CN106520693B - Memory B cell precursor and application thereof - Google Patents

Memory B cell precursor and application thereof Download PDF

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CN106520693B
CN106520693B CN201610916356.9A CN201610916356A CN106520693B CN 106520693 B CN106520693 B CN 106520693B CN 201610916356 A CN201610916356 A CN 201610916356A CN 106520693 B CN106520693 B CN 106520693B
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cell
memory
antagonist
functional analogue
precursor
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CN106520693A (en
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祁海
王毅峰
史静雯
晏家骢
侯小筱
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Tsinghua University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/206—IL-9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2309—Interleukin-9 (IL-9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention proposes memory B cell precursor and application thereof, memory B cell precursor has following marker: CD19+GL7+Fas+Bcl6‑, for preferred pin to marker CD38, the memory B cell precursor has the expression opposite with centrum germinativum, and the optional memory B cell precursor is G0 phase cell.The memory B cell precursor has the potential for being divided into memory B cell.

Description

Memory B cell precursor and application thereof
Technical field
The present invention relates to biological field, in particular it relates to memory B cell precursor and application thereof, more specifically Ground, the present invention relates to memory B cell precursor, the method for acquisition memory B cell precursor, promotion memory B cell precursors Method that cell is converted to memory B cell, the method for inhibiting memory B cell precursor to convert to memory B cell, reagent are being made The method and vaccine of purposes, acquisition memory B cell in standby drug.
Background technique
When body is by extraneous pathogen infection, it is antiviral to support that immune system will start a series of protection mechanisms Invasion, including inherent immunity and acquired immunity.Certain specific pathogen simply can only pass through inherent immunity machine System is removed, and the pathogen of the overwhelming majority can break through inherent immunity defence line, to start acquired immunity defense mechanism.It participates in The main cell of acquired immunity process includes the T cell and B cell of antigentic specificity, wherein the B cell quilt of antigentic specificity This special construction of centrum germinativum can be formed after Antigen-activated with the help of helper T lymphocyte, and experience is a large amount of wherein The process of the affinity maturations such as proliferation, somatic hypermutation, isotype conversion, and constantly output is a large amount of outward in this process Generate the thick liquid cell and memory B cell of high-affinity antibody.And memory B cell can meet with same or like cause of disease in next time Body-sensing responds rapidly to when dye, is divided into the thick liquid cell that can produce a large amount of specific antibodies, faster and better removing rapidly Virion.It is worth noting that the effective vaccine of most clinical uses is all by eliciting protective antibody even to this day It is realized with memory B cells.
Generally, it is considered that when the B cell of antigentic specificity will form centrum germinativum, further hair tonic after receiving antigenic stimulus Center can be as the main source of memory B cell, and the memory B cell that centrum germinativum relies on is due to by affinity maturation Screening process has higher affinity and functionality compared with the memory B cell in non-centrum germinativum source.
However, still being needed to be studied about the formation of memory B cell at present.
Summary of the invention
The application is to be proposed based on inventor to following problems and true discovery:
Generally, it is considered that centrum germinativum is the main source of memory B cell, inventor has found that not in hair tonic All cells of the heart can be converted into memory B cell.Further, inventor has been separated and can effectively have been converted by further investigation For the cell mass of memory B cell, and it is named as memory B cell precursor.Although inventor is it was unexpectedly observed that memory B The ratio of cell-progenitor cells Jin Zhan centrum germinativum very little, but can be effectively under the action of cell factor such as IL-9 It is converted into memory B cell.Specifically, the present inventor uses fluorescence ubiquitination cell cycle indicating gage (Fluorescence Ubiquitin Cell Cycle Indicator, Fucci) technology (Sakaue-Sawano, A., et al.(2008)."Visualizing spatiotemporal dynamics of multicellular cell-cycle progression."Cell132 (3): 487-498.), Germinal center B cell is observed by Fucci transgenic mice, is identified Memory B cell precursor, then by bioinformatics discovery, pass through physiological function experimental verification cytokine interleukin The important function that plain -9 pairs of memory B cells are formed.
In the first aspect of the present invention, the invention proposes a kind of memory B cell precursors.Implementation according to the present invention Example, the memory B cell precursor have following marker: CD19+GL7+Fas+Bcl6-.According to an embodiment of the invention, For marker CD38, the memory B cell precursor has the expression opposite with centrum germinativum.It is according to the present invention Another embodiment, the memory cell precursor are G0 phase cell.Inventor is found through experiments that, before the memory B cell Body cell, which has, is divided into the potential of memory B cell, such as can be effectively converted under the action of IL-9 memory B thin Born of the same parents.
In the second aspect of the present invention, mentioned-above memory B cell precursor is obtained the invention proposes a kind of Method.According to an embodiment of the invention, the described method includes: based on CD19, GL7, Fas, Bcl6 and optional CD38 are utilized Fluidic cell separating method, sorts cell.As described in preceding invention, inventors have found that in centrum germinativum, memory B cell Precursor has CD19+GL7+Fas+Bcl6-Marker, it is preferable that be directed to marker CD38, the memory B cell precursor With the expression opposite with centrum germinativum.Inventor utilize fluidic cell separating method, be based on CD19, GL7, Fas, Bcl6 with And optional CD38 accurately can sort and obtain memory B cell precursor in the expression on B cell surface.
In the third aspect of the present invention, the invention proposes it is a kind of promote mentioned-above memory B cell precursor to The method of memory B cell conversion.According to an embodiment of the invention, the described method includes: make the memory B cell precursor with Selected from least one of following contact: (a) IL-9 or its functional analogue；
(b) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(c) IL-9 agonist or Its functional analogue；(d) STAT-1 agonist or its functional analogue；(e) IL-9 receptor stimulating agent or its functional analogue；With (f) IL-9 or its expression of receptor promotor.Inventor is found through experiments that, the mentioned-above high table of memory B cell precursor Up to interleukin 9 (IL-9) receptor (IL9R)；Inventor is further found through experiments that, before mentioned-above memory B cell Body cell can receive the regulation of IL-9, STAT1 signaling molecule expression quantity height；And inventor is further found through experiments that, Under the stimulation of IL-9, mentioned-above memory B cell precursor can be converted into memory B cell, in the conversion process, JAK-STAT signal path is activated.In turn, inventors have found that when the memory B cell precursor with selected from it is following extremely When one of few contact: (a) IL-9 or its functional analogue；(b) IL-9 or the cell of its functional analogue are secreted, is preferably secreted The Th9 cell of IL-9；(c) IL-9 agonist or its functional analogue；(d) STAT-1 agonist or its functional analogue；(e) IL-9 receptor stimulating agent or its functional analogue；(f) IL-9 or its expression of receptor promotor, can effectively facilitate mentioned-above Memory B cell precursor is converted to memory B cell.
In the fourth aspect of the present invention, the invention proposes it is a kind of inhibit mentioned-above memory B cell precursor to The method of memory B cell conversion.According to an embodiment of the invention, the described method includes: make the memory B cell precursor with Selected from least one of following contact: (i) IL-9 antagonist or its functional analogue；(ii) STAT-1 antagonist or its function class Like object；(iii) IL-9 receptor antagonist or its functional analogue；(iv) IL-9 or its expression of receptor inhibitor.As previously mentioned, hair Bright people is found through experiments that mentioned-above memory B cell precursor height expresses interleukin 9 (IL-9) receptor (IL9R)；Inventor is further found through experiments that mentioned-above memory B cell precursor can receive the tune of IL-9 Control, STAT1 signaling molecule expression quantity are high；And inventor is further found through experiments that, noted earlier under the stimulation of IL-9 Memory B cell precursor can be converted into memory B cell, in the conversion process, JAK-STAT signal path is activated.Into And inventor is further verified by experiment, when the memory B cell precursor is contacted with selected from least one of following When: (i) IL-9 antagonist or its functional analogue；(ii) STAT-1 antagonist or its functional analogue；(iii) IL-9 receptor is short of money Anti-agent or its functional analogue；(iv) IL-9 or its expression of receptor inhibitor, before can effectively preventing mentioned-above memory B cell Body cell is converted to memory B cell.
In the fifth aspect of the invention, the purposes the invention proposes reagent in medicine preparation, the drug is for promoting It is converted into mentioned-above memory B cell precursor to memory B cell, the reagent is selected from least one of following: (a) IL-9 or its functional analogue；(b) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(c) IL-9 agonist or its functional analogue；(d) STAT-1 agonist or its functional analogue；(e) IL-9 receptor stimulating agent or its Functional analogue；(f) IL-9 or its expression of receptor promotor.Inventor is found through experiments that, using selected from it is following at least One of reagent: (a) IL-9 or its functional analogue；(b) IL-9 or the cell of its functional analogue, preferably secretion IL-9 are secreted Th9 cell；(c) IL-9 agonist or its functional analogue；(d) STAT-1 agonist or its functional analogue；(e) IL-9 by Body agonist or its functional analogue；(f) drug prepared by IL-9 or its expression of receptor promotor, can be effectively used for promoting Conversion of the mentioned-above memory B cell precursor to memory B cell.
In the sixth aspect of the present invention, the invention proposes the purposes of reagent in medicine preparation, the drug is for pressing down It makes mentioned-above memory B cell precursor to convert to memory B cell, the reagent is selected from least one of following: (i) IL-9 antagonist or its functional analogue；(ii) its functional analogue of STAT-1 antagonist；(iii) IL-9 receptor antagonist or its Functional analogue；(iv) IL-9 or its expression of receptor inhibitor.Inventor is found through experiments that, using selected from it is following at least it One reagent: (i) IL-9 antagonist or its functional analogue；(ii) its functional analogue of STAT-1 antagonist；(iii) IL-9 by Body antagonist or its functional analogue；(iv) drug prepared by IL-9 or its expression of receptor inhibitor, can be effectively used for inhibiting Mentioned-above memory B cell precursor is converted to memory B cell.
In the seventh aspect of the present invention, the invention proposes a kind of methods for obtaining memory B cell.Reality according to the present invention Apply example, which comprises contact mentioned-above memory B cell precursor with selected from least one of following: (a) IL-9 or its functional analogue；(b) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(c) IL-9 agonist or its functional analogue；(d) STAT-1 agonist or its functional analogue；(e) IL-9 receptor stimulating agent or its Functional analogue；(f) IL-9 or its expression of receptor promotor.According to an embodiment of the invention, thin using above-mentioned acquisition memory B The method of born of the same parents can efficiently obtain memory B cell.
In the eighth aspect of the present invention, the invention proposes the purposes of reagent in medicine preparation, the drug is for adjusting Memory B cell precursor/memory B cell ratio in centrum germinativum is saved, the reagent is selected from least one of following: (1) IL-9 or its functional analogue；(2) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(3) IL-9 agonist or its functional analogue；(4) STAT-1 agonist or its functional analogue；(5) IL-9 receptor stimulating agent or its Functional analogue；(6) IL-9 or its expression of receptor promotor；(7) IL-9 antagonist or its functional analogue；(8) STAT-1 is short of money Its functional analogue of anti-agent；(9) IL-9 receptor antagonist or its functional analogue；(10) IL-9 or its expression of receptor inhibit Agent.Inventor is found through experiments that, using selected from least one of following reagent: (1) IL-9 or its functional analogue；(2) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(3) IL-9 agonist or its function are similar Object；(4) STAT-1 agonist or its functional analogue；(5) IL-9 receptor stimulating agent or its functional analogue；(6) IL-9 or its Expression of receptor promotor；(7) IL-9 antagonist or its functional analogue；(8) its functional analogue of STAT-1 antagonist；(9)IL- 9 receptor antagonists or its functional analogue；(10) drug prepared by IL-9 or its expression of receptor inhibitor, can be effectively used for Adjust memory B cell precursor/memory B cell ratio in centrum germinativum.
