CN110229813A - Oligonucleotides with vaccine adjuvant effect and oncotherapy effect - Google Patents

Oligonucleotides with vaccine adjuvant effect and oncotherapy effect Download PDF

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CN110229813A
CN110229813A CN201810178426.4A CN201810178426A CN110229813A CN 110229813 A CN110229813 A CN 110229813A CN 201810178426 A CN201810178426 A CN 201810178426A CN 110229813 A CN110229813 A CN 110229813A
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王丽颖
于永利
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Suzhou Send Biotechnology Co Ltd
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Abstract

The present invention provides two kinds of single-stranded deoxy-oligonucleotides, they are used as the adjuvant of microorganism vaccine and tumor vaccine, show anti-infective and antineoplastic action, can be used alone or treat tumour with other anti-tumor agent use in conjunction.

Description

Oligonucleotides with vaccine adjuvant effect and oncotherapy effect
Technical field:
The present invention relates to the purposes of two kinds of oligonucleotides as well as vaccine adjuvant and treatment tumour.
Background technique
Transforminggrowthfactor-β2 (transforming growth factor beta 2, TGF-β 2) be conversion growth because The member of son-β cell factor superfamily (TGF-β Cytokine Superfamily).TGF-β superfamily have 40 cells because Son has TGF-β 1, TGF-β 2 and TGF-β 3 [Rider CC, et in these cell factors with TGF name al.Molecules.2017 Apr 29；22 (5) .pii:E713；Travis MA, et al.Annu Rev Immunol.2014；32:51-82], they are also usual signified TGF-β.TGF-β 1 after translation synthesis, 2 He of TGF- β TGF-β 3 is precursor molecule, by signal peptide, N-terminal segment (latency-associated peptide, LAP) and the end C- Segment (short C-terminal fragment) composition.The C- terminal fragment released forms dimer, has for mature Biological activity cell factor TGF-β.In amino acid levels, TGF-β 1, TGF-β 2 and TGF-β 3 have the homology of 71-79% [Travis MA, et al.Annu Rev Immunol.2014；32:51-82].
The TGF-β receptor (T β R) of the TGF-β that can transduce signal is the tetramer, contains 2 T β RI and 2 T β RII molecules, T β RI It is serine/threonine kinase with T β RII.Once being formed, T β RII kinase activation, identification T β RI's is specific for tetramer compound Serine residue makes it activate [Ahmed S, et al.J Clin Med.2017 Jan 5；6 (1) .pii:E5].TGF-β2 The signal transduction of activation relies on β-glycan (β-glycan), and β-glycan is also referred to as TGF-β RIII.β-glycan assists TGF-β 2 In conjunction with TGF-β RII [Travis MA, et al.Annu Rev Immunol.2014；32:51-82].After TGF-β receptor activation Pass through Smad protein transduction signal.Smad albumen has a three classes, regulation Smads (receptor-regulated Smads, R-Smad), common mediator Smad (co-Smad) and inhibition Smad (inhibitory Smad, I-Smad). Smad2 and Smad3 is R-Smads, is the direct substrate of T β RI.TGF-β combines activation TGF-β receptor, the TGF-β RI of activation Cell inner end raise Smad2 and Smad3, and directly by its phosphorylation.Smad2/3 and Smad4 (co-Smad) shape of phosphorylation At tripolymer (Smad compound).The indexing of Smad compound regulates and controls the expression of several genes to nucleus.The I-Smad of activation (Smad6/7) by promoting T β RI degradation, inhibit Smad2/Smad3 phosphorylation, Smad compound combination chromatin is inhibited to bear Regulate and control signal transduction [Ahmed S, the et al.J Clin Med.2017 Jan 5 of TGF-β；6 (1) .pii:E5；Travis MA, et al.Annu Rev Immunol.2014；32:51-82].TGF-β can pass through classical pathway (canonical ) and non-classical approach (noncanonical pathway) transduction activation signal [Huang JJ, et al. pathway Biochem Soc Trans.2016 Oct 15；44 (5): 1441-1454].In people and mouse, T β RIII is mainly expressed huge Phagocyte, neutrophil leucocyte, megacaryocyte, dendritic cells and T cell [Ortega-Francisco S, et al.Biochem Biophys Res Commun.2017 Dec 9；494 (1-2): 82-87], it prompts, these cells are also that the target of TGF-β 2 is thin Born of the same parents.
TGF-β has down regulation to immune response, adjusts a variety of adaptive immune response cells and innate immune response Cell, including dendritic cells, T cell, B cell, NK cell, inherent immunity lymphoid cell (innate lymphoid ) and granulocyte [Kelly A, et al. Adv Immunol.2017 cells；134:137-233].TGF-β 2 lowers main group Knit the expression of compatible compound (major histocompatibility complex, MHC) molecule.Condition of culture in vitro Under, TGF-β 2 (1,5, or 10ng/ml) significantly lowers horse mescenchymal stem cell (mesenchymal stem cells, MSCs) The MHC-I class molecule and MHC-II class molecule on surface, can also partial agonist IFN-γ-to MSCs MHC-I class molecule and MHC- The up-regulation of II class molecule acts on [Berglund AK, et al.Front Vet Sci.2017 Jun 12；4:84].Experimental Allergic encephalitis (experimental allergic encephalomyelitis, EAE) mouse is turned down under TGF-β 2 is significant Spongiocyte expresses MHC-II class molecule [De Feo D, et al.J Clin Invest.2017Nov 1；127 (11): 3937-3953].TGF-β 2 can antagonism IFN-γ and TNF-d to the inducing action of rat astrocytes expression MHC-II class molecule [Schluesener HJ.J Neuroimmunol (1990) 27:41-7].The function of the interference antigen presenting cell of TGF-β 2.Bone The Dendritic Cells (BMDCs) in marrow source expresses T β R.In vitro under condition of culture, TGF-β 2 significantly inhibits CD40L stimulation BMDCs expression, secretion IL-23 [De Feo D, et al.J Clin Invest.2017Nov 1；127 (11): 3937- 3953].This inhibition almost can completely eliminate [De Feo by TGFBRI or TGFBRII tyrosine kinase inhibitor SB431542 D, et al.J Clin Invest.2017 Nov 1；127 (11): 3937-3953].IL-23 is a kind of mucosa-immune induction Agent.In mouse, it is added to full influenza virus vaccine (whole influenza virus vaccine, WIV) (IL- of IL-23 23-WIV) effect for inducing virus-specific nasal cavity IgA through mucosa-immune significantly improves.The serum of mouse is immunized in IL-23-WIV With may occur in which high-caliber resisiting influenza virus IgA in nasal cavity eluate.This prompt inhibits TGF-β 2 may be by releasing IL-23 The inhibition of secretion acts on to play adjuvant.The expression of the inhibition cell factor of TGF-β 2.Under basis and inflammatory conditions, TGF-β 2 Inhibit horn cell express IL8, CXCL5, CXCL1, chemokine (C-C motif) ligand 20 (CCL20), IL1A, IL1B [Li D, et al.J Clin Invest.2015 Aug 3；125 (8): 3008-26].In immature small intestine, TGF-β 2, which weaken LPS-, induces IL-6, biological activity [Nguyen DN, the et al.Am J Physiol of IL-1 β and TNF-α Gastrointest Liver Physiol.2014Oct 1；307 (7): G689-99].2 suppressor inducer T lymphocyte of TGF-β (regulatory T cell, Treg).Antigen presenting cell (the antigen of TGF-β 2 is expressed in mucous membrane and ocular tissue Presenting cells, APCs) it can induce Treg [Mir FA, et al.Immunology.2015 Dec；146 (4): 547- 56].It is proved with anti-T β RIII enclosed experiment, the signal that T β RIII is mediated promotes TGF β-dependence CD4+T cell is to differentiation [Ortega-Francisco S, et al.Biochem Biophys Res Commun.2017 Dec 9；494 (1-2): 82- 87]。
TGF-β 2 is to promote the tumor factor.95% high-grade glioma cell expresses high-caliber TGF-β 2, expression and The invasiveness of glioma cell is positively correlated [Zhang C, et al.J Exp Clin Cancer Res.2017 Nov 16；36 (1): 162].TGF-β 2 is named as T cytostatic factor (the glioblastoma-derived T-cell in glioma source suppressor factor).In glioma patient, TGF-β 2 induces immunosuppressive condition and immune surveillance function missing.Blood Progress, poor prognosis [Hau P, the et al. Curr Pharm of the horizontal high gliomatosis patient disease of middle TGF-β 2 Biotechnol.2011Dec；12 (12): 2150-7].The horizontal muscle with bladder cancer of TGF-β 2 in Bladder Cancer infiltrates Degree is positively correlated.TGF-β 2 can be used as biomarker [the Mahdavinezhad A, et of early diagnosis of tumor al.Investig Clin Urol.2017 Mar；58 (2): 140-145].In carcinoma of mouth, cancer associated fibroblast cell The TGF-β 2 of (Cancer-associated fibroblasts) secretion may be maintained by the adherency weakened between spongiocyte Cancer cell of oral cavity canceration phenotype [Cirillo N, et al.Carcinogenesis.2017 Jan；38 (1): 76-85].TGF- β -2 induces epithelial cell interstitial conversion.Epithelial cell interstitial conversion (epithelial-mesenchymal transition, It EMT) is tumor cell invasion [Cirillo N, et al.Carcinogenesis.2017 Jan；38 (1): 76-85] and migrate Critical process.
Summary of the invention:
1, the present invention provides two kinds of single-stranded deoxy-oligonucleotides (abbreviation oligonucleotides), sequence such as sequence tables<400>1 Shown in<400>2.They can reduce transforminggrowthfactor-β2 (transforming growth factor beta 2, TGF-β 2) mRNA, inhibit 2 albumen of TGF-β expression and weaken, inhibits TGF-β 2 mediation immunosuppressive action, Jin Erzeng Strong individual shows anti-infective and antitumor effect to the immune response of pathogenic microorganisms and tumour cell.
2, both oligonucleotides can pass through various chemical modifications or be changed structure.
3, both oligonucleotides are used as the adjuvant of pathogenic microorganisms vaccine and tumor vaccine, have anti-infective and anti- Function of tumor.
4, both oligonucleotides can be used for oncotherapy.
5, it is right can to enhance individual with other adjuvants or inherent immunity reinforcing agent use in conjunction for both oligonucleotides The protective immune response of pathogenic microorganisms and tumour cell.
6, both oligonucleotides can treat tumour with other anti-tumor agents and oncotherapy cell use in conjunction.
Term in invention:
Except non-specifically emphasizing, term in the present invention has and can be understood by technical staff in fields of the present invention Ordinary meaning.If there is the conflict in meaning, the explanation in the present invention should be deferred to, defines or illustrates.
" oligonucleotides ": the molecule that oligonucleotides is made of multiple nucleotide, the quantity of nucleotide can be several Or tens.Nucleotide (nucleotide) is the basic composition unit of nucleic acid and oligonucleotides.Nucleotide is by nucleosides (nucleoside) it is formed with phosphoric acid.Nucleosides is made of pentose (pentose) and base (base).Pentose includes ribose and takes off Oxygen ribose.Pentose molecule connects to form nucleosides (nucleoside) with base.Nucleosides is connected by phosphate group and forms nucleotide. Nucleotide is keyed to form oligonucleotides by di-phosphate ester.The base for forming nucleosides includes pyrimidine and purine.Pyrimidine includes chest Gland pyrimidine (thymine, be abbreviated as T or t) and cytimidine (cytosine is abbreviated as C or c).Purine includes adenine (adenine, be abbreviated as A or a) and guanine (guanine is abbreviated as G or g).Base in oligonucleotides can be rare Base.Rare bases include but is not limited to 5-hydroxymethyl cytosine, 7- methyl guanine and 5-hydroxymethyl cytosine.Oligonucleotides It can be single-stranded, double-strand, annular or the molecule with ring structure.In the present invention, oligonucleotides (Oligonucleotide, ODN it) can be replaced with its english abbreviation ODN.Nucleotide arrangement sequence sequence in oligonucleotides constitutes its primary structure, This sequence is also referred to as nucleotide sequence.Nucleotide sequence can be represented with base sequence, and therefore, the sequence of nucleotide is also referred to as alkali Basic sequence.The sequence of deoxy-oligonucleotide can indicate that T or t represent thymidine, and C or c represent born of the same parents with the english abbreviation of base Pyrimidine, A or a represent adenine, and G or g represent guanine.
" oligonucleotides provided by the invention ": referring to two oligonucleotides as described in Example 1, be named as TIO1 and TIO3.TIO1 has the sequence as shown in sequence table<400>1, and TIO3 has the sequence as shown in sequence table<400>2.This hair The oligonucleotides of bright offer is single-stranded deoxy-oligonucleotide, referred to as oligonucleotides (ODN).
" chemical modification ": compared with natural DNA, oligonucleotides provided by the invention can be modified by sulphation (chemical modification).The chemical modification of oligonucleotides is to send out its covalent structure and introducing or removing any chemical gene Raw the phenomenon that changing or method.The chemical modification position of oligonucleotides can occur in phosphodiester bond, ribose and base.Few core The chemical modification of thuja acid can occur at 5 ' ends or 3 ' ends, can carry out in synthesis or after synthesis.Chemical modification of the present invention Including but not limited to the modification of oligonucleotide backbone, such as thio-modification (the non-bridge of phosphoric acid in internucleotide phosphate diester linkage Oxygen atom is replaced by sulphur atom) and substitution modification (substitution including alkyl, aromatic radical or other any chemical groups).Few core The chemical modification of thuja acid further includes base substitution and base modification, and the base of substitution can be spreading out for rare bases or various bases Biology.The chemical modification of oligonucleotides further includes connecting one or more nucleotide and/or other at its 5 ' end and/or 3 ' ends What chemical group.
" UTR ": referring to the non-translational region (untranslated region, UTR) of mRNA molecule, is located at mRNA molecule and compiles The both ends in code area, be located at 5 ' ends is referred to as 5 ' end UTR, and be located at 3 ' ends is referred to as 3 ' end UTR.
" 2 mediated immunity inhibiting effect of TGF-β ": oligonucleotides provided by the invention, which can inhibit, weaken the mediation of TGF-β 2 exempts from Epidemic disease inhibiting effect.The beta 2 mediated immunosuppressive action of TGF- refers to the suppression for the immune response that TGF-β 2 induces invasive organism Production is used, and also refers to the inhibiting effect to tumour cell immune response.It reduces, interference TGF-β 2mRNA can inhibit TGF-β 2 to mediate Immunosuppressive action；Inhibit, reduce the generation of 2 albumen of TGF-β can also inhibit 2 mediated immunity inhibiting effect of TGF-β.TGF-β2 It is immunosuppressive factor.Inhibit 2 mediated immunity inhibiting effect of TGF-β that can enhance the individual immune response to invasive organism, Generate anti-infectious function.Inhibit 2 mediated immunity inhibiting effect of TGF-β that can break individual to the immune tolerance of tumour cell, enhancing The anti-tumor capacity of individual.By inhibiting 2 mediated immunity inhibiting effect of TGF-β to weaken, inhibiting the beta 2 mediated immunosupress of TGF- Effect can produce antitumor action [Kim S, et al.Tumour Biol.2016Aug；37 (8): 11397-407].
" level for reducing TGF-β 2mRNA ": oligonucleotides provided by the invention can reduce immunocyte TGF-β 2mRNA's It is horizontal." reduction " and " reduction " has the same meaning.The level for reducing TGF-β 2mRNA can weaken, TGF-β 2 is inhibited to mediate Immunosuppressive action, thus show the humidification to pathogenic microorganisms and tumour cell immune response.With reduction TGF-β The horizontal preparation of 2mRNA can become the adjuvant of pathogenic microorganisms vaccine or tumor vaccine, can also be utilized separately for controlling for tumour It treats.
