析。
3.5. 结果：
A1 和A2抑制P蛋白再刺激诱导的小鼠免疫细胞CTLA-4 mRNA的表达（图-3）和行使其功能。这说明，A1和A2可抑制CTLA-4 mRNA的表达和抑制其行使其功能，因而促进、维持T细胞的激活。该结果说明，具抑制P蛋白再刺激诱导的小鼠免疫细胞CTLA-4 mRNA的表达和行使其功能的A1或A2可被用于个体来增强其对微生物抗原（疫苗）、肿瘤抗原（疫苗）的免疫应答，也可被用于个体来增强其抗肿瘤反应。
实施例4 寡核苷酸（A1，A2）对抗原再刺激诱导的CD4+T细胞 CTLA-4 表达的抑制作用：
采用如实施例2所述P蛋白按实施例2所述免疫ICR小鼠，在免疫后取小鼠脾细胞用P蛋白做回忆反应（recall）实验,在细胞培养体系中加入寡核苷酸A1或寡核苷酸A2。培养48小时后，收取细胞，用荧光标记CTLA-4抗体染细胞做流式分析。CTLA-4 是对T细胞有负调节作用的膜信号分子。在TCR激活(T cell receptor activation) （识别抗原肽的激活） 后, Ｔ细胞迅速表达CTLA-4[Sakaguchi S, et al. Science. 2011 Apr 29;332(6029):542-3]。CD4+ 和 CD8+ T细胞表达CTLA-4。和CD8+ T 细胞相比，CD4+ T 细胞可表达更高水平的 CTLA-4 。抑制CTLA-4表达可促进、维持T细胞激活增生。在这种情况下，CD4+ T 细胞比 CD8+ T细胞激活增生的更明显[Chan DV, et al. Genes Immun. 2014 Jan; 15(1): 25-32]。
4.1 材料
P 蛋白（简称P，见2.1.1.2）。
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
RPIM 1640 培养基和胎牛血清（FBS）购自Gibco公司。
FITC 标记的抗鼠CD4抗体和PE标记的抗鼠CTLA4抗体购自BD 公司。
4.2 小鼠
为P-ISA 35免疫ICR小鼠（见2.1.3）。
4.3. 主要实验器材
24 孔培养板、平皿、毛玻璃片、300目滤网、小镊子、细胞计数板、滴管、加样器。CO2细胞培养孵箱（日本SANYO公司）、细胞培养倒置显微镜（日本Olympus公司）、离心机（德国Biofuge Fresco）、ACEA NovoCyte流式细胞仪（美国艾森公司）。
4.4 实验
牺牲P-ISA 35加强免疫ICR小鼠，按3.4所述的方法获得小鼠脾细胞悬液。计数细胞，用10％FBS 1640培养基调细胞浓度为5×106个/ml。将细胞加入24孔培养板，每孔1ml。加入P蛋白(终浓度10μg/ml)，或加入P蛋白(终浓度10μg/ml)和A1(终浓度10μg/ml)，或加入P蛋白(终浓度10μg/ml)和A2(终浓度10μg/ml)。培养48 小时后，收取细胞。用加样器轻吹细胞，置于1.5ml EP管中，2000rpm，离心5min。弃上清，将细胞弹匀，加入1ml PBS清洗细胞，2000rpm，离心2min。用40μl PBS重悬细胞，分别置于2个1.5 EP管中，20μl/管。加FITC标记的抗鼠CD4荧光抗体，加PE标记的抗鼠CTLA4 荧光抗体，冰上，避光孵育30min。加入1ml PBS清洗细胞，2000rpm，离心2min。用200μl PBS重悬，用300目滤网过滤后，用流式细胞仪检测。
4.5 结果
A1 能抑制P蛋白再刺激诱导的小鼠CD4+ T 细胞表达CTLA4（图-4）。这说明，A1可抑制CTLA-4 的表达，因而促进、维持T细胞的激活；具有这一活性的A1可被用于个体来增强其对微生物抗原（疫苗）、肿瘤抗原（疫苗）的免疫应答，也可被用于个体来增强其抗肿瘤反应。
实施例5 寡核苷酸（A1和A2）对重组蛋白抗原再刺激小鼠抗原提呈细胞CD80和CD86的影响：