In the ninth aspect of the present invention, the invention proposes a kind of vaccines.According to an embodiment of the invention, the vaccine contains There is antigen and selected from least one of following: (a) IL-9 or its functional analogue；(b) IL-9 or its functional analogue are secreted Cell, preferably secrete IL-9 Th9 cell；(c) IL-9 agonist or its functional analogue；(d) STAT-1 agonist or its Functional analogue；(e) IL-9 receptor stimulating agent or its functional analogue；(f) IL-9 or its expression of receptor promotor.According to this The embodiment of invention, the vaccine of the application can effectively facilitate mentioned-above memory B cell precursor to note in body Recall B cell conversion, so as to generate long-term immanoprotection action in body, induction protection mechanism has long-term effect Feature.
Detailed description of the invention
Fig. 1 is the result figure of the identification of transition state subgroup in Germinal center B cell according to embodiments of the present invention；
Fig. 2 is CD19 according to an embodiment of the present invention+GL7+Fas+CD38+Bcl6-mKO2hiBe memory B cell precursor it is thin The result figure of born of the same parents；
Fig. 3 is that IL-9 according to an embodiment of the present invention (IL-9) promotes the development of GC-MP cell and to memory B cell Differentiation result figure；
Fig. 4 is the result figure that IL-9 according to an embodiment of the present invention promotes memory B cell to be formed；And
Fig. 5 is the main adjusting of signal path analysis chart and GC-MP and GC-MPP cell according to an embodiment of the present invention The RNA- sequence of son analyzes data.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair It is bright, and be not considered as limiting the invention.
Definition and general terms
The invention is intended to cover all replacement, modification and equivalent technical solutions, they are included in as claim is fixed In the scope of the invention of justice.Those skilled in the art will appreciate that many and similar or equivalent method and material described herein It can be used in the practice present invention.The present invention is not limited to method described herein and material.In document, patent and the class combined One or more or contradict in the case where (including but not limited to defined in terms, term different from the application like material Using, described technology, etc.), it is subject to the application.
It will further be appreciated that certain features of the invention, be it is clearly visible, carry out in a number of independent embodiments Description, but can also provide in combination in a single embodiment.Conversely, various features of the invention, for brevity, It is described in a single embodiment, but can also be individually or with the offer of any suitable sub-portfolio.
Unless otherwise stated, all scientific and technical terminologies used in the present invention have with those skilled in the art of the invention's It is generally understood identical meaning.All patents of the present invention and public publication are integrally incorporated this hair by reference It is bright.
There is apparent conflict unless otherwise indicated or in context, the article " one " used herein, " one (kind) " " described " is intended to include "at least one" or " one or more ".Therefore, these articles used herein refer to one or The article of more than one (i.e. at least one) object.For example, " component " refers to one or more components, it is possible to have more than one Component be taken into account in the embodiment of the embodiment and use or use.
Term " study subject " used in the present invention refers to animal.The typically described animal is mammal.It is tested right As, such as also refer to primate (such as mankind, sex), ox, sheep, goat, horse, dog, cat, rabbit, rat, small Mouse, fish, bird etc..In certain embodiments, the study subject is primate.In other embodiments, it is described by Trying object is people.
Term " patient " used in the present invention refers to people (including adult and children) or other animals.In some implementations In scheme, " patient " refers to people.
Term "comprising" is open language, that is, includes content specified by the present invention, but be not precluded otherwise Content.
Term " functional analogue " refers to the substance with identity function, structure do not limit it is identical, as long as having identical Or similar function.
Term " agonist " refers in conjunction with the receptor of certain bioactive substance, and the substance of the effect of the active material is presented Or drug.
Term " antagonist " refers to can be in conjunction with receptor, but does not have a substance of intrinsic activity.Antagonist is divided into competing Striving property antagonist and noncompetitive antaganist.
Term " receptor stimulating agent " is also known as full agonist (full agonist), refer to have receptor compared with strong affinity and Intrinsic activity can play the drug of ceiling effect by receptor agonism.And there is enough affinity with receptor, but intrinsic activity is not By force, weaker effect is only generated, the drug that can but fight excitomotor part effect is known as partial agonist (partial agonist)。
Term " specific system of defense " refers to that the component of immune system reacts to the presence of specific antigen.Result from spy The example of the inflammation of anisotropic system of defense reaction includes that classical response to exotic antigen, autoimmune disease and Delayed onset are super quick It reacts response (cell-mediated by T-).(such as kidney and marrow move for the repulsion of chronic inflammatory disease, transplanting solid tissue and organ The repulsion of plant) and graft versus host disease (GVHD) be other examples of specific system of defense inflammatory reaction.
" autoimmune disease " used in the present invention refers to and body fluid or cell-mediated to body itself component response The set of any disease of relevant tissue damage.
Any disease of term " treatment " used in the present invention or illness, refer to improvement disease in some of these embodiments Or illness (development for slowing down or prevent or mitigate disease or its at least one clinical symptoms).In other embodiments, " treatment " refers to mitigation or improves at least one body parameter, including the body parameter that may not be discovered by patient.At other In embodiment, " treatment " refers to from body (such as stablizing perceptible symptom) or physiologically (such as the ginseng of stable body Number) or above-mentioned two aspect adjust disease or illness.In other embodiments, " treatment ", which refers to, prevents or delays disease or illness Breaking-out, generation or deterioration.
Memory B cell precursor
In the first aspect of the present invention, the invention proposes a kind of memory B cell precursors.Implementation according to the present invention Example, the memory B cell precursor have following marker: CD19+GL7+Fas+Bcl6-, it is preferable that it is directed to marker CD38, The memory B cell precursor has the expression opposite with centrum germinativum.Inventor is in an experiment it was unexpectedly observed that In There is the cell mass that a group is special in centrum germinativum, the cell surface marker molecule CD19 of this group of cells is raised, GL7 is raised, Fas up-regulation, and the key transcription factor Bcl6 of Germinal center B cell (GC B cell) is maintained to lower, it, should for marker CD38 Group's cell has the expression opposite with centrum germinativum.Inventor is further experimentally confirmed, which has differentiation For the potential of memory B cell, and then in this application, this group of cells are named as memory B cell precursor by inventor.
It should be noted that statement used herein above " is directed to marker CD38, the memory B cell precursor tool Have the expression opposite with centrum germinativum " refer to when other cell height express CD38 in centrum germinativum, memory B cell precursor The CD38 expression lowered is presented in cell, refers to as other cells low expression CD38 in centrum germinativum, memory B cell precursor The CD38 expression of up-regulation is presented.According to a particular embodiment of the invention, in Mice Body, CD38 is in memory B cell precursor Middle up-regulation (CD38+)；In human body, CD38 lowers (CD38 in memory B cell precursor-)。
In addition, it is necessary to explanation, statement " up-regulation " used herein above refer to other cells relative to centrum germinativum, The cell surface marker molecule in B cell precursor cell high expression and in the other cells of centrum germinativum low expression or this is thin Cellular surface marker molecule is expressed in B cell precursor cell and is not expressed in the other cells of centrum germinativum；Similarly, made here Statement " downward " refers to other cells relative to centrum germinativum, and the cell surface marker molecule is in B cell precursor cell Middle low expression and in the other cells of centrum germinativum high expression or the cell surface marker molecule in B cell precursor cell not It expresses and is expressed in the other cells of centrum germinativum.
According to a particular embodiment of the invention, inventors have found that CD19+GL7+Fas+CD38+Bcl6-Cell is that the G0 phase is thin Born of the same parents, under non-Fucci transgenic mice background, inventor uses the CD19 of G0 phase+GL7+Fas+CD38+Bcl6-To define memory B Cell-progenitor cells, and under Fucci transgenic mice background, inventor uses CD19+GL7+Fas+CD38+Bcl6-mKO2hi Define memory B cell precursor, wherein mKO2hiCharacterization cell is in the G0 phase.
The method for obtaining memory B cell precursor
In the second aspect of the present invention, mentioned-above memory B cell precursor is obtained the invention proposes a kind of Method.According to an embodiment of the invention, this method comprises: based on CD19, GL7, Fas, Bcl6 and optional CD38 utilize stream Formula cell sorting methods, sort cell.As described in preceding invention, as described in preceding invention, inventors have found that in centrum germinativum In, memory B cell precursor has CD19+GL7+Fas+Bcl6-Marker, it is preferable that be directed to marker CD38, memory B Cell-progenitor cells have the expression opposite with centrum germinativum.Inventor utilizes fluidic cell separating method, is based on CD19, GL7, Fas, Bcl6 and optional CD38 accurately can sort and obtain memory B cell precursor in the expression on B cell surface Cell.
The method for promoting memory B cell precursor to convert to memory B cell
In the third aspect of the present invention, the invention proposes it is a kind of promote mentioned-above memory B cell precursor to The method of memory B cell conversion.According to an embodiment of the invention, this method comprises: making the memory B cell precursor and choosing It is contacted from least one of following: (a) IL-9 or its functional analogue；(b) IL-9 or the cell of its functional analogue are secreted, it is excellent The Th9 cell of choosing secretion IL-9；(c) IL-9 agonist or its functional analogue；(d) STAT-1 agonist or its function are similar Object；(e) IL-9 receptor stimulating agent or its functional analogue；(f) IL-9 or its expression of receptor promotor.
Inventor is found through experiments that mentioned-above memory B cell precursor height expresses interleukin 9 (IL- 9) receptor (IL9R)；Inventor is further found through experiments that mentioned-above memory B cell precursor can receive IL-9 Regulation, STAT1 signaling molecule expression quantity it is high；And inventor is further found through experiments that, under the stimulation of IL-9, before The memory B cell precursor can be converted into memory B cell, and in the conversion process, JAK-STAT signal path is swashed It is living.In turn, inventors have found that when the memory B cell precursor is with selected from least one of following contact: (a) IL-9 Or its functional analogue；(b) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(c)IL-9 Agonist or its functional analogue；(d) STAT-1 agonist or its functional analogue；(e) IL-9 receptor stimulating agent or its function Analog；(f) IL-9 or its expression of receptor promotor can effectively facilitate mentioned-above memory B cell precursor to note Recall B cell conversion.
Specific example according to the present invention, IL-9 functional analogue include but is not limited to that (it has combines truncated IL-9 And IL9R is activated, and activate the activity of downstream signaling pathway).IL-9 or its functional analogue can combine and activate IL9R, into And STAT1 signal path is further activated, under the mediation of STAT1 signal path signal path, realize that memory B cell precursor is thin Conversion of the born of the same parents to memory B cell.
Specific example according to the present invention is such as assisted when memory B cell precursor and the cell for preferably secreting IL-9 When T cell Th9 cell co-cultures, for memory B cell precursor under the collective effect for the IL-9 that Th9 cell is secreted, memory B is thin Born of the same parents' precursor can Efficient Conversion be memory B cell.
Specific example according to the present invention, IL-9 is under the action of IL-9 agonist or its functional analogue, IL-9 activity It improves, further under the IL-9 effect of activation, memory B cell precursor Efficient Conversion is memory B cell.
The type of specific example according to the present invention, STAT-1 agonist is not particularly limited, and is related to JAK-STAT1 signal The jak kinase and STAT1 kinases of access can be used as STAT-1 agonist.In STAT-1 agonist or its functional analogue Under effect, JAK-STAT1 signal path is effectively activated, it can be achieved that memory B cell precursor efficiently turns to memory B cell Change.
