CN110464841A - The pharmaceutical composition and its application of Immune-enhancing effect - Google Patents
The pharmaceutical composition and its application of Immune-enhancing effect Download PDFInfo
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Abstract
The present invention discloses the pharmaceutical composition and its application of Immune-enhancing effect.The pharmaceutical composition of Immune-enhancing effect of the invention includes vaccine and regulator, wherein vaccine includes at least one nucleic acid and/or at least one polypeptide, the at least one nucleic acid includes that at least one encodes at least one antigen open reading frame, and at least one polypeptide includes at least one epitope.Regulator includes selected from least one of BcL2, CD40L, IL-15 and IL-15Ra or its code nucleic acid and transforming growth factor signal pathway inhibitor.The pharmaceutical composition of Immune-enhancing effect of the invention can provide the protective immunity from pathogenic infection, and can be used for preventing and/or treating kinds of tumors.
Description
Technical field
The present invention relates to pharmaceutical products, and in particular to antitumor in the pharmaceutical composition and raising lymphocyte of Immune-enhancing effect
The method of the special T lymphocyte ratio of antigen.The special T lymphocyte of the antitumor antigens have expression TNF-a or/and
The characteristics of IFN-r.
Background technique
The immune response of body is to capture antigen by antigen presenting cell (APC) first, will be resisted after processing and processing
Prime information submission then causes a series of specific immune response to lymphocyte.Dendritic Cells (DC) is presently considered to be
The strongest APC of function, maximum feature be can stimulate primary tape T cell (T cells) proliferation and work
Change, the key link for being starting, regulating and controlling and maintaining specific immune response.In antitumor immunity of organism, mediated by T cell
Cellular immunity plays an important role.
The study found that there is the reduction of DC quantity and functional defect, tumor tissues and its surrounding wetting in tumor patient
The quantity of DC, the generation of function and tumour, development, transfer, prognosis have close relationship.The Tumor Differentiation journey that DC intensively infiltrates
Degree is high, and prognosis is preferable；And the tumour of DC mild infiltration is often accompanied by that differentiation degree is low and malignant progression.Tumour cell has compared with Gao Shui
Flat Fas expression such as can induce the Lymphocyte Apoptosis of FasL expression, and can secrete TGF-β, IL-10 at the inhibitive abilities of immunity cell
The factor causes HLA-II antigen to decline, to escape immune attack.
In recent years, understood that immune system identifies tumour antigen really, but while there are tumour antigen, T cell
Ensure to hold stationary state.Based on this phenomenon, there is such hypothesis: the antigen presenting cell of patient's body, cannot correctly identify swollen
It is presented to T lymphocyte by tumor antigen, causes the immune response of tomour specific.In recent years, how to increase antigen presenting cell
Quantity, improve antigen presenting cell, especially DC cellular uptake, transhipment, present antigen, stimulate T cell ability, be current
An emphasis in tumour immunity research.
A large number of studies show that the signal that CD40-CD40L is mediated can induce the activation of APC.The latter is in activation tomour specific
Property immunologic cytotoxicity T cell in play an important role: provide antigenic stimulus signal and second signal, the two synergistic effect starting is exempted from
Epidemic disease cascade reaction plays tumour immunity function.Meanwhile research find CD40L can also maintain DC cell activation, existence and
The amplification of CD8+T cell, and energy antagonism cell factor IL-10 is to the depression effect of DC cell differentiation, maturation and function.
CD40L molecule can with DC surface C D40 interact activation DC, promote APC costimulatory molecules expression and cell because
Son secretion.The DC of activation is the bridge between tumour cell and tumor-specific immunity killer T cell, can present tumour antigen to
T cell and activation T cell, generate tumour-specific killer T cell.Therefore, pass through CD40-CD40L signaling molecule pathway activation
The submission to weak immunogene tumor-cell antigen, inducing tumor specific immune can be enhanced in DC.
IL-15 is a kind of cell factor similar with IL-2 structure, and wide expression is in various cells and tissue, such as monokaryon
Cell, macrophage, DC cell, fibrocyte etc..IL-15 by conjunction with IL-15 receptor alpha, activate downstream JAK1,
JAK3, leads to the phosphorylation of downstream STAT3 and STAT5 and the activation of signal path, induction BCL2, map kinase access, Lck and
The phosphorylation of syk leads to the proliferation and maturation of cell.
IL-15 is capable of the activation and proliferation of regulatory T-cell and NK cell, is able to maintain that the Memorability in the case where lacking antigenic stimulus
The survival of T cell.There are some researches prove in the lymphocyte of rodent, IL-15 passes through induction BCL2L1/BCL-x (L)
And inhibit apoptosis.Likewise, it has also been found that IL-15 inhibits withering for T lymphocyte by induction BCL2 and/or Bcl-xL in human body
It dies.
Recently, there is researcher to use newcastle disease virus (NDV) as carrier, construct a kind of expression recombination IL-15 albumen
DNA tumor vaccine.It is preclinical the results show that it is raw that the tumor vaccine shows control melanoma in mouse model
Long potentiality.Similarly, the recombined vaccinia virus of expression of influenza virus A albumen and IL-15 can promote the intersection of CD4+T cell
Protection.Another include IL-15 gene recombination brucella DNA vaccination shown in mouse enhancing CD8+T cell exempt from
The ability of epidemic disease response.
Transforming growth factor-β (TGF-β) is a kind of multi-functional cell factor, can influence the growth of cell, differentiation,
Apoptosis etc..In addition, there are also important immunoregulation effects for TGF-β.TGF-β can effectively inhibit T cell function and DC cell
Antigen presentation capability.Nearest research has illustrated TGF-β in up-regulation Plasmacytoid DC (pDC) immunological regulation enzyme indoles amine
Direct effect in 2,3- dioxygenases (IDO) expression, and long-term T cell is caused to be resistant to.By being catalyzed essential amino acid color
The degradation of propylhomoserin, the activity of IDO depression effect T cell and promotion Treg differentiation and activation.
Transforming growth factor-β (TGF-β) carries out letter by bind to transforming growth factor-beta receptor (TGFBR) compound
Number transduction.According to the difference of molecular structure and functional character, TGFBR family includes I receptor (TGFBR1), II receptor
(TGFBR2) and type III receptor (TGFBR3).TGFBR1/2 belongs to transmembrane receptor serine/threonine kinase enzyme family, born of the same parents
Inside contain serine/threonine kinase area.After transforming growth factor-β is in conjunction with TGFBR1/2 heterodimer, downstream is activated
Signaling molecule, and then activation signal conducts.Intracellular section of TGFBR3 does not contain kinase activity area, does not participate in signal transduction directly, main
If adjusting the combination of TGF-β and frizzled receptor, be otherwise known as cooperative expert systems.
TGFBR3 is the coreceptor of typical TGF-β signaling pathways, participates in mediating the non-with SMAD of SMAD dependence
The downstream signaling pathway of dependence.Research finds the early stage in many tumours, such as in breast cancer, the expression of TGFBR3
There is apparent downward with patient's Carcinoma side normal tissue ratio.It data show, in some tumor models, TGFBR3 inhibits cell
Migration and invasion show that TGFBR3 has the function of inhibiting tumour progression and transfer.
In recent years, there is a large amount of report using the DC vaccine therapy tumour for having loaded antigen, from the data reported at present
From the point of view of, DC vaccine seems to represent a kind of new very potential method for improved immunotherapy of tumors.However, single
The use of only DC vaccine generally can not lead to the improvement of expected immunotherapeutic effects, cannot obtain satisfactory clinic
Effect.
Summary of the invention
To solve at least partly technical problem in the prior art, the present invention is had found after extensive studies it by being applied in combination
Different regulators can cooperate with enhancing or improve the immunity of vaccine.It is based at least partially on this and completes the present invention.Specifically,
The present invention includes the following contents.
The first aspect of the present invention provides a kind of pharmaceutical composition of Immune-enhancing effect, it includes:
(1) vaccine, the vaccine include at least one nucleic acid and/or at least one polypeptide, and the nucleic acid includes at least one
At least one antigen open reading frame of a coding, the polypeptide include at least one epitope；
(2) regulator, the regulator include selected from least one of BcL2, CD40L, IL-15 and IL-15Ra or its
Code nucleic acid and transforming growth factor signal pathway inhibitor.
In certain embodiments, the transforming growth factor signal pathway inhibitor be selected from for TGF-β, TGFBR1,
The inhibitor of at least one of TGFBR2 and TGFBR3.