抗原激活的T细胞表达CTLA-4，这种CTLA-4分子能结合抗原提呈细胞表面的CD80或CD86分子并将其内化，使抗原提呈细胞缺少这两种共刺激分子。缺少共刺激分子的抗原提呈细胞不能通过结合CD28向T细胞提供第二激活信号，因此限制T细胞的激活[ Sakaguchi S et al. Science. 2011 Apr 29;332(6029):542-3]。调节性T细胞（Treg）表达的CTLA-4也能和抗原提呈细胞表面的CD80或CD86分子结合并将其内化，因而使T细胞处于免疫耐受状态[ Sansom DM.Science.2015 Jul 24;349(6246):377-8]。因此，A1和A2使抗原提呈细胞表面的CD80或CD86减少是CTLA-4表达受到抑制的一种反应。
5.1 免疫小鼠
5.1.1 材料
5.1.1.1 小鼠
ICR 小鼠（吉林大学医学部动物室），雌性，体重为17-18克。
5.1.1.2 抗原
采用大肠杆菌表达的PCV2b衣壳蛋白（简称D蛋白），此蛋白能组装成类病毒颗粒（virus like particls, VLP）。
在SDS-PAGE检测，D蛋白(D)被显示为分子量为 28 KD的区带，在电镜下观察（4万倍放大），D蛋白形成典型的大小约为17 nM 的VLP，见图-5。
5.1.1.3 佐剂
ISA 35 乳化剂（Seppic）
5.1.2 疫苗的配制
将D蛋白用PBS稀释为100μg/ml,将此D蛋白溶液和ISA 35 乳化剂按1:1(体积：体积)混合制成疫苗，此疫苗被命名为D-ISA 35。100μl D-ISA 35中含5μg D蛋白。
5.1.3 小鼠免疫
于第0天对ICR小鼠进行初次免疫，方法是注射100μl D-ISA 35，注射部位是右后肢肌肉，单点注射。于第14天按初次免疫的抗原和方法对ICR小鼠进行加强免疫。
5.2 寡核苷酸（A1，A2）对D蛋白再激活抗原提呈细胞表达的 CD80 和CD86 的影响
5.2.1 试剂
RPIM 1640 培养基和胎牛血清（FBS）购自Gibco公司。FITC标记的抗鼠CD4抗体，PE标记的抗鼠CD80抗体和PE标记的抗鼠86 抗体均购自BD 公司。
D 蛋白（见5.1.1.2）,也用D表示。
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
5.2.2. 器材和仪器
24 孔培养板、平皿、毛玻璃片、300目滤网、小镊子、细胞计数板、滴管、加样器。CO2细胞培养孵箱（日本SANYO公司）、细胞培养倒置显微镜（日本Olympus公司）、离心机（德国Biofuge Fresco）、ACEA NovoCyte流式细胞仪（美国艾森公司）。
5.2.3. 实验
在加强免疫后第48天牺牲D-ISA 35免疫小鼠，按3.4所述的方法获得小鼠脾细胞悬液。计数细胞，用10％FBS 1640调细胞浓度为5×106个/ml。将细胞加入24孔培养板，每孔1ml。加入D蛋白(终浓度10μg/ml),或加入D蛋白(终浓度10μg/ml)和A1(终浓度5μg/ml)，或加入D蛋白(终浓度5μg/ml)和A2(终浓度5μg/ml)。培养24 小时后，收取细胞。用加样器轻吹细胞，置于1.5ml EP管中，2000rpm，离心5min。弃上清，将细胞弹匀，加入1ml PBS清洗细胞，2000rpm，离心2min。用40μl PBS重悬细胞，分别置于2个1.5 EP管中，20μl/管。加FITC标记的抗鼠CD11c抗体，PE标记的抗鼠CD80抗体或PE标记的抗鼠CD86抗体。冰上，避光孵育30min。加入1ml PBS清洗细胞，2000rpm，离心2min。用200μl PBS重悬，用300目滤网过滤后，用流式细胞仪检测。
5.2.4. 结果
A1
或A2处理可使CD11c细胞CD80（图-6）和CD86 (图-7）增多。这说明,应用A1或A2可通过抑制CTLA使抗原提呈细胞表面的共刺激分子增多，因而通过提供第二激活信号促进微生物抗原和肿瘤抗原激活T淋巴细胞。具有这一活性的A1或A2可被用于个体来增强其对微生物抗原（疫苗）、肿瘤抗原（疫苗）的免疫应答，也可被用于个体来增强其抗肿瘤反应。