" expression for inhibiting 2 albumen of TGF-β ": oligonucleotides provided by the invention can inhibit the expression of 2 albumen of TGF-β.Suppression System, the identical meaning that has for reducing 2 albumen of TGF-β inhibit the translation of TGF-β 2mRNA and inhibit the table of 2 albumen of TGF-β Up to also there is identical meaning.The reduction of 2 albumen of TGF-β can weaken, inhibit 2 mediated immunity inhibiting effect of TGF-β, thus show To the humidification of pathogenic microorganisms and tumour cell immune response.Preparation with the expression for inhibiting TGF-β 2 can be used as cause The adjuvant of sick microorganism vaccine or tumor vaccine can also be utilized separately for the treatment of tumour.
" immune response ": oligonucleotides provided by the invention can be by enhancing to pathogenic microorganisms antigen and tumour antigen Immune response.Immune response (immune response) and immune response meaning having the same.Immune response is individual Immunocyte include bone-marrow-derived lymphocyte, T lymphocyte, NK cell, gamma delta T cells, NKT cell, dendritic cells, macrophage and Granulocyte etc. is to antigen or other stimulation [the relevant model molecule of such as pathogen (pathogen associated Molecular pattern, PAMP) and relevant model molecule (the damage associated molecular of damage Pattern, DAMP)] reaction made.Immune response the result is that selective destruction or remove invasion pathogenic microorganisms or The tumour cell of interior life.Immune response includes innate immune response and adaptive immune response.Adaptive immune response includes cell Immune response and humoral immune response.The immune response excited using vaccine made of pathogenic microorganisms antigen can make by Immune individual obtains the ability for resisting pathogenic microorganism infection.It immune is answered using what vaccine made of tumour antigen was excited Oncotherapy effect can be occurred in the individual being immunized by answering.Promote individual that anti-sense can occur to the immune response of pathogenic microorganisms Dye effect.Promote individual that antitumor action can occur to the immune response of tumour cell.Tool can be generated during immune response There is the cell factor of immunosuppressive action.TGF-β 2 is a kind of cell factor with immunosuppressive action.TGF-β 2 can inhibit Immune response.The expression for reducing the level, inhibition TGF-β 2 of TGF-β 2mRNA, which can promote, resists pathogenic microorganisms antigen or tumour Former immune response shows immunoprophylaxis or Immunotherapy.Oligonucleotides provided by the invention can be by enhancing to micro- life The immune response of object antigen and tumour antigen and be used as the adjuvant of microorganism vaccine and tumor vaccine or be used for controlling for tumour It treats.
" lymphocyte ": lymphocyte (lymphocyte), which refers to present in blood, lymph and the lymphoid tissue, not to be had The mono-nuclear leukocytes of phagocytic activity, including bone-marrow-derived lymphocyte (also referred to as B cell) and T lymphocyte (also referred to as T cell).T cell T cell (the CD4 of surface expression CD4 molecule can be divided into+T cell) and for surface expression CD8 molecule T cell (CD8+T is thin Born of the same parents).
“CD4+T cell ": oligonucleotides provided by the invention can increase specific C D4 in draining lymph node+T cell Quantity.CD4+T cell is the T cell of surface expression CD4 molecule, is helper lymphocyte T (Th).Th Help B Cells produce Raw antibody, also assists CD8+T cell removes killing virus infected cell and tumour cell.Stimulate CD4+T cell hyperplasia (is shown as Cell number increases) preparation can enhance individual to the immune response of pathogenic microorganisms and tumour cell.
“CD19+Lymphocyte ": oligonucleotides provided by the invention can increase specific C D19 in draining lymph node+Lymph The quantity of cell.CD19+Lymphocyte is the lymphocyte of surface expression CD19 molecule, is also referred to as CD19+Bone-marrow-derived lymphocyte Or CD19+Cell.Stimulate CD19+The preparation of lymphocytosis (showing as cell number to increase) can enhance individual to micro- life of causing a disease The humoral immune response of object and the adjuvant for being used as pathogenic microorganisms vaccine.
" antigen presenting cell " oligonucleotides provided by the invention can increase draining lymph node antigen presenting cell The quantity of (antigen presenting cells, APC).APC is can be by the peptide fragment from microbial antigen or tumour antigen It is presented on a kind of immunocyte that activation antigen specific T-cells are carried out on its surface, including dendritic cells (dendritic cell, DC), macrophage and bone-marrow-derived lymphocyte etc..CD11c is the surface marker of DC.The quantity for increasing draining lymph node APC promotes to resist The activation of former specific T-cells, and then enhance the immune response to pathogenic microorganisms and tumour cell.
" CD86 ": oligonucleotides provided by the invention can raise, dendritic cells is maintained to express CD86.CD86 is a kind of expression In antigen presenting cell (antigen presenting eells, APC) or the costimulatory molecules of tumor cell surface (costimulatory molecules).During immune response, CD86 can start the second letter of activated T lymphocytes Number.T lymphocyte activation is key condition of the excitation individual to pathogenic microorganisms and tumour cell immune response.T lymphocyte Activation needs to obtain two activation signals.First activation signal passes through T cell antigen receptor (T cell from T lymphocyte Antigen receptor, TCR) identification APC or target cell surface Antigenic Peptide-MHC compound.Second activation signal comes from T Lymphocyte passes through CD28 molecular recognition, in conjunction with the costimulatory molecules including CD86 of APC or target cell surface.Second Activation signal is also referred to as costimulatory signal (costimulatory signals).Only obtain the T lymphocyte of the first activation signal It cannot sufficiently activate, or even immune tolerance state can be entered.After only obtaining the first and second activation signals simultaneously, T lymph is thin Born of the same parents could sufficiently be activated and [the Sharma P.et al.Science.2015Apr 3 that functions；348 (6230): 56-61； Greenwald RJ, et al.Annu Rev Immunol.2005；23:515-48].TGF-β 2 can reduce the costimulation of APC Molecule, thus weaken the second signal of T lymphocyte activation.The level of TGF-β 2mRNA inhibits the expression of TGF-β 2 that can maintain Or the level of CD86 is improved, enhance the second signal of t cell activation, and then promote to exempt from pathogenic microorganisms and tumour cell Epidemic disease response enhances the anti-infective and antitumor ability of individual.
" CD28 ": CD28 is the albumen expressed on T cell surface, can recognize the CD86 molecule of Antigen Presenting Cell surface. After identifying CD86 molecule, CD28 provides costimulatory signal (co-stimulatory signals) to T cell, and also referred to as second Signal.
" T cell receptor ": T cell receptor (T cell receptor, TCR) be express antigen on T cell surface by Body is heterodimer transmembrane protein.TCR by the variable area (V) and constant (C) district's groups at.The area V is the knot of TCR identification epitope Structure domain.TCR is unable to Direct Recognition epitope, can only identify that the Antigenic Peptide-MHC of antigen presenting cell or target cell surface is (main Want histocompatibility complex) molecular complex.
" MHC molecule ": MHC molecule is major histocompatibility complex (major histocompatibiltiy Complex, MHC) gene coding proteinaceous molecule.MHC molecule includes MHC I class molecule and MHC II class molecule.
" activation of T lymphocyte ": the activation of T lymphocyte needs to come two signals.First signal is identified by its TCR APC or target cell (can be by CD8+T cell killing cell) surface Antigenic Peptide-MHC molecule compound obtain；Second signal, Also referred to as costimulatory signal is obtained by the B7 molecule of its CD28 molecular recognition APC or target cell surface.In the effect of dual signal Under, T cell starts activation signal Signal Transduction Pathways, and then proliferation, differentiation function.Only the first signal and without second activate Signal can make T cell enter not response status.T lymphocyte reaction occurs after T lymphocyte activation.
" T lymphocyte reaction ": T lymphocyte reacts (T lymphocyte response) and " T lymphocyte activity " (T lymphocyte activity) or " function that T lymphocyte is exercised " is the term that can be interchanged in the present invention.T leaching Bar cell effect includes T lymphocyte hyperplasia and/or is divided into helper lymphocyte T (Th), cytotoxic T lymphocyte It (Tc) or Autoimmune disease (Treg), also include providing signal to bone-marrow-derived lymphocyte from Th it is assisted to generate antibody, by Tc Killing target cell adjusts the function of other immunocytes with soluble factor such as cell factor is discharged.Th is CD4+T cell, Tc is CD8+T cell.Upon activation, CD4+T cell Help B Cells generate antibody, assist CD8+T cell kills target cell such as Virus infected cell and tumour cell.Upon activation, CD8+T cell can kill virus infected cell and tumour cell.Promote, It maintains t cell activation that can enhance individual and anti-infectious function is generated to the immune response of microbial antigen, can also individual be promoted to exempt from The immune response of epidemic disease cells against tumor cells generates oncotherapy effect.It reduces the level of TGF-β 2mRNA, inhibit TGF-β 2 Expression can enhance T lymphocyte reaction.Oligonucleotides provided by the invention can be by enhancing T lymphocyte increased response to cause The immune response of sick microorganism and tumour cell and be used as the adjuvant of pathogenic microorganisms vaccine and tumor vaccine or be used to swell The treatment of tumor.
" individual ": individual (subject or individual) in the present invention refers to people and inhuman vertebrate.
" target cell ": target cell (Target cell) refer to individual can by immune cells attack, killing or the cell of effect, It can be tumour cell, virus infected cell, can also refer to the cell acted on by oligonucleotides provided by the invention.
" tumour ": the tumour that defines of " tumour " i.e. modern medicine in the present invention can be divided into benign tumour and pernicious swollen Tumor.Tumour (tumor) and cancer (cancer) may be used interchangeably, and have the same meaning.Tumour includes solid tumor, soft group Knit sarcoma and marrow sample or lymphoid system tumour.Oligonucleotides provided by the invention can enhance individual and tumour antigen is immunized Response generates antitumor action, and the tumour being related to includes but is not limited to: cancer of the esophagus, gallbladder cancer, bladder cancer, breast cancer, colon Cancer, colorectal cancer, adrenal cortical tumor, kidney, liver cancer, lung cancer, oophoroma, cervix cancer, uterine cancer, carcinoma of vagina, pancreas Cancer, the carcinoma of the rectum, prostate cancer, gastric cancer, cutaneum carcinoma, melanoma, brain tumor, glioma, bone tumour, sarcoma, carcinoma of penis, view Nethike embrane blastoma, leukaemia, lymthoma and myeloma.
" treatment ": the symptom and simultaneously of disease (such as tumour) is prevented or delayed including application oligonucleotides provided by the invention Send out the appearance of disease.Treatment is also possible to preventative.The treatment of tumour is also referred in the progress of individual control tumour, tumour is extended The life cycle of patient, quality of making the life better mitigate symptom, even are eliminated tumor regression, contain metastases.? " oncotherapy " and " antitumor action " or " treatment tumour " has identical meaning in the present invention.Antitumor action includes to swollen The treatment of tumor also includes the prevention for occurring to tumour, recurring and shifting.Oligonucleotides provided by the invention can be used for tumour Treatment.
" antigen ": oligonucleotides provided by the invention can enhance individual to the immune response of antigen.Antigen is can be by B cell Receptor or T cell receptor identify and excite the substance of individual adaptability immune response (adaptive immune response) Or molecule.Antigen can come from the outside of individual, such as microbial antigen；Also the inside of individual, such as tumour antigen be may be from.It is micro- Biological antigens are can be identified by B-cell receptor or T cell receptor on microorganism and excite individual adaptability immune response The substance or molecule of (adaptive immune response).It is answered using the vaccine that microbial antigen is prepared into individual It can be made to obtain the immunity to the microorganism after, be protected it when being contacted again same or like pathogen. It can be excited after to individual applications using vaccine made of tumour antigen to the adaptive immune response of tumour cell and then is produced Raw oncotherapy effect.Antigen can be produced from microorganism or tumour cell extraction or using recombinant DNA technology or it The synthesis of its method.Tumor cell lysate contains kinds of tumors antigen, the antigen being used as in tumor vaccine.
" vaccine ": it is to be used for made of antigen that vaccine (vaccine), which is with attenuation or the causal organism killed or its component, The biological products of artificial active immunity.Antigen and adjuvant are the main components of vaccine.The purpose of vaccine inoculation is to make individual acquisition It is protected it when touching corresponding pathogen again in turn the immunity of pathogen.Oligonucleotides provided by the invention Its immune efficacy can be enhanced with vaccine for man use in conjunction, these vaccines include but is not limited to that can prevent following infectiousness diseases Vaccine [Stanley A.Plotkin, Walter A.Orenstein, the Paul A.Offit, Vaccines, Sixth of disease Edition, An imprint of Elsevier Inc.2013, ISBN-13:9781455700905]: diphtheria, tetanus, Huang Pyreticosis, pertussis, haemophilus influenzae b infection, polio, measles,mumps,rubella, typhus, rabies, wheel Shape virus infection, hepatitis A, hepatitis B, Hepatitis E, influenza, tuberculosis, malaria, cholera, varicella, popular B-mode brain Inflammation, tick-borne encephalitis, cholera, Lyme disease, pneumococcal infection, meningococcal infection, typhoid fever, smallpox, anthrax, ebola disease Poison infection, green monkey virus (Marburg viruses, MARV) infection and Venezuelan equine encephalitis virus (Venezuelan Equine encephalitis virus, VEEV) infection.Vaccine of the present invention also includes tumor vaccine.Tumour antigen It is the main component of tumor vaccine with adjuvant.Tumor vaccine can treat or prevent tumour.Antigen in tumor vaccine can be swollen Tumor antigen, the cell for offering tumour antigen, the cell or tumor cell lysate for expressing tumour antigen.Widow provided by the invention Nucleotide can enhance the effect of its prevention and/or treatment tumour with tumor vaccine use in conjunction, and this kind of vaccine includes but unlimited In the Human-papilloma Vaccine of: cervix cancer prevention, the Provenge vaccine for treating prostate cancer, with various tumours Vaccine made of antigen, the vaccine made of various tumour cells and the vaccine made of tumor cell lysate.The present invention mentions The oligonucleotides of confession can enhance its immune efficacy with animal vaccines use in conjunction, these vaccines include but is not limited to can be pre- Prevent the vaccine of following communicable diseases: Schweineseuche, pig blue-ear disease (porcine reproductive and respiratory syndrome), swine fever, pseudorabies Disease, Infection of Porcine circovirus, porcine parvovirus infection, Streptococcus suis, transmissible gastroenteritis of swine, swine enzootic pneumonia, the bloodthirsty bar of secondary pig Bacterium infection, brickpox, swine plague, atrophic rhinitis, transmissible gastroenteritis of swine, pig encephalitis, swine flu, pig cloth Lu Shi disease, son Diarrhea of pigs, pig parainfluenza virus infection, swine flu, mycoplasma hyopneumoniae infection, hydropsy for baby pigs, Salmonella choleraesuls, pig Epidemic diarrhea, swine plague；Ox blackleg, ox anthrax, Bovine Ephemeral Fever, bovine pasteurellosis, B.abortus infection, ox are broken Wind, the infection of ox clostridieum welchii, the infection of ox clostridium botulinum, ox aftosa, ox paratyphoid, cowpox, ox diarrhea；Sheep braxy, sheep are sudden Subcutaneous ulcer, sheep enterotoxemia, lamb dysentery, sheep black disease (infectious necrotic hepatitis), goatpox disease, sheep tetanus, braxy disease, Lamb colibacillosis, Ovine abortion Chlamydia infection, sore mouth virus, infectious pleuropneumonia in sheep, Brucella ovis disease, mutton poison Clostridium nosotoxicosis, sheep pox, sheep hammer seedling diseases, sheep rabies, sheep aftosa, sore mouth, ovine blue tongue；Contagious equine abortion, Equine influenza；Pig horse cattle and sheep dog encephalitis；Newcastle disease, infectious bursal disease, Marek's disease, bird flu, avian cholera, Chicken pox, chicken metachromia bronchitis, chicken viral hepatitis, chicken egg drop syndrome；Duck influenza, riemerella anatipestifer seedling, duck plague, Duck infectious serositis, duck E. coli；Gosling plague, goose paramyxovirus, goose influenza；Rabies, canine distemper, dog are tiny Virosis, canicola fever, canine infectious hepatitis, Infectious Tracheobroncheitis, dog parainfluenza virus, dog encephalitis and dog nest cough Vaccine.Oligonucleotides provided by the invention can enhance the effect of vaccine.