In certain embodiments, the transforming growth factor signal pathway inhibitor is selected from group consisting of the following: short of money
Resistance antibody encodes the nucleic acid of antagonistic antibodies, siRNA, antisense RNA, turns comprising that can combine TGF-β and can block
Change the albumen of the amino acid sequence of growth factor signal access or the nucleic acid of the coding amino acid sequence, competed with TGF-β
To its receptor TGFBR1, TGFBR2 or TGFBR3 in conjunction with soluble protein or the nucleic acid of the coding soluble protein and small
Molecule inhibitor.
In certain embodiments, the transforming growth factor signal pathway inhibitor is the core of antibody or encoding antibody
Acid, the antibody are directed to TGF-β or its receptor.
In certain embodiments, described comprising in conjunction with TGF-β and transforming growth factor signal path can be blocked
Amino acid sequence albumen be selected from TGFBR1, TGFBR2 or TGFBR3 segment.
In certain embodiments, the segment is the extracellular domain containing TGFBR1, TGFBR2 or TGFBR3
Fusion protein.
In certain embodiments, the fusion protein be TGFBR1, TGFBR2 or TGFBR3 extracellular domain with
The fusion protein of the part Fc of immunoglobulin.
The second aspect of the present invention provides a kind of method for improving TNF-a and/or IFN-r content in lymphocyte, packet
Include the step of pharmaceutical composition is acted on into lymphocyte.
The third aspect of the present invention provides a kind of ratio for improving TNF-a+ and/or IFN-r+ cell in lymphocyte populations
Method comprising pharmaceutical composition according to claim 1-7 is acted on to the step of the lymphocyte populations
Suddenly.
In other raising lymphocyte populations of the invention the method for the ratio of TNF-a+ and/or IFN-r+ cell include with
Lower step:
1) the step of expression cell obtains transfection cell is introduced the reagents into；With
2) the transfection cell and the lymphocyte populations are co-cultured, or makes at least partly secretion of the transfection cell
The step of object and the lymphocyte populations contact；
Wherein the reagent include as regulator coding in BcL2, CD40L, IL-15 and IL-15Ra at least
A kind of nucleic acid and coding is with TGF-β competition to the nucleic acid of the soluble protein in conjunction with its receptor TGFBR1, FBR2 or TGFBR3；
Or
The reagent includes (a) as at least one nucleic acid of vaccine and/or at least one polypeptide, and the nucleic acid encode is extremely
A kind of few antigen open reading frame, the polypeptide include at least one epitope；(b) it is selected from as the coding of regulator
The nucleic acid of at least one of BcL2, CD40L, IL-15 and IL-15Ra and coding and TGF-β competition to its receptor TGFBR1,
The nucleic acid for the soluble protein that TGFBR2 or TGFBR3 is combined.
The pharmaceutical composition of Immune-enhancing effect of the invention can greatly enhance the immunity of vaccine, can provide from cause of disease
The protective immunity of body-sensing dye can be especially useful for the effect of enhancing prevention and/or treatment kinds of tumors.
Detailed description of the invention
Fig. 1 is the survivorship curve of DC cell after the nucleic acid molecules for having transfected coding present composition albumen, with control group
Than having transfected the DC cell of the present composition, hence it is evident that have higher survival rate.
Fig. 2 is DC cell phenotype after the nucleic acid molecules for transfecting coding present composition albumen, thin with immature dendron
Born of the same parents (iDC) compare, the CD80 on mature DC cell (mDC-survivin and mDC-survivin/ regulator nucleic acid group) surface,
CD86, CD83 are obviously raised, and show significant maturation DC cell characteristic；With the DC cell phase for only having transfected survivin antigen
Than the DC cell phenotype for having transfected survivin/ regulator nucleic acid is consistent with the only transfection DC cell of survivin antigen.Fig. 2
In, the left side of every group of column is mDC control, and right side is the mDC cell for having transfected the present composition.
Fig. 3 is influence of the immunomodulatory agents to t cell response.Compared with only transfecting survivin antigen group, transfection
The DC cell of survivin/ regulator nucleic acid molecules can cause stronger t cell response, by the T cell of DC cytositimulation
It is expressed with higher proportion of TNF-a and IFN-r.In the column diagram of Fig. 3, the column on the left of in the of every group is IFN-r, the column on the right side of in the of every group
For TNF-a.
Specific embodiment
The existing various exemplary embodiment that the present invention will be described in detail, the detailed description are not considered as to limit of the invention
System, and it is understood as the more detailed description to certain aspects of the invention, characteristic and embodiment.
It should be understood that it is to describe special embodiment that heretofore described term, which is only, it is not intended to limit this hair
It is bright.In addition, for the numberical range in the present invention, it is thus understood that specifically disclose the range upper and lower bound and they it
Between each median.Median and any other statement value in any statement value or stated ranges or in the range
Lesser range is also included in the present invention each of between interior median.These small range of upper and lower bounds can be independent
Ground includes or excludes in range.
Unless otherwise stated, all technical and scientific terms used herein has the routine in field of the present invention
The normally understood identical meanings of technical staff.Although the present invention only describes preferred method and material, of the invention
Implement or also can be used and similar or equivalent any method and material described herein in testing.The institute mentioned in this specification
There is document to be incorporated by reference into, to disclosure and description method relevant to the document and/or material.It is incorporated to any
When document conflicts, it is subject to the content of this specification.Unless otherwise stated, " % " is the percentage based on weight.
In the present invention, term " antigen " refers to and can be identified by immune system, and can be special by forming antibody or/and antigen
Specific T cell and the substance for causing the immune response of antigen-specific.Generally, antigen can be comprising at least one epitope
And the albumen or polypeptide on T cell surface can be presented to by major histocompatibility complex (MHC).In the present invention,
Antigen can be the product of mRNA translation, the product after being also possible to DNA transcription and translation.
In the present invention, term " nucleic acid " includes DNA (i.e. DNA) and ribonucleic acid (i.e. RNA).In the feelings of RNA
Under condition, the degradation of the unstability and number of ways of RNA in order to prevent can be with according to more kinds of Natural Degradation approach of known RNA
A variety of optimizations are carried out to nucleic acid molecules.For example, end structure is vital to the stabilization of mRNA.For example, naturally occurring
MRNA 5 ' ends, there are the guanosine nucleotide of modification, referred to as 5 ' cap sequences, and 3 ' ends have a Duan Changyue 200~
Adenosine nucleoside acid (i.e. poly A tract bar) structure of 300 bases, the UTR sequence at 5 ' and 3 ' ends, such as the beta- globulin of people
UTR sequence.
[pharmaceutical composition of Immune-enhancing effect]
The first aspect of the present invention provides the pharmaceutical composition of Immune-enhancing effect, includes at least:
(1) vaccine, the vaccine include at least one nucleic acid and/or at least one polypeptide, and the nucleic acid includes at least one
At least one antigen open reading frame of a coding, the polypeptide include at least one epitope；With
(2) regulator, the regulator include selected from least one of BcL2, CD40L, IL-15 and IL-15Ra or its
Code nucleic acid and transforming growth factor signal pathway inhibitor.
Vaccine
Vaccine of the invention is generally understood as providing one or more antigens (preferably immunogene) for preventing or treating
Substance.For example, vaccine may include antigen, or encode the nucleic acid of the antigen.The nucleic acid can be DNA or RNA.Vaccine is also
It may include the cell for expressing the antigen, such as DC cell or PBMC cell.Antigen or immunogene can be derived from any be suitable for
The substance of inoculation.
Vaccine of the invention can be any kind of vaccine, and the example includes but is not limited to tumor vaccine, infectious disease epidemic disease
Seedling.Present invention preferably uses tumor vaccines as vaccine.
In certain embodiments, vaccine of the invention includes nucleic acid.Nucleic acid of the invention can be one, be also possible to
It is multiple.Each nucleic acid codified at least one antigen open reading frame.Nucleic acid at this time is also referred to as antigen nucleic acid or antigen core
Acid molecule.
In one embodiment, antigen nucleic acid molecule encoding bacterium, virus, fungi or other pathogens has
The peptide fragment of immunogenicity.It includes HAV, HBV, HCV, cytomegalovirus that wherein pathogen, which includes but is not limited to the hepatitis virus of people,
CMV, human immunodeficiency virus HIV, Epstein-Barr virus, dengue fever virus, human papilloma virus (HPV), Respiratory Syncytial Virus(RSV), rhinopathy
Poison, I type of human T-cell lymphotropic virus (HTLV-1), influenza, bovine leukemia virus (BLV), pertussis, gray nucleus
Inflammation, measles,mumps,rubella, smallpox, shingles zoster, anthrax, tetanus, rotavirus, rabies viruses, fowl pox, meningitis ball
Bacterium, encephalitis, pneumococcus, streptococcus, staphylococcus, Neisseria, Escherichia coli, Shigella, Leishmania, exhales at anthrax
Inhale road syncytial virus, parainfluenza, adenovirus, varicella, flavivirus, mycobacterium tuberculosis and malaria etc..