实施例6 寡核苷酸（A1和A2）对重组蛋白疫苗（圆环病毒疫苗）免疫效力的增强作用：
6.1 免疫小鼠及采集血清
6.1.1 材料
6.1.1.1 小鼠
ICR 小鼠（吉林大学医学部动物室），雌性，体重为17-18克。
6.1.1.2 抗原
D 蛋白（D），如实施例5中5.1.2所述。
6.1.1.3 佐剂
ISA 35 乳化剂（Seppic）
6.1.1.4 寡核苷酸
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
6.1.2 疫苗的配制
将D蛋白的浓度用D蛋白组装液调节为600μg/ml,将此D蛋白溶液和ISA 35 乳化剂按1:1(体积：体积)混合制成疫苗，此疫苗被命名为D-ISA 35。100μl D-ISA 35中含30μg D蛋白。分别配制含A1和A2的D-ISA 35，分别被称为 D-ISA 35 A1和D-ISA 35 A2。每100μl D-ISA 35 A1或D-ISA 35 A2中的D蛋白为30μg，A1和A2均为5μg。
6.1.3 免疫前采血
在初次免疫前一天采集待免疫小鼠血清。小鼠尾静脉采血，采血量≥100μl。将采集的全血（1.5mlEP管中）室温放置30分钟。11000rpm 离心15分钟，收集血清，分装，-20℃保存。
6.1.4 小鼠免疫
于第0天对ICR小鼠进行初次免疫，方法是注射100μl D-ISA 35或D-ISA 35 A1或D-ISA 35 A2，注射部位是右后肢肌肉，单点注射。于第14天对ICR小鼠进行加强免疫，方法同初次免疫。
6.1.5. 免疫后采血
在二次免疫后7天和14天，采集免疫小鼠血清。方法如免疫前采血（6.1.3）。
6.2. 小鼠血清特异性抗体检测
采用ELISA方法检测小鼠血清中的圆环病毒（PCV2b）特异性抗体 。
6.2.1 材料
6.2.1.1 灭活圆环病毒（PCV2b）（天津瑞普生物技术股份有限公司，TCID50/ml=7.0）。
6.2.1.2 试验器材
酶标条（组合式酶标板），0.5mlEP管，1.5mlEP管，加样器头，移液器，多道移液器，平皿（直径9 cm），刻度玻璃瓶。
6.2.1.3 试剂
碳酸钠（国药集团化学试剂有限公司），NaCl（国药集团化学试剂有限公司），KCl（北京化工厂），Na2HPO4·12H2O（国药集团化学试剂有限公司），KH2PO4（天津市永大化学试剂开发中心），吐温20（天津市光复精细化工研究所），脱脂奶粉（Biotopped），柠檬酸（国药集团化学试剂有限公司），OPD（上海三浦化工有限公司），30%H2O2（北京化工厂），浓硫酸（北京化工厂）和戊二醛（天津市福晨化学试剂厂）。
6.2.1.4 液体
包被液 （25%戊二醛 PBS），PBS （7.3 mol/L NaCl,3 mmol/L KCl,10 mmol/L Na2HPO4·12H2O和17.6 mmol/L KH2PO4水溶液），洗涤液（0.05%吐温20的PBS），封闭液（5%脱脂奶粉的洗涤液），0.1 Mol/L柠檬酸水溶液，0.2 Mol/L Na2HPO4.12H2O水溶液，10×甲乙液[0.1 Mol/L柠檬酸水溶液（体积）：0.2 Mol/L Na2HPO4.12H2O水溶液（体积）=94.5：100]，底物缓冲液[含OPD（1μg/ml）和0.045% H2O2的甲乙液]和终止液(20%浓硫酸)。
6.2.2 实验
用灭活PCV2b在包被液中包被酶标条，100μl/孔，4℃过夜。甩干液体，用封闭液于37℃ 封闭2小时，200μl/孔。加待测血清，37℃，1小时。甩干液体，加洗涤液（300μl/孔），室温3min，甩干液体，重复洗涤两次。加HRP标记二抗（羊抗小鼠）IgG（用洗涤液做1:5000稀释）。100 μl/孔，37℃ 1小时。甩干液体，用洗涤液洗涤，300μl/孔，室温3min，甩干液体，重复洗涤两次。加入底物液，100 μl/孔，室温避光(包锡纸)显色15min。加终止液（稀硫酸2mmol/L,50μl/孔。用酶标仪（波长549 nm）检测。