" tumour antigen ": oligonucleotides provided by the invention may be used as the adjuvant of tumour antigen, enhance to tumour antigen Immune response.In the present invention, tumour antigen (Tumor antigen), tumor-cell antigen (tumor cell Antigen) and tumor associated antigen (tumor associated antigen, TAA) can exchange application, there is identical contain Justice.Tumour antigen can excite anti-tumor immune response, can produce oncotherapy effect for the immune response of tumour antigen.Tumor Antigen includes but is not limited to: BAGE (B melanoma antigen), GAGE (G antigen 12B/C/D/E), MAGE (melanoma antigen-encoding gene),NY-ESO-1；CEA(carcinoembryonic antigen), gp100(glycoprotein 100)、Melan-A(melanoma antigen recognized by T cells 1)、PSA (prostate-specific antigen), tyrosinase, HER2 (human epidermal growth factor Receptor 2, hTERT (telomerase transcriptase), p53, survivin, β-catenin-m, HSP70-2/ m(heat shock-related 70kDa protein 2mutated)、KRAS、GM2(ganglioside GM2)、MUC1 (mucin-1), antigen, the human papilloma virus base of hepatitis B virogene coding for antigens, hepatitis c virus gene coding Antigen (the Epstein Barr Virus encoded by coding for antigens (such as L1 albumen, E6 albumen and E7 albumen) and Epstein-Barr virus gene Peptides) [Zielinski C, et al.Nat Rev Clin Oncol.2014Sep；11 (9): 509-24；AdamsJL, et al.Nat Rev Drug Discov.2015Sep；14 (9): 603-22].Tumour antigen further includes swelling entirely through inactivation treatment Oncocyte and tumor cell lysate.Tumour antigen can be the neoantigen (neo-antigen) expressed by gene mutation, The heat shock protein tumour cell peptide complexes for being also possible to the idiotype antigen of B cell tumour and being extracted from tumour cell [Suot, R&Srivastava, P (1995) Science 269:1585-1588；Tamura, Y.et al. (1997) Science 278:117-120].
" tumor vaccine " oligonucleotides provided by the invention is used as the adjuvant of tumor vaccine, enhances its oncotherapy Effect.Tumor vaccine, which refers to, can induce the biological products that individual generates antitumor adaptive immune response.It is of the present invention swollen Tumor vaccine includes using tumour antigen, tumor cell lysate, tumour cell and offering the cell of tumour antigen with certain agent Vaccine made of type.This kind of including but not limited to following [Zielinski C, the et al.Nat Rev Clin of vaccine Oncol.2014 Sep；11 (9): 509-24]: the tumor vaccine of prostate cancer is treated, the tumour antigen of use includes prostate Acid phosphatase (being used for sipuleucel-T),And PSA；The tumor vaccine of breast cancer is treated, use is swollen Tumor antigen includes polypeptide, MUC1 and WT1 (the Wilms tumour protein) antigen in the source HER2；The tumour epidemic disease for treating lung cancer Seedling, the tumour antigen of use include MUC1, melanoma-associated antigen-3 (MAGE-A3) polypeptide, Telomerase reverse transcriptase and EGF；The vaccine of melanoma is treated, the antigen of use includes β- catenin、gp100、MAGE-A3、MART1 (melanoma antigen recognized by T cells 1)、NY- ESO-1 and survivin；The tumor vaccine of pancreatic tumor vaccine is treated, the tumour antigen of use includes telomere enzyme peptide, allogeneic Tumour cell and mutation RAS synthetic peptide；The tumor vaccine of colorectal cancer is treated, the tumour antigen of use includes carcinomebryonic antigen (carcinoembryonic antigen, CEA), MUC1 and full tumour cell；The tumor vaccine of clear-cell carcinoma is treated, is used Tumour antigen include tumour cell RNA；Neoplastic hematologic disorder treatment use vaccine, the tumour antigen of use include WT1, MAGE, MUC1, PRAME (preferentially expressed antigen of melanoma) and idiotype antigen.It is provided by the invention Oligonucleotides can enhance the immune response to tumor vaccine.
" tumor cell lysate " oligonucleotides provided by the invention can enhance tumor cell lysate inducing antitumor and exempt from The effect of epidemic disease response is used as using tumor cell lysate as the adjuvant of the tumor vaccine of antigen.Tumor cell lysate can It is obtained by broken tumour cell, contains various tumour antigens.The method of broken tumour cell include but is not limited to multigelation, Ultrasonic treatment and Mechanical Crushing.Primary tumo(u)r, secondary or metastatic tumour cell may be used to prepare tumor cell lysate. Primary tumo(u)r and secondary or metastatic tumour cell are cultivated in vitro, it is thin to can get tumor cell line (tumor cell line) Born of the same parents, these tumor cell line cells can also be used to prepare tumor cell lysate.It is used to prepare tumor cell lysate Tumour cell can come from the individual of self, allogeneic or xenogenesis.
" adjuvant " oligonucleotides provided by the invention can be used in combination anti-to enhance microorganism with one or more adjuvants Former and tumour antigen immune efficacy.Adjuvant (adjuvant) is the substance that in vaccine and antigen is applied together, is had following Active (1) reduces the inoculation times of vaccine；(2) extend the immune duration of vaccine；(3) promoted by exciting innate immune response Into humoral immune response and cellullar immunologic response；(4) the cross protection immune response of antigen induction is extended；(5) enhance weak immune Immune response of such as aged individual or immunocompromised subject of response individual to antigen；(6) dosage of antigen is reduced.Can and this Adjuvant [Stanley A.Plotkin, Walter the A. Orenstein, Paul of the oligonucleotides use in conjunction provided are provided A.Offit, Vaccines, Sixth Edition, An imprint of Elsevier Inc.2013, ISBN-13: 9781455700905] include but is not limited to: Alum adjuvant (by aluminium hydroxide or aluminum phosphate vaccine adjuvant as main component), AS04 adjuvant [Aluminium phosphate adjuvant of absorption MPL (the Gram-negative bacteria lipopolysaccharides through chemical detoxification)], MF59 adjuvant are (a kind of Water-in-oil emulsifier is oily phase with squalene), AS03 adjuvant (a kind of take squalene as the oil-in-water adjuvant of oil phase), AF03 Adjuvant (a kind of take squalene as the oil-in-water emulsifiers of oily phase), 51 adjuvant of Montanide ISA (be oil phase with mineral oil Water-in-oil emulsifier), Freund's adjuvant, incomplete Freund's adjuvant, virosomes adjuvant (virosome adjuvant), N- oxygen Change polyethylene piperazidine derivative (polyoxidonium) adjuvant, Toll-like receptor agonist, pathogen associative mode Molecule and its analogies (analog), damage associative mode molecule and its analogies (analog), ring dinucleotides and its simulation Object (analog), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-12, oil Property emulsifier (AS02, AS03, AF03, MF59 and MontanideTMISA-51), white-oil adjuvant, MontanideTM ISA- 206 adjuvants, QS21, polylactide co-glycolide, virus (Adenovirus, vaccinia, fowlpox) carrier Adjuvant, BCG vaccine (BCG, bacillus Calmette-Gu é rin), the double-stranded RNA including poly (I:C) and its similar Object (analogies), the lipoid A analog including MPL, flagellin, including Imiquimod and R848 Imidazoquinolines, CpG ODN, the saponin including QS21, the C- type agglutinin ligand including TDB, packet Include CD1d ligand including α-galactosylceramide, AS01 (containing MPL, QS21 and liposome), AS02 (containing MPL, QS21 and emulsifier), AS15 (contain MPL, QS21, CpG ODN and liposome), GLA-SE (containing GLA and emulsifier), IC31 (contain CpG ODN and cationic polypeptide), CAF01 (containing TDB and cationic-liposome) and ISCOMs (containing saponin and phosphatide) [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].
" inherent immunity activator ": oligonucleotides provided by the invention can be played with inherent immunity activator use in conjunction Anti-infective and antitumor action.Inherent immunity activator include relevant model molecule of pathogen and the like, Toll-like by Body agonist and the like, the relevant model molecule of damage and the like and ring dinucleotides and the like.Analog There is identical meaning with analogies.
" the relevant model molecule of pathogen ": oligonucleotides provided by the invention can model molecule relevant with pathogen (pathogen-associated molecular patterns, PAMPs) and the like use in conjunction come enhance cause a disease it is micro- The immune efficacy of biological antigens and tumour antigen plays anti-infective and antitumor action.PAMPs is the various conservative of microorganism Ingredient, including bacterium and fungal cell wall ingredient and viral nucleic acid.Inherent immunity cell passes through pattern recognition receptors (pattern-recognition receptors, PRRs) identifies PAMPs and is activated.PRR includes Toll-like Receptors (TLRs), nucleotide-binding oligomerization domain (Nod)-, leucine-rich repeat-containing receptors(NLRs)、RIG-I-like receptors(RLRs)、C-type lectin Receptors (CLRs) and AIM-2 like receptors；It also include the intracellular nucleic acid receptor of intracellular identification nucleic acid (intracellular sensors of nucleic acids), OAS albumen and cGAS [Iwasaki A et al.Nat Immunol.2015 Apr；16 (4): 343-53].
" Toll-like receptor agonist ": oligonucleotides provided by the invention can be with one or more Toll-like receptors (Toll-like receptors, TLRs) agonist use in conjunction enhances the immune of pathogenic microorganisms antigen and tumour antigen Effect shows anti-infective or anti-tumor activity.Can include with the TLR agonist of oligonucleotides use in conjunction provided by the invention But it is not limited to: TLR7 the and TLR8 agonist including Imiquimod and imidazoquinolines；Including 852A TLR7 agonist；TLR8 agonist including VTX-2337；Including IMO-2055, CPG 7909, MGN1703 and its TLR9 agonist including its CpG ODN (deoxy-oligonucleotide containing CpG)；TLR2/TLR4 including BCG vaccine (BCG) Agonist；Including OM-174, monophosphoryl lipid A, aminoalkyl glucosamine phosphates and TLR4 agonist including other lipoid A (lipid A) analogs；TLR9 excitement including viral nucleic acid, bacterial nucleic acid Agent；TLR5 agonist including bacterial flagellin, the TLR2/TLR6 agonist including zymosan；Including poly- TLR3 agonist including inosinicacid cytidylic acid (poly IC), viral double-stranded RNA or its analogies and include virus singly TLR7/TLR8 agonist [the Adams JL et al Nat Rev Drug Discov.2015 of chain RNA or its analogies Sep；14 (9): 603-22].
" damaging relevant model molecule ": oligonucleotides provided by the invention can with damage relevant model molecule (damage-associated molecular patterns, DAMPs) and the like use in conjunction is anti-to enhance microorganism Former and tumour antigen immune efficacy.DAMPs be discharged by damaging cells, can excite innate immune response own cells at Point.DAMPs can excite the innate immune response of body by PRR.These DAMP include but is not limited to: heat shock protein, HMGB1 (high-mobility group box 1), hyaluronan segment, glycans, glycoconjugates, ATP, (5 '-triphosphate of Adenosine), adenylate, uric acid, S100 albumen, heparin sulfate, Galectins, Nucleus DNA, N-formylated peptides, antimicrobial peptide, mitochondrial DNA and calreticulin [KryskoDV et al.Nat Rev Cancer.2012 Dec；12 (12): 860-75；Pouwels SD et al.Mucosal Immunol.2014 Mar；7 (2): 215-26].
" ring dinucleotides " this oligonucleotides provided by the invention can be with ring dinucleotides and its analogies use in conjunction To enhance the immune effect of microbial antigen and tumour antigen.Ring dinucleotides (Cyclic dinucleotides, CDNs) can From bacterium, can also be generated in mammalian cell.Bacterium CDNs includes but is not limited to c-di-GMP (cyclic Di-GMP, cdG), two adenylate of ring (cyclic di-AMP, cdA) and cyclic adenosine monophosphate-guanylic acid (cyclic AMP-GMP, cAMP-GMP).Bacterium CDN is a kind of PAMP, can exciting innate immune response.It also may occur in which CDN in mammalian cells, As cyclic guanylic acid-adenylate (cyclic guanosine monophosphate-adenosine monophosphate, cGAMP)[Wu J et al.Science.2013 Feb 15；339 (6121): 826-3].
" anti-tumor agent ": anti-tumor agent (anti-tumor agent) is applied to the system that can treat tumour after individual Agent, these preparations include but is not limited to tumor vaccine, immune stuck point mortifier, costimulation receptor activators, chemotherapeutics, put Treat preparation, hormone inhibitor or hormone, cell factor, oncotherapy antibody, small molecule kinase inhibitors, PARP mortifier, Angiogenesis inhibitors and oncolytic virus etc..
" immune stuck point molecule ": immune stuck point molecule (immune checkpoint molecules) is expression immune Protein molecular on cell starts the activation of inhibition signal path thus inhibition immunocyte after identifying its ligand.It is immune Stuck point molecule includes but is not limited to CTLA-4 molecule, PD-1 molecule, PD-L1/2 molecule, lymphocyte activator gene 3 (lymphocyte-activation gene 3, LAG-3), TIM-3 (T cell immunoglobulin and mucin domain-containing 3)、TIGIT(T cell immunoreceptor with immunoglobul in and ITIM domains) and BTLA (B and T lymphocyte attenuator).Close immune stuck point (immune Checkpoint blockade) refer to that inhibiting to be immunized stuck point molecule promotes in the function of immunocyte transmission inhibition signal, ties up The method for holding t cell activation.Immune stuck point enhancing individual is closed to the immune response of microbial antigen, shows anti-infectious function, Also it can promote the anti-tumor activity of individual immunity cell, performance oncotherapy acts on [Melero I, et al.Nat Rev Cancer.2015 Aug；15 (8): 457-72].
" immune stuck point mortifier ": oligonucleotides provided by the invention can do micro- with immune stuck point inhibitor use in conjunction The adjuvant of biovaccine or tumor vaccine can also treat tumour with immune stuck point mortifier use in conjunction.Immune stuck point mortifier (immune checkpoint inhibitor) is substance [the Melero I et that can inhibit immune stuck point molecular function al.Nat Rev Cancer.2015Aug；15 (8): 457-72].Inhibiting the function of immune stuck point molecule makes immune cell activation Signal highlighted, thus immunocyte is made to be in lasting state of activation.CD4 in sustained activation state+T cell Meeting Help B Cells generate antibody, also assist CD8+T cell kills target cell such as virus infected cell and tumour cell.In holding The CD8 of continuous state of activation+T cell can kill virus infected cell and tumour cell.Therefore, the system of t cell activation state is maintained Such as immune mortifier of adding some points of agent can improve antigen or vaccine in the immune efficacy of individual, can also occur at cancer patient (individual) Oncotherapy effect.The immune stuck point molecule that immune stuck point inhibitor inhibits includes but is not limited to CTLA4, PD1, LAG3 (Lymphocyte activation gene 3)、2B4(CD244)、BTLA (B and T lymphocyte Attenuator), TIM3 (T cell membrane protein 3) and A2aR (adenosine A2a receptor) [Pardoli DM.Nat Rev Cancer.2012 Mar 22；12 (4): 252-64].It can inhibit the antibody of immune stuck point function Belong to immune stuck point mortifier, including but not limited to CTLA-4 antibody, CD-1 antibody and CD-L antibody.