Specific example according to the present invention, in the work of the functional analogue of IL-9 receptor stimulating agent or IL-9 receptor stimulating agent Under, IL-9 receptor is activated, and the IL-9 receptor of activation further activates downstream signaling pathway, such as JAK-STAT1 signal path, Under the mediation for the signal path with mediated cell transformation of memory B cell precursor being activated, memory B cell Precursor can Efficient Conversion be memory B cell.
Specific example according to the present invention, under the action of IL-9 expresses promotor or IL-9 expression of receptor promotor, IL- 9 expression or secretory volume increase or IL-9 expression of receptor amount improves, and mentioned-above memory B cell precursor can Efficient Conversion For memory B cell.
Memory B cell precursor is inhibited to convert to memory B cell
In the fourth aspect of the present invention, the invention proposes it is a kind of inhibit mentioned-above memory B cell precursor to The method of memory B cell conversion.According to an embodiment of the invention, this method comprises: making the memory B cell precursor and choosing It is contacted from least one of following: (i) IL-9 antagonist or its functional analogue；(ii) STAT-1 antagonist or its function are similar Object；(iii) IL-9 receptor antagonist or its functional analogue；(iv) IL-9 or its expression of receptor inhibitor.
As previously mentioned, inventor is found through experiments that, mentioned-above memory B cell precursor height expression leucocyte is situated between Plain -9 (IL-9) receptors (IL9R)；Inventor is further found through experiments that mentioned-above memory B cell precursor can Regulation, the STAT1 signaling molecule expression quantity for receiving IL-9 are high；And inventor is further found through experiments that, in the thorn of IL-9 Under swashing, mentioned-above memory B cell precursor can be converted into memory B cell, in the conversion process, JAK-STAT signal Access is activated.In turn, inventor is by experiment further verifying, when the memory B cell precursor and selected from following At least one contact when: (i) IL-9 antagonist or its functional analogue；(ii) STAT-1 antagonist or its functional analogue； (iii) IL-9 receptor antagonist or its functional analogue；(iv) IL-9 or its expression of receptor inhibitor, can effectively prevent front institute The memory B cell precursor stated is converted to memory B cell.
The functional analogue of specific example according to the present invention, IL-9 antagonist or IL-9 antagonist can by competitive or The activity of Noncompetition inhibition IL-9, and then mentioned-above memory B cell precursor is effectively prevented to turn to memory B cell Change.
Specific example according to the present invention, the type of STAT-1 antagonist are related to JAK-STAT1 signal not by particularly significant The jak kinase antagonist and STAT1 kinase antagonists of access can be used as STAT-1 antagonist.In STAT-1 antagonist or Under the action of STAT-1 antagonist functional analogue, memory B cell precursor can effectively be prevented to turn to the height of memory B cell Change.
Specific example according to the present invention, under the action of IL-9 receptor antagonist or its functional analogue, contestable Inhibit the activity of the activation downstream signaling pathway of IL-9 receptor, and then can effectively prevent memory B cell precursor thin to memory B The conversion of born of the same parents.
Specific example according to the present invention, under the action of the agent of IL-9 expression inhibiting or IL-9 expression of receptor inhibitor, IL- 9 expression or secretory volume substantially reduce, and the expression quantity of IL-9 receptor significantly reduces, and then can effectively prevent mentioned-above memory Conversion of the B cell precursor cell to memory B cell.
The method for obtaining memory B cell
In another aspect of the invention, the invention proposes a kind of methods for obtaining memory B cell.Reality according to the present invention Example is applied, this method comprises: contacting mentioned-above memory B cell precursor with selected from least one of following: (a) IL-9 Or its functional analogue；(b) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(c)IL-9 Agonist or its functional analogue；(d) STAT-1 agonist or its functional analogue；(e) IL-9 receptor stimulating agent or its function Analog；(f) IL-9 or its expression of receptor promotor.Specific example according to the present invention, IL-9 functional analogue, IL-9 swash Dynamic agent or its functional analogue, STAT-1 agonist or its functional analogue, IL-9 receptor stimulating agent or its functional analogue, IL-9 or its expression of receptor promotor as previously mentioned, according to an embodiment of the invention, using memory B cell precursor with it is above-mentioned At least one contact, memory B cell precursor Efficient Conversion memory B cell can be made, and then effectively obtain memory B cell.
Purposes
According to an embodiment of the invention, the purposes the invention proposes reagent in medicine preparation, the drug is for promoting Mentioned-above memory B cell precursor is converted to memory B cell, and the reagent is selected from least one of following: (a) IL-9 or its functional analogue；(b) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(c) IL-9 agonist or its functional analogue；(d) STAT-1 agonist or its functional analogue；(e) IL-9 receptor stimulating agent or its Functional analogue；(f) IL-9 or its expression of receptor promotor.Inventor is found through experiments that, using selected from it is following at least One of reagent: (a) IL-9 or its functional analogue；(b) IL-9 or the cell of its functional analogue, preferably secretion IL-9 are secreted Th9 cell；(c) IL-9 agonist or its functional analogue；(d) STAT-1 agonist or its functional analogue；(e) IL-9 by Body agonist or its functional analogue；(f) drug prepared by IL-9 or its expression of receptor promotor, can be effectively used for promoting Conversion of the mentioned-above memory B cell precursor to memory B cell.
According to another embodiment of the present invention, the purposes the invention proposes reagent in medicine preparation, the drug For inhibiting mentioned-above memory B cell precursor to convert to memory B cell, the reagent be selected from it is following at least it One: (i) IL-9 antagonist or its functional analogue；(ii) its functional analogue of STAT-1 antagonist；(iii) IL-9 receptor antagonist Agent or its functional analogue；(iv) IL-9 or its expression of receptor inhibitor.Inventor is found through experiments that, using selected from following At least one reagent: (i) IL-9 antagonist or its functional analogue；(ii) its functional analogue of STAT-1 antagonist；(iii) IL-9 receptor antagonist or its functional analogue；(iv) drug prepared by IL-9 or its expression of receptor inhibitor, can be effective It is converted in the mentioned-above memory B cell precursor of inhibition to memory B cell.
Still another embodiment according to the present invention, the invention proposes the purposes of reagent in medicine preparation, the drugs For adjusting memory B cell precursor/memory B cell ratio in centrum germinativum, the reagent be selected from it is following at least it One: (1) IL-9 or its functional analogue；(2) IL-9 or the cell of its functional analogue are secreted, the Th9 of preferably secretion IL-9 is thin Born of the same parents；(3) IL-9 agonist or its functional analogue；(4) STAT-1 agonist or its functional analogue；(5) IL-9 receptor agonism Agent or its functional analogue；(6) IL-9 or its expression of receptor promotor；(7) IL-9 antagonist or its functional analogue；(8) Its functional analogue of STAT-1 antagonist；(9) IL-9 receptor antagonist or its functional analogue；(10) IL-9 or its by body surface Up to inhibitor.Inventor is found through experiments that using selected from least one of following reagent: (1) IL-9 or its function are similar Object；(2) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(3) IL-9 agonist or its function It can analog；(4) STAT-1 agonist or its functional analogue；(5) IL-9 receptor stimulating agent or its functional analogue；(6)IL- 9 or its expression of receptor promotor；(7) IL-9 antagonist or its functional analogue；(8) its functional analogue of STAT-1 antagonist； (9) IL-9 receptor antagonist or its functional analogue；(10) drug prepared by IL-9 or its expression of receptor inhibitor, can have Effectiveness memory B cell precursor/memory B cell ratio in adjusting centrum germinativum.
The present invention provides the purposes of reagent in medicine preparation, the drug include (1) IL-9 disclosed by the invention or Its functional analogue；(2) IL-9 or the cell of its functional analogue are secreted, the Th9 cell of IL-9 is preferably secreted；(3) IL-9 swashs Dynamic agent or its functional analogue；(4) STAT-1 agonist or its functional analogue；(5) IL-9 receptor stimulating agent or its function class Like object；(6) IL-9 or its expression of receptor promotor；(7) IL-9 antagonist or its functional analogue；(8) STAT-1 antagonist its Functional analogue；(9) IL-9 receptor antagonist or its functional analogue；(10) IL-9 or its expression of receptor inhibitor and medicine Acceptable auxiliary material, excipient, carrier, solvent or their combination on.Above-mentioned agonist disclosed by the invention, promotor, The amount of antagonist or inhibitor, which refers to, can effectively change the amount that memory B cell precursor is converted to B cell.
It will also be appreciated that above-mentioned agonist, promotor, antagonist or inhibitor disclosed by the invention or functional analogue can To exist in a free form for treating, or if can suitably exist in the form of its pharmaceutically acceptable derivates. Some non-limiting embodiments of pharmaceutically acceptable derivative include pharmaceutically acceptable prodrug, salt, ester, these esters Salt, or when patient in need is administered above-mentioned agonist of the present invention, promotor, antagonism can be directly or indirectly provided Any other adduct or derivative of agent or inhibitor or functional analogue or its metabolite or residue.
Above-mentioned agonist, promotor, antagonist or inhibitor disclosed by the invention or its functional analogue can be prepared and be wrapped Dress is (bulk) form in bulk, wherein extractable safe and effective amount, then gives patient with powder or syrup form.Alternatively, this Above-mentioned agonist, promotor, antagonist or the inhibitor of disclosure of the invention can prepare and be packaged as unit dosage forms, wherein each physics Upper discrete unit contains safe and effective amount.When being prepared with unit dosage forms, above-mentioned agonist disclosed by the invention, promotor, Antagonist or inhibitor can usually contain, for example, 0.5mg to 1g or 1mg to 700mg or 5mg to 100mg.
" pharmaceutically acceptable auxiliary material " used in the present invention means relevant to form of administration or pharmaceutical composition consistency Pharmaceutically acceptable material, mixture or solvent.Every kind of auxiliary material must be other at split-phase with pharmaceutical composition in mixing Hold, to avoid will be greatly reduced above-mentioned agonist disclosed by the invention, promotor, antagonist or inhibitor when administering to a patient The interaction of effect and will lead to be not pharmaceutically acceptable pharmaceutical composition interaction.In addition, every kind of auxiliary material must It must be pharmaceutically acceptable, for example, having sufficiently high purity.
Suitable pharmaceutically acceptable auxiliary material can be different according to selected specific dosage form.In addition, can combined according to them Specific function in object selects pharmaceutically acceptable auxiliary material.For example, may be selected to can help to produce the certain of equal one dosage type low temperature Pharmaceutically acceptable auxiliary material.The certain pharmaceutically acceptable auxiliary materials that can help to produce stabilizer type may be selected.It may be selected Facilitate to carry or transport disclosed compound of present invention when administering to a patient from an organ of body or partially to the another of body One organ or partial certain pharmaceutically acceptable auxiliary materials.The certain pharmaceutically acceptable of enhancing patient compliance may be selected Auxiliary material.
Suitable pharmaceutically acceptable auxiliary material includes following kind of auxiliary material: diluent, filler, adhesive, disintegration Agent, lubricant, glidant, granulating agent, coating agent, wetting agent, solvent, cosolvent, suspending agent, emulsifier, sweetener, flavoring Agent, odor mask, colorant, anticaking agent, moisturizer, chelating agent, plasticiser, tackifier, antioxidant, preservative, stabilizer, Surfactant and buffer.Technical staff can be appreciated that certain pharmaceutically acceptable auxiliary materials can provide more than one function, And alternative function is provided, this is depended in preparation in the presence of there are which other auxiliary materials in how much auxiliary materials and preparation.