6.2.3 结果
A1 或A2可增强重组蛋白疫苗（圆环病毒疫苗）的免疫效力（图-8）。这说明 A1或A2可被用于个体来增强其对微生物抗原（疫苗）的免疫应答而成为一种新型微生物抗原或微生物疫苗的佐剂。
实施例7 寡核苷酸（A1和A2）对乙型肝炎疫苗免疫效力的增强作用:
7.1 免疫小鼠及采集血清
7.1.1 材料
7.1.1.1 小鼠
Balb/c 小鼠，雌性，体重为17-18g,购自北京维通利华实验动物有限公司。
7.1.1.2 疫苗
采用铝佐剂配制的乙型肝炎表面抗原（HBsAg）疫苗（沃森生物技术有限公司），简称HBsAg疫苗或HBsAg。
7.1.1.3 寡核苷酸
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )]
和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ],
如实施例1所述。
7.1.1.4 含寡核苷酸疫苗的配制
在HBsAg疫苗中加入A1或A2配成含寡核苷酸HBsAg疫苗，分别被命名为HBsAg-A1和HBsAg-A2。每100μl HBsAg-A1和HBsAg-A2中的HBsAg为1μg，A1和A2均为5μg。
7.2. 免疫前采血
在初次免疫前一天采集待免疫小鼠血清。方法如实施例6的6.1.3所述。
7.3. 小鼠免疫
于第0天对Balb/c小鼠进行初次免疫，方法是注射100μl HBsAg疫苗或HBsAg-A1或HBsAg-A2。注射部位是右后肢肌肉，单点注射。于第14天对Balb/c小鼠进行加强免疫，方法同初次免疫。
7.4. 免疫后采血
在二次免疫后7天和14天，采集免疫小鼠血清。方法如实施例6的6.1.3所述。
7.5. 免疫小鼠血清特异性抗体检测
采用ELISA方法检测小鼠血清中的乙型肝炎表面抗原（HBsAg）特异性抗体。
7.5.1 材料
7.5.1.1 重组乙型肝炎表面抗原（HBsAg）（沃森生物技术有限公司），简称HBsAg。
7.5.1.2 试验器材
同实施例6。
7.5.1.3 试剂
同实施例6。
7.5.1.4 液体
同实施例6。
7.5.2 试验方法
用HBsAg在包被液中包被酶标条，100μl/孔（每孔HBsAg为0.1μg），4℃过夜。余下操作步骤同实施例6。
7.5.3 结果
A1 或A2可增强重组蛋白疫苗（乙型肝炎病毒疫苗）的免疫效力（图-9）。这说明, A1或A2可被用于个体来增强其对微生物抗原（疫苗）的免疫应答而成为一种新型微生物抗原或微生物疫苗的佐剂。
实施例8 寡核苷酸（A1和A2）对狂犬疫苗免疫效力的增强作用:
8.1 免疫小鼠及采集血清
8.1.1 材料
8.1.1.1 小鼠
ICR/c 小鼠，体重18－22克，来自吉林大学医学部动物室。
8.1.1.2 疫苗
狂犬疫苗（长春生物制品研究所）。
8.1.1.3 寡核苷酸
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
8.1.1.4 含寡核苷酸狂犬疫苗的配制
在狂犬疫苗中加入A1或A2配成含寡核苷酸狂犬疫苗，分别被命名为狂犬疫苗-A1和狂犬疫苗-A2。每500μl含寡核苷酸疫苗含A1和A2 10μg。
8.1.2 免疫前采血
在初次免疫前两天采集待免疫小鼠血清，方法如实施例6的6.1.3所述。
8.1.3. 免疫小鼠
于第0天、3天、7天、14天、28天经小鼠腹腔注射0.5ml疫苗（狂犬疫苗或狂犬疫苗-A1或狂犬疫苗-A2)，每组8只小鼠，雌雄各半。
8.1.5 免疫前采血
于第35天采集免疫小鼠血清，方法如实施例6的6.1.3所述。
8.2 血清狂犬病毒中和抗体检测
采用快速狂犬疫苗荧光灶抑制实验(RFFIT)方法检小鼠血清中狂犬疫病毒中和抗体。
8.3. 结果
A1