In one embodiment, antigen nucleic acid molecule encoding tumour antigen.In this case, tumour antigen can express
In positions such as the surface of tumour cell, cytoplasm or nucleus.Tumour antigen can also be selected from compared with normal cell swollen
The albumen being overexpressed in oncocyte.Tumour antigen can be further divided into tumor associated antigen (TAA) and tumour specific antigen
(TSA).TAA is a kind of in tumour cell and the existing antigen molecule of normal cell, and the example includes: Embryo albumen, sugar
Proteantigen, squamous cell antigen etc..TAA and non-tumor cell institute is peculiar, normal cell can also be with microsynthesis, and in tumour
Height is expressed when cell Proliferation.TSA, which refers to only to express, may be not present the neoantigen in normal cell in tumor cell surface.It is this kind of anti-
Original may be present in the tumour of the same organization type of Different Individual, and the melanoma as people's malignant mela noma gene encodes is special
Property antigen, may be present in the melanoma cells of Different Individual, but normal melanocyte is not expressed.TSA may be not
Common to tumour with histological type, such as the Ras gene product of mutation can be common in lung cancer, tumor in digestive tract, but due to
The expression product of its amino acid sequence and normal proto-oncogene Ras is inconsistent, can be identified by the immune system of body, excitation
The immune response of body.In general, this kind of antigen generated by mutation, referred to as neoantigen (neoantigen).These antigens are all
It can be identified by cytotoxic T lymphocyte, and the cell for presenting the antigen can be killed by T lymphocyte.
In certain embodiments, vaccine of the invention includes at least one polypeptide.Polypeptide herein includes at least one
Epitope.Therefore, polypeptide herein can be understood as a kind of antigen.
The preferred tumour antigen of antigen of the invention, can be selected from least one of group consisting of the following: TDO2,
MAGEC1、HMOX1、WT1、LY6H、AIM2、IDO1、CHI3L1、IL13RA2、LCK、GFAP、KIF20A、CNTN2、MUC16、
PEG10、TNC、SOX11、IGF2BP3、S100A8、AKAP4、TTK、CHI3L2、PTHLH、CDC45、PMEL、TOP2A、PTTG1、
NRCAM、HMMR、MUC4、LY6K、SOX10、FOSL1、PRAME、FOLR1、BIRC5、KIF2C、ITGAV、ART4、PROM1、
CT83、S100A9、PPIB、S100A12、STAT1、EPCAM、ROR1、MLANA、KAAG1、KLK3、NT5E、PTPRZ1、SPAG4、
MET、RGS1、CSPG4、PDCD1LG2、MUC1、CD274、PSCA、WDHD1、FABP7、PLIN2、PTGER2、HAVCR2、
TPD52、ABCC3、TSSK6、ERBB2、CCDC110、TERT、CTAG2、SEC61G、ADORA2A、AURKB、ACPP、EPHX1、
PTGS1、EZH2、PTGS2、PLK4、DDX43、PA2G4、PAX8、IDH1、SFMBT1、EPHA2、NAPSA、LPGAT1、NUF2、
SPAG5、STAT3、MELK、ST8SIA1、EBAG9、KIFC3、CEACAM5、RPL19、SYCP2、DSE、ANKRD30A、
TRAPPC1、RGS5、MGAT5、KRT19、B3GALNT1、CAGE1、AGER、ACRBP、LAG3、NELFA、RAB38、CCND1、
SART1、UBE2V1、SLAMF7、KCNMA1、MUM1、HSPH1、GUCY1A3、AKAP13、SQSTM1、BCAN、CCNB1、TP53、
SUGT1、AURKA、RAN、LY6D、NLGN4X、SART3、PRKDC、FOXP3、HBEGF、PIK3R1、SLC1A3、PCNA、KIF1C、
BSG、ATP2A3、SPAG9、RPSA、NFYC、LRRC8A、IQGAP1、LY6E、TRIOBP、ART1、BAGE、BIRC7、CA9、
CCDC54、DCT、IDO2、MAGED4、SOX2、SYCP1、TYR、5T4、BAGE2、BORIS、CALR3、CSAG2、CSH1、
CTAG1A、CTAG1B、CTAGE1、E6、FMR1NB、GAGE1、GAGE2A、GAGE3、GAGE4、GAGE5、GAGE6、GAGE7、
GAGE8、GRDX、HNPRL、IGHD、MAGEA1、MAGEA10、MAGEA12、MAGEA2、MAGEA3、MAGEA4、MAGEA6、
MAGEA9、MAGEB1、MAGEB2、MAGEC2、PAGE4、PAP、SAGE1、SPANXB1、SPO11、SSX1、SSX2、SSX4、
Brachyury/TFT、TTR、TYRP1、VSIR、XAGE1B、XAGE1E、ENAH、AKR1B10、GPC3、AFP、FLVCR1、
FGF19、PSPH、ABL2、CCT3、SMYD3、TMEM106C、ZNF260、EPCAM、ICK、PHF20L1、ANXA3、ZNF623、
CASK、FAM122B、IRS1、OTUD6B、TMEM68、VASH2、FGF3、TERF1、TSHZ2、TTC13、UTP14A、Survivin。
In addition, tumour antigen can also include tumor patient individual it is special, generated by gene mutation in tumour cell
Neoantigen.The gene of the mutation can be any one intracellular gene, and expression product can be expressed in cell surface,
Portion in the cell can be expressed.
Regulator
Regulator of the invention is included (a) selected from least one of BcL2, CD40L, IL-15 and IL-15Ra or its volume
Code nucleic acid, and (b) transforming growth factor signal pathway inhibitor.
In certain embodiments, (a) of the invention ingredient is albumen comprising BCL2 albumen, CD40L albumen, IL-15
At least one of albumen, IL-15Ra albumen.Preferably, BCL2 albumen has the amino acid sequence of SEQ ID NO:1.It is preferred that
Ground, CD40L albumen have the amino acid sequence of SEQ ID NO:2.Preferably, IL-15 albumen has the amino of SEQ ID NO:3
Acid sequence.IL-15Ra albumen has the amino acid sequence of SEQ ID NO:4.(a) ingredient of the invention can be in above-mentioned albumen
One kind, be also possible to two or more combinations.In the case where two or more combinations, two or more ingredients can be mixed
Object form exists, can also individual two kinds of ingredients exist, can also be the shape that two or more ingredients passes through chemical bonds
Formula exists.In certain embodiments, (a) ingredient is the fusion egg of two kinds of albumen of BcL2, CD40L, IL-15 and IL-15Ra
It is white.Such as the fusion protein of IL-15 and IL-15Ra.
In certain embodiments, (a) of the invention ingredient is nucleic acid comprising encodes nucleic acid, the coding CD40L of BcL2
Nucleic acid, encode the nucleic acid of IL-15 and encode at least one of the nucleic acid of IL-15Ra.Preferably, the nucleic acid of CD40L is encoded
Sequence with SEQ ID NO:5.Preferably, the nucleic acid for encoding BCL2 albumen has the sequence of SEQ ID NO:6.Preferably,
The nucleic acid for encoding IL-15 has sequence shown in SEQ ID NO:12.Preferably, the nucleic acid for encoding IL-15Ra has SEQ ID
Sequence shown in NO:13.(a) nucleic acid of the invention can be one of above-mentioned nucleic acid, be also possible to two or more combinations.
It is further preferred that nucleic acid of the invention is while encoding the nucleic acid of two or more albumen.Herein, " encode simultaneously " refer to it is same
Nucleic acid molecules can encode two or more albumen.Albumen two or more at this time, which can merge form, to be existed, but it is highly preferred that
Same nucleic acid molecule encoding generates the albumen of two or more individualisms.Two or more eggs is generated simultaneously in same nucleic acid molecules
It, can be by making to connect such as ribosome entry site (IRES) Lai Shixian between two adjacent genes in the case where white.Ribose
The sequence of body entry site can be as shown in SEQ ID NO:7.It optionally, can also be by making to connect between two adjacent genes
The nucleic acid sequence of self cleavage polypeptide sequence is encoded to realize.As illustrative examples, nucleic acid of the invention can be while encode IL-
The nucleic acid of 15 and IL-15Ra.The example of such nucleic acid includes but is not limited to nucleic acid shown in SEQ ID NO:8, which can be same
When coding generate individual IL-15 albumen and IL-15Ra albumen.