" immune stuck point molecular antibody ": oligonucleotides provided by the invention can be controlled with immune stuck point molecular antibody use in conjunction Treat tumour or the adjuvant as microorganism vaccine and tumor vaccine.Immune stuck point molecular antibody includes but is not limited to CTLA-4 anti- Body, PD-1 molecular antibody and PD-L1/2 antibody.CTLA-4 antibody is the antibody of energy specific bond CTLA4, can close CTLA- The inhibition signal of 4 transductions, activates T lymphocyte sufficiently by tumour antigen, can extend the life cycle of tumor patient. Ipilimumab is a kind of IgG1 CTLA-4 monoclonal antibody [Lipson EJ., the et al.Clin of full-length human Cancer Res.2011 Nov 15；17 (22): 6958-62], it is approved by the FDA in the United States 2011 to treat advanced melanoma Drug [Sharma P.et al.Science.2015 Apr 3；348 (6230): 56-61].Tremelimumab is another IgG2 CTLA-4 monoclonal antibody [Ribas A., the et al.Oncologist.2007 Jul of kind humanization；12 (7): 873-83].In clinical test, Tremelimumab be used to treat hepatocellular carcinoma [Sangro B., et al.J Hepatol.2013 Jul；59 (1): 81-8], gastric cancer and the cancer of the esophagus [Ralph C.et al.Clin Cancer Res.2010 Mar 1；16 (5): 1662-72].The antibody (Anti-PD-1 Antibodies) of PD-1 is the monoclonal antibody of PD1 molecule, tool There is the function for the immunocyte negative regulator signal transduction for inhibiting PD-1 to mediate, for stuck point mortifier is immunized.2014, two kinds of PD- 1 antibody (pembrolizumab and nivolumab) is approved by the fda in the United States for oncotherapy.Nivolumab is full humanization IgG4 monoclonal antibody, function [Topalian SL et al.Curr Opin that is combinable and inhibiting PD-1 Immunol.2012Apr；24 (2): 207-12], it be used to treat melanoma, lung cancer in non-cellule type, oophoroma and kidney [Ito A et al.Biomed Res Int.2015；2015:605478].Pidilizumab (CT-011) is humanization IgG- 1 κ monoclonal antibody, function that is combinable and inhibiting PD-1, be used to treat dispersivity large B cell lymphoid tumor and folliculus lymph Tumor.In addition, the antibody of PD-1 further includes Pembrolizumab (MK-3475), it is combinable and inhibits PD-1 function IgG-4 κ monoclonal antibody [Ito A et al. Biomed Res Int.2015；2015:605478].Anti- PD-L antibody is It identifies, in conjunction with the antibody of PD-L1 or PD-L2.It can be blocked to combine the PD-1 on immunocyte surface after combining PD-L, thus Blocking immunity cell-stimulating inhibits the transduction of signal, maintains the state of activation of immunocyte, and then enhances individual to antigen or swell The immune response of oncocyte.A variety of anti-PD-L1 antibody (Anti-PD-L1 Antibodies) are demonstrated by oncotherapy effect, These antibody include but is not limited to BMS-936559, MPDL3280A, MEDI4736 and MSB0010718 [Ito A, et al.Biomed Res Int.2015；2015:605478].BMS-936559 is that the anti-PD-L1 monoclonal of full humanization IgG4 is anti- Body be used to treat melanoma, non-small cell lung cancer, oophoroma and kidney.MPDL3280A is the anti-PD- of humanization IgG-1 κ L1 monoclonal antibody be used to treat melanoma and bladder cancer.MEDI4736 is humanization IgG-1 κ monoclonal antibody, can Extend the life cycle of lotus knurl individual.MSB0010718 is humanization IgG1 PD-L1 monoclonal antibody [Ito A, et al.Biomed Res Int.2015；2015:605478].
" costimulation receptor activators ": oligonucleotides provided by the invention can enhance with co-receptor activator use in conjunction Individual enhances the anti tumor immune response of individual to the immune response of vaccine or antigen.Costimulation receptor is expression immune thin The receptor of cellular surface, the transduction of mediated immunity cell activation signal after being activated by activator, thus promote individual to antigen Or vaccine immune response, enhancing individual antineoplastic immune activity.Costimulation receptor activators be by combine costimulation by The preparation of immune cell activated after body.Costimulation receptor activation monoclonal antibody (Co-stimulatory receptor Activating monoclonal antibody) it is costimulation receptor activators, it can enhancement antigen or effect, the increasing of vaccine The anti-tumor immune response of strong individual.Costimulation receptor (the Co-stimulatory of this kind of activity monoclonal antibody target It receptor) include but is 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.2015Aug；42 (4): 640-55].
" chemotherapeutics " oligonucleotides provided by the invention can be with chemotherapy drugs in combination application for the treatment of tumour.Chemotherapeutics It is the chemicals that tumour can be treated by inhibition, killing tumor cell.Chemicals of the invention signified include but not It is limited to alkylating agents, anti-metabolism, anti-micro-pipe preparation class, topoisomerase enzyme inhibitor class and cytotoxic antibiotics class drug. Alkylating agents drug includes nitrogen mustards, nitrosoureas, tetrazine class, aziridine class, cis-platinum and its derivative species and non-warp Allusion quotation alkylating agents.Mustargen (nitrogen mustards) class drug include mechlorethamine, cyclophosphamide, Chlorambucil, ifosfamide and busulfan.Nitrosoureas include N-Nitroso-N-methylurea, Carmustine, lomustine, semustine, fotemustine and streptozotocin.Tetrazine (tetrazines) Class drug includes dacarbazine (dacarbazine), mitozolomide and temozolomide.Aziridine (aziridines) class drug includes thiotepa, mytomycin and diaziquone.Cis-platinum and its derivative species include cis-platinum (cisplatin), carboplatin (carboplatin) and oxaliplatin (oxaliplatin).Nonclassic alkylating agents class includes methyl Benzyl hydrazine (procarbazine) and hexamethylmelamine.Anti-metabolism includes anti-folic acid class, fluorouracil, deoxidation Nucleoside analog and thiopurine medicine.Anti- folic acid class includes methopterin (methotrexate) and pemetrexed.Fluorine urine Miazines drug includes 5-fluor-uracil (5-fluorouracil).Deoxynucleoside analog drug includes cytarabine, Ji His shore (gemcitabine) of west, decitabine, vidaza, fludarabine, nelarabine, cladribine, Clofarabine and pentostatin.Thiopurine (thiopurines) drug include thioguanine and mercaptopurine.Anti- micro-pipe preparation medicine includes vinca alkaloids, taxanes and podophyllinic acid lactone class.Catharanthus roseus Alkaloid (vinca alkaloids) class includes that vincristine (vincristine), vincaleukoblastinum (vinblastine), Changchun are auspicious Guest (vinorelbine), vindesine and vinflunine.Taxanes (Taxanes) include taxol (paclitaxel) and Docetaxel (Docetaxel).Podophyllinic acid lactone (Podophyllotoxin) class includes relying on pool Glycosides (etoposide) and teniposide.Topoisomerase enzyme inhibitor (topoisomerase inhibitors) includes Topoisomerase I inhibitor and topoisomerase II inhibitor.Topoisomerase I inhibitor includes Irinotecan and topotecan.Topoisomerase II inhibitors include topoisomerase II poisons and catalysis suppression Object processed.Topoisomerase II poisons include etoposide, doxorubicin, mitoxantrone and teniposide.Being catalyzed mortifier includes novobiocin, merbarone and aclarubicin.Cytotoxic antibiotics packet Include adriamycin, daunorubicin, epirubicin (epirubicin), idarubicin, pirarubicin, Aclarubicin, mitoxantrone, gactinomycin, bleomycin (bleomycin), plicamycin and mitomycin。
" radiotherapy agents ": oligonucleotides provided by the invention can be with chemotherapy combined radiotherapy application for the treatment of tumour.Radiotherapy agents are It can produce the substance of ray.Method using radiotherapy agents treatment tumour is referred to as chemotherapy.Substance for radiotherapy is to generate α, β, gamma-ray radioactive isotope.Ray for radiotherapy can also be generated by machine, and this kind of machine includes x-ray treatment Machine or accelerator.
" hormone inhibitor or hormone ": oligonucleotides provided by the invention can be with the hormone inhibitor for oncotherapy Or hormons application for the treatment of tumour.Hormone inhibitor include but is not limited to hormone sensitive lipase gene inhibitor, hormone receptor antagonists and Supplement uses hormone.Hormone sensitive lipase gene inhibitor includes arimedex and gonadotropin-releasing hormone (GRH) (gonadotropin-releasing hormone, GnRH) analog.Arimedex includes Letrozole, Anastrozole and Aminoglutethimide.GnRH analog includes Leuprolide and gosereli.Hormone receptor is short of money Anti-agent includes selective estrogen receptor modulators and androgen receptor antagonists.Selective estrogen receptor modulators includes Tamoxifen, Raloxifene, Toremifene and fulvestrant.Androgen receptor antagonists include Flutamide and bicalutamide.The supplement side that hormone is using hormone supplemented (Hormone supplementation) treatment tumour Method, the hormone of use include progestational hormone, androgen, estrogen, progesterone sample drug, stosterone sample drug, estrogen agonist and Somat analog.Progesterone sample drug includes megestrol acetate and medroxyprogesterone acetate.Stosterone sample drug includes Fluoxymesterone.Estrogen antagonist includes diethylstilbestrol, Estrace and Polyestradiol phosphate.Somat analog includes Octraotide.
" cell factor ": oligonucleotides provided by the invention can treat tumour, enhancing vaccine with cell factor use in conjunction Immune effect.These cell factors include but is not limited to: interleukins (IL) -2, granulocyte colony stimulating factor (G- CSF), granulocyte-monocyte colony-stimulating factor (GM-CSF) and interferon-' alpha '.
" oncotherapy antibody " oligonucleotides provided by the invention can be with oncotherapy antibody (tumor Therapeutic antibodies) use in conjunction treatment tumour.Oncotherapy is to after suffering from tumour individual applications with antibody Can extend the antibody of its life cycle, the molecule of these antibody targets include but is not limited to CD20, ErbB2, epidermal growth because Sub- receptor, immune stuck point molecule, vascular endothelial growth factor receptor, CD30, CD52 including CTLA-4 and PD-1 And CD33.These antibody of involved anti-tumor therapeutic antibody include but is not limited to the Tositumomab for targeting CD20 (Bexxar), Rituximab (Rituxan) and Ofatumumab (Arzerra；Genmab)；Target ErbB2's Trastuzumab(Herceptin)；The Panitumumab (Vectibix) of targeting epidermal growth factor acceptor and Cetuximab (Erbitux)；Target the humanized antibody of PD-1；Target the humanized antibody of CTLA-4；Target vascular therapy endothelium The Bevacizumab (Avastin) of growth factor acceptor；Target the Brentuximab (vedotin) of CD30；Targeting The Alemtuzumab (Campath) of the CD52 and Gemtuzumab ozogamicin (Mylotarg for targeting CD33；Wyeth) [Scott AM et al.Nat Rev Cancer.2012 Mar 22；12 (4): 278-87].
" small molecule kinase mortifier ": oligonucleotides provided by the invention can be with small molecule kinase mortifier (small- Molecule kinase inhibitors) use in conjunction treatment tumour.Small molecule kinase mortifier is that one kind can pass through inhibition Protein kinase activity plays the small molecule compound of oncotherapy effect, including but not limited to following: to target Bcr-Abl's Imatinib；Targeting EGFR/ErbB2 Afatinib；Target VEGFR1/VEGFR2/VEGFR3/PDGFRB/c-KIT's Axitinib；Target the Bosutinib of BcrAbl/SRC；Target the Crizotinib of ALK/Met；Target ErbB1's Erlotinib；Target the Fostamatinib of Syk；The Gefitinib of targeting EGFR；Target the Ibrutinib of BTK；Targeting The Lapatinib of ErbB1/ErbB2；Target the Lenvatinib of VEGFR2/VEGFR2；Target the Nilotinib of Bcr-Abl； Target the Pazopanib of VEGFR2/PDGFR/c-kit；Target the Ruxolitinib of JAK；Targeting mutation BRAF's Vemurafenib (Zelboraf) and dabrafenib (Tafinlar) and the trametinib (Mekinist) for targeting MEK.It is small Molecule protein kinase inhibitor further includes all small molecule compounds that tumour is treated by the following protein kinases of inhibition: 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].
" polyadenosine diphosphate ribose polymerase inhibitors ": oligonucleotides provided by the invention can be with two phosphorus of polyadenosine Sour ribose polymerase mortifier (poly ADP ribose polymerase, PARP) use in conjunction treatment tumour is used for The adjuvant of microorganism vaccine and tumor vaccine.Polyadenosine diphosphate ribose polymerase inhibitors, abbreviation PARP mortifier are The preparation of inhibition PARP active treatment tumour can be passed through.PARP mortifier includes but is not limited to: treatment breast cancer and lung squamous are thin The Iniparib of born of the same parents' cancer；The Talazoparib (BMN-673) for treating breast cancer；Treat breast cancer, colon cancer, oophoroma and evening The Olaparib of phase prostate cancer；The Rucaparib for treating mammary gland and oophoroma；Treat metastasis melanin tumor and breast cancer Veliparib and treatment non-small cell lung cancer CEP 9722 [Nature Reviews Clinical Oncology 12, 27-41,2015].
" angiogenesis inhibitors ": oligonucleotides provided by the invention can treat swollen with angiogenesis inhibitors use in conjunction Tumor.Angiogenesis inhibitors are can be by preparation [the Albini A et al.Nat Rev of inhibition angiogenesis treatment tumour Clin Oncol.2012 Sep；9 (9): 498-509], including but not limited to: including Avastin (Avastin or Bevacizumab) in the Humanized monoclonal antibodies of interior vascular endothelial growth factor (VEGF)；Including rhEndostatin (ENDOSTAR) The aptamer of vascellum esoderma inhibin inside and the anti-vascular endothelial cell growth factor including pegaptinib (aptamer)。
" oncolytic virus ": oligonucleotides provided by the invention can treat tumour with oncolytic virus use in conjunction.Oncolytic virus It is the virus that tumour can be treated by cracking tumour cell, including but not limited to newcastle disease virus, herpes simplex virus, gland Virus, poxvirus, gram Sa virus, reovirus, measles virus, poliovirus, stomatitis follicularis virus, Seneca Valley virus, parvovirus and retrovirus [Kaufman HL et al.Nat Rev Drug Discov.2015 Sep 1；14 (9): 642-62].
" oncotherapy cell ": oligonucleotides provided by the invention can be treated with oncotherapy cell use in conjunction Tumour.Oncotherapy is applied to the cell that can exercise anti-tumor effect after individual with cell, these cells include but is not limited to Dendritic cells, T lymphocyte and NK cell.
" dendritic cells ": oligonucleotides provided by the invention can be swollen to treat with autologous fibroblasts (DC) use in conjunction Tumor.DC is the immunocyte for expressing CD11c.Can offer tumour antigen DC be applied to individual after can excite antineoplastic immune Response, this cell are used as DC vaccine.Oncotherapy DC includes but is not limited to load single tumour antigen (albumen Antigen or Antigenic Peptide, such as prostatic acid phosphatase) DC, load tumor cell lysate DC, load tumour cell RNA DC and load autologous tumor cell elution peptide DC [Nestle, F.et al. (1998) Nature Medicine 4:328- 332；Palucka K et al.Nat Rev Cancer.2012 Mar 22；12 (4): 265-77].Oncotherapy DC DC is transfected including gene.Gene for transfecting DC include but is not limited to tumour antigen encoding gene, cell factor (such as IL-2, GM-CSF) encoding gene and costimulatory molecules encoding gene.Oncotherapy also includes DC and tumour cell fused cell with DC [Kugler, A.et al. (2000) Nature Medicine 6:332-336].
" oncotherapy T cell ": oligonucleotides provided by the invention can be controlled with oncotherapy with T cell use in conjunction Treat tumour.Oncotherapy T cell includes tumor-infiltrated T cell and the T cell (Genetically through genetic modification engineered T cells).The composable Chimeric antigen receptor for having identification tumour antigen of T cell through genetic modification (chimeric antigen receptor, CAR), the T cell for expressing the receptoroid is referred to as CAR-T.Tumor-infiltrated T is thin Born of the same parents and CAR-T are fed back in tumor patient body [Kershaw MH et al.Nat Rev after being expanded in vitro with IL-2 Cancer.2013 Aug；13 (8): 525-41].
" oncotherapy natural killer cell ": oligonucleotides provided by the invention can be with oncotherapy natural killer Cell [Natural killer (NK) cells] use in conjunction treats tumour.Oncotherapy natural killer (NK) cell can be with From peripheral blood or the Cord blood separation of individual, can also be induced from hematopoietic precursor cells cell, embryonic stem cell or multipotential stem cell It generates.Separation or inductive formation NK cell can in vitro with IL-2 and IL-15 amplification after feed back to individual [Childs RW, et al.Nat Rev Drug Discov.2015Jul；14 (7): 487-98].