或A2可增强灭活病毒疫苗（狂犬疫苗）的免疫效力（图-10）。这说明,应 A1或A2可被用于个体来增强其对微生物抗原（疫苗）的免疫应答而成为一种新型微生物抗原或微生物疫苗的佐剂。
实施例9 寡核苷酸（A1和A2）对流感病毒疫苗免疫效力的增强作用:
9.1 免疫小鼠及采集血清
9.1.1 材料
9.1.1.1 小鼠
Balb/c 小鼠（吉林大学实验动物中心）。雌性，体重为 18克左右。
9.1.1.2 疫苗
禽流感病毒H5亚型二价灭活疫苗（H5N1，Re-1株+Re-4株）（哈尔滨维科生物技术开发公司），此疫苗被简称为H5疫苗。
9.1.1.3 寡核苷酸
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
9.1.1.4 含寡核苷酸疫苗
在H5疫苗中加入A1或A2配成含寡核苷酸H5疫苗，分别被命名为H5疫苗-A1和H5疫苗-A2。每100μl H5疫苗-A1和H5疫苗-A2中的A1和A2均为10μg。
9.2. 免疫及采血
免疫两次，初次免疫后22天加强免疫一次。采用后腿肌肉注射，注射疫苗的体积为100μl。在初次免疫的-2和29天经尾静脉采血，50 ml/ 只。采集的血液在37°C环境放置30分钟，4°C放置3小时后使其血清充分析出，4000 r/m离心10分钟，吸出血清，1:400倍稀释后用于ELISA检测。
9.3. 免疫小鼠血清特异性抗体检测
采用ELISA方法检测小鼠血清中的流感病毒特异性抗体。用提取自禽流感病毒H5亚型二价灭活疫苗的抗原作为包被抗原（见下述）用包被液（Na2CO3 1.59 g，NaHCO3 2.93 g，pH 9.6，定容至1 L）1:2稀释，用100μl/孔包被酶标板，密封， 4°C过夜；用洗涤液（含0.05%Tween-20的PBS）洗涤酶标板，300μl/孔，洗3次；加入封闭液（含5%FBS的PBS），200μl /孔，37°C 2小时；用 PBS稀释小鼠血清（1:400稀释），将稀释血清加入酶标板，100μl/孔加入，37°C放置1小时；用洗涤液洗涤3次，然后加辣根过氧化酶标记羊抗小鼠二抗（用封闭液1:1000稀释），100μl /孔，37°C放置1小时；用洗涤液洗涤3次，加入即配即用底物液（10 mL中柠檬酸0.01M，Na2HPO4 0.02 M，超纯水9 mL，30% H2O2 15μl，OPD 4 mg），100 m L/ 孔，室温避光显色20分钟；加入终止液（20%硫酸），50μl/孔；于A492测各孔的OD值。
注：从禽流感病毒H5亚型二价灭活疫苗提取包被抗原。此疫苗采用的是油包水型佐剂，冷冻以及高速离心都可以使其破乳、分层。将此疫苗于-70°C/室温冻融2次，11000 r/m离心20分钟，分离下层水相，室温融化后11000r/m离心，可见清晰分层，上层为白色乳糜状（油相），下层清澈透明（水相）。用Bradford方法检测，上层不含蛋白，下层含有蛋白。下层水相即为含H5流感病毒抗原的包被液。
9.4. 结果
A1 或A2可增强病毒疫苗（流感病毒疫苗）的免疫效力（图-11）。这说明, A1或A2可被用于个体来增强其对微生物抗原（疫苗）的免疫应答而成为一种新型微生物抗原或微生物疫苗的佐剂。
实施例10、寡核苷酸（A1，A2）对胶质瘤细胞裂解物疫苗的增效作用 :
10.1 材料
10.1.1 小鼠
C57BL/6 小鼠，购自北京维通利华实验动物有限公司。
10.1.2 GL261 细胞
GL261 细胞是C57BL/6小鼠来源的胶质瘤细胞，来自北京军事研究院野战血研所
10.1.3 佐剂
ISA 35 乳化剂（Seppic, Cedex, Paris, France）
10.1.4 寡核苷酸
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
10.2. GL261 细胞胶质瘤肿瘤细胞裂解物的制备