In certain embodiments, (a) of the invention ingredient can also be the combination of above-mentioned albumen or nucleic acid.Albumen and core
The group composition and division in a proportion of acid is not particularly limited, and can be readily determined as needed by those skilled in the art.
In certain embodiments, (b) of the invention transforming growth factor signal pathway inhibitor be selected from for TGF-β,
The inhibitor of at least one of TGFBR1, TGFBR2 and TGFBR3.Preferably, for the inhibitor of TGFBR3.It is highly preferred that
The core of the soluble protein is competed to the soluble protein in conjunction with its receptor TGFBR1, FBR2 or TGFBR3 or encoded with TGF-β
Acid or micromolecular inhibitor.It is further preferred that transforming growth factor signal pathway inhibitor be selected from TGFBR1, TGFBR2 or
The segment of TGFBR3 or its code nucleic acid.For example, the fusion egg of the extracellular domain containing TGFBR1, TGFBR2 or TGFBR3
White or its code nucleic acid, the example include but is not limited to the soluble T GFBR3 segment with sequence shown in SEQ ID NO:9.Make
Example for such fusion protein includes but is not limited to the extracellular domain and immune globulin of TGFBR1, TGFBR2 or TGFBR3
The fusion protein or its code nucleic acid of the white part Fc.As the such nucleic acid shown in SEQ ID NO:10 point of enumerating of example
Son, fusion protein shown in SEQ ID NO:11 etc..
The amount ratio of (a) ingredient and (b) ingredient is not particularly limited in regulator of the invention.Under normal circumstances, (a) and
(b) molar ratio is 0.1~10:1, preferably 0.2~5:1, further preferably 0.2~1:1.
The combination of vaccine and regulator
Pharmaceutical composition of the invention includes at least the combination of both vaccine and regulator.The form of the two combination is not special
It limits.The combination that can be both albumen as vaccine and the albumen as regulator, can be albumen as vaccine with
The combination of both nucleic acid as regulator, can also be the combination of both the nucleic acid as vaccine and the albumen as regulator,
It is also possible to the combination of both the nucleic acid as vaccine and the nucleic acid as regulator.
In the present invention, vaccine and regulator can exist in the form of individual, can also exist as a mixture.Vaccine with
The group composition and division in a proportion of regulator, is not particularly limited, such as can be 1:1~0.3:1.Those skilled in the art can be according to pharmaceutical composition
Dosage form, type of vaccine and regulator of object etc. and be readily determined.
Pharmaceutical composition of the invention can be administered by methods known in the art.Preferably, by passing in vivo
It is sent to host cell.In one embodiment, by viral vectors such as adenovirus (AdV), adeno-associated virus (AAV), reverse transcription
Virus, slow virus, herpes simplex virus etc. are by nucleic acid molecules to subject in need pharmaceutical composition incorporated in the present invention.This
Outside, it can also be transfected from elaioplast nanometer particle and enter host cell in turn to subject's pharmaceutical composition incorporated in the present invention
Object.In one embodiment, it is thin can to imported into subject itself DC in the method for electroporation to pharmaceutical composition of the invention
In born of the same parents, using DC cell as in vector introduction subject's body.In one embodiment, pharmaceutical composition of the invention can be with electricity
The method of perforation is imported into the self PBMC cell of the subject either PBMC cell of xenogenic origin, with self PBMC cell or
Person is that the PBMC cell of xenogenic origin is in vector introduction subject body.
[method for improving TNF-a and/or IFN-r content in lymphocyte]
The second aspect of the present invention provides the method for improving TNF-a and/or IFN-r content in lymphocyte comprising make
The step of pharmaceutical composition of the invention acts on lymphocyte.
Lymphocyte of the invention is the target cell of pharmaceutical composition effect comprising the cell of in vitro culture or subject
Intracorporal cell.The effect of pharmaceutical composition and lymphocyte can carry out in several ways.It include albumen in pharmaceutical composition
In the case of, lymphocyte can be acted on and contacting pharmaceutical composition directly with lymphocyte.Include in pharmaceutical composition
It, can be by making the corresponding albumen of the expression of nucleic acid in pharmaceutical composition, the albumen then made and leaching in the case where nucleic acid
The contact of bar cell.For example, expressing that the nucleic acid in pharmaceutical composition in expression cell first, then make expression cell and conduct
The lymphocyte of target cell contacts.Expression cell herein includes PBMC cell and/or DC cell.
[method for improving the ratio of TNF-a+ and/or IFN-r+ cell in lymphocyte populations]
The third aspect of the present invention provides the side for improving the ratio of TNF-a+ and/or IFN-r+ cell in lymphocyte populations
Method comprising the step of pharmaceutical composition is acted on into lymphocyte populations.The mode of action of pharmaceutical composition and lymphocyte populations
It can be identical as the mode of TNF-a and/or IFN-r content in above-mentioned raising lymphocyte.Details are not described herein.
In certain embodiments, the method packet of the ratio of TNF-a+ and/or IFN-r+ cell in lymphocyte populations is improved
Include following steps:
1) it introduces the reagents into such as DC cell or PBMC cell expression cell obtains the step of transfecting cell；With
2) the transfection cell and the lymphocyte populations are co-cultured, or makes at least partly secretion of the transfection cell
The step of object and the lymphocyte populations contact；
Wherein the reagent include as regulator coding in BcL2, CD40L, IL-15 and IL-15Ra at least
A kind of nucleic acid and coding is with TGF-β competition to the nucleic acid of the soluble protein in conjunction with its receptor TGFBR1, FBR2 or TGFBR3；
Or
The reagent includes (a) as at least one nucleic acid of vaccine and/or at least one polypeptide, nucleic acid encode at least one
Kind antigen open reading frame, polypeptide include at least one epitope；(b) as the coding of regulator be selected from BcL2,
The nucleic acid of at least one of CD40L, IL-15 and IL-15Ra and coding and TGF-β competition to its receptor TGFBR1, FBR2 or
The nucleic acid for the soluble protein that TGFBR3 is combined.
Embodiment 1
The present embodiment is the DNA and mRNA for preparing coding for antigens and immunity inspection point inhibitor
1. preparing DNA and mRNA construct
Building is used for the DNA sequence dna of encoding tumor-antigens survivin protein mRNA, while is constructed respectively for encoding
The DNA sequence dna of BCL2, CD40L, IL-15, IL-15Ra and TGF-beta inhibitor and for it is subsequent be transcribed in vitro it is anti-
It answers.High GC sequence is introduced by codon optimization to stablize the mRNA of synthesis, followed by 3 ' UTR of the beta-globin from people
Sequence prepares construct followed by one section of polyadenosine segment.The amino acid sequence of these nucleic acid sequences and its coding is as follows
Shown in table 1.
Table -1
| Title | Protein sequence number | Nucleic acid sequence number |
| BCL2 | SEQ ID NO:1 | SEQ ID NO:6 |
| CD40L | SEQ ID NO:2 | SEQ ID NO:5 |
| IL-15 | SEQ ID NO:3 | - |
| IL-15Ra | SEQ ID NO:4 | - |
| IL-15-IRES-IL-15Ra | - | SEQ ID NO:8 |
| TGF-beta-Fc | SEQ ID NO:11 | SEQ ID NO:10 |
2. being transcribed in vitro
Corresponding DNA plasmid prepared by step 1 uses speI restriction endonuclease by plasmid linearization first, with the matter of linearisation
Grain is template, and preparation mRNA is transcribed in vitro using t7 rna polymerase.Then with the lithium chloride precipitation method purify preparation mRNA.
Embodiment 2
The present embodiment is used to verify the influence combined as the mRNA of pharmaceutical composition to dendritic cells phenotype and existence.
The external evoked culture of 1.DC cell
Aseptic aspiration Healthy People venous blood 50ml separates peripheral blood mononuclear with lymphocyte separation medium in superclean bench
Monocyte is added in AIM-V culture medium, is put into 37 DEG C, 5%CO by cell2It is incubated in incubator, makes adherent mononuclear cells.
After 2h, remove non-attached cell, attached cell is added iDC culture medium and (is added final concentration of 800U/mL's in AIM-V culture medium
The IL-4 of GM-CSF, 500U/mL), 37 DEG C are put into, 5%CO2It is cultivated 6 days in incubator.By half cell culture medium be transferred to from
In heart pipe, cell is collected by centrifugation in 500g, removes supernatant, and isometric fresh mDC culture medium is added.The mDC fresh culture
It configures as follows: 1600U/mLGM-CSF and 1000U/mLIL-4, TNF-a (5ng/ml), IL-1 β (5ng/ml), IL-6 (150ng/
) and prostaglandinE2 (PGE2) (1ug/ml) ml.It after cell is resuspended, is added in culture bottle, cultivates 8~18 hours,
Induce DC cell maturation.