Technical staff grasps the knowledge and skills of this field, so that they can select for the suitable of appropriate amount of the invention Pharmaceutically acceptable excipient.Additionally, there are resources obtained by a large amount of technical staff, they describe pharmaceutically acceptable Excipient, and for selecting suitable pharmaceutically acceptable excipient.Example includes Remington's Pharmaceutical Sciences(Mack Publishing Company),The Handbook of Pharmaceutical Additives(Gower Publishing Limited),and The Handbook of Pharmaceutical Excipients(the American Pharmaceutical Association and the Pharmaceutical Press)。
In Remington:The Science and Practice of Pharmacy, 21st edition, 2005, ed.D.B.Troy,Lippincott Williams&Wilkins,Philadelphia,and Encyclopedia of Pharmaceutical Technology,eds.J.Swarbrick and J.C.Boylan,1988-1999,Marcel The various carriers for configuring pharmaceutically acceptable composition are disclosed in Dekker, New York, and for its preparation Well-known technique, the respective content of these documents are incorporated by reference into the present invention.Except any such as because generating any undesirable life Object effect, or with interaction occurs for any other ingredient in harmful way and pharmaceutically acceptable composition and with the present invention Outside the incompatible any commonly employed carrier of open compound, pays close attention to its application and belong to the scope of the present invention.
Drug disclosed by the invention is prepared using technology and methods well known by persons skilled in the art.This field is some often It can be found in Remington's Pharmaceutical Sciences (Mack Publishing with the description of method Company)。
Therefore, on the other hand, the present invention relates to the technique for preparing drug, the drug includes disclosed by the invention above-mentioned sharp Dynamic agent, promotor, antagonist or inhibitor and pharmaceutically acceptable auxiliary material, excipient, carrier, solvent or their combination, The technique includes mixing various composition.The drug of above-mentioned agonist, promotor, antagonist or inhibitor is disclosed comprising the present invention, It can mix under such as environment temperature and atmospheric pressure to prepare.
Above-mentioned agonist, promotor, antagonist or inhibitor disclosed by the invention or functional analogue are usually formulated as It is suitable for the dosage form administered to a patient by required approach.For example, dosage form includes those dosage forms for being suitable for following administration route: (1) be administered orally, such as tablet, capsule, caplet agent, pill, contain tablet, pulvis, syrup, elixir, suspension, solution Agent, emulsion, granule and cachet；(2) parenteral, such as sterile solution agent, suspension and freeze-dried powder agent；(3) thoroughly Skin administration, such as transdermal patch tablet；(4) rectally, such as suppository；(5) it sucks, such as aerosol, solution and dry powder doses； (6) local administration, such as cream, ointment, lotion, solution, paste, spray, foaming agent and gelling agent.
In some embodiments, above-mentioned agonist, promotor, antagonist or inhibitor disclosed by the invention can be prepared At peroral dosage form.In other embodiments, above-mentioned agonist, promotor, antagonist or inhibitor disclosed by the invention can To be configured to inhalant dosage form.In other embodiments, above-mentioned agonist, promotor, antagonist or suppression disclosed by the invention Preparation can be configured to nose administration dosage form.In other embodiment, above-mentioned agonist disclosed by the invention, promotor, Antagonist or inhibitor can be configured to transdermal administration.Also in some embodiments, above-mentioned excitement disclosed by the invention Agent, promotor, antagonist or inhibitor can be configured to Topical dosage forms.
Drug provided by the invention can be with compressed tablets, development piece, chewable pastille, rapidly dissolving tablet, multiple compressed tablet or enteric Piece, sugar-coat or Film coated tablets provide.Enteric coatel tablets are that gastric acid acts on but the substance for dissolving or being disintegrated in intestines is coated with being resistant to Compressed tablets, to prevent the acidic environment of active ingredient contacts stomach.Enteric coating includes, but are not limited to fatty acid, fat, water Poplar acid phenenyl ester, wax, shellac, ammonification shellac and cellulose acetate phthalate ester.Sugar coated tablet is the compressed tablets that sugar-coat surrounds, It can be conducive to cover taste or smell beastly and can prevent tablet from aoxidizing.Thin membrane coated tablet is with water-soluble substances The compressed tablets of thin layer or film covering.Film coating includes, but are not limited to hydroxyethyl cellulose, sodium carboxymethylcellulose, poly- second Glycol 4000 and cellulose acetate phthalate ester.Film coating possesses general characteristic identical with sweet tablet.Multiple compressed tablet is By the compressed tablets prepared more than a press cycles, including multilayer tablet and pressed coated or dry coating tablet.
Tabules can be by one kind that powder, crystallization or granular active constituent are individual or describe with the present invention Or prepared by variety carrier or excipient composition, the carrier and excipient include adhesive, disintegrating agent, controlled release polymer, profit Lubrication prescription, diluent and/or colorant.Fumet and sweetener are particularly useful when forming chewable tablets and pastille.
Drug provided by the invention can be provided with soft capsule or hard capsule, can by gelatin, methylcellulose, shallow lake It is prepared by powder or calcium alginate.The hard gelatin capsule is also referred to as dry-filled capsules (DFC), is formed by two sections, and a slug enters separately In one section, therefore active constituent is enclosed completely.Soft elastic capsules (SEC) are soft, spherical shell, such as gelatin shell, are passed through Glycerol, sorbierite or the plasticizing of similar polyalcohol is added.Soft gelatin shell may include the pre- micro-organism growth of preservative.Properly Preservative be as described in the present invention those, including methylparaben and propylben and sorbic acid.The present invention provides Liquid, semisolid and solid dosage forms can be encapsulated in capsule.Suitable liquid and semisolid dosage form are included in propylene carbonate Solution and suspension in ester, vegetable oil or triglycerides.Capsule comprising such solution can be such as in United States Patent (USP) U.S.Pat.Nos.4,328,245；It is prepared described in 4,409,239 and 4,410,545.The capsule can also be using such as Coating well known by persons skilled in the art, so as to improve or maintain active constituent dissolution.
Drug provided by the invention can be provided with liquid and semisolid dosage form, including emulsion, solution, suspension, the wine made of broomcorn millet Agent and syrup.Emulsion is two-phase system, and one of liquid is thoroughly dispersed in pellet form in another liquid, can be with It is oil-in-water type or water-in-oil type.Emulsion may include pharmaceutically acceptable on-aqueous liquid and solvent, emulsifier and preservative. Suspension may include pharmaceutically acceptable suspending agent and preservative.Aqueous alcohol solutions may include pharmaceutically acceptable contracting Aldehyde, such as two (low alkyl group) acetals of low alkyl group aldehyde, such as acetaldehyde diethyl acetal；With there are one or more hydroxyls Water-soluble solvent, such as propylene glycol and ethyl alcohol.Elixir is transparent, sweet taste water-alcohol solution.Syrup is dense sugared such as sugarcane The aqueous solution of sugar, and can also include preservative.For liquid dosage form, for example, the solution in polyethylene glycol can use foot The pharmaceutically acceptable liquid-carrier of amount such as water dilutes, to be accurately, conveniently administered.
Other useful liquid and semisolid dosage form include, but are not limited to comprising above-mentioned agonist provided by the invention, rush Into agent, antagonist or inhibitor and second levelization be mono- or those of poly- alkylene glycol dosage form, described mono- or poly- alkylene glycol packet It includes: 1,2- dimethoxymethane, diethylene glycol dimethyl ether, triglyme, tetraethylene glycol dimethyl ether, polyethylene glycol -350- diformazan Ether, polyethylene glycol -550- dimethyl ether, polyethylene glycol -750- dimethyl ether, wherein the approximation of 350,550,750 finger polyethylene glycol is flat Average molecular weight.These preparations may further include one or more antioxidant, such as Butylated Hydroxytoluene (BHT), Butylated Hydroxyanisole (BHA), propylgallate, vitamin E, quinhydrones, Hydroxycoumarin, ethanol amine, lecithin, cephalin, ascorbic acid, apple Acid, sorbierite, phosphoric acid, bisulfites, sodium pyrosulfite, thio-2 acid and its ester and dithiocarbamate.
Where appropriate, can be by the dosage unit preparations microencapsulation of oral administration.It can also be prepared into extending or tie up Hold the composition of release, such as by being coated by microparticle material or be embedded in polymer, wax or the like.
Oral drugs provided by the invention can also be provided in the form of liposome, micella, microballoon or nanometer system.Micella Dosage form can be prepared with the method that U.S.Pat.No.6,350,458 is described.
Drug provided by the invention can be provided with the granule and pulvis of non-effervesce or effervesce, to be reconstructed into liquid agent Type.The pharmaceutically acceptable carrier used in non-effervescent or pulvis and excipient may include diluent, sweet taste Agent and wetting agent.The pharmaceutically acceptable carrier used in effervescent or pulvis and excipient may include organic acid And carbon dioxide source.
Colorant and flavoring agent can be used in all above-mentioned dosage forms.
Above-mentioned agonist, promotor, antagonist or inhibitor disclosed in this invention or functional analogue can also be with works It is combined for the soluble polymer of target medicine carrier.Such polymer includes polyvinylpyrrolidone, pyran co-polymer, gathers The poly- bad ammonia of the polyoxyethylene that hydroxypropyhnethacrylamide-phenol, polyhydroxyethylaspart or palmitoyl residues replace Acid.In addition, above-mentioned agonist, promotor, antagonist or inhibitor disclosed in this invention can be with the controls in realization drug One kind Biodegradable polymeric used in release combines, for example, polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyrate, poly- original Acid esters, polyacetals, poly- dihydropyran, polybutylcyanoacrylate and hydrogel crosslinking or amphiphilic block copolymer.
Drug provided by the invention can be configured to immediately or Modified release dosage forms, including delay-, sustained release-, pulse-, control System-, targeting-and sequencing releasing pattern.
Drug provided by the invention can with the other active constituent co-formulations that will not damage expected therapeutic effect, or Person and the expected substance co-formulation acted on of supplement.
Drug provided by the invention can be by injection, infusion or implantation parenteral administration, for being locally or systemically administered. As the parenteral administration that uses of the present invention includes in intravenous, intra-arterial, peritonaeum, in intrathecal, intra-ventricle, urethra, in breastbone, cranium Interior, intramuscular, intrasynovial and subcutaneous administration.
Drug provided by the invention can be configured to any dosage form suitable for parenteral administration, including solution, suspension, cream Agent, micella, liposome, microballoon, nanometer system and the solid form suitable for solution or suspension is made in a liquid before the injection. Such dosage form can be prepared according to conventional method known to the technical staff in pharmaceutical science field (referring to Remington: The Science and Practice of Pharmacy, ibid).
Be intended for parenteral administration pharmaceutical composition may include one or more pharmaceutically acceptable carriers and Excipient includes, but are not limited to containing transporter, water miscibility carrier, non-transporter, antimicrobial or resists micro- life Preservative, stabilizer, dissolution enhancers, isotonic agent, buffer, antioxidant, local anesthetic, suspending agent and the dispersion of object growth Agent, wetting agent or emulsifier, complexing agent, sequestering agent or chelating agent, antifreezing agent, cryoprotector, thickener, pH adjusting agent And inert gas.