采用含10%（v/v）胎牛血清，100 IU青霉素/ml和100 IU 的链霉素/ml的RPIM 1640培养基（Gibco），在37℃，5% CO2条件下培养培养GL261细胞。将1ml生长状态良好的GL261细胞（1×107/ml）接种于健康C57BL/6小鼠的腹腔。待小鼠腹部膨隆后，处死小鼠，置于75%乙醇浸泡2-3min消毒。在超净台中打开小鼠腹腔，可见形成的GL261细胞实体瘤（图-12）。按无菌操作取出GL261细胞实体瘤组织。将其在取无菌平皿中用手术剪成组织小块，用生理盐水反复冲洗去除肿瘤组织和残留的血液。将洗净的组织块在组织研磨器中反复研磨，收集研磨液。将研磨液置于液氮和室温反复冻融5次。2000 rmp 离心10 min 后收集上清。此上清为GL261细胞胶质瘤肿瘤细胞裂解物（GTL）。用Bradford法对GTL进行蛋白含量测定。用SDS-PAGE对GTL做进一步的分析(图-12)。 将CTL置-80℃冰箱保存。
10.3 GTL 疫苗及含寡核苷酸GTL疫苗的制备
用PBS将GTL的蛋白浓度调节为4 mg/ml。将此GTL和ISA 35 乳化剂按1:1(体积：体积)混合制成疫苗，此疫苗被命名为GTL疫苗。在GTL疫苗中加入A1或A2配制的含A1或A2的GTL疫苗，这两种疫苗分别被称为 GTL-A1疫苗和GTL-21-A2疫苗，其中A1和A2的浓度均为10μg/100μl。
10.4 小鼠免疫
于第0天和第14天在小鼠小鼠前肢左右腋窝下分别注射100μl GTL疫苗或GTL-A1疫苗或GTL-21-A2疫苗。
10.5 接种胶质瘤细胞
在第21天，注射1×104 GL261细胞到小鼠颅内，注射体积为2μl，使小鼠发生胶质瘤，方法如文献（Prins RM, et al.Cancer Res. 2003 Dec 1;63(23):8487-91）所述。自接种肿瘤后开始观察并记录小鼠的生存期，在小鼠死亡后做尸解确认小鼠颅内发生了胶质瘤。
10.6 结果（见表-1）
表-1、寡核苷酸（A1，A2）对胶质瘤肿瘤细胞裂解物疫苗的增效作用
组别 小鼠数（n） 生存期（天）
A1+GL261 疫苗 8 81±13
GL261 疫苗 8 34±6
A2+GL261 疫苗 8 78±9
结果表明，A1或A2可增强胶质瘤肿瘤细胞裂解物（肿瘤抗原疫苗）的免疫效力（表-1），是荷瘤小鼠的生存期延长（p<0.05）。这说明, A1或A2可被用于个体来增强其对肿瘤抗原（疫苗）的免疫应答而成为一种新型肿瘤疫苗的佐剂。
实施例11、寡核苷酸（A1，A2）对肺癌细胞裂解物疫苗的增效作用 :
11.1 材料
11.1.1 小鼠
C57BL/6 雄性小鼠（北京维通利华实验动物技术有限公司），6周龄。
11.1.2 Lewis 肺癌细胞
Lewis 肺癌（Lewis lung carcinoma，LLC）细胞（源自美国ATCC）。在37℃，5% CO2的条件下培养。使用的培养基为含10%（v/v）灭活的胎牛血清，100 IU青霉素/ml和100 IU 的链霉素/ml的IMDM培养基。
11.1.3 佐剂
ISA 35 乳化剂（Seppic, Cedex, Paris, France）。
11.1.4 寡核苷酸
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
11.2. Lewis 肺癌细胞（LLC）细胞裂解物的制备
将体外传代培养的LLC细胞（1×106/0.1 ml）接种于C57BL/6J雄性小鼠背部皮下长成实体肿瘤。牺牲小鼠，取出肿瘤，按实施例10所述的方法制备、鉴定和保存LLC细胞裂解物（ LTL）。每100μl LTL由2×106 LLC细胞制成。
11.3 LTL 疫苗及含寡核苷酸LTL疫苗的制备
将制备的GTL和ISA 35 乳化剂按1:1(体积：体积)混合制成疫苗，此疫苗被命名为LTL疫苗。在LTL疫苗中加入A1或A2配制的含A1或A2的LTL疫苗，这两种疫苗分别被称为 LTL-A1疫苗和LTL-A2疫苗，其中A1和A2的浓度均为10μg/100μl。