2. pharmaceutical composition transfects DC cell
On the transfection same day, DC cell dissociation is washed carefully after centrifugation with PBS at cell suspension with the cell dissociation reagent of non-enzyme
Cell is resuspended twice, with PBS in born of the same parents, adjusts cell density in 25-30 × 106DCs/ml.According to every 106DC cell transfecting~4ug
The ratio of mRNA mixes DC cell and nucleic acid molecules of the invention (BCL2, CD40L and IL-15-IRES-IL-15Ra) mRNA,
Electric revolving cup is added in cell-mRNA mixture antigen mRNA is transfected into DC cell using ECM630 electroporation.After electricity turns
Cell, be resuspended with the AIM-V culture medium of the cell-free factor, adjustment cell density to 1 × 106DCs/ml, with every hole 200ul's
Volume kind is put into 37 DEG C, 5%CO into 96 porocyte culture plates2Continue to cultivate in cell incubator.With the transfection of same condition
In GFP mRNA to DC cell, as a control group.The quantity of DC cell in culture plate, continuous record 5 days are recorded daily.
3. the measurement of transfection efficiency
After transfection 24 hours, the ratio of all DC cells is accounted for the DC cell of flow cytometry analysis expressing green fluorescent protein
Example.
The identification of 4.DC cell phenotype
Using direct immunofluorescence labelling method, by transfection DC cell centrifugation, with FACS buffer, (PBS containing 2%FBS is molten
Liquid) DC cell is resuspended, cell concentration is 1 × 106Cells/ml takes 100ul transfection DC cell suspension that fluidic cell pipe is added, point
It Jia Ru not 5ul corresponding antibody CD80, CD83, CD86 and corresponding Isotype control.4 DEG C are protected from light dyeing 30min.Every pipe adds
Enter 3ml FACS Buffer and wash cell, abandon supernatant, be added 500ul FACS buffer, flow cytometer showed detect CD80, CD83,
The expression of CD86.
As shown in Figure 1, having transfected the DC cell of immune modulator composition compared with the DC cell controls of untransfected, have
There is better cell survival rate.
As shown in Fig. 2, the DC cell of immune modulator composition has been transfected compared with the DC cell controls of untransfected,
Cell surface molecule CD80, CD83, CD86 stablize expression, without apparent difference.
Embodiment 3
The present embodiment is influence of the verifying composition of the invention to t cell response
1. the external evoked culture of DC cell
Aseptic aspiration Healthy People venous blood 50ml separates peripheral blood mononuclear with lymphocyte separation medium in superclean bench
Monocyte is added in AIM-V culture medium, is put into 37 DEG C, 5%CO by cell2It is incubated in incubator, makes adherent mononuclear cells.
After 2h, remove non-attached cell, attached cell is added iDC culture medium and (is added final concentration of 800U/mL's in AIM-V culture medium
The IL-4 of GM-CSF, 500U/mL), 37 DEG C are put into, 5%CO2It is cultivated 6 days in incubator.By half cell culture medium be transferred to from
In heart pipe, cell is collected by centrifugation in 500g, removes supernatant, and isometric fresh mDC culture medium is added.The mDC fresh culture
It configures as follows: 1600U/mL GM-CSF and 1000U/mLIL-4, TNF-a (5ng/ml), IL-1 β (5ng/ml), IL-6
(150ng/ml) and prostaglandinE2 (PGE2) (1ug/ml).It after cell is resuspended, is added in culture bottle, culture 8~18
A hour induces DC cell maturation.
2. pharmaceutical composition transfects DC cell
On the transfection same day, DC cell dissociation is washed carefully after centrifugation with PBS at cell suspension with the cell dissociation reagent of non-enzyme
Cell is resuspended twice, with PBS in born of the same parents, adjusts cell density in 25-30 × 106DCs/ml.According to every 106DC cell transfecting 4ug
The ratio of mRNA mixes DC cell and antigen mRNA and composition (Bcl2, CD40L and IL-15-IRES-IL- of the invention
15Ra) mRNA is combined, and electric revolving cup is added in cell-mRNA mixture, antigen mRNA is transfected into DC using ECM630 electroporation
In cell.Cell after electricity turn, is resuspended in 1640 culture mediums of the cell-free factor, adjustment cell density to 2 × 105DCs/
Ml is put into 37 DEG C, 5%CO2Continue culture 6 hours in cell incubator.
3. by the MNC cell for recovering overnight with 2 × 106The concentration of/ml is inoculated into 96 orifice plates, carries out T lymphocyte
Activation.Test grouping situation are as follows: MNC blank control group, MNC+ antigen mRNA-DC vaccine group, MNC+ antigen+regulator mRNA
(including Bcl2, CD40L, IL-15/IL-15Ra and TGF-beta inhibitor TGFBR3)-DC vaccine group, MNC+PMA/
Ionomycin positive controls.The DC cell that load has corresponding mRNA, MNC:DC=are added in different holes according to grouping situation
10:1；In positive control the concentration of Anti-CD3/anti-CD28 be 1 μ g/ml or PMA/Ionomycin concentration be 50ng/ml and
1ug/ml；37 DEG C are cultivated 10~12 days.
4. final concentration of 2 μM of monensin or 3 μ g/ml are added in the 5-8h before collecting cell in cell culture fluid
Brefeldin A, mixes well；(the blocking agent that Monensin and Brefeldin A is transported as albumen, in cell liquid
12h is not to be exceeded in time).
5. cell is transferred in streaming pipe, after CD3, CD4, CD8 antibody staining cell of fluorescent marker, fixes and lead to
Saturating cell carries out dyeing intracellular with TNF-a the and IFN-r antibody of fluorescent marker.
6. with the ratio of TNF-a+ and IFN-r+ cell in flow cytomery lymphocyte.
As a result as shown in figure 3, can be drawn using individual antigen mRNA or antigen/immune modulator composition mRNA
Play the antitumor special immune response of T lymphocyte.In the control group for only having transfected survivin antigen mRNA, IFN-r sun
Property T lymphocyte ratio be 0.35%, and transfected in antigen/immune modulator composition mRNA group, IFN-r is positive
T lymphocyte ratio is 1.96%, is 5.60 times of control group；In the control group for only having transfected survivin antigen mRNA,
The T lymphocyte ratio of the TNF-a positive is 1.86%, and has been transfected in antigen/immune modulator composition mRNA group, TNF-
The T lymphocyte ratio of a positive is 3.26%, is 1.75 times of control group.It should be the experiment results show that combination of the present invention
Object can significantly increase DC cell antigen presentation and activate the ability of its T lymphocyte, preferably stimulate T lymphocyte, generate
Stronger antitumor special immune response.
Without departing substantially from the scope or spirit of the invention, the specific embodiment of description of the invention can be done more
Kind improvements and changes, this will be apparent to those skilled in the art.Other realities obtained by specification of the invention
Applying mode for technical personnel is apparent obtain.Present specification and embodiment are merely exemplary.