Suitably include, but are not limited to containing transporter: water, salt water, physiological saline or phosphate buffered saline (PBS) (PBS), Sodium chloride injection, Ringers injection, isotonic glucose injection, Sterile Water Injection, glucose and Lactated Ringers injection.Non- transporter includes, but are not limited to fixed oil, castor oil, corn oil, the cottonseed of plant origin Oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soya-bean oil, hydrogenated vegetable oil, hydrogenated soybean oil and coconut oil middle chain Triglycerides and palm seed oil.Water miscibility carrier includes, but are not limited to the poly- second two of ethyl alcohol, 1,3-BDO, liquid Alcohol (such as Liquid Macrogol and polyethylene glycol 400), propylene glycol, glycerol, n-methyl-2-pyrrolidone, N, N- dimethylacetamide Amine and dimethyl sulfoxide.
Suitable antimicrobial or preservative include, but are not limited to phenol, cresols, mercurial, benzyl alcohol, chlorobutanol, Methyl p-hydroxybenzoate and propylparaben, thimerosal, benzalkonium chloride (such as benzethonium chloride), methylparaben and Propylben and sorbic acid.Suitable isotonic agent includes, but are not limited to sodium chloride, glycerol and glucose.Suitable buffer Include, but are not limited to phosphate and citrate.Suitable antioxidant is such as those of present invention description, including sulfurous acid Hydrogen salt and sodium metabisulfite.Suitable local anesthetic includes, but are not limited to procaine hydrochloride.Suitable suspending agent and point Powder is such as those of present invention description, including sodium carboxymethylcellulose, hydroxypropyl methyl cellulose and polyvinylpyrrolidone. Suitable emulsifier includes those of present invention description, including polyoxyethylene sorbitan monolaurate.Polyoxyethylene is de- Water sorbitol monooleate 80 and triethanolamine oleate ester.Suitable sequestering agent or chelating agent include, but are not limited to EDTA. Suitable pH adjusting agent includes, but are not limited to sodium hydroxide, hydrochloric acid, citric acid and lactic acid.Suitable complexing agent includes, but unlimited In cyclodextrin, including alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-β-cyclodextrin, Sulfobutylether-beta-cyclodextrin and sulfobutyl group Ether 7- beta-cyclodextrin (CyDex,Lenexa,KS)。
Drug provided by the invention can be configured to single dose or multiple dose administration.The single-dose preparations are packaged in peace In small jar agent, bottle or syringe.The multi-dose parenteral administration must include antibacterial or fungistatic concentrations antimicrobial. All parenteral administrations all must be it is sterile, as known in the art and practice.
In some embodiments, drug is provided with instant sterile solution.In other embodiments, drug with Sterile dried soluble product provides, including freeze-dried powder agent and hypodermic tablet, is reconstructed using preceding with carrier.In In other embodiment, drug is formulated into instant sterile suspensions.In other embodiment, drug is formulated into The sterile dry insolubility product reconstructed before use with carrier.Also in some embodiments, drug is formulated into i.e. With type without bacterial emulsion.
Drug disclosed in this invention can be configured to immediately or Modified release dosage forms, including delay-, sustained release-, pulse-, Control-, targeting-and sequencing releasing pattern.
Drug can be configured to suspension, solid, semisolid or thixotropic liquid, the reservoir administration as implantation.Some In embodiment, drug disclosed in this invention is dispersed in solid interior matrix, is insoluble to body fluid but is allowed in drug The external polymeric membrane that diffuses through of active constituent surrounded.
Suitable internal matrix include polymethyl methacrylate, poly- butyl methacrylate, plasticising or it is unplasticizied Polyvinyl chloride, the nylon of plasticising, the polyethylene terephthalate of plasticising, the polyethylene terephthalate of plasticising, natural rubber, Polyisoprene, polyisobutene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, silicone rubber, poly- diformazan silicon oxygen Alkane, silicone carbonate copolymer, the hydrogel of the ester of hydrophilic polymer such as acrylic acid and methacrylic acid, collagen, crosslinking The polyvinyl acetate of the partial hydrolysis of polyvinyl alcohol and coach.
Suitable external polymeric membrane includes polyethylene, polypropylene, ethylene/propene copolymer, ethylene/ethyl acrylate copolymerization Object, ethylene/vinyl acetate copolymer, silicone rubber, dimethyl silicone polymer, neoprene, haloflex, polychlorostyrene second Alkene, the copolymer of ethlyene dichloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer are poly- to benzene two Formic acid second diester, butyl rubber chlorohydrin rubber, ethylene/vinyl alcohol copolymer, Ethylene/vinyl acetate/vinyl alcohol trimer and Ethylene/vinyl ethoxy-ethanol copolymer.
On the other hand, drug disclosed in this invention can be configured to be suitable for any dosage form to patient's inhalation, example Such as dry powder doses, aerosol, suspension or liquid composite.In some embodiments, drug disclosed in this invention can match The dosage form being suitable for dry powder doses to patient's inhalation is made.In other embodiment, drug disclosed in this invention can To be configured to be suitable for the dosage form by sprayer to patient's inhalation.It is usually wrapped by the dry powder composite of inhalation delivery to lung Containing fine powdered above-mentioned agonist, promotor, antagonist or inhibitor disclosed in this invention and one or more fine Powdered pharmaceutically acceptable excipient.Pharmaceutically acceptable excipient be especially suitable for dry powder doses is this field Technical staff is known comprising lactose, starch, mannitol and mono-, two- and polysaccharide.Fine powder can be for example, by micro mist Change and grinding is prepared.In general, (as being micronized) compound that size reduces can be by about 1 to 10 micron of D50 Value (for example, measured with laser diffractometry) Lai Dingyi.
Aerosol can by by above-mentioned agonist disclosed in this invention, promotor, antagonist or inhibitor suspend or It is dissolved in liquefied propellant and prepares.Suitable propellant includes chlorohydrocarbon, hydro carbons and other liquefied gas.Representative propulsion Agent includes: trichlorofluoromethane (propellant 11), dichlorofluoromethane (propellant 12), dichlorotetra-fluoroethane (propellant 114), tetrafluoro Ethane (HFA-134a), 1,1- Difluoroethane (HFA-152a), difluoromethane (HFA-32), pentafluoroethane (HFA-12), seven fluorine Propane (HFA-227a), perfluoropropane, perfluorinated butane, perflenapent, butane, iso-butane and pentane.Comprising presently disclosed The aerosol of compound usually pass through metered dose inhaler (MDI) and administer to a patient.Such device is art technology Personnel are known
Aerosol may include pharmaceutically acceptable excipient that is additional, being used by MDIs, such as surface-active Agent, lubricant, cosolvent and other excipient, with improve preparation physical stability, improve valve characteristic, improve dissolubility, Or improve taste.
The pharmaceutical composition for being suitable for cutaneous penetration can be prepared into discontinuous patch agent, it is intended that keep with the epidermis of patient It is in close contact the time of an elongated segment.For example, the delivering active ingredients from patch agent can be permeated by ion, such as Pharmaceutical Research, 3 (6), the general description in 318 (1986).
The drug for being suitable for local administration can be formulated into ointment, cream, suspension, lotion, pulvis, solution Agent, paste, gelling agent, spray, aerosol or finish.For example, ointment, cream and gelling agent can use water or oil base Matter, and suitable thickener and/or gelling agent and/or solvent configure.Such matrix may include water, and/or oil is for example Liquid-liquid paraffin and vegetable oil (such as peanut oil or castor oil) or solvent such as polyethylene glycol.It is used according to medium property Thickener and gelling agent include soft paraffin, aluminum stearate, cetostearyl alcohol, polyethylene glycol, lanolin, beeswax, carbopol And cellulose derivative and/or single stearic acid glycerine lipoprotein and/or nonionic emulsifier.
Lotion can be prepared with water or oil matrix, and generally also contain one or more emulsifying agents, stabilizer, dispersion Agent, suspending agent or thickener.
Externally-applied powder can form in the presence of any suitable powder matrix such as talcum powder, lactose or starch.Drops It can be formulated with the water comprising one or more dispersing agents, solubilizer, suspending agent or preservative or non-aqueous matrix.
Topical formulations can be by being administered using one or many daily in affected part；The impermeable plastic wound dressing for covering skin is preferential It is used.Adhesiveness store system can realize continuous or extended administration.
When treating eyes or other organs such as mouth and skin, the combination as topical ointment or cream can be applied Object.When being formulated as ointment, compound disclosed in this invention can be used together with paraffin or water-soluble ointment matrix.Or Person, compound disclosed in this invention can be configured to cream together with Oil-in-water emulsifiable paste agent matrix or oil-in-water base.
Present invention offer is used to prepare using above-mentioned agonist, promotor, antagonist or inhibitor disclosed in this invention The drug for promoting or memory B cell precursor being inhibited to convert to memory B cell is used to prepare to adjust in centrum germinativum and remembers B Cell-progenitor cells/memory B cell ratio drug.
In some embodiments, the present invention provide a kind of above-mentioned agonist disclosed in this invention, promotor or comprising The drug of presently disclosed above-mentioned agonist, promotor, for treating, preventing or improve by less memory B cell influence Disease or disorder one or more symptoms.In other embodiments, the disease, disorder or disease or disorder One or more symptoms are related to less memory B cell behavior.Also in some embodiments, the disease, disorder or Disease or one or more symptoms of disorder are related to less memory B cell behavior.
In some embodiments, the present invention provide a kind of above-mentioned antagonist disclosed in this invention or inhibitor or comprising The drug of presently disclosed above-mentioned antagonist or inhibitor, for treating, preventing or improve by more memory B cell influence Disease or disorder or the disease or disorder that are influenced by unsuitable more memory B cell one or more symptoms.
In some embodiments, the present invention provides a kind of above-mentioned agonist disclosed in this invention, promotor, antagonist Or inhibitor or its functional analogue or include presently disclosed above-mentioned agonist, promotor, antagonist or inhibitor or function The drug of energy analog, for treating, preventing or improve by memory B cell precursor/memory B in unsuitable centrum germinativum The disease or disorder or by memory B cell precursor in unsuitable centrum germinativum/memory B cell ratio that cell proportion influences One or more symptoms of disease or disorder that example influences.In other embodiments, the disease, disorder or disease or Memory B cell precursor/memory B cell ratio behavior phase in one or more symptoms of disorder and unsuitable centrum germinativum It closes.Also in some embodiments, the disease, disorder or disease or one or more symptoms of disorder and unsuitable life Memory B cell precursor/memory B cell ratio behavior is related in hair center.
" memory B cell precursor/memory B cell ratio in unsuitable centrum germinativum " refers to generation in particular patient Deviate memory B cell precursor in normal centrum germinativum/memory B cell ratio behavior with it.In unsuitable centrum germinativum Memory B cell precursor/the behavior of memory B cell ratio can show memory B cell is excessive or very few.Therefore, the present invention mentions Method for treating these diseases and disorder.
Consistent with above description, such disease or disorder include but is not limited to: immunologic function disorder immune lacks Fall into, the related diseases associated with inflammation of immunological regulation or disorder, autoimmune disease, tissue transplantation rejection, graft versus host disease(GVH disease), Wound healing, nephrosis, multiple sclerosis, thyroiditis, type-1 diabetes mellitus, sarcoidosis, psoriasis, allergic rhinitis, inflammatory bowel Disease includes Crohn disease and ulcerative colitis (UC), systemic loupus erythematosus (SLE), arthritis, osteoarthritis, rheumatoid Arthritis, osteoporosis, asthma and chronic obstructive pulmonary disease (COPD) and dry eye syndrome (or keratoconjunctivitis sicca (KCS))。
On the one hand, the present invention provide a kind of above-mentioned agonist, promotor, antagonist or inhibitor disclosed in this invention or Its functional analogue includes presently disclosed above-mentioned agonist, promotor, antagonist or inhibitor or its functional analogue Drug, for prevent and/or treat the autoimmune disease of mammal (including the mankind), anaphylactia, inflammatory disease, Or graft rejection.