11.4 小鼠免疫
于第0天和第14天在6周龄C57BL/6J雄性小鼠，右侧背部皮下注射100μl LTL疫苗或LTL-A1疫苗或LTL-A2疫苗。每组8只小鼠。
11.5 接种Lewis 肺癌（LLC）细胞
于第21天，小鼠左侧背部皮下注射100μl LLC细胞悬液。LLC细胞悬液的制备方法是：将体外传代培养的LLC细胞（1×107/100μl）接种于C57BL/6J雄性小鼠背部皮下长成实体肿瘤。牺牲小鼠，按无菌操作方法取出肿瘤组织，将其用0.25%胰蛋白酶-0.04%EDTA 消化30 min，制成单细胞悬液。在PBS中调细胞浓度为 每1×106/100μl。在接种肿瘤后的第18天牺牲小鼠，剖出瘤块，称其重量。
11.6 结果（见表-2）
表-2、寡核苷酸（A1，A2）对Lewis 肺癌细胞裂解物疫苗的增效作用
组别 动物数（n） 瘤重（g）
LTL-A1 疫苗 8 0.257±0.040
LTL 疫苗 8 0.663±0.140
LTL-A2 疫苗 8 0.301±0.128
结果表明，A1或A2可增强肺癌肿瘤细胞裂解物（肿瘤抗原疫苗）的免疫效力（表-2），使肿瘤缩小（p<0.05）。这说明, A1或A2可被用于个体来增强其对肿瘤抗原（疫苗）的免疫应答而成为一种新型肿瘤疫苗的佐剂。
实施例12 寡核苷酸（A1，A2）对黑色素瘤的治疗作用：
12.1 材料
12.1.1 小鼠
C57BL/6 雌性小鼠（北京维通利华实验动物技术有限公司），18-22g。
12.1.2 B16 细胞
B16 细胞是C57BL/6小鼠来源的黑色素瘤细胞（源自美国ATCC）。在37℃，5% CO2的条件下培养。使用的培养基为含10％胎牛血清，100 IU青霉素/ml和100 IU 的链霉素/ml的RPMI1640培养基（GIBCO）。
12.1.3 寡核苷酸
寡核苷酸（A1）[5'ttctttgggc tgtgccattc cctaa 3'( 序列表 <400>1 )] 和寡核苷酸（A2） [5'tttcatggaa aatattgagt taaaa 3'( 序列表 <400> 2 ], 如实施例1所述。
12.2 主要实验器材和仪器
100ml 细胞培养瓶、1ml注射器、细胞计数板。CO2细胞培养孵箱（日本SANYO公司）、细胞培养倒置显微镜（日本Olympus公司）、离心机（德国Biofuge Fresco）。
12.3 实验
将体外培养的B16细胞于第0天接种于小鼠左后肢背侧皮下，注射体积为200μl，其中含1X106B16细胞。于接种B16细胞后的第10天开始给小鼠注射A1，然后每个两天再注射一次，共注射6次（图-13）。注射部位为左后肢接种肿瘤淋巴结引流区皮下。每次的注射体积为100μl，其中含25μg A1。对照组小鼠按同样的程序注射PBS。观察并记录小鼠的生存时间。
12.4 结果
单独应用A1可在小鼠发挥抗黑色素瘤的作用（图-13）,使荷瘤小鼠生存期延长。这说明, A1或A2可被用于个体来治疗包括黑色素瘤在内的肿瘤而成为一种新型的抗肿瘤制剂。
 序列表自由内容
<110> 苏州派动生物技术有限公司
<120> 具有抗原增效作用和肿瘤治疗作用的寡核苷酸
<130> 2015
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> 人工序列
<400> 1
ttctttgggc tgtgccattc cctaa 25
<210> 2
<211> 25
<212> DNA
<213> 人工序列
<400> 2
tttcatggaa aatattgagt taaaa 25

Claims (1)

  1. 1、 单链脱氧寡核苷酸，具有如序列表 <400> 1 和 <400> 2 所示的序列。
    2、 按照权利要求 1 所述的单链脱氧寡核苷酸，它们可以被化学修饰。