" anti-infective ": oligonucleotides provided by the invention is used alone or can play anti-infective work as vaccine adjuvant application With.Treatment and prevention of the anti-infective finger to disease caused by pathogenic microorganisms.Oligonucleotides provided by the invention is to micro- life of causing a disease Object has anti-infectious function.
" pathogenic microorganisms ": oligonucleotides provided by the invention is used alone or can play anti-cause as vaccine adjuvant application The effect of sick microorganism infection.Pathogenic microorganisms includes pathogenic virus and pathogenic bacteria.
" pathogenic virus ": oligonucleotides provided by the invention can be used alone or play anti-cause as vaccine adjuvant application The effect of characteristic of disease virus (pathogenic viruses) infection.These pathogenic virus include hepatitis A virus, B-mode liver Scorching virus, Hepatitis C Virus, varicella virus (VZV), I herpes simplex virus type (HSV-1), type herpe simplex Viral (HSV-II), cytomegalovirus (CMV), EB virus, adenovirus, influenza virus, arboviruse, echovirus, rhinopathy Poison, Coxsackie virus, coronavirus, Respiratory Syncytial Virus(RSV), mumps virus, rotavirus, measles virus, rubella virus, Parvovirus, circovirus, foot and mouth disease virus, hand-foot-and-mouth disease virus, the thermophilic T cell of people is viral (HTLV), dengue fever virus, cream Head tumor virus, mollascus contagiosum virus, poliovirus, rabies viruses, polyomavirus and arboviral encephalitides disease Poison.
" pathogenic bacteria ": oligonucleotides provided by the invention can be used alone or play anti-cause as vaccine adjuvant application The effect of characteristic of disease bacterium (pathogenic bacteria) infection.These pathogenic bacterias include Chlamydia, and Richettsia is thin Bacterium, mycobacteria, staphylococcus, streptococcus, pneumococcus, meningococcus, Klebsiella pneumoniae, Serratieae, false unit cell Bacterium, corynebacterium diphtheriae, salmonella, comma bacillus, clostridium tetani, clostridium botulinum, bacillus anthracis, yersinia pestis and Lyme disease Bacterium.
" pharmaceutical composition ": oligonucleotide drug provided by the invention can be with pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) forms pharmaceutical composition (pharmaceutical compositions).The dosage of oligonucleotides in the composition is effective dose (Effective Dosages).It should Composition can combine with antigen, vaccine, adjuvant and preparation with oncotherapy effect or cell answers.The pharmaceutical composition can It is made into certain dosage form, these dosage forms include but is not limited to solution, emulsion, liposome and freeze-dried powder etc..
" pharmaceutically acceptable carrier ": oligonucleotide drug provided by the invention can be with pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) forms pharmaceutical composition (pharmaceutical compositions).Pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) refers to one Filler, diluent or the encapsulating substance of kind or many kinds of solids or liquid.This kind of carrier is suitble to few nucleosides provided by the invention Acid is applied to individual.The carrier can be it is organic, inorganic, it is natural or synthesis.Pharmaceutically acceptable carrier can be with It is pharmaceutically acceptable solvent (aqueous solution and non-aqueous solution), dispersing agent, suspension, emulsifier, pulvis, diluent, lipid Body, antibacterial agent, antifungal agent, isotonic preparation, delayed absorption preparation, freeze drying protectant and others are suitble to widow provided by the invention The immune efficacy of nucleotide vaccine or antigen, the preparation for generating treatment function of tumor.Aqueous solution includes singly being not limited to water, physiology salt Water, PBS buffer solution, balanced salt solution and glucose solution.Solvent or dispersing agent may include water, ethyl alcohol, polyalcohol (such as glycerol, Propylene glycol, polyethylene glycol etc.), it also include the mixture being made of these solvents or dispersing agent.In order to maintain pharmaceutical composition Mobility can be using the lipid including lecithin.It is applied to allow pharmaceutical composition to be in ideal graininess Surfactant.In order to there is suitable osmotic pressure, sugar can be added in pharmaceutical composition, including mannitol and sorbierite Polyalcohol and sodium chloride etc..In order to extend action time, sustained release agent such as stearate and bright can be added in pharmaceutical composition Glue.Emulsifier may include oil-water emulsifiers, water-in-oil emulsifier or W/O/W emulsifier.Pharmaceutically acceptable load Body also includes pharmaceutically acceptable antioxidant (pharmaceutically-acceptable antioxidants), these are anti- Oxidant includes: water soluble antioxidant, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, Sodium Metabisulfite and Sodium sulfite etc.；Oil-soluble inhibitor, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene, ovum Phosphatide, propylgallate and alpha-tocopherol etc.；Metal-chelator, such as citric acid, EDTA, D-sorbite, tartaric acid and Phosphoric acid.
" effective dose ": the effective dose (Effective Dosages) of oligonucleotides provided by the present invention includes " increasing Strong antigen immune efficacy effective dose " and " oncotherapy effective dose ".Enhancement antigen immune efficacy effective dose is to individual The oligonucleotides dosage of antigen or vaccine immunity effect can be significantly increased after, also refer to can produce ideal after giving individual Prevention or treatment communicable disease or tumour oligonucleotides dosage.Oncotherapy effective dose is to energy after individual applications Generate the dosage of the oligonucleotides of oncotherapy effect.The number of dosage is decided by the mark that those skilled in the art should be known Standard, referring also to other factors, these factors include being not limited to the serious journey of the size and health condition and disease of individual Degree.Oligonucleotides provided by the invention single or multiple can be applied to individual, and each dosage range can be 1 μ g to 1000mg's Range.In order to reach ideal effect, those skilled in the art can be adjusted the dosage of this oligonucleotides, agent Amount range can be 10 times to 1000 times of aforementioned range.When giving individual applications, oligonucleotides provided by the invention be can be used Dosage unit.Per unit includes quantitative prevention or the therapeutic effect oligonucleotides and required pharmaceutically acceptable of can produce Composition.What dosage unit defined is receiving to be somebody's turn to do according to the peculiar living features and individual for being oligonucleotides generation therapeutic effect Widow closes sensibility when sweet acid is treated to the oligonucleotides.If desired, daily can be with the oligonucleotides of dosage unit application By certain time interval using 2 times, 3 times, 4 times, 5 times or repeatedly.Oligonucleotides provided by the invention can be by individual unit Weight (host body weight) application, dosage range are 0.0001 to 100mg/kg, and the interval of application can be every two weeks Once or monthly or every 3 to 6 months primary or other suitable time intervals for generating the effect that prevents, treats.And antigen Or when vaccine use in conjunction, the dosage of the oligonucleotides can be 1-1000 μ g/ml.Effective dose includes treatment effective dose (therapeutically effective dose) and prevention effective dose (prophylactically-effective dose)。
" administration route ": oligonucleotides provided by the invention be used alone or with other preparations such as antigen, adjuvant, intrinsic The administration route of parenteral, external application or sucking can be used when immunoactivator and anti-tumor agent use in conjunction.Parenteral administration route Including through lymph node, tumor tissues direct injection and lymph node by vein, peritonaeum, intrathecal, muscle, subcutaneous, intradermal, part, tumor Interior injection.Topical administration approach includes through skin, mouth, eye, ear and nose.Sucking can be through schneiderian membrane and lung.
" therapeutic device ": the pharmaceutical composition containing oligonucleotides provided by the invention can be answered with those skilled in the art The therapeutic device known is applied to individual.Therapeutic device includes but is not limited to Needleless injection device, implanted device, warehouse device (modules), the micro- infusion pump of implantable (implantable micro-infusion pump), infusion pump and osmotic drug are passed Send system (osmotic drug delivery system).
" delivery vector ": the oligonucleotides in the present invention can be through delivery vector application.Delivery vector includes but is not limited to: class Sterol (such as cholesterol), network and object, emulsion, immunostimulating complex (ISCOMs), lipid (such as cation lipid and yin from Sub- lipid), liposome, bacteria carrier (such as salmonella, Escherichia coli, mycobacteria will congratulate (family name) bacillus, Bacillus acidi lactici), Viral vectors (such as bovine vaccine, adenovirus, herpes simplex virus), virosomes, virus-like particle, microballoon, nucleic acid vaccine, height Molecular material (such as carboxymethyl cellulose, chitosan) and cyclic polymer.Delivery vector is also possible to specific receptor Ligand or cell targeted molecular.
Detailed description of the invention:
Synergistic effect of Fig. 1 TIO1 and TIO3 to recombinant protein vaccine (circovirus vaccine)
In Fig. 1, CP represents CP-ISA35, and TIO1, TIO2 and TIO3 respectively represent CP-ISA35+TIO1, CP-ISA35+ TIO2 or CP-ISA35+TIO3, ODN represent single-stranded deoxy-oligonucleotide, and ns represents not statistically significant, the height of antibody level It is low to be indicated with light absorption value (A492 OD value), each circle symbol, square symbol, positive triangle symbol and inverted triangle symbol Respectively represent a mouse.
Fig. 2 TIO1 and TIO3 is to the inhibiting effect for inducing immune anamnestic reaction mouse immune cell TGF β -2 expression
In Fig. 2, CP represents CP-ISA35, TIO1 or TIO3 and respectively represents CP-ISA35+TIO1 or CP-ISA35+ TIO3, ODN represent single-stranded deoxy-oligonucleotide, and it is positive thin that TGF-β 2relative expression represents film combination TGF-β 2 Born of the same parents' percentage shared in draining lymph node cells, each circle symbol, square symbol, positive triangle symbol respectively represent One mouse.
Fig. 3 TIO1 and TIO3 acts on the reduction of the IL-4 immunocyte TGF β -2mRNA level induced
In Fig. 3, ODN represents single-stranded deoxy-oligonucleotide, the height relative of TGF β -2mRNA level Expression indicates, each circle symbol, square symbol, positive triangle symbol, inverted triangle symbol and diamond symbols generation respectively One sample of table.
Fig. 4 TIO1 and TIO3 acts on the reduction of the LPS immunocyte TGF β -2mRNA level induced
In Fig. 4, ODN represents single-stranded deoxy-oligonucleotide, the height relative of TGF β -2mRNA level Expression indicates, each circle symbol, square symbol, inverted triangle symbol, diamond symbols and positive triangle symbol generation respectively One sample of table.
Fig. 5 TIO1 and TIO3 is to the dendritic cells for expressing CD86 in the immune anamnestic reaction mouse lymph nodal cell of induction Increase effect
In Fig. 5, CP represents CP-ISA35, TIO1, TIO2 or TIO3 and respectively represents CP-ISA35+TIO1, CP-ISA35+ TIO2 or CP-ISA35+TIO3, ODN represent single-stranded deoxy-oligonucleotide, each circle symbol, square symbol, positive triangle symbol Number symbol respectively represents a mouse.
Fig. 6 TIO1 and TIO3 is to the T lymphocyte for expressing CD4 in the immune anamnestic reaction mouse lymph nodal cell of induction Increase effect
In Fig. 6, CP represents CP-ISA35, TIO1 or TIO3 and respectively represents CP-ISA35+TIO1 or CP-ISA35+ TIO3, ODN represent single-stranded deoxy-oligonucleotide, and each circle symbol, square symbol, positive triangle symbol respectively represent one Mouse, CD4+% represents CD4+Cell quantity shared in the draining lymph node cells analyzed.
Fig. 7 TIO1 and TIO3 increases the bone-marrow-derived lymphocyte for expressing CD19 in the immune anamnestic reaction mouse lymph nodal cell of induction Effect
In Fig. 7, CP represents CP-ISA35, TIO1, TIO2 or TIO3 and respectively represents CP-ISA35+TIO1, CP-ISA35+TIO2 Or CP-ISA35+TIO3, ODN represent single-stranded deoxy-oligonucleotide, each circle symbol, square symbol, positive triangle and three Angle symbol respectively represents a mouse, CD19+Cell% represents CD19+Cell is shared in the draining lymph node cells analyzed Quantity.
Synergistic effect of Fig. 8 TIO1 and TIO3 to Hepatitis B virus vaccine
In fig. 8, HBsAg represents HBsAg vaccine；TIO1, TIO2, TIO3, which are respectively represented, joined TIO1 in HBsAg vaccine, TIO2 or TIO3；ODN represents single-stranded deoxy-oligonucleotide；Each circle symbol, square symbol, positive triangle and inverted triangle symbol Number respectively represent a mouse；OD Value (A492nm) represents the level of HBsAg in serum specific antibody to be checked.
The synergistic effect of Fig. 9 TIO1 or TIO3 infected by influenza vaccine
In Fig. 9, FM1 represents FM1 inactivated influenza virus vaccine；TIO1, TIO2, TIO3 are respectively represented joined in FM1 TIO1 or TIO3；ODN represents single-stranded deoxy-oligonucleotide；Each circle symbol, square symbol, positive triangle symbol generation respectively One mouse of table；OD Value (A492nm) represents the level of influenza virus specific antibody in serum to be checked.
Synergistic effect of Figure 10 TIO1 or TIO3 to hydrophobia
In Figure 10, TIO1, TIO2, TIO3 is respectively represented joined TIO1, TIO2 or TIO3 in rabies vaccine；ODN is represented Single-stranded deoxy-oligonucleotide；Each circle symbol, square symbol, positive triangle and inverted triangle symbol respectively represent a mouse. The effect of vaccine is indicated with IU/ml.
Scheme synergistic effect of -11 TIO3 to glioma cell lysate vaccine
In Figure 11, GTL represents glioma cell lysate vaccine；GTL+TIO3 represents the glioma cell cracking containing TIO3 Object vaccine；Days, the number of days after being inoculated with glioma cell；Percent survival represents the percentage of existence mouse.
Scheme therapeutic effect of -12 TIO3 to gastric cancer
In Figure 12, PBS represents injection PBS；PBS+TIO3 represents the TIO3 that injection is dissolved in PBS；Days represents inoculation gastric cancer Number of days after cell；Percent survival represents the percentage of existence mouse.
Specific implementation method:
The design and synthesis of 1 oligonucleotides of embodiment (TIO1, TIO3)
According to the sequence of 3 ' UTR of people and mouse transforming growth factor-beta 2 (TGF-β 2) mRNA, three kinds of single-stranded deoxidations are devised Oligonucleotides, these three oligonucleotides are named as TIO1, TIO2 and TIO3 respectively, and TIO1 has such as<400>1 institute of sequence table The sequence shown, TIO3 have the sequence as shown in sequence table<400>2.
The sequence of TIO1 is 5 '-tggcaaagtatttggtctcca-3 ' (sequence table<400>1).
The sequence of TIO3 is 5 '-ttaccactagagcaccaca-3 ' (sequence table<400>2).
The sequence of TIO2 is 5 '-tccttaagccatccatgagttt-3 '
The sequence of the 3 ' UTR of sequence and TGF-β 2mRNA of TIO1, TIO2 and TIO3 are complementary.
TIO1, TIO2 and TIO3 are synthesized by precious bioengineering (Dalian) Co., Ltd (Takara Bio), and skeleton is complete Thio-modification.
In use, handling sterile PBS or other solvents dissolution TIO1 or TIO3 with through no heat source.- 20 DEG C freeze.
Contain TIO1 using the detection of horseshoe crab ameboid cell dissolution method, the endotoxin in TIO2 and TIO3 solution.
The content that TIO1, TIO2 and TIO3 in solution are determined using spectrophotometer (260nm wavelength), can also be used fine jade Content (is estimated) in the estimation of sepharose (3%) electrophoresis according to the single-stranded deoxy-oligonucleotide standard items of known content.
Humidification of embodiment 2 TIO1 and TIO3 to recombinant protein vaccine (circovirus vaccine) immune efficacy:
2.1 material
2.1.1 mouse
ICR mouse (medical board animal housing, Jilin University), female, weight are 17-18 grams.
2.1.2 antigen
Use recombination circovirus 2 b capsid protein (CP albumen or CP) for antigen [Vaccine.2016 Dec 7；34 (50): 6358-6366].