On the other hand, the present invention provides a kind for the treatment of and suffers from or the risky mammal for suffering from disease disclosed herein Method, the method includes give effectively treatment illness amount or effectively prevention illness amount one or more medicines disclosed herein Object or above-mentioned agonist, promotor, antagonist or inhibitor or its functional analogue.On the other hand, provided herein is one kind to control Treatment is suffered from or the method for the risky mammal for suffering from autoimmune disease, anaphylactia, inflammatory disease or graft rejection. On the other hand, provided herein is the method for removing pathogen, the pathogen includes but is not limited to gram-positive bacteria, gram Negative bacterium, fungi, helminth and virus.
On the other hand, the side of the mammal of autoimmune disease is susceptible or suffering from provided herein is treatment and/or prevention Method, the method includes giving one or more drugs disclosed herein of effective therapeutic dose or effective preventive dose or above-mentioned excitement Agent, promotor, antagonist or inhibitor or its functional analogue.In particular instances, autoimmune disease is selected from COPD, roars Asthma, systemic loupus erythematosus, skin lupus erythematosus, lupus nephritis, dermatomyositis, Sjogren syndrome, psoriasis, I type glycosuria Disease and inflammatory bowel disease.
On the other hand, provided herein is a kind of above-mentioned agonist, promotor, antagonist or inhibitor disclosed herein or its function Energy analog, for treating and/or preventing autoimmune disease.In certain embodiments, autoimmune disease is selected from COPD, asthma, systemic loupus erythematosus, skin lupus erythematosus, lupus nephritis, dermatomyositis, Sjogren syndrome, psoriasis, I Patients with type Ⅰ DM and inflammatory bowel disease.
On the other hand, provided herein is a kind of above-mentioned agonist, promotor, antagonist or inhibitor disclosed herein or its function Energy analog, or the drug comprising above-mentioned agonist, promotor, antagonist or inhibitor or its functional analogue is disclosed herein, It is used to prepare the drug for treating or preventing autoimmune disease.In certain embodiments, autoimmune disease be selected from COPD, Asthma, systemic loupus erythematosus, skin lupus erythematosus, lupus nephritis, dermatomyositis, Sjogren syndrome, psoriasis, I type sugar Urine disease and inflammatory bowel disease.
On the other hand, the method that the mammal of anaphylactia is susceptible or suffering from provided herein is treatment and/or prevention, The method includes giving the one or more pharmaceutical compositions disclosed herein or chemical combination of effective therapeutic dose or effective preventive dose Object.In certain embodiments, anaphylactia is selected from respiratory anaphylactic disease, nasosinusitis, eczema and morbilli, food mistake Quick and insect venom allergies.
On the other hand, provided herein is a kind of above-mentioned agonist, promotor, antagonist or inhibitor disclosed herein or its function Energy analog, for treating and/or preventing anaphylactia.In certain embodiments, anaphylactia is selected from respiratory tract Anaphylactia, nasosinusitis, eczema and morbilli, food hypersenstivity and insect venom allergies.
On the other hand, provided herein is a kind of above-mentioned agonist, promotor, antagonist or inhibitor disclosed herein or its function Energy analog, or the drug comprising above-mentioned agonist, promotor, antagonist or inhibitor or its functional analogue is disclosed herein, It is used to prepare the drug for treating or preventing anaphylactia.In certain embodiments, anaphylactia is selected from respiratory tract mistake Quick property disease, nasosinusitis, eczema and morbilli, food hypersenstivity and insect venom allergies.
On the other hand, the method that the mammal of inflammatory disease is susceptible or suffering from provided herein is treatment and/or prevention, institute The method of stating includes giving the one or more drugs disclosed herein or above-mentioned agonist, rush of effective therapeutic dose or effective preventive dose Into agent, antagonist or inhibitor or its functional analogue.In certain embodiments, inflammatory disease be selected from inflammatory bowel disease, gram Sieve grace disease, rheumatoid arthritis, juvenile arthritis and psoriasis arthropathica.
On the other hand, provided herein is a kind of above-mentioned agonist, promotor, antagonist or inhibitor disclosed herein or its function Energy analog, for treating and/or preventing inflammatory disease.In certain embodiments, inflammatory disease be selected from inflammatory bowel disease, gram Sieve grace disease, rheumatoid arthritis, juvenile arthritis and psoriasis arthropathica.
On the other hand, provided herein is a kind of above-mentioned agonist, promotor, antagonist or inhibitor disclosed herein or its function Energy analog, or the pharmaceutical composition comprising compound is disclosed herein, are used to prepare the drug for treating or preventing inflammatory disease.In In specific embodiment, inflammatory disease be selected from inflammatory bowel disease, Crohn disease, rheumatoid arthritis, juvenile arthritis and Psoriasis arthropathica.
On the other hand, the method that the mammal of graft rejection is susceptible or suffering from provided herein is treatment and/or prevention, institute The method of stating includes giving the one or more drugs disclosed herein or above-mentioned agonist, rush of effective therapeutic dose or effective preventive dose Into agent, antagonist or inhibitor or its functional analogue.In particular instances, graft rejection is organ-graft refection, tissue shifting Plant repulsion and cell transplant rejection.
On the other hand, provided herein is a kind of above-mentioned agonist, promotor, antagonist or inhibitor disclosed herein or its function Energy analog, for treating and/or preventing graft rejection.In certain embodiments, graft rejection be organ-graft refection, Tissue transplantation rejection and cell transplant rejection.
On the other hand, provided herein is a kind of above-mentioned agonist, promotor, antagonist or inhibitor disclosed herein or its function Energy analog, or the drug comprising above-mentioned agonist, promotor, antagonist or inhibitor or its functional analogue is disclosed herein, It is used to prepare the drug for treating or preventing graft rejection.In particular instances, graft rejection is organ-graft refection, tissue transplantation Repulsion and cell transplant rejection.
On the other hand, it is used as drug provided herein is one kind to be especially used as treating and/or preventing disease medicament noted earlier Above-mentioned agonist, promotor, antagonist or inhibitor disclosed herein or its functional analogue.It is also provided with disclosed herein Disease noted earlier is treated and/or is prevented in above-mentioned agonist, promotor, antagonist or inhibitor or the manufacture of its functional analogue Drug.
One special projects of this method include giving a effective amount of present invention of study subject with inflammation to disclose chemical combination For a period of time, the time is enough to reduce the level of inflammation of study subject object, and preferably terminates the process of the inflammation.The party The special embodiment of method includes giving a effective amount of present invention public affairs of tested patients for suffering from or being susceptible to suffer from bone rheumatoid arthritis Open compound for a period of time, the time is enough to reduce or prevent the arthritis of the patient respectively, and preferably terminates institute State the process of inflammation.
Above-mentioned agonist, promotor, antagonist or inhibitor disclosed in this invention or its functional analogue can be used as Individual active agent administration, or can be administered with other therapeutic agents, including simultaneously with same or similar therapeutic activity And safe and efficient other compounds are determined as such administering drug combinations.
On the one hand, the present invention provides treatment, prevention or the method for improving disease or illness, including giving safe and effective amount Comprising the present invention, above-mentioned agonist, promotor, antagonist or inhibitor or its functional analogue and one or more treatments are disclosed The combination medicine of activating agent.In some embodiments, combination medicine includes one or two kinds of other therapeutic agents.
The example of other therapeutic agents includes including but is not limited to: anti-inflammatory agent；With immunity regulatin remedy agent or immunosuppressor.
On the other hand, the present invention provides the product including the compounds of this invention and at least one other therapeutic agent, can prepare At the combination simultaneously, separately or sequentially applied in the treatment.In some embodiments, treatment is to be directed in centrum germinativum to remember The treatment of disease or symptom that B cell precursor cell/memory B cell ratio mediates.The product that joint preparation provides includes existing Comprising above-mentioned agonist, promotor, antagonist or inhibitor or its functional analogue is disclosed herein in same pharmaceutical composition Composition, or existing in different forms above-mentioned agonist, promotor, antagonist or inhibitor or its function is disclosed herein Analog and other therapeutic agents, for example, medicine box.
On the other hand, the present invention provide it is a kind of comprising be disclosed herein above-mentioned agonist, promotor, antagonist or inhibitor or Its functional analogue and the drug of another or a variety of therapeutic agents.In some embodiments, drug may include institute as above Pharmaceutically acceptable excipient, carrier, adjuvant or the solvent stated.
On the other hand, the present invention provides the medicine box of the drug alone comprising two kinds or more, wherein at least one drug packet Containing above-mentioned agonist, promotor, antagonist or inhibitor disclosed by the invention or its functional analogue.In some embodiments In, medicine box includes the tool for individually keeping the composition, such as container, separated bottle or separated foil box.This kind of medicine box Example is blister package, is commonly used for package troche, capsule etc..
The present invention also provides the above-mentioned agonist of the present invention, promotor, antagonist or inhibitor or its functional analogue to exist The purposes in the disease or symptom that memory B cell precursor in centrum germinativum/memory B cell ratio mediates is treated, wherein suffering from Person previously (such as in 24 hours) has been treated with other therapeutic agents.
Above-mentioned agonist, promotor, antagonist or inhibitor is disclosed herein or its functional analogue can be used as single work Property component application or as such as adjuvant, be co-administered with other therapeutic agents.
In some embodiments, above-mentioned agonist, promotor, antagonist or inhibitor is disclosed herein or its function is similar Object can also be co-administered with other medicines.The other medicines include immunosuppressor, immunomodulator, other anti-inflammatory agents, Such as treat or prevent allogeneic or xenograft acute or chronic rejection, inflammatory, autoimmune disease medicine Object.For example, the present invention discloses above-mentioned agonist, promotor, antagonist or inhibitor or its functional analogue can live with following Property component in conjunction: with immunosuppressive properties ascosin, such as ABT-281, ASM981 etc.；Corticosteroid；Ring phosphinylidyne Amine；Imuran；Methotrexate (MTX)；Leflunomide；Mizoribine；Mycophenolic Acid or salt；Mycophenolate mofetil；15- deoxidation Spergualin or its immunosupress homologue, analog or derivative；Pkc inhibitor, such as WO 02/38561 or WO 03/ Described in 82859, such as the compound of embodiment 56 or 70；Immunosupress monoclonal antibody, such as the list of leukocyte receptors Clonal antibody, for example, MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD52, CD58, CD80, CD86 Or its ligand；Other immunomodulatory compounds, for example, at least partly extracellular domain with CTLA4 recombination binding molecule or it is prominent Variant, such as at least extracellular portion or its mutant of the CTLA4 that are connected with non-CTLA4 protein sequence, such as CTLA4Ig (such as being named as ATCC 68629) or its mutant, such as LEA29Y；Adhesion molecule inhibitor, such as LFA-1 antagonist, The antagonist of ICAM-1 or -3, VCAM-4 antagonist or VLA-4 antagonist.