Sequence table
<110>Qi Chensheng biotechnology (Zhuhai) Co., Ltd
<120>pharmaceutical composition and its application of Immune-enhancing effect
<130> BH1900066-1
<141> 2019-08-19
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cgacaggugc cucugcggcc aaaagccacg uguauaagau acaccugcaa aggcggcaca 960
accccagugc cacguuguga guuggauagu uguggaaaga gucaaauggc ucuccucaag 1020
cguauucaac aaggggcuga aggaugccca gaagguaccc cauuguaugg gaucugaucu 1080
ggggccucgg ugcacaugcu uuacaugugu uuagucgagg uuaaaaaacg ucuaggcccc 1140
ccgaaccacg gggacguggu uuuccuuuga aaaacacgau gauaauaugg ccacaacguc 1200
gacgccacca uggcuccuag gagagccaga ggguguagga cacugggacu gccagcucug 1260
cugcugcugc ugcugcugag accuccagcu acaaggggaa ucaccugccc uccuccuaug 1320
agcguggagc acgccgacau uugggugaag agcuacagcc uguacagccg ggagcgcuac 1380
auuugcaaca gcggcuucaa gaggaaggcc ggaacaagcu cucucaccga gugcgugcug 1440
aacaaggcca ccaacguggc ccauuggaca accccuagcc ugaagugcau cagggaccca 1500
gcacuggugc accagagacc agcuccuccu agcacaguga ccacagccgg agugacaccu 1560
cagccagaaa gccugagccc uagcggaaaa gaaccagccg ccucuagccc cagcagcaau 1620
aauaccgccg ccacaacagc cgcuauugug ccaggaagcc agcugaugcc uagcaagagc 1680
ccuagcaccg gcacaacaga gaucagcagc cacgagagca gccacggaac accuagccag 1740
accacagcca agaauuggga gcugaccgcc agcgccagcc accagccucc aggaguguac 1800
ccucagggac acagcgauac caccguggcc aucucuacca gcacagugcu gcugugcgga 1860
cugucagcug ugucccugcu ggcuugcuac cugaagagca gacagacccc uccucuggcc 1920
agcguggaaa uggaggcuau ggaggcccug ccagugacuu ggggaaccuc uagcagagac 1980
gaggaccugg agaauugcag ccaccaccug uaggaauucg cuggagccuc gguagccguu 2040
ccuccugccc gcugggccuc ccaacgggcc cuccuccccu ccuugcaccg gcccuuccug 2100
gucuuuggcu ggagccucgg uagccguucc uccugcccgc ugggccuccc aacgggcccu 2160
ccuccccucc uugcaccggc ccuuccuggu cuuugaaaaa aaaaaaaaaa aaaaaaaaaa 2220
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2316
<210> 9
<211> 646
<212> PRT
<213> Homo sapiens
<400> 9
Met Thr Ser His Tyr Val Ile Ala Ile Phe Ala Leu Met Ser Ser Cys
1 5 10 15
Leu Ala Thr Ala Gly Pro Glu Pro Gly Ala Leu Cys Glu Leu Ser Pro
20 25 30
Val Ser Ala Ser His Pro Val Gln Ala Leu Met Glu Ser Phe Thr Val
35 40 45
Leu Ser Gly Cys Ala Ser Arg Gly Thr Thr Gly Leu Pro Gln Glu Val
50 55 60
His Val Leu Asn Leu Arg Thr Ala Gly Gln Gly Pro Gly Gln Leu Gln
65 70 75 80
Arg Glu Val Thr Leu His Leu Asn Pro Ile Ser Ser Val His Ile His
85 90 95
His Lys Ser Val Val Phe Leu Leu Asn Ser Pro His Pro Leu Val Trp
100 105 110
His Leu Lys Thr Glu Arg Leu Ala Thr Gly Val Ser Arg Leu Phe Leu
115 120 125
Val Ser Glu Gly Ser Val Val Gln Phe Ser Ser Ala Asn Phe Ser Leu
130 135 140
Thr Ala Glu Thr Glu Glu Arg Asn Phe Pro His Gly Asn Glu His Leu
145 150 155 160
Leu Asn Trp Ala Arg Lys Glu Tyr Gly Ala Val Thr Ser Phe Thr Glu
165 170 175
Leu Lys Ile Ala Arg Asn Ile Tyr Ile Lys Val Gly Glu Asp Gln Val
180 185 190
Phe Pro Pro Lys Cys Asn Ile Gly Lys Asn Phe Leu Ser Leu Asn Tyr
195 200 205
Leu Ala Glu Tyr Leu Gln Pro Lys Ala Ala Glu Gly Cys Val Met Ser
210 215 220
Ser Gln Pro Gln Asn Glu Glu Val His Ile Ile Glu Leu Ile Thr Pro
225 230 235 240
Asn Ser Asn Pro Tyr Ser Ala Phe Gln Val Asp Ile Thr Ile Asp Ile
245 250 255
Arg Pro Ser Gln Glu Asp Leu Glu Val Val Lys Asn Leu Ile Leu Ile
260 265 270
Leu Lys Cys Lys Lys Ser Val Asn Trp Val Ile Lys Ser Phe Asp Val
275 280 285
Lys Gly Ser Leu Lys Ile Ile Ala Pro Asn Ser Ile Gly Phe Gly Lys
290 295 300
Glu Ser Glu Arg Ser Met Thr Met Thr Lys Ser Ile Arg Asp Asp Ile
305 310 315 320
Pro Ser Thr Gln Gly Asn Leu Val Lys Trp Ala Leu Asp Asn Gly Tyr
325 330 335
Ser Pro Ile Thr Ser Tyr Thr Met Ala Pro Val Ala Asn Arg Phe His
340 345 350
Leu Arg Leu Glu Asn Asn Ala Glu Glu Met Gly Asp Glu Glu Val His
355 360 365
Thr Ile Pro Pro Glu Leu Arg Ile Leu Leu Asp Pro Gly Ala Leu Pro
370 375 380
Ala Leu Gln Asn Pro Pro Ile Arg Gly Gly Glu Gly Gln Asn Gly Gly
385 390 395 400
Leu Pro Phe Pro Phe Pro Asp Ile Ser Arg Arg Val Trp Asn Glu Glu
405 410 415
Gly Glu Asp Gly Leu Pro Arg Pro Lys Asp Pro Val Ile Pro Ser Ile
420 425 430
Gln Leu Phe Pro Gly Leu Arg Glu Pro Glu Glu Val Gln Gly Ser Val
435 440 445
Asp Ile Ala Leu Ser Val Lys Cys Asp Asn Glu Lys Met Ile Val Ala
450 455 460
Val Glu Lys Asp Ser Phe Gln Ala Ser Gly Tyr Ser Gly Met Asp Val
465 470 475 480
Thr Leu Leu Asp Pro Thr Cys Lys Ala Lys Met Asn Gly Thr His Phe
485 490 495
Val Leu Glu Ser Pro Leu Asn Gly Cys Gly Thr Arg Pro Arg Trp Ser
500 505 510
Ala Leu Asp Gly Val Val Tyr Tyr Asn Ser Ile Val Ile Gln Val Pro
515 520 525
Ala Leu Gly Asp Ser Ser Gly Trp Pro Asp Gly Tyr Glu Asp Leu Glu
530 535 540
Ser Gly Asp Asn Gly Phe Pro Gly Asp Met Asp Glu Gly Asp Ala Ser
545 550 555 560
Leu Phe Thr Arg Pro Glu Ile Val Val Phe Asn Cys Ser Leu Gln Gln
565 570 575
Val Arg Asn Pro Ser Ser Phe Gln Glu Gln Pro His Gly Asn Ile Thr
580 585 590
Phe Asn Met Glu Leu Tyr Asn Thr Asp Leu Phe Leu Val Pro Ser Gln
595 600 605
Gly Val Phe Ser Val Pro Glu Asn Gly His Val Tyr Val Glu Val Ser
610 615 620
Val Thr Lys Ala Glu Gln Glu Leu Gly Phe Ala Ile Gln Thr Cys Phe
625 630 635 640
Ile Ser Pro Tyr Ser Asn
645
<210> 10
<211> 2765
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gagaccggcc ucgagcagcu gaagcuuccu gcaggucgac ucuagagcca ccaugaccag 60
ccacuacgug aucgccaucu ucgcccugau gagcagcugu cuggccacag caggaccaga 120
gccaggcgcc cugugugaac ucagcccagu guccgcuucu cauccagugc aggcccugau 180
ggagagcuuc acagugcuga gcggcugcgc cagcagaggc acaacaggac ugccucagga 240
ggugcacgug cugaaccuga gaaccgcagg acagggacca ggacagcugc agagggaagu 300
gacccugcac cugaacccca ucagcagcgu gcacauccac cacaagagcg ugguguuccu 360
gcugaacagc ccucacccac uggucuggca ccugaagacc gagagacugg cuacaggcgu 420
guccagacug uuccuggugu ccgaaggcag cguggugcag uuuagcagcg cuaacuucag 480
ccugaccgcc gaaaccgagg agagaaacuu cccccacggc aacgagcacc ugcugaauug 540
ggccaggaag gaguacggag ccgugaccag cuucaccgag cugaagaucg cccggaacau 600
cuacaucaag gucggcgagg accagguguu cccacccaag ugcaacaucg gcaagaacuu 660
ccugagccug aacuaccugg ccgaguaucu gcagccuaaa gccgcagagg gcugcgugau 720
gucuagccag ccccagaacg aggaggugca caucaucgag cugaucaccc ccaacagcaa 780
ccccuacagc gccuuccagg uggacaucac caucgacauc cggccuagcc aggaggaucu 840
ggaggucgug aagaaccuga uccugauccu caagugcaag aagagcguga auugggucau 900
caagagcuuc gacgugaagg gcagccugaa gaucaucgcc cccaacagca ucggcuuugg 960
caaagagagc gagcggagca ugaccaugac caagagcauc cgggacgaca uccccucuac 1020
acagggcaac cucgucaagu gggcacugga uaacggcuac agcccuauca ccagcuacac 1080
cauggcccca guggccaaca gauuccaccu gcggcuggag aacaacgccg aagagauggg 1140
cgacgaggaa gugcacacca ucccucccga gcugagaauc cugcuggacc ccggcgcccu 1200
gccagcucug cagaauccuc cuauuagagg cggcgaggga cagaacggag gacugccuuu 1260
cccuuucccc gacaucagca ggagagugug gaacgaggag ggcgaagacg gacugccuag 1320
accuaaggac cccgugaucc cuagcaucca gcuguuccca ggccugagag agccagagga 1380
agugcaggga agcguggaca ucgcucugag cgucaagugc gacaacgaga agaugaucgu 1440