2.1.3 adjuvant
35 emulsifier of ISA (Seppic)
2.1.4 oligonucleotides
TIO1 (has the sequence as shown in sequence table<400>1), and TIO2 (sequence is as described in Example 1) and TIO3 (have The sequence as shown in sequence table<400>2), as described in Example 1.
The preparation of 2.2 vaccines
CP is dissolved with PBS, it is mixed and made into vaccine in 1: 1 (volume: volume) ratio with ISA35 emulsifier, this vaccine It is named as CP-ISA35.Contain 10 μ g CP in 100 μ l CP-ISA35.The CP- for containing TIO1, TIO2 and TIO3 is prepared respectively The dosage of ISA35, TIO1, TIO2 and TIO3 are 10 μ g/100 μ l.CP-ISA35 containing TIO1, TIO2 and TIO3 respectively by It is named as CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3.
2.3 immune mouse
Initial immunity is carried out to mouse in the 0th day.100 μ l CP-ISA35, CP-ISA35+TIO1, CP- are injected respectively ISA35+TIO2 or CP-ISA35+TIO3, injection site are right hind muscle, single-point injection.Mouse is added in the 14th day It is strong immune, the same initial immunity of method.
2.4. it takes a blood sample after being immunized
The 28th day acquisition immune serum after booster immunization.Tail vein blood, blood sampling volume >=100 μ l.By acquisition Whole blood (in 1.5mlEP pipe) is placed at room temperature for 30 minutes.11000rpm is centrifuged 15 minutes, collects serum, packing, -20 DEG C of preservations.
2.5. mice serum detection of specific antibody
Using circovirus (PCV2b) specific antibody in ELISA method detection mice serum.
2.5.1 material
2.5.1.1 circovirus (PCV2b) (Tianjin Ruipu Biotechnology Co., Ltd, TCID50/ml=is inactivated 7.0)。
2.5.1.2 test equipment
Enzyme mark strip (combined type ELISA Plate), 0.5mlEP pipe, 1.5mlEP pipe, sample injector head, pipettor, multichannel pipettor, Plate (diameter 9cm), graduated glass bottle.
2.5.1.3 reagent
Sodium carbonate (Sinopharm Chemical Reagent Co., Ltd.), NaCl (Sinopharm Chemical Reagent Co., Ltd.), KCl (Beijing Chemical Plant), Na2HPO4·12H2O (Sinopharm Chemical Reagent Co., Ltd.), KH2PO4(Tianjin big chemical reagent forever Development centre), polysorbas20 (fine chemistry industry research institute is recovered in Tianjin), skimmed milk power (Biotopped), citric acid (traditional Chinese medicines collection Chemical reagent Co., Ltd, group), OPD (Shanghai San Pu Chemical Co., Ltd.), 30%H2O2 (Beijing Chemical Plant), the concentrated sulfuric acid (north Capital chemical plant) and glutaraldehyde (Tianjin good fortune morning chemical reagent factory).
2.5.1.4 liquid
Coating buffer (25% glutaraldehyde PBS), PBS (7.3mol/L NaCl, 3mmol/L KCl, 10mmol/L Na2HPO4· 12H2O and 17.6mmol/L KH2PO4 aqueous solution), cleaning solution (PBS of 0.05% polysorbas20), confining liquid be (5% skimmed milk power Cleaning solution), 0.1Mol/L aqueous citric acid solution, 0.2Mol/L Na2HPO4.12H2O aqueous solution, 10 × the first and second liquid [0.1Mol/L Aqueous citric acid solution (volume): 0.2Mol/L Na2HPO4.12H2O aqueous solution (volume)=94.5: 100], substrate buffer solution [contains The first and second liquid of OPD (1 μ g/ml) and 0.045%H2O2] and terminate liquid (20% concentrated sulfuric acid).
2.5.2 experiment
Enzyme mark strip is coated in coating buffer with inactivation PCV2b, 100 holes μ l/, 4 DEG C overnight.Dry liquid, with confining liquid in 37 DEG C are closed 2 hours, 200 holes μ l/.Add test serum (1: 100 dilution), 37 DEG C, 1 hour.Liquid is dried, cleaning solution is added (300 hole μ l/), room temperature 3min dry liquid, and repeated washing is twice.Add HRP label secondary antibody (sheep anti-Mouse) IgG (with washing Liquid does 1: 5000 dilution).100 holes μ l/, 37 DEG C 1 hour.Liquid is dried, is washed with cleaning solution, 300 holes μ l/, room temperature 3min, Liquid is dried, repeated washing is twice.Substrate solution, 100 holes μ l/ is added, room temperature is protected from light (tinning paper) colour developing 15min.Add terminate liquid (dilute sulfuric acid 2mmol/L, 50 holes μ l/.It is detected with microplate reader (wavelength 492nm).
2.5.3 result
TIO1 and TIO3 can enhance the immune efficacy (figure -1) of recombinant protein vaccine (circovirus vaccine).The result explanation TIO1 and TIO3 can be used for individual to enhance it to the immune of the pathogenic microorganisms antigen (vaccine) including circovirus Response and the adjuvant for being used as pathogenic microorganisms vaccine.
Embodiment 3 TIO1 and TIO2 is to the inhibiting effect for inducing immune anamnestic reaction mouse immune cell TGF β -2 expression
3.1 material
3.1.1 antigen
CP albumen (abbreviation CP), as described in 2.1.2 in embodiment 2.
3.1.2 oligonucleotides
TIO1 (has the sequence as shown in sequence table<400>1), and T1O3 (has the sequence as shown in sequence table<400>2 Column), as described in embodiment 1.
3.1.3 culture medium
RPMI1640 culture medium (Gibco company).Complete RPMI1640 culture medium contains 10% fetal calf serum (FBS) (Gibco Company), 2mM L-Glutamine, 100U/ml penicillin, 100 μ g/ml streptomysins.Serum-free RPMI1640 culture medium is free of tire Cow's serum.
3.1.4 fluorescence antibody
2 antibody of anti-mouse TGF-β (BD company) of FITC label.
3.1.5 mouse
ICR mouse (medical board animal housing, Jilin University), female, weight are 17-18 grams.
3.1.6 vaccine
CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 are prepared as described in the 2.2 of embodiment 2.
3.2. major experimental equipment
24 well culture plates, plate, frosted glass plate, 300 mesh filter screens, pincet, cell counting board, dropper, sample injector.CO2 Cell culture incubator (Japanese SANYO company), cell culture inverted microscope (Japanese Olympus company), centrifuge (Germany Biofuge Fresco), flow cytometer be Accuri C6 flow cytometer (BD Bioscience), fluidic cell Instrument Analysis of test results software is NovoExpress software (ACEA Biosciences).
3.3 experiment
Initial immunity is carried out to mouse in the 0th day.100 μ l CP-ISA35, CP-ISA35+TIO1 or CP- are injected respectively ISA35+TIO3, injection site are right hind muscle, single-point injection.Booster immunization is carried out to mouse in the 14th day, method is the same as just It is secondary immune.It carries out within 120 days after booster immunization induction anamnestic reaction (recall) to be immunized, method is infused respectively to each group mouse 100 μ l CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 are penetrated, injection site is right hind muscle, single-point injection. Mouse is sacrificed after 6 hours.
The mouse of sacrifice is immersed in 70% ethyl alcohol, speed is taken out.Skin and other groups are cast aside using sterile surgical instruments It knits, aseptically takes draining lymph node, in the PBS for being placed 4 DEG C of pre-coolings of 0.5ml sterilizing.Set carry out on ice it is following Operation: lymph node is shredded, with abrasive glass slice lapping, filter screen obtains single cell suspension.By cell be suspended in 4 DEG C pre-cooling PBS or Fluorescence activated cell separates (fluorescence-activated cell sorting, FACS) buffer and (contains 2% tire ox blood Clearly, the PBS of 5mM EDTA and 1mM Sodium azide).
Cell is washed in PBS or the FACS buffer solution centrifugation (277g, 5 minutes, 4 DEG C) being pre-chilled with 4 DEG C of 5ml.It is pre-chilled with 4 DEG C PBS or FACS buffer (200-1,000 μ l) hanged cell.Remember cell number, adjusts cell concentration.It excludes to test with platform phenol indigo plant Detect the vigor of cell.
By 1-10X106Lymph node cells are transferred in 5ml round bottom polystyrene tube.With FACS buffer solution centrifugation (277g, 5 points Clock, 4 DEG C) washing cell.Supernatant is abandoned, cell is suspended in the FACS of 50-100 μ l 2 antibody of anti-mouse TGF-β marked containing FITC In buffer.The content of every kind of fluorescence antibody is 0.5-1 μ g, acts on 30-45min on ice.Sample is protected from light.Use FACS buffer solution It is centrifuged (277g, 5 minutes, 4 DEG C) and washs cell.Cell is suspended in 50-100 μ l FACS buffer solution, flow cytometer showed is done.
3.4 result
TIO1 and TIO3 can make TGF β -2 egg for inducing the lymph node cells of immune anamnestic reaction (recall) mouse to express It is white to substantially reduce (figure -2), show as film mating type TGF β -2 albumen [Yang ZZ, et al PLoS One.2013；8 (3): J.2014 18 Jan e59456Miller MM, et al.Virol；11:7.] substantially reduce.This explanation, TIO1 and TIO3 can Inhibit the translation of TGF β -2mRNA；TIO1 and TIO3 can inhibit, weaken TGF β -2 by reducing immunocyte expression TGF β -2 The immunosupress of mediation, thus enhance individual to the immune response of pathogenic microorganisms and tumour cell, it generates anti-infective and anti-swollen Tumor effect.
Embodiment 4 TIO1 and TIO2 acts on the reduction of immunocyte TGF β -2mRNA level
4.1 material
4.1.1 cell
264.7 cell of RAW: mouse macrophage cell [Cell 1978 Sep, 15 (1) 261-7] comes from ATCC.
4.1.2 culture medium
RPMI1640 culture medium (Gibco company).Complete RPMI1640 culture medium contains 10% fetal calf serum (FBS) (Gibco Company), 2mM L-Glutamine, 100U/ml penicillin, 100 μ g/ml streptomysins.Serum-free RPMI1640 culture medium is free of tire Cow's serum.
4.1.3 2 inducer of TGF-β
Recombined small-mouse IL-4 (IL-4) is purchased from PeproTech.In vitro under condition of culture, IL-4 stimulates RAW 264.7 thin Born of the same parents generate a large amount of 2 [cell Biol Int.2017 Sep of TGF-β；41 (9): 960-968].
LPS (lipopolysaccharide), extract from E.coli serotype O111:B4 (Sigma, St.Louis, MI, USA).In vitro under condition of culture, LPS stimulates 264.7 cell of RAW to express [the PLoS One.2015 Dec of TGF-β 2 14；10 (12): e0144954].
4.1.4 oligonucleotides
TIO1 (has the sequence as shown in sequence table<400>1), and TIO2 (sequence is as described in Example 1) and TIO3 (have The sequence as shown in sequence table<400>2) as described in Example 1.
4.1.5 PCR reagent
TRIzol reagent is purchased from U.S. Invitrogen company.EasyScript First-Strand cDNA Synthesis SuperMix, Top Green qPCR SuperMix (TransStartTM) kit are purchased from Quan Shijin (Transgen) company；
GAPDH specific primer (being house-keeping gene for expanding GAPDH specificity cDNA, GAPDH), the sequence of upstream primer Column are: 5 '-ATCACCATCTTCCAGGAGCGA-3 ', and the sequence of downstream primer is 5 '-TCTCGTGGTTCACACCCATCA- 3’。
2 specific primer of TGF-β (for expanding 2 specificity cDNA of TGF-β), the sequence of upstream primer are as follows: 5 '- The sequence of ttgtgaaaaccagagcggagg-3 ' downstream primer are as follows: 5 '-agaggtgccatcaatacctgc-3 '.
GAPDH specific primer and 2 specific primer of TGF-β are by precious bioengineering (Dalian) Co., Ltd (Takara Bio it) synthesizes.
4.2. major experimental equipment and instrument
24 well culture plates, plate, frosted glass plate, 300 mesh filter screens, pincet, cell counting board, dropper, sample injector.CO2 Cell culture incubator (Japanese SANYO company), cell culture inverted microscope (Japanese Olympus company), centrifuge (Germany Biofuge Fresco), fluorescence quantitative PCR instrument (U.S. Applied Biosystems, model: ABI Prism 7300).
4.3 experiment
Using complete 1640 culture medium, at 37 DEG C, 5%CO2Under the conditions of cultivate 264.7 cell of RAW.5×106RAW 264.7 cells/ml is added to 24 well culture plates, every hole 1ml culture medium.It is added IL-4 (25ng/ml), TIO1, TIO2 is being added Or TIO3 (10 μ g/ml of final concentration) collects cell after culture 48 hours.Total serum IgE is extracted with Trizol method.Using EasyScript First-Strand cDNA Synthesis SuperMix、Top Green qPCR SuperMix (TransStartTM) kit is qPCR and is expanded GAPDH respectively with GAPDH specific primer and 2 specific primer of TGF-β Specific cDNA and TGF-β 2mRNA specificity cDNA.Quantitative fluorescence analysis is carried out by kit specification.
The culture medium of 1ml 264.7 cell containing RAW is added to 24 well culture plates, every hole cell number is 5 × 106.It is added LPS (2 μ g/mL) is cultivated 2 hours, is added TIO1, TIO2 or TIO3 (10 μ g/ml of final concentration).After culture 48 hours, collect thin Born of the same parents.Total serum IgE is extracted with Trizol method.Using EasyScript First-Strand cDNA Synthesis SuperMix, Top Green qPCR SuperMix (TransStartTM) kit, with 2 specificity of GAPDH specific primer and TGF-β Primer is qPCR and expands GAPDH specificity cDNA and TGF-β 2mRNA specificity cDNA respectively.It is carried out by kit specification glimmering Light quantitative analysis.
4.4. result
TIO1 or TIO3 substantially reduces the TGF-β 2mRNA (figure -3) of the mouse macrophage expression of IL-4 stimulation, also significantly Reduce the level (figure -4) of the TGF-β 2mRNA of the mouse macrophage expression of LPS stimulation.These results explanation, TIO1 or TIO3 can significantly reduce the level of the TGF-β 2mRNA of the immunocyte including macrophage, thus reduce TGF-β 2 Synthesis release, and then the immunosupress for weakening or TGF-β 2 being inhibited to mediate.The result also illustrates that TIO1 or TIO3 can be by subtracting The TGF-β 2mRNA of few immunocyte enhances the immune response to microbial antigen (vaccine) or tumour antigen (vaccine), because And the adjuvant for being used as pathogenic microorganisms vaccine and tumor vaccine generates anti-infectious function and antitumor action, can also by with In oncotherapy.
Influence of embodiment 5 TIO1 and TIO3 to immune anamnestic reaction murine antigen presenting cells expression CD86 is induced:
5.1 material
ICR mouse (medical board animal housing, Jilin University), female, weight are 17-18 grams.
5.1.1.2 antigen
Use recombination circovirus 2 b capsid protein (CP) for antigen [Vaccine.2016 Dec 7；34 (50): 6358- 6366]。
5.1.1.3 adjuvant
35 emulsifier of ISA (Seppic)
5.1.1.4 oligonucleotides
TIO1 (has the sequence as shown in sequence table<400>1), and TIO2 (sequence is as described in Example 1) and TIO3 (have The sequence as shown in sequence table<400>2), as described in Example 1.
5.1.1.5 fluorescence antibody
The anti-mouse CD11c antibody of FITC label and the anti-mouse CD86 antibody of PE label are purchased from BD company.
5.1.1.6 culture medium
RPMI1640 culture medium (Gibco company).Complete RPMI1640 culture medium contains 10% fetal calf serum (FBS) (Gibco Company), 2mM L-Glutamine, 100U/ml penicillin, 100 μ g/ml streptomysins.Serum-free RPMI1640 culture medium is free of tire Cow's serum.