On the other hand, it includes that the present invention discloses above-mentioned agonist, promotor, antagonist or inhibitor that the present invention, which provides a kind of, Or the joint of its functional analogue and corticosteroid.Suitable corticosteroid refers to that those oral and sucking cortex classes are solid Alcohol, and its have the prodrug of anti-inflammatory activity.Example includes methylprednisolone, prednisolone (prednisolone), dexamethasone (dexamethasone), fluticasone propionate (fluticasone propionate), -16 α of 6 alpha, 9 alpha-difluoro-11 beta-hydroxy - Methyl-17-alpha-[(4- methyl-1,3-thiazole -5- carbonyl) oxygroup] -17 β of -3- oxo-androst -1,4- diene-thiocarboxylic acid S- fluorine Methyl esters, 6 α, -16 Alpha-Methyl -3- oxo-androst -1,4- two of fluoro- 17 α-of 9 α-two [(2- furanylcarbonyl) oxygroup] -11 beta-hydroxy - 17 β of alkene-thiocarboxylic acid S- fluorine methyl esters (fluticasone furoate), -16 Alpha-Methyl -3- oxo -17 of 6 alpha, 9 alpha-difluoro-11 beta-hydroxy α--17 β of propionyloxy-androsta -1,4- diene-thiocarboxylic acid S- (2- oXo-tetrahydro furans -3S- base) ester, 6 α, 9 α-two are fluoro- - 17 α of -16 Alpha-Methyl -3- oxo of 11 beta-hydroxy-(2,2,3,3- tetramethyl cyclopropyl carbonyl) oxygroup-androstane -1,4- diene -17 - 17 α of β-thiocarboxylic acid S- cyano methyl ester and -16 Alpha-Methyl of 6 alpha, 9 alpha-difluoro-11 beta-hydroxy-(1- ethyl cyclopropyl carbonyl) oxygen - 17 β of base -3- oxo-androst -1,4- diene-thiocarboxylic acid S- methyl fluoride ester, beclomethasone ester (such as 17- propionic ester or 17,21- Dipropionic acid rouge), budesonide (budesonide), flunisolide (flunisolide), Mometasone ester (such as momestasone furoate), Triamcinolone acetonide (triamcinolone acetonide), sieve fluoronaphthalene moral (rofleponide), ciclesonide (ciclesonide) (16 α, 17- [[(R)-cyclohexylmethylene] is bis- (oxygroup)] -11 β, 21- dihydroxy-pregnant steroid -1,4- diene -3,20- diketone), Butixocort propionate (butixocort propionate), RPR-106541 and ST-126.Preferred corticosteroid includes third Sour fluticasone (fluticasone propionate), -17 α of -16 Alpha-Methyl of 6 alpha, 9 alpha-difluoro-11 beta-hydroxy-[(4- methyl - 1,3- thiazole -5- carbonyl) oxygroup] -17 β of -3- oxo-androst -1,4- diene-thiocarboxylic acid S- methyl fluoride ester, 6 α, 9 α-two are fluoro- - 16-17 β of Alpha-Methyl-3- oxo-androst-1,4- diene of 17 α-[(2- furanylcarbonyl) oxygroup]-11 beta-hydroxy-thiocarboxylic acid S- Methyl fluoride ester, -17 α of -16 Alpha-Methyl -3- oxo of 6 alpha, 9 alpha-difluoro-11 beta-hydroxy-(2,2,3,3- tetramethyl cyclopropyl carbonyl) oxygen Base--17 β of androstane -1,4- diene-thiocarboxylic acid S- cyano methyl ester and -16 Alpha-Methyl of 6 alpha, 9 alpha-difluoro-11 beta-hydroxy -17 α-(1- Methylcyclopropyl groups carbonyl) -17 β of oxygroup -3- oxo-androst -1,4- diene-thiocarboxylic acid S- fluorine methyl esters.In some embodiments In, corticosteroid is 6 α, -16 Alpha-Methyl -3- oxo of fluoro- 17 α-of 9 α-two [(2- furanylcarbonyl) oxygroup] -11 beta-hydroxy-hero - 17 β of steroid -1,4- diene-thiocarboxylic acid S- methyl fluoride ester.
On the other hand, it includes that the present invention discloses above-mentioned agonist, promotor, antagonist or inhibitor that the present invention, which provides a kind of, Or the joint of its functional analogue and nonsteroidal GR agonist.To Transcription inhibition have selectivity (compared with transcriptional activation), The nonsteroidal compound with glucocorticoid agonist activity that can be used for combination therapy includes that those covered in following patent In compound: WO 03/082827, WO 98/54159, WO 04/005229, WO 04/009017, WO 04/018429, WO 03/104195、WO 03/082787、WO 03/082280、WO 03/059899、WO 03/101932、WO 02/02565、WO 01/16128, WO 00/66590, WO 03/086294, WO 04/026248, WO 03/061651 and WO 03/08277.More Nonsteroidal compound be included in WO 2006/000401, WO 2006/000398 and WO 2006/015870.
On the other hand, it includes that the present invention discloses above-mentioned agonist, promotor, antagonist or inhibitor that the present invention, which provides a kind of, Or the joint of its functional analogue and nonsteroidal anti-inflammatory drug (NSAID's).The example of NSAID's includes nasmil, how more Sieve rice sodium (nedocromil sodium), phosphodiesterase (PDE) inhibitor (such as theophylline, PDE4 inhibitor or mixed type PDE3/PDE4 inhibitor), leukotriene antagonist, leukotriene synthesis inhibitors (such as montelukast), iNOS inhibitor, tryptose Enzyme and elastatinal, Beta 2 integrin antagonist and adenosine receptor agonist or antagonist (e.g., adenosine 2a receptor Agonist), cytokine antagonist (such as chemokine receptor anagonists, including CCR3 antagonist), cell factor synthesis inhibit Agent or 5-LO inhibitor.Wherein, iNOS (iNOS) inhibitor is preferably administered orally.INOS suppression The example of preparation includes those in WO 93/13055, WO 98/30537, WO 02/50021, WO 95/34534 and WO 99/ Compound disclosed in 62875.CCR3 inhibitor includes those compounds disclosed in WO 02/26722.
In some embodiments, the present invention relates to the present invention to disclose above-mentioned agonist, promotor, antagonist or inhibitor Or its functional analogue with the application in the combining of phosphodiesterase 4 (PDE4) inhibitor, especially in inhalant dosage form Using.PDE4 specific inhibitor for this aspect of the present invention can be known inhibition PDE4 enzyme or be found to be used as PDE4 Any compound of inhibitor, they are only PDE4 inhibitor, are not to inhibit other members in PDE family, such as PDE3 and The compound of PDE5.Compound includes cis- -4- cyano -4- (3- cyclopentyloxy -4- methoxyphenyl) hexamethylene -1- carboxylic Acid, 2- carbomethoxy -4- cyano -4- (3- cyclo propyl methoxy -4- difluoro-methoxy phenyl) hexamethylene -1- ketone and cis--[4- cyanogen Base -4- (3- cyclo propyl methoxy -4- difluoro-methoxy phenyl) hexamethylene -1- alcohol]；It also include cis- -4- cyano -4- [3- (ring Propoxyl group) -4- methoxyphenyl] hexamethylene -1- carboxylic acid (also referred to as Xi Luosi) and its salt, ester, prodrug or physical form, It is disclosed in 09 month 1996 No. 03 United States Patent (USP) US 5,552,438 authorized, this patent and its disclosed compound are by drawing Be integrally incorporated herein.
On the other hand, it includes that the present invention discloses above-mentioned agonist, promotor, antagonist or inhibitor that the present invention, which provides a kind of, Or the joint of its functional analogue and H1 antagonist.The example of H1 antagonist includes, but are not limited to Amlexanox (amelexanox), this western imidazoles (astemizole), azatadine (azatadine), azelastine (azelastine), Ah Cut down sting (acrivastine), Brompheniramine (brompheniramine), cetirizine (cetirizine), levocetirizine (levocetirizine), Efletirizine (efletirizine), chloropheniramine (chlorpheniramine), clemastine (clemastine), marezine (cyclizine), Carebastine (carebastine), cyproheptadine (cyproheptadine), carbinoxamine (carbinoxamine), descarboethoxyloratadine (descarboethoxyloratadine), doxylamine (doxylamine), diformazan indenes (dimethindene), Ebastine (ebastine), epinastine (epinastine), Efletirizine (efletirizine), fexofenadine (fexofenadine), hydroxyzine (hydroxyzine), Ketotifen (ketotifen), Loratadine (loratadine), Zuo Ka Bath spit of fland (levocabastine), Mizolastine (mizolastine), mequitazine (mequitazine), Mianserin (mianserin), the primary sting of promise (noberastine), meclizine (meclizine), Tecastemizole (norastemizole), olopatadine (olopatadine), piperacetazine (picumast), than Lamine (pyrilamine), Phenergan (promethazine), RMI 9918 (terfenadine), Tripelennamine (tripelennamine), for beauty this Spit of fland (temelastine), nedeltran (trimeprazine) and triprolidine (triprolidine), preferably cetirizine (cetirizine), levocetirizine (levocetirizine), Efletirizine (efletirizine) and fexofenadine (fexofenadine).In other embodiments, the present invention provides a kind of comprising disclosed compound of present invention and H3 antagonism The joint of agent (and/or inverse agonist).The example of H3 antagonist includes those in WO 2004/035556 and WO 2006/ Compound disclosed in 045416.Can be used for include H4 with other united histamine receptor antagonists of disclosed compound of present invention by Body antagonist (and/or inverse agonist), such as in Jablonowski et al., J.Med.Chem., 2003,46:3957- Compound disclosed in 3960.
It is above-described combine therefore, including defined above group be prepared into pharmaceutical composition with can be convenient come using It closes and represents another aspect of the present invention with the pharmaceutical composition of pharmaceutically acceptable excipient or carrier.
These united each compounds with alone or in combination pharmaceutical preparation form order of administration or can be administered simultaneously. In one embodiment, each component is administered simultaneously with combined pharmaceutical preparation form.The suitable dosage of known treatment agent is easy In being understood by the person skilled in the art.
Therefore, on the other hand, the present invention provides a kind of drug, comprising above-mentioned agonist disclosed by the invention, promotor, short of money Anti-agent or inhibitor or its functional analogue are combined with other treatment activating agent.
In other embodiments, pharmaceutical composition provided by the invention includes that the present invention discloses above-mentioned agonist, rush Into combining for agent, antagonist or inhibitor or its functional analogue and corticosteroid.
In other embodiments, pharmaceutical composition provided by the invention includes that the present invention discloses above-mentioned agonist, rush Into combining for agent, antagonist or inhibitor or its functional analogue and nonsteroidal GR agonist.
In other embodiments, pharmaceutical composition provided by the invention includes that the present invention discloses above-mentioned agonist, rush Into combining for agent, antagonist or inhibitor or its functional analogue and anticholinergic drug.
In other embodiment, pharmaceutical composition provided by the invention includes that the present invention discloses above-mentioned agonist, rush Into combining for agent, antagonist or inhibitor or its functional analogue and antihistamine.
In other embodiment, pharmaceutical composition provided by the invention includes that the present invention discloses above-mentioned agonist, rush Into combining for agent, antagonist or inhibitor or its functional analogue and anti-inflammatory agents.
In other embodiment, pharmaceutical composition provided by the invention includes that the present invention discloses above-mentioned agonist, rush Into combining for agent, antagonist or inhibitor or its functional analogue and immunomodulator.