    3、 按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们 可通过抑制CTLA-4 mRNA的表达和干扰CTLA-4 mRNA的翻译而解除或减弱由CTLA-4介导的免疫应答的负调节，进而表现免疫增强作用。
    4、 按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可促进对微生物抗原或疫苗的免疫应答，因而被应用于增强微生物抗原或疫苗的免疫效力，产生抗感染作用。
    5、 按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可以和包括圆环病毒抗原或圆环病毒疫苗在内的病毒抗原或疫苗联合应用增强其免疫效力，产生抗感染作用。
    6、 按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可以和包括乙型肝炎病毒表面抗原或乙型肝炎病毒疫苗在内的病毒抗原或疫苗联合应用增强其免疫效力，产生抗感染作用。
    7、 按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可以和包括狂犬病毒抗原或狂犬病毒疫苗在内的病毒抗原或疫苗联合应用增强其免疫效力，产生抗感染作用。
    8、 按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可以和包括流感病毒抗原或流感病毒疫苗在内的病毒抗原或疫苗联合应用增强其免疫效力，产生抗感染作用。
    9 、按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可以和肿瘤抗原或肿瘤疫苗联合应用来增强个体对肿瘤抗原的免疫应答，因而表现抗肿瘤作用。
    10 、按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可以和包括脑胶质瘤肿瘤抗原在内的肿瘤抗原联合应用对包括脑胶质瘤在内的肿瘤产生治疗作用。
    11 、按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可以和肺癌肿瘤抗原在内的肿瘤抗原联合应用对包括肺癌在内的肿瘤产生治疗作用。
    12 、按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可以被用于个体来增强其抗肿瘤免疫反应而产生肿瘤治疗作用。
    13 、按照权利要求 1 ， 2 所述的单链脱氧寡核苷酸，它们可以被来治疗包括黑色素瘤在内的肿瘤。
    14 、按照权利要求 1 ， 2 ， 4, 5, 6 ， 7, 8 ， 9 ， 10, 11 所述的单链脱氧寡核苷酸，它们可以和各种佐剂联合应用来增强个体对微生物抗原和肿瘤抗原的免疫应答。
    15 、按照权利要求 1 ， 2 ， 9, 10, 11 ， 12 ， 13 所述的单链脱氧寡核苷酸，它们可以和各种抗肿瘤制剂联合应用治疗肿瘤。
    16 、 按照权利要求 1 ， 2 ， 9, 10, 11 ， 12 ， 13 所述的单链脱氧寡核苷酸，它们可以和肿瘤治疗用细胞联合应用治疗肿瘤。
    17 、 按照权利要求 1 ， 2 ， 4, 9 ， 12 所述的单链脱氧寡核苷酸，它们能 与 药物学可接受的载体组成药物 组合物应用并采用 有效剂量。
    18 、按照权利要求 1 ， 2 ， 4, 9 ， 12 所述的单链脱氧寡核苷酸，它们通过给药途径应用于个体。
    19 、按照权利要求 1 ， 2 ， 4, 9 ， 12 所述的单链脱氧寡核苷酸，它们可以通过 治疗装置或 递送载体 应用于个体。
    20、 按照权利要求 1 ， 2 ， 3 所述的单链脱氧寡核苷酸以及与 CTLA-4 mRNA 3'UTR 序列互补并具有干扰抑制 CTLA-4 功能的寡核苷酸。
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