5.2 major experimental equipment
Plate, frosted glass plate, 300 mesh filter screens, pincet, cell counting board, dropper, sample injector.CO2 cell culture incubator (Japanese SANYO company), cell culture inverted microscope (Japanese Olympus company), centrifuge (German Biofuge Fresco), flow cytometer is Accuri C6 flow cytometer (BD Bioscience), flow cytomery knot It is NovoExpress software (ACEA Biosciences) that fruit, which analyzes software,.
5.3 experiment
5.3.1 the preparation of vaccine
CP is dissolved with PBS, it is mixed and made into vaccine in 1: 1 (volume: volume) ratio with ISA35 emulsifier, this vaccine It is named as CP-ISA35.Contain 10 μ g CP in 100 μ l CP-ISA35.The CP- for containing TIO1, TIO2 and TIO3 is prepared respectively The dosage of ISA35, TIO1, TIO2 and TIO3 are 10 μ g/100 μ l.Vaccine containing TIO1, TIO2 and TIO3 is named respectively For CP-ISA35+TIO1, CP-ISA35+TIO2 and CP-ISA35+TIO3.
5.3.2 mouse is immunized
Initial immunity is carried out to mouse in the 0th day.100 μ l CP-ISA35, CP-ISA35+TIO1, CP- are injected respectively ISA35+TIO2 or CP-ISA35+TIO3, injection site are right hind muscle, single-point injection.Mouse is added in the 14th day It is strong immune, the same initial immunity of method.It carries out within 120 days after booster immunization inducing immune anamnestic reaction (recall) immune, method It is to inject 100 μ l CP-ISA35, CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+ respectively to each group mouse TIO3, injection site are right hind muscle, single-point injection.
5.3.3. separation drainage mouse lymph nodal cell
6 hours sacrifice mouse after inducing immune anamnestic reaction immune, separating mouse draining lymph node cells (such as embodiment Described in 3,3.3).
5.3.4. the fluorescent antibody staining and flow cytometer showed of draining lymph node cells
By 1X106Lymph node cells are transferred in 5ml round bottom polystyrene tube.With FACS buffer solution centrifugation (277g, 5 minutes, 4 DEG C) washing cell.Supernatant is abandoned, cell is suspended in 50-100 μ l and is resisted containing what the FITC anti-mouse CD11c antibody marked and PE marked In the FACS buffer solution of mouse CD86 antibody.The content of every kind of fluorescence antibody is 0.5-1 μ g, acts on 30-45min on ice.Sample is kept away Light.Do flow cytometer showed.
5.4 result
TIO1 or TIO3 can be such that the CD11c positive cell (dendritic cells) for expressing CD86 in mouse lymph nodal cell significantly increases More (figures -5).The result shows can make antigen presenting cell table by reducing the expression of TGF-β 2 using TIO1 or TIO3 The costimulatory molecules in face significantly increase, thus it is micro- to promote antigen presenting cell to more second activation signals of T cell offer The adaptive immune response of biological antigens and tumour antigen.It can be used for individual with this active TIO1 or TIO3 to increase Its strong immune response to microbial antigen (vaccine), tumour antigen (vaccine), it is antitumor to enhance its to may be alternatively used for individual Reaction.
Embodiment 6 TIO1 and TIO3 increases the immune anamnestic reaction mouse draining lymph node CD4+T lymphocyte of induction Effect
6.1 material
6.1.1 mouse
ICR mouse (medical board animal housing, Jilin University), female, weight are 17-18 grams.
6.1.2 antigen
Use recombination circovirus 2 b capsid protein (CP) for antigen [Vaccine.2016 Dec 7；34 (50): 6358- 6366]。
6.1.3 adjuvant
35 emulsifier of ISA (Seppic)
6.1.4 oligonucleotides
TIO1 (with the sequence as shown in sequence table<400>1) and TIO3 (have the sequence as shown in sequence table<400>2 Column) as described in embodiment 1.
6.1.5 fluorescence antibody
The anti-mouse CD4 antibody of FITC label is purchased from BD company.
6.1.6 culture medium
RPMI1640 culture medium (Gibco company).Complete RPMI1640 culture medium contains 10% fetal calf serum (FBS) (Gibco Company), 2mM L-Glutamine, 100U/ml penicillin, 100 μ g/ml streptomysins.Serum-free RPMI1640 culture medium is free of tire Cow's serum.
6.2 major experimental equipment
Plate, frosted glass plate, 300 mesh filter screens, pincet, cell counting board, dropper, sample injector.CO2 cell culture incubator (Japanese SANYO company), cell culture inverted microscope (Japanese Olympus company), centrifuge (German Biofuge Fresco), flow cytometer is Accuri C6 flow cytometer (BD Bioscience), flow cytomery knot It is NovoExpress software (ACEA Biosciences) that fruit, which analyzes software,.
6.3 experiment
6.3.1 the preparation of vaccine
CP is dissolved with PBS, it is mixed and made into vaccine in 1: 1 (volume: volume) ratio with ISA35 emulsifier, this vaccine It is named as CP-ISA35.Contain 10 μ g CP in 100 μ l CP-ISA35.The CP- for containing TIO1, TIO2 and TIO3 is prepared respectively The dosage of ISA35, TIO1, TIO2 and TIO3 are 10 μ g/100 μ l.Vaccine containing TIO1, TIO2 and TIO3 is named respectively For CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3.
6.3.2 mouse is immunized
Initial immunity is carried out to mouse in the 0th day.100 μ l CP-ISA35, CP-ISA35+TIO1 or CP- are injected respectively ISA35+TIO3, injection site are right hind muscle, single-point injection.Booster immunization is carried out to mouse in the 14th day, method is the same as just It is secondary immune.It carries out within 120 days after booster immunization inducing immune anamnestic reaction (Fecall) immune, method is to each group mouse point 100 μ l CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 are not injected, and injection site is right hind muscle, single-point Injection.
6.3.3. separation drainage mouse lymph nodal cell
It is immunized in induction anamnestic reaction and sacrifices mouse after 6 hours afterwards, separating mouse draining lymph node cells (such as embodiment 3, Described in 3.3).
6.3.4. fluorescent antibody staining and flow cytometer showed
By 1X106Lymph node cells are transferred in 5ml round bottom polystyrene tube.With FACS buffer solution centrifugation (277g, 5 minutes, 4 DEG C) washing cell.Supernatant is abandoned, cell is suspended in the FACS buffer solution of 50-100 μ l anti-mouse CD4 marked containing FITC.Fluorescence The content of antibody is 0.5-1 μ g, acts on 30-45min on ice.Sample is protected from light.Do flow cytometer showed.
6.4 result
TIO1 or TIO3 can make the lymphocyte (CD4 of the expression CD4 in mouse lymph nodal cell+Cell) significantly increase (figure -6).The result shows can make the complementary of antigentic specificity by reducing the expression of TGF-β 2 using TIO1 or TIO3 T cell significantly increases, thus promotes the adaptive immune response of microbial antigen and tumour antigen.It is active with this TIO1 or TIO3 can be used for individual to enhance its immune response to microbial antigen (vaccine), tumour antigen (vaccine), It can be used for individual to enhance its antitumor reaction.
Embodiment 7 TIO1 and TIO3 is to CD19 in the immune anamnestic reaction mouse draining lymph node of induction+Bone-marrow-derived lymphocyte Increase effect
7.1 material
ICR mouse (medical board animal housing, Jilin University), female, weight are 17-18 grams.
7.1.2 antigen
Use recombination circovirus 2 b capsid protein (CP) for antigen [Vaccine.2016 Dec 7；34 (50): 6358- 6366]。
7.1.3 adjuvant
35 emulsifier of ISA (Seppic)
7.1.4 oligonucleotides
TIO1 (has the sequence as shown in sequence table<400>1), and TIO2 (sequence is as described in Example 1) and TIO3 (have The sequence as shown in sequence table<400>2) as described in Example 1.
7.1.5 fluorescence antibody
The anti-mouse CD19 antibody of FITC label, is purchased from BD company.
7.1.6 culture medium
RPMI1640 culture medium (Gibco company).Complete RPMI1640 culture medium contains 10% fetal calf serum (FBS) (Gibco Company), 2mM L-Glutamine, 100U/ml penicillin, 100 μ g/ml streptomysins.Serum-free RPMI1640 culture medium is free of tire Cow's serum.
7.2 major experimental equipment
Plate, frosted glass plate, 300 mesh filter screens, pincet, cell counting board, dropper, sample injector.CO2 cell culture incubator (Japanese SANYO company), cell culture inverted microscope (Japanese Olympus company), centrifuge (German Biofuge Fresco), flow cytometer is Accuri C6 flow cytometer (BD Bioscience), flow cytomery knot It is NovoExpress software (ACEA Biosciences) that fruit, which analyzes software,.
7.3 experiment
7.3.1 the preparation of vaccine
CP is dissolved with PBS, it is mixed and made into vaccine in 1: 1 (volume: volume) ratio with ISA35 emulsifier, this vaccine It is named as CP-ISA35.Contain 10 μ g CP in 100 μ l CP-ISA35.The CP- for containing TIO1, TIO2 and TIO3 is prepared respectively The dosage of ISA35, TIO1, TIO2 and TIO3 are 10 μ g/100 μ l.Vaccine containing TIO1, TIO2 and TIO3 is named respectively For CP-ISA35+TIO1, CP-ISA35+TIO2 or CP-ISA35+TIO3.
7.3.2 mouse is immunized
Initial immunity is carried out to mouse in the 0th day.100 μ l CP-ISA35, CP-ISA35+TIO1, CP- are injected respectively ISA35+TIO2 or CP-ISA35+TIO3, injection site are right hind muscle, single-point injection.Mouse is added in the 14th day It is strong immune, the same initial immunity of method.It carries out within 120 days after booster immunization inducing immune anamnestic reaction (recall) immune, method It is to inject 100 μ l CP-ISA35, CP-ISA35+TIO1 or CP-ISA35+TIO3 respectively to each group mouse, injection site is right Hindlimb muscle, single-point injection.
7.3.3. separation drainage mouse lymph nodal cell
It is immunized in induction anamnestic reaction and sacrifices mouse after 6 hours afterwards, separating mouse draining lymph node cells (such as embodiment 3, Described in 3.3 experiments).
7.3.4. fluorescent antibody staining and flow cytometer showed
By 1X106Lymph node cells are transferred in 5ml round bottom polystyrene tube.With FACS buffer solution centrifugation (277g, 5 minutes, 4 DEG C) washing cell.Supernatant is abandoned, cell is suspended in the FACS buffer solution of 50-100 μ l anti-mouse CD19 antibody marked containing FITC In.The content of every kind of fluorescence antibody is 0.5-1 μ g, acts on 30-45min on ice.Sample is protected from light.Do flow cytometer showed.
7.4 result
TIO1 or TIO3 can make expression CD19 cell (bone-marrow-derived lymphocyte) in mouse lymph nodal cell significantly increase (figure -7). CD19 is the surface marker of bone-marrow-derived lymphocyte, and the bone-marrow-derived lymphocyte that specificity increases due to anamnestic reaction can be divided into rapidly generation The thick liquid cell of specific antibody.The result shows can be increased anti-using TIO1 or TIO3 by the expression of reduction TGF-β 2 The quantity of the bone-marrow-derived lymphocyte of former specificity.It can be used for individual with this active TIO1 or TIO3 to enhance it to pathogenic The humoral immune response of the duration length of microbial antigen (vaccine).
Humidification of embodiment 8 TIO1 and TIO3 to hepatitis b vaccination effect:
8.1 material
8.1.1. mouse
Balb/c mouse, female, weight 17-18g tie up experimental animal Co., Ltd, tonneau China purchased from Beijing.
8.1.2 vaccine
Hepatitis B surface antibody (HBsAg) vaccine (Watson Bioisystech Co., Ltd) prepared using aluminium adjuvant, letter Claim HBsAg vaccine or HBsAg.
8.1.3 oligonucleotides
Oligonucleotides
TIO1 (has the sequence as shown in sequence table<400>1), and TIO2 (sequence is as described in Example 1) and TIO3 (have The sequence as shown in sequence table<400>2) as described in Example 1.
8.1.4 the preparation of the vaccine containing oligonucleotides
TIO1, TIO2 or TIO3 are added in HBsAg vaccine and is made into the vaccine of HBsAg containing oligonucleotides, is named as respectively HBsAg-TIO1, HBsAg-TIO2 and HBsAg-TIO3.Contain 1 μ g HBsAg in every 100 μ l vaccine.In HBsAg-TIO1, In HBsAg-TIO2 and HBsAg-TIO3, the content of oligonucleotides is 10 μ g.
8.1.5
HbsAg (HBsAg) (Watson Bioisystech Co., Ltd), abbreviation HBsAg.
8.2. mouse immune
In the 0th day to Balb/c mouse carry out initial immunity, method be injection 100 μ l HBsAg vaccines, HBsAg-TIO1, HBsAg-TIO2 or HBsAg-TIO3.Injection site is right hind muscle, single-point injection.In the 14th day to Balb/c mouse into Row booster immunization, the same initial immunity of method.
8.3. it takes a blood sample after being immunized
The 28th day acquisition immune serum after booster immunization.Tail vein blood, blood sampling volume >=100 μ l.By acquisition Whole blood (in 1.5mlEP pipe) is placed at room temperature for 30 minutes.11000rpm is centrifuged 15 minutes, collects serum, packing, -20 DEG C of preservations.
8.4. immune serum detection of specific antibody
Using hepatitis B surface antibody (HBsAg) specific antibody in ELISA method detection mice serum.With HBsAg is coated with enzyme mark strip in coating buffer, and 100 holes μ l/ (every hole HBsAg is 0.1 μ g), 4 DEG C overnight.The dilution of test serum Degree is 1: 6400.
Its remaining operating procedure of serum is the same as described in embodiment 2 2.5.
8.5. result
TIO1 or TIO3 can enhance the immune efficacy (figure -8) of recombinant protein vaccine (Hepatitis B virus vaccine).This says Bright, TIO1 or TIO3 can be used for individual to enhance it to the immune response of pathogenic microorganisms antigen (vaccine) and as one kind The adjuvant of novel microbial antigen or microorganism vaccine.
The synergistic effect of 9 TIO1 or TIO3 infected by influenza vaccine of embodiment
9.1. material
9.1.1 mouse
ICR mouse (Laboratory Animal Center of Jilin University).Female, weight are 18 grams or so.
9.1.2 oligonucleotides
TIO1 (with the sequence as shown in sequence table<400>1) and TIO3 (have the sequence as shown in sequence table<400>2 Column), as described in embodiment 1.
9.1.3 vaccine
With 11 days instar chicken embryo amplification FM1 (mouse adapts to influenza virus, comes from biology department, the Massachusetts Institute of Technology), allantois is taken Liquid.With hemagglutination-inhibition test (hemagglutination, HA) measurement allantoic fluid FM1 blood clotting primitive unit cell (HA unit, HAU).With the Formalin inactivation FM1 of 10%V/V, prepare full inactivation of viruses (whole-inactivated viruses, WIV)[Vaccine 2016 Jan 20；34(4)495-502].FM1 epidemic disease is matched using ISA 35 emulsifier (Seppic) emulsifier Seedling (abbreviation FM1).Inactivation FM1 containing 700HAU in every 100 μ l FM1 vaccine.TIO1 or TIO3 is added in FM1 vaccine to be made into The vaccine of FM1 containing oligonucleotides, is named as FM1-TIO1 and FM1-TIO3 respectively, and oligonucleotides content therein is 10 μ g.
9.2. it is immunized and takes a blood sample
It is immunized twice, 22 days booster immunizations are primary after initial immunity.It is injected using hind leg muscle, the volume vaccinated is 100μl.Through tail vein blood, 50 μ l/ are only within the 28th day after booster immunization.The blood of acquisition is placed 30 minutes in 37 DEG C of environment, 4 DEG C place 3 hours after its serum is precipitated sufficiently, 4000r/m be centrifuged 10 minutes, be sucked out serum, 1: 400 times dilution after be used for ELISA detection.