ggccguggag aaggacagcu uccaggcuag cggauacagc ggaauggacg ugacccugcu 1500
ggacccuacu ugcaaggcca agaugaacgg cacccacuuc gugcuggagu ccccccugaa 1560
cgguugcggc acaagaccua gguggagcgc ucuggacgga gugguguacu acaacuccau 1620
cgugauccag gugcccgcuc ugggagauuc uagcgguugg ccagacggcu acgaggaucu 1680
ggagagcgga gacaacggcu ucccaggcga uauggacgag ggagacgcuu cucuguucac 1740
caggcccgag aucguggugu ucaauugcag ccugcagcag guccgcaacc cuucuagcuu 1800
ccaggagcag ccucacggca acaucaccuu caacauggag cuguacaaca ccgaccuguu 1860
ccuggugcca ucacagggag uguucagcgu gcccgagaac ggacacgugu acguggaggu 1920
guccgugacc aaggcagaac aggagcuggg cuucgccauc cagacuugcu ucaucagccc 1980
cuacagcaac gagcccaaau cuugugacaa aacucacaca ugcccaccgu gcccagcacc 2040
ugaacuccug gggggaccgu cagucuuccu cuucccccca aaacccaagg acacccucau 2100
gaucucccgg accccugagg ucacaugcgu ggugguggac gugagccacg aagacccuga 2160
ggucaaguuc aacugguacg uggacggcgu ggaggugcau aaugccaaga caaagccgcg 2220
ggaggagcag uacaacagca cguaccgugu ggucagcguc cucaccgucc ugcaccagga 2280
cuggcugaau ggcaaggagu acaagugcaa ggucuccaac aaagcccucc cagcccccau 2340
cgagaaaacc aucuccaaag ccaaagggca gccccgagaa ccacaggugu acacccugcc 2400
cccaucccgg gaugagcuga ccaagaacca ggucagccug accugccugg ucaaaggcuu 2460
cuaucccagc gacaucgccg uggaguggga gagcaauggg cagccggaga acaacuacaa 2520
gaccacgccu cccgugcugg acuccgacgg cuccuucuuc cucuacagca agcucaccgu 2580
ggacaagagc agguggcagc aggggaacgu cuucucaugc uccgugaugc augaggcucu 2640
gcacaaccac uacacgcaga agagccucuc ccugucuccg gguaaaugag aauucuuaau 2700
uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760
aaaaa 2765
<210> 11
<211> 878
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 11
Met Thr Ser His Tyr Val Ile Ala Ile Phe Ala Leu Met Ser Ser Cys
1 5 10 15
Leu Ala Thr Ala Gly Pro Glu Pro Gly Ala Leu Cys Glu Leu Ser Pro
20 25 30
Val Ser Ala Ser His Pro Val Gln Ala Leu Met Glu Ser Phe Thr Val
35 40 45
Leu Ser Gly Cys Ala Ser Arg Gly Thr Thr Gly Leu Pro Gln Glu Val
50 55 60
His Val Leu Asn Leu Arg Thr Ala Gly Gln Gly Pro Gly Gln Leu Gln
65 70 75 80
Arg Glu Val Thr Leu His Leu Asn Pro Ile Ser Ser Val His Ile His
85 90 95
His Lys Ser Val Val Phe Leu Leu Asn Ser Pro His Pro Leu Val Trp
100 105 110
His Leu Lys Thr Glu Arg Leu Ala Thr Gly Val Ser Arg Leu Phe Leu
115 120 125
Val Ser Glu Gly Ser Val Val Gln Phe Ser Ser Ala Asn Phe Ser Leu
130 135 140
Thr Ala Glu Thr Glu Glu Arg Asn Phe Pro His Gly Asn Glu His Leu
145 150 155 160
Leu Asn Trp Ala Arg Lys Glu Tyr Gly Ala Val Thr Ser Phe Thr Glu
165 170 175
Leu Lys Ile Ala Arg Asn Ile Tyr Ile Lys Val Gly Glu Asp Gln Val
180 185 190
Phe Pro Pro Lys Cys Asn Ile Gly Lys Asn Phe Leu Ser Leu Asn Tyr
195 200 205
Leu Ala Glu Tyr Leu Gln Pro Lys Ala Ala Glu Gly Cys Val Met Ser
210 215 220
Ser Gln Pro Gln Asn Glu Glu Val His Ile Ile Glu Leu Ile Thr Pro
225 230 235 240
Asn Ser Asn Pro Tyr Ser Ala Phe Gln Val Asp Ile Thr Ile Asp Ile
245 250 255
Arg Pro Ser Gln Glu Asp Leu Glu Val Val Lys Asn Leu Ile Leu Ile
260 265 270
Leu Lys Cys Lys Lys Ser Val Asn Trp Val Ile Lys Ser Phe Asp Val
275 280 285
Lys Gly Ser Leu Lys Ile Ile Ala Pro Asn Ser Ile Gly Phe Gly Lys
290 295 300
Glu Ser Glu Arg Ser Met Thr Met Thr Lys Ser Ile Arg Asp Asp Ile
305 310 315 320
Pro Ser Thr Gln Gly Asn Leu Val Lys Trp Ala Leu Asp Asn Gly Tyr
325 330 335
Ser Pro Ile Thr Ser Tyr Thr Met Ala Pro Val Ala Asn Arg Phe His
340 345 350
Leu Arg Leu Glu Asn Asn Ala Glu Glu Met Gly Asp Glu Glu Val His
355 360 365
Thr Ile Pro Pro Glu Leu Arg Ile Leu Leu Asp Pro Gly Ala Leu Pro
370 375 380
Ala Leu Gln Asn Pro Pro Ile Arg Gly Gly Glu Gly Gln Asn Gly Gly
385 390 395 400
Leu Pro Phe Pro Phe Pro Asp Ile Ser Arg Arg Val Trp Asn Glu Glu
405 410 415
Gly Glu Asp Gly Leu Pro Arg Pro Lys Asp Pro Val Ile Pro Ser Ile
420 425 430
Gln Leu Phe Pro Gly Leu Arg Glu Pro Glu Glu Val Gln Gly Ser Val
435 440 445
Asp Ile Ala Leu Ser Val Lys Cys Asp Asn Glu Lys Met Ile Val Ala
450 455 460
Val Glu Lys Asp Ser Phe Gln Ala Ser Gly Tyr Ser Gly Met Asp Val
465 470 475 480
Thr Leu Leu Asp Pro Thr Cys Lys Ala Lys Met Asn Gly Thr His Phe
485 490 495
Val Leu Glu Ser Pro Leu Asn Gly Cys Gly Thr Arg Pro Arg Trp Ser
500 505 510
Ala Leu Asp Gly Val Val Tyr Tyr Asn Ser Ile Val Ile Gln Val Pro
515 520 525
Ala Leu Gly Asp Ser Ser Gly Trp Pro Asp Gly Tyr Glu Asp Leu Glu
530 535 540
Ser Gly Asp Asn Gly Phe Pro Gly Asp Met Asp Glu Gly Asp Ala Ser
545 550 555 560
Leu Phe Thr Arg Pro Glu Ile Val Val Phe Asn Cys Ser Leu Gln Gln
565 570 575
Val Arg Asn Pro Ser Ser Phe Gln Glu Gln Pro His Gly Asn Ile Thr
580 585 590
Phe Asn Met Glu Leu Tyr Asn Thr Asp Leu Phe Leu Val Pro Ser Gln
595 600 605
Gly Val Phe Ser Val Pro Glu Asn Gly His Val Tyr Val Glu Val Ser
610 615 620
Val Thr Lys Ala Glu Gln Glu Leu Gly Phe Ala Ile Gln Thr Cys Phe
625 630 635 640
Ile Ser Pro Tyr Ser Asn Glu Pro Lys Ser Cys Asp Lys Thr His Thr
645 650 655
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
660 665 670
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
675 680 685
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
690 695 700
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
705 710 715 720
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
725 730 735
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
740 745 750
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
755 760 765
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
770 775 780
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
785 790 795 800
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
805 810 815
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
820 825 830
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
835 840 845
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
850 855 860
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
865 870 875
<210> 12
<211> 489
<212> DNA
<213> Homo sapiens
<400> 12
atgagaattt cgaaaccaca tttgagaagt atttccatcc agtgctactt gtgtttactt 60
ctaaacagtc attttctaac tgaagctggc attcatgtct tcattttggg ctgtttcagt 120
gcagggcttc ctaaaacaga agccaactgg gtgaatgtaa taagtgattt gaaaaaaatt 180
gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag tgatgttcac 240
cccagttgca aagtaacagc aatgaagtgc tttctcttgg agttacaagt tatttcactt 300
gagtccggag atgcaagtat tcatgataca gtagaaaatc tgatcatcct agcaaacaac 360
agtttgtctt ctaatgggaa tgtaacagaa tctggatgca aagaatgtga ggaactggag 420
gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 480
acttcttga 489
<210> 13
<211> 804
<212> DNA
<213> Homo sapiens
<400> 13
atggctccta ggagagccag agggtgtagg acactgggac tgccagctct gctgctgctg 60
ctgctgctga gacctccagc tacaagggga atcacctgcc ctcctcctat gagcgtggag 120
cacgccgaca tttgggtgaa gagctacagc ctgtacagcc gggagcgcta catttgcaac 180
agcggcttca agaggaaggc cggaacaagc tctctcaccg agtgcgtgct gaacaaggcc 240
accaacgtgg cccattggac aacccctagc ctgaagtgca tcagggaccc agcactggtg 300
caccagagac cagctcctcc tagcacagtg accacagccg gagtgacacc tcagccagaa 360