9.3. immune serum detection of specific antibody
Using the influenza virus specific antibody in ELISA method detection mice serum.With the antigen of the full inactivation of viruses of FM1 It as envelope antigen, is diluted, is used with coating buffer (Na2CO3 1.59g, NaHCO3 2.93g, pH 9.6, be settled to 1L) 1: 2 100 hole μ l/ coated elisa plates, sealing, 4 DEG C overnight；ELISA Plate is washed with cleaning solution (PBS containing 0.05%Tween-20), 300 holes μ l/, are washed 3 times；Be added confining liquid (PBS containing 5%FBS), 200 holes μ l/, 37 DEG C 2 hours；Mouse blood is diluted with PBS ELISA Plate is added in dilute serum by (1: 4000 dilution) clearly, and 100 holes μ l/ are added, and 37 DEG C are placed 1 hour；3 are washed with cleaning solution Secondary, then plus horseradish peroxidase marks sheep anti-Mouse secondary antibody (being diluted with confining liquid 1: 1000), and 100 holes μ l/, 37 DEG C are placed 1 Hour；Washed 3 times with cleaning solution, be added i.e. with i.e. with substrate solution (citric acid 0.01M in 10 mL, Na2HPO4 0.02M, it is ultrapure 15 μ l, OPD 4mg of water 9mL, 30%H2O2), 100 holes μ L/, room temperature is protected from light colour developing 20 minutes；Terminate liquid (20% sulphur is added Acid), 50 holes μ l/；The OD value in each hole is surveyed in A492.
9.4. result
TIO1 or TIO3 can enhanced virus vaccine (influenza virus vaccine) immune efficacy (figure -9).This explanation, TIO1 or TIO3 can be used for individual enhancing it to the immune response of the pathogenic microorganisms antigen (vaccine) including influenza virus and A kind of adjuvant as novel microbial vaccine.
Synergistic effect of embodiment 10 TIO1 or TIO3 to rabies vaccine
10.1 materials
10.1.1 mouse
ICR/c mouse, comes from Jilin University's medical board animal housing by 18-22 grams of weight.
10.1.2 vaccine
Rabies vaccine (Changchun Biological Products Institute).
10.1.3 oligonucleotides
TIO1 (has the sequence as shown in sequence table<400>1), and TIO2 (sequence is as described in Example 1) and TIO3 (have The sequence as shown in sequence table<400>2), as described in Example 1.
10.1.4 vaccine
TIO1, TIO2 or TIO3 are added in rabies vaccine and is made into rabies vaccine containing oligonucleotides, is named as respectively mad Canine vaccines-TIO1, rabies vaccine-TIO2 or rabies vaccine-TIO3.Few nucleosides in every 500 μ l rabies vaccine containing oligonucleotides The content of acid is 10 μ g.
10.2 immune preceding blood samplings
It a few days ago acquires in initial immunity to immune serum.Through tail vein blood, 50 μ l/ are only.The blood of acquisition exists 37 DEG C of environment place 30 minutes, 4 DEG C place 3 hours after its serum is precipitated sufficiently, 4000r/m is centrifuged 10 minutes, and blood is sucked out Clearly, -20 DEG C of preservations are set.
10.3. immune mouse
In the 0th day, 3 days, 7 days, 14 days, 28 days through mouse peritoneal injection 0.5ml rabies vaccine, rabies vaccine-TIO1, mad Canine vaccines-TIO2 or rabies vaccine-TIO3.Every group of mouse, half male and half female.
10.4 blood samplings
In the 35th day acquisition immune serum, method was as described in 10.2.
The detection of 10.5 serum rabies viruses neutralizing antibodies
It is neutralized using mad dog epidemic disease poison in quick rabies vaccine fluorescence stove Inhibition test (RFFIT) method inspection mice serum anti- As a result body indicates [Virol Sin.2012 Jun with international unit/milliliter (IU/ml)；27 (3): 187-93].
10.6. result
TIO1 or TIO3 can enhance the immune efficacy (figure -10) of inactivated virus vaccine (rabies vaccine).This explanation, TIO1 or TIO3 can enhance the immunity of the rabies poison infection of individual, the pathogenic microorganisms being used as including rabies vaccine The adjuvant of vaccine.
The synergistic effect of embodiment 11, TIO3 to glioma cell lysate vaccine
11.1 materials
11.1.1 mouse
C57BL/6 mouse ties up experimental animal Co., Ltd, tonneau China purchased from Beijing.
11.1.2GL261 cell
GL261 cell is the glioma cell (ATCC) in C57BL/6 mouse source.
11.1.3 oligonucleotides
TIO3 (has the sequence as shown in sequence table<400>2), as described in Example 1.
11.1.4 culture medium
RPMI1640 culture medium (Gibco company.Complete RPMI1640 culture medium contains 10% fetal calf serum (FBS) (Gibco Company), 2mM L- glutamine, 100U/ml penicillin, 100 μ g/ml streptomysins.Serum-free RPMI1640 culture medium is free of tire Cow's serum
11.2.GL261 the preparation of cell lysate
Using complete RPMI1640 culture medium at 37 DEG C, 5%CO2Under the conditions of cultivate GL261 cell.By 1ml growth conditions Good GL261 cell (1 × 107/ ml) it is inoculated in the abdominal cavity of healthy C57BL/6 mouse.After mouse web portion bulge, put to death Mouse is placed in 75% ethyl alcohol and impregnates 2-3min disinfection.Mouse peritoneal is opened in super-clean bench, it is seen that the GL261 cell of formation is real Body tumor.GL261 cellular entities tumor is taken out by sterile working.It is cut into small pieces in plate with operating scissors, it is anti-with physiological saline Tumor tissues and remaining blood are washed off again.Clean tissue block is ground repeatedly in tissue grinder, collects grinding Liquid.Lapping liquid is placed in liquid nitrogen and room temperature multigelation 5 times.Supernatant is collected after 2000rmp centrifugation 10min.This supernatant is GL261 cell colloid struma oncocyte lysate (GTL).Determining the protein quantity is carried out to GTL with Bradford method.Use SDS- PAGE is further analyzed GTL.GTL is set -80 DEG C of refrigerators to save.
The preparation of 11.3 GTL vaccines and the vaccine of GTL containing oligonucleotides
The protein concentration of GTL is adjusted to 4mg/ml with PBS.This GTL and emulsifier are mixed by 1: 1 (volume: volume) Vaccine is made, this vaccine is named as GTL vaccine.The GTL vaccine containing TIO-3 that TIO-3 is prepared is added in GTL vaccine, this Kind vaccine is referred to as GTL-TIO-3 vaccine, and wherein the concentration of TIO-3 is 10 μ g/100 μ l.
11.4 immune mouse
100 μ l GTL vaccines or GTL-TIO-3 vaccine were subcutaneously injected in mouse neck in the 0th day and the 9th day.
11.5 being inoculated with glioma cell
At the 14th day, injection 1 × 104For GL261 cell to mouse intracranial, volume injected is 2 μ l, makes mouse that colloid occur Tumor, method such as document (Prins RM, et al.Cancer Res.2003 Dec 1；63 (23): 8487-91) it is described.From connecing Start to observe and record the life cycle of mouse after kind tumour, becomes celestial after dead mouse and confirm that colloid has occurred in mouse intracranial Tumor.
11.6 results
TIO3 can significantly increase the immune efficacy (Figure 11) of glioma cell lysate (tumour antigen vaccine), keep lotus knurl small The life cycle of mouse significantly extends.Should the result shows that, TIO3 can enhance individual anti-glioma immune response, be used as include The adjuvant of tumor vaccine including glioma vaccine.
The synergistic effect of embodiment 12, TIO1 or TIO3 to lung carcinoma cell lysate vaccine:
12.1 materials
12.1.1 mouse
C57BL/6 male mice (Beijing Vital River Experimental Animals Technology Co., Ltd.), 6 week old.
12.1.2 cell
Lewis lung cancer (Lewis lung carcinoma, LLC) cell (ATCC) originates from C57BL/6 mouse.It uses Complete RPMI1640 culture medium (as described in 11.1.4), at 37 DEG C, 5%CO2Under conditions of cultivate the cell.
12.1.3 oligonucleotides
TIO1 (with the sequence as shown in sequence table<400>1) and TIO3 (have the sequence as shown in sequence table<400>2 Column), as described in embodiment 1.
12.2.Lewis the preparation of lung carcinoma cell (LLC) lysate
By the LLC cell (1 × 10 of subculture in vitro separately culture6/ 0.1ml) to be inoculated in C57BL/6J male mice dorsal sc long At entity tumor.Mouse is sacrificed, tumour is taken out, is split by the preparation of method described in embodiment 11 11.2, identification and preservation LLC It solves object (LTL).Every 1 × 106LLC cell prepares the LTL of 100 μ l.
The preparation of 12.3 LTL vaccines and the vaccine of LTL containing oligonucleotides
The LTL of preparation and emulsifier are mixed and made into vaccine by 1: 1 (volume: volume), this vaccine is named as LTL epidemic disease Seedling.The LTL vaccine containing TIO1 or TIO3 that TIO1 or TIO3 is prepared is added in LTL vaccine, both vaccines are known respectively as LTL-TIO1 vaccine and LTL-TIO3 vaccine, wherein the concentration of TIO1 or TIO3 is 10 μ g/100 μ l.
12.4 mouse immunes
In the 0th day and the 14th day in 6 week old C57BL/6J male mices, 100 μ l LTL vaccine of right side dorsal sc injection, LTL-TIO1 vaccine or LTL-TIO3 vaccine.Every group of 10 mouse.
12.5 inoculation Lewis lung cancer (Lewis lung cancer, LLC) cells
The 100 μ l LLC cell suspension of dorsal sc injection on the left of the 21st day, mouse.The preparation method of LLC cell suspension It is: by the LLC cell (1 × 10 of subculture in vitro separately culture7/ 100 μ l) it is inoculated in C57BL/6J male mice dorsal sc and grows up to reality Body tumour.Mouse is sacrificed, tumor tissues is taken out by sterile working method, it is disappeared with 0.25% trypsase -0.04%EDTA Change 30min, single cell suspension is made.It is every 1 × 10 that cell concentration is adjusted in PBS6/100μl.The 18th after inoculated tumour It sacrifices mouse, cuts tumor mass, claims its weight.
12.6 results (table -1)
The synergistic effect of table -1, TIO1 or TIO3 to lung carcinoma cell lysate vaccine
The result shows that TIO-1 or TIO-3 can enhance the immune efficacy (table -1) of lung carcinoma cell lysate, make tumor regression (p < 0.05).This explanation, TIO-1 or TIO-3 can enhance the anti-lung cancer immune response of individual, be used to include that lung cancer vaccine exists The adjuvant of interior tumor vaccine.
Therapeutic effect of 13 TIO3 of embodiment to gastric cancer:
13.1 materials
13.1.1 mouse
Balb/c female mice (Beijing Vital River Experimental Animals Technology Co., Ltd.), 18-22g.
13.1.2 stomach cancer cell
MFC cell (mouse gastric cancer cell) is that the stomach cancer cell in Balb/c mouse source (is originated from the U.S. ATCC).With complete RPMI1640 culture medium (as described in 11.1.4), at 37 DEG C, 5%CO2Under conditions of cultivate the cell.
13.1.3 oligonucleotides
TIO3 (has the sequence as shown in sequence table<400>2), as described in Example 1.
13.2 major experimental equipment and instrument
100ml Tissue Culture Flask, 1ml syringe, cell counting board.CO2Cell culture incubator (Japanese SANYO company), Cell culture inverted microscope (Japanese Olympus company), centrifuge (German Biofuge Fresco).
13.3 experiments
The MFC subcutaneous transplantation knurl model cell of in vitro culture was inoculated in mouse left hind dorsal subcutaneous in the 0th day, is injected Volume is 200 μ l, wherein containing 1X106MFC cell.Start within the 10th day after being inoculated with MFC cell (molten to mouse injection TIO3 In PBS), it then injects again within every two days once, co-injection 6 times.Injection site is that left hind inoculated tumour lymph node drains area's skin Under.Each volume injected is 100 μ l, wherein containing 25 μ g TIO3.Control group mice presses same programmed injection PBS.Observation And record the life span of mouse.
13.4 results
TIO3, which is used alone, can make tumor-bearing mice life cycle be obviously prolonged (figure -12).This explanation, TIO3 can enhance individual Anti-gastric cancer cellullar immunologic response can be used to treat the tumour including gastric cancer.

Claims (20)

1. single-stranded deoxy-oligonucleotide has the sequence as shown in sequence table<400>1 and<400>2.
2. single-stranded deoxy-oligonucleotide described in accordance with the claim 1, they can be modified by sulphation.
3. single-stranded deoxy-oligonucleotide described in 2, they can be by inhibiting transforming grouth factor beta 2 (TGF- according to claim 1 β 2) expression and inhibit or weaken the immunosupress mediated by TGF-β 2, and then enhance to pathogenic microorganisms and tumour cell Immune response.
4. single-stranded deoxy-oligonucleotide described in 2, they can be by reducing transforming grouth factor beta 2 (TGF- according to claim 1 β 2) mRNA level and inhibit or weaken the immunosupress mediated by TGF-β 2, and then enhance it is thin to pathogenic microorganisms and tumour The immune response of born of the same parents.
5. single-stranded deoxy-oligonucleotide described in 2, they can promote to pathogenic microorganisms antigen or vaccine according to claim 1 Immune response, thus be used as adjuvant to enhance the effect of pathogenic microorganisms vaccine, generate anti-infectious function.
6. single-stranded deoxy-oligonucleotide described in 2, they can be used as including circovirus vaccine according to claim 1 The adjuvant of pathogenic virus vaccine enhance its immune efficacy, generate anti-infectious function.
7. single-stranded deoxy-oligonucleotide described in 2, they can be used as including Hepatitis B virus vaccine according to claim 1 The adjuvant of pathogenic virus vaccine inside enhances its immune efficacy, generates anti-infectious function.
8. single-stranded deoxy-oligonucleotide described in 2, they can be used as including influenza virus vaccine according to claim 1 The adjuvant of pathogenic virus vaccine enhance its immune efficacy, generate anti-infectious function.
9. single-stranded deoxy-oligonucleotide described in 2, they can be used as including hydrophobia according to claim 1 The adjuvant of pathogenic virus vaccine enhance its immune efficacy, generate anti-infectious function.
10. single-stranded deoxy-oligonucleotide described in 2, they can be used as the adjuvant of tumor vaccine to enhance according to claim 1 Individual generates therapeutic effect to tumour to the immune response of tumour cell.
11. single-stranded deoxy-oligonucleotide described in 2, they can be used as including brain glioblastoma cell cracking according to claim 1 The adjuvant of tumor vaccine including object vaccine generates therapeutic effect to the tumour including glioma.
12. single-stranded deoxy-oligonucleotide described in 2, they can be used as including lung carcinoma cell lysate epidemic disease according to claim 1 The adjuvant of tumor vaccine including seedling generates therapeutic effect to the tumour including lung cancer.
13. single-stranded deoxy-oligonucleotide described in 2, they can be used for oncotherapy according to claim 1.
14. single-stranded deoxy-oligonucleotide described in 2, they can be by swollen including gastric cancer to treat according to claim 1 Tumor.
15. according to claim 1, single-stranded deoxy-oligonucleotide described in 2,3,4,5,6,7,8,9,10,11,12,13,14, it Can enhance with various adjuvants, inherent immunity activator use in conjunction individual pathogenic microorganisms and tumour cell are immunized Response generates anti-infectious function and oncotherapy effect.
16. single-stranded deoxy-oligonucleotide described in 2,10,11,12,13,14, they can resist with various according to claim 1 Antineoplastic agents use in conjunction treats tumour.
17. single-stranded deoxy-oligonucleotide described in 2,10,11,12,13,14, they can be controlled with tumour according to claim 1 It treats and treats tumour with cell use in conjunction.
18. single-stranded deoxy-oligonucleotide described in 2, they can be with pharmaceutically acceptable vehicle group patent medicine according to claim 1 Compositions are applied and use effective dose.
19. single-stranded deoxy-oligonucleotide described in 2, they can be applied to a by a variety of administration routes according to claim 1 Body.
20. single-stranded deoxy-oligonucleotide described in 2, they can pass through therapeutic device or delivery vector application according to claim 1 In individual.
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