agcctgagcc ctagcggaaa agaaccagcc gcctctagcc ccagcagcaa taataccgcc 420
gccacaacag ccgctattgt gccaggaagc cagctgatgc ctagcaagag ccctagcacc 480
ggcacaacag agatcagcag ccacgagagc agccacggaa cacctagcca gaccacagcc 540
aagaattggg agctgaccgc cagcgccagc caccagcctc caggagtgta ccctcaggga 600
cacagcgata ccaccgtggc catctctacc agcacagtgc tgctgtgcgg actgtcagct 660
gtgtccctgc tggcttgcta cctgaagagc agacagaccc ctcctctggc cagcgtggaa 720
atggaggcta tggaggccct gccagtgact tggggaacct ctagcagaga cgaggacctg 780
gagaattgca gccaccacct gtag 804
Claims (10)
1. a kind of pharmaceutical composition of Immune-enhancing effect, characterized by comprising:
(1) vaccine, the vaccine include at least one nucleic acid and/or at least one polypeptide, and the nucleic acid includes at least one volume
At least one antigen open reading frame of code, the polypeptide include at least one epitope；With
(2) regulator, the regulator include selected from least one of BcL2, CD40L, IL-15 and IL-15Ra or its coding
Nucleic acid and transforming growth factor signal pathway inhibitor.
2. pharmaceutical composition according to claim 1, which is characterized in that the transforming growth factor signal pathway inhibitor
Selected from the inhibitor for being directed at least one of TGF-β, TGFBR1, TGFBR2 and TGFBR3.
3. pharmaceutical composition according to claim 1, which is characterized in that the transforming growth factor signal pathway inhibitor
Selected from group consisting of the following: antagonistic antibodies or encode the nucleic acid of antagonistic antibodies, siRNA, antisense RNA, comprising can
In conjunction with TGF-β and can block transforming growth factor signal path amino acid sequence albumen or the coding amino acid sequence
The nucleic acid of column, with TGF-β competition can described in the soluble protein or coding in conjunction with its receptor TGFBR1, TGFBR2 or TGFBR3
The nucleic acid and micromolecular inhibitor of dissolubility albumen.
4. pharmaceutical composition according to claim 1, which is characterized in that the transforming growth factor signal pathway inhibitor
It is the nucleic acid antibody of antibody perhaps encoding antibody for TGF-β or its receptor.
5. pharmaceutical composition according to claim 3, which is characterized in that described comprising that in conjunction with TGF-β and can hinder
The albumen of the amino acid sequence of disconnected transforming growth factor signal path is selected from the segment of TGFBR1, TGFBR2 or TGFBR3.
6. pharmaceutical composition according to claim 5, which is characterized in that the segment be containing TGFBR1, TGFBR2 or
The fusion protein of the extracellular domain of TGFBR3.
7. pharmaceutical composition according to claim 3, which is characterized in that the fusion protein be TGFBR1, TGFBR2 or
The fusion protein of the part Fc of the extracellular domain and immunoglobulin of TGFBR3.
8. a kind of method for improving TNF-a and/or IFN-r content in lymphocyte, which is characterized in that including that will be wanted according to right
The step of asking the described in any item pharmaceutical compositions of 1-7 to act on lymphocyte.
9. a kind of method for improving the ratio of TNF-a+ and/or IFN-r+ cell in lymphocyte populations, which is characterized in that including inciting somebody to action
The step of pharmaceutical composition according to claim 1-7 acts on the lymphocyte populations.
10. a kind of method for improving the ratio of TNF-a+ and/or IFN-r+ cell in lymphocyte populations, which is characterized in that including
Following steps:
1) the step of expression cell obtains transfection cell is introduced the reagents into；With
2) the transfection cell and the lymphocyte populations are co-cultured, or make at least partly secretion of the transfection cell with
The step of lymphocyte populations contact；
Wherein the reagent includes the coding as regulator selected from least one of BcL2, CD40L, IL-15 and IL-15Ra
Nucleic acid and coding compete with TGF-β to the nucleic acid of the soluble protein in conjunction with its receptor TGFBR1, FBR2 or TGFBR3；Or
The reagent includes (a) as at least one nucleic acid of vaccine and/or at least one polypeptide, and the nucleic acid includes at least one
At least one antigen open reading frame of a coding, the polypeptide include at least one epitope；(b) as the volume of regulator
Nucleic acid and coding of the code selected from least one of BcL2, CD40L, IL-15 and IL-15Ra are competed with TGF-β to its receptor
The nucleic acid for the soluble protein that TGFBR1, FBR2 or TGFBR3 are combined.
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| CN201910764395.5A CN110464841B (en) | 2019-08-19 | 2019-08-19 | Immunity-enhancing pharmaceutical composition and application thereof |
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|---|---|---|---|
| CN201910764395.5A CN110464841B (en) | 2019-08-19 | 2019-08-19 | Immunity-enhancing pharmaceutical composition and application thereof |
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| Publication Number | Publication Date |
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| CN110464841A true CN110464841A (en) | 2019-11-19 |
| CN110464841B CN110464841B (en) | 2020-05-22 |
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ID=68511159
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110694061A (en) * | 2019-11-21 | 2020-01-17 | 启辰生生物科技(珠海)有限公司 | Composition for enhancing T lymphocyte immunity, immune cell and application |
| CN110732021A (en) * | 2019-11-21 | 2020-01-31 | 北京启辰生生物科技有限公司 | Composition for relieving tumor immunosuppression and application thereof |
| CN110743006A (en) * | 2019-11-22 | 2020-02-04 | 北京启辰生生物科技有限公司 | Composition for synergistically relieving immune cell failure and application |
| CN110777115A (en) * | 2019-11-21 | 2020-02-11 | 北京启辰生生物科技有限公司 | Composition for enhancing capacity of antigen presenting cells to sensitize T cells and application |
| CN110841058A (en) * | 2019-11-21 | 2020-02-28 | 北京启辰生生物科技有限公司 | Composition for synergistically improving immune response and use |
| CN110859952A (en) * | 2019-12-13 | 2020-03-06 | 启辰生生物科技(珠海)有限公司 | Composition for relieving immune cell immunosuppression, antigen presenting cell and preparation method thereof |
| CN110904133A (en) * | 2019-12-13 | 2020-03-24 | 北京启辰生生物科技有限公司 | Composition for synergistically relieving T cell failure and application |
| CN110917342A (en) * | 2019-12-17 | 2020-03-27 | 北京启辰生生物科技有限公司 | Immunomodulatory compositions and uses thereof |
| CN111337678A (en) * | 2020-02-21 | 2020-06-26 | 杭州凯保罗生物科技有限公司 | Biomarker related to tumor immunotherapy effect and application thereof |
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