Above-mentioned agonist, promotor, antagonist or inhibitor disclosed by the invention or its functional analogue can also be advantageous Ground be used for in the combination of other compounds or the combination of other therapeutic agents, such as treatment leishmaniasis, trypanosomiasis, toxoplasm The other therapeutic agents of disease and cerebral cysticercosis.Such medicament includes, but are not limited to nivaquin, atovaquone-proguanil, Hao Jia Ether-lumefantrine, quinine sulfate, Artesunate, quinine, fortimicin (doxycycline), clindamycin (clindamycin), Meglumine antimony (meglumine antimoniate), stibii natrii gluconas (sodium stibogluconate), Miltefosine (miltefosine), ketoconazole (ketoconazole), pentamidine (pentamidine), amphotericin B (AmB), AmB lipid Body, paromomycin (paromomycine), Eflornithine (eflornithine), nifurtimox (nifurtimox), Ursula Bright (suramin), melarsoprol (melarsoprol), prednisolone (prednisolone), benzimidazole, sulphadiazine, second Amic metadiazine, synergistic sulfonamide methylisoxazole, radonil, azithromycin (azitromycin), Atovaquone, dexamethasone, pyrrole quinoline Ketone, albendazole (albendazole), beta-lactam, fluoroquinolones medicine, macrolides medicine, aminoglycoside medicine, sulfanilamide (SN) Pyrimidine and pyrimethamine.
The structure of the active constituent determined by code name, common name or trade name and its preparation can be from classic " The Merck Index (Merck index) " current edition (such as M.J.O ' Neil et al. compile ' The MerckIndex ', the 13rd Version, Merck Research Laboratories, 2001) or from database (such as Patents International (example Such as IMS World Publications)) in know.
It is above-described, can with the present invention above-mentioned agonist, promotor, antagonist or inhibitor or its function class be disclosed Like the compound that object is applied in combination, it can be prepared and be administered according to above-mentioned method recorded in the literature by those skilled in the art.
Compound disclosed by the invention can also combine with therapeutic process, improve curative effect.For example, giving hormone therapy." connection Conjunction " indicates the medicine box of the fixing joint in single dose unit form or the part for administering drug combinations, wherein the present invention is public Above-mentioned agonist, promotor, antagonist or the inhibitor or its functional analogue and joint companion opened can be only in the same time Vertical application can be applied respectively at a certain time interval, and joint companion is especially made to show cooperation, for example cooperate with Effect.Term " co-administered " or " administering drug combinations " etc. as used herein, which are intended to include to be applied to selected joint companion, to be needed Want its single individual (such as patient), and be intended to include wherein substance without going through identical administration route or be administered simultaneously Therapeutic scheme.More than one active constituents are mixed or are combined obtained production by " medication combined " expression of term as used herein Product, and both included the fixing joint of active constituent or combined including on-fixed.Term " fixing joint " indicates active constituent such as Compound disclosed by the invention, and joint companion are administered simultaneously in the form of single entities or dosage in patient.Term " on-fixed Joint " indicates that active constituent above-mentioned agonist, promotor, antagonist or inhibitor for example disclosed by the invention or its function are similar Object, and joint companion are used as corpus separatum successively to administer to a patient simultaneously, jointly or without specific time limitation ground, and wherein this is given Prescription formula provides the treatment effective level of two kinds of compounds in patient's body.The latter applies also for cocktail therapy, such as applies With three or more active constituents.
Vaccine
In terms of last of the invention, the invention proposes a kind of vaccines.According to an embodiment of the invention, the vaccine contains There is antigen and selected from least one of following: (a) IL-9 or its functional analogue；(b) IL-9 or its functional analogue are secreted Cell, preferably secrete IL-9 Th9 cell；(c) IL-9 agonist or its functional analogue；(d) STAT-1 agonist or its Functional analogue；(e) IL-9 receptor stimulating agent or its functional analogue；(f) IL-9 or its expression of receptor promotor.According to this The embodiment of invention, the vaccine of the application can effectively facilitate mentioned-above memory B cell precursor to note in body Recall B cell conversion, so as to generate long-term immanoprotection action in body, induction protection mechanism has long-term effect Feature.
According to a particular embodiment of the invention, vaccine disclosed in the present application can also combine with other specificity vaccines and make With making body generate strong and lasting specific immune response.
According to a particular embodiment of the invention, vaccine disclosed by the invention can be used for enhancing immunity of organism, produce in body The immanoprotection action in growth period enhances the resistance of body.
According to another embodiment of the present invention, vaccine disclosed by the invention can also be used for preventing, treating autoimmunity Property disease (IDD) includes primary immunodeficiency disease and secondary immunodeficiency disease.According to a particular embodiment of the invention, this hair Bright disclosed vaccine, which can be used, prevents and treats the autoimmune disorder disease that primary disease be caused, including infection (rubeola, morbilli, Leprosy, tuberculosis, cytomegalovirus infection, coccidioides immitis infection etc.), malignant tumour (Hodgkin's disease, acute and chronic white blood Disease, myeloma etc.), autoimmune disease (SLE, rheumatoid arthritis etc.), albumen lose (nephrotic syndrome, albumen funeral Lose enteropathy), immunoglobulin synthesis is insufficient, lymphocyte loses (because of drug, system infections etc.) and certain other diseases are (such as Diabetes, cirrhosis, subacute sclerosing panencephalitis) and immunosuppressive therapy etc..
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair It is bright, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according to text in the art It offers described technology or conditions or is carried out according to product description.Reagents or instruments used without specified manufacturer, For can be with conventional products that are commercially available.In quantitative test in the examples below, three repeated experiments are respectively provided with, are tied Fruit is averaged.
In the examples below, related key technology is as described below:
Fluorescence ubiquitination cell cycle indicating gage (Fluorescence Ubiquitin Cell Cycle Indicator, Fucci): bibliography: Sakaue-Sawano, A., et al. (2008) " Visualizing spatiotemporal dynamics of multicellular cell-cycle progression."Cell132(3):487-498.Inventor's benefit Two fluorescent marker fusion proteins in bibliography are connected with 2A peptide fragment, and have cultivated transgenic mice.
Immunization method: every mouse uses 100 μ g NP-KLH (to buy from Biosearch Technologies) and 1 μ (LPS, purchase is dissolved in equal volume aluminium adjuvant from Sigma) (to be bought from Thermo Scientific) and carries out abdominal cavity g lipopolysaccharides It is immune.Bibliography: Liu, D., et al. (2015) " T-B-cell entanglement and ICOSL-driven feed-forward regulation of germinal centre reaction."Nature517(7533):214-218。
Cell factor and its neutralizing antibody: (IL-9 is bought from PeproTech) mouse interleukin -9,5-6 week old B6 mouse every each dosage is 1.33 μ g, passes through intraperitoneal injection.- 9 neutralizing antibody of mouse interleukin (aIL-9, purchase It buys from BioXCell), 200 μ g are injected intraperitoneally in every mouse every time).Bibliography: Nakatsukasa, H., et al. (2015).“The DNA-binding inhibitor Id3regulates IL-9production in CD4(+)T cells.”Nat Immunol 16(10):1077-1084。
Airflow classification and mRNA sequencing: the spleen cell of mouse after separating immune is marked different using fluorescent labeled antibody Cell surface marker, sort each 100-200 cell of different monoids using flow cell sorter, and with reference to hereafter It offers construction cDNA library and carries out deep sequencing.Tang,F.,et al.(2010)."RNA-Seq analysis to capture the transcriptome landscape of a single cell."Nat Protoc 5(3):516-535。
The sequencing of 100 cell RNAs: inventor improves Peking University's biodynamic optical imagery center Tang Fuchou study group and opens The unicellular RNA sequencing approach of hair, makes it be suitable for 100-200 lymphocyte populations well using two volumes reaction system Body.It is specific to get on very well, 13 or 8 days after NP-KLH SRBC peritoneal immunity, it is unicellular outstanding to take out mouse spleen grinding collection Liquid is incubated for after splitting erythrocyte using biotin-GL7 or biotin-NIP, is then incubated for Streptavidin MagneSphere GC B cell or NP antigen-specific b cells are enriched with by magnetic frame afterwards.96 orifice plate of flow sorter is used after antibody dyeing Different cell populations is sorted to enter in the PCR tubule equipped with lysate.Since cell concentration is in 100-200 or so, so invention The lysate volume that people has used twice unicellular RNA sequencing used is sufficiently to crack, and first cDNA chain of reverse transcription, Excess primers remove and remain twice of reaction system in end the step of adding A, until in the step of synthesizing the second chain by one The volume of part water replaces, to restore normal reaction system.Pass through each sample of quantitative PCR detection after one wheel PCR amplification The cDNA of product expands quality (marker gene etc. including house-keeping gene and Different groups).Repurity, repurity after two wheel amplifications Obtain final cDNA library.Obtained cDNA library send to Texas, USA southwest medical research center microarray dataset into The Illumina TruSeq of rower standard builds library and the single-ended sequencing process of 50 bases.Sequencing data passes through the processing life of CASAVA software At fastq file, then compares to mouse reference genome via TopHat2 software and refer to transcript, obtain different genes Reads number information.Differential expression calculating is analyzed to obtain by DESeq2 software.
Genetic enrichment analyze GSEA: be MIT and harvard broad institute exploitation it is a set of for sequencing and The calculation and analysis methods of micro- array data, by provide a certain specific gene set (usually each signal path or certain Related gene involved by special cellular localization and certain cell function), the gene in gene set is analyzed in two kinds of samples Expression between product whether there is certain aggregation tendency, i.e., most of expression high all in certain sample or low expression, so that it may To understand whether the gene set has apparent expression difference in two kinds of samples.
Memory B cell second set response: turn base using 100 μ g NP-KLH/Alum/LPS proteantigen peritoneal immunity dsRed Because of mouse, go out within 13 days the CD38 of NP specificity after immune by airflow classification+GL7+FAS+CD19+Bcl6-GC B cell (about 2x104) donor source as B cell.The intracorporal CD4 sun of mouse of the protein isolate antigen OVA after peritoneal immunity 3-4 weeks simultaneously Property donor source of the T cell as T cell, in the Recipient mice body that their adoptive transfers simultaneously are irradiated to half lethal dose (4.5Gy can kill most of periphery lymphocyte) carries out abdominal cavity to Recipient mice followed by proteantigen NP-OVA and exempts from Epidemic disease.After second set response 7 days, the differentiation destiny of Mouse spleen cells detection dsRed positive B-cells is taken.The also needle of sorting simultaneously To the MD4B cell or polyclonal B progenitor cells of another specific antigen, same NP special CD38-GL7+FAS+GC B it is thin Born of the same parents, and real NP specific memory B-cell is as positive control.
The identification of transition state subgroup in 1 Germinal center B cell of embodiment
In the present embodiment, inventor describes transition state subgroup-memory B cell precursor in Germinal center B cell in detail Cell mKO2hiQualification process and result.
In order to identify the nondividing GC B cell of separation, inventor constructs a kind of cell cycle fluorescence indicator mouse UBP-2A-Fucci(Sakaue-Sawano,A.,et al.(2008)."Visualizing spatiotemporal dynamics of multicellular cell-cycle progression."Cell132 (3): 487-498.), the mouse Red fluorescent protein mKO2 can be expressed in tranquillization state G0/G1 phase cell cycle, phase division stage G2/S/M expresses green protein mAG.With NP-KLH, (NP is a kind of haptens to inventor, is linked on this large protein of KLH as immunogene, NP right and wrong are usually Immune means can determine the specificity for NP antigen by tracking BCR (B-cell receptor)) it is small to Fucci transgenosis Mouse carries out peritoneal immunity, finds GL7+FAS+The GC B cell of (centrum germinativum common label) can be divided into according to fluorescence it is several (such as Shown in Fig. 1 a), wherein the cell of the mAG green fluorescent protein positive is just the not no cell of fluorescin in the cell of division stage For the cell for just completing division, the cell of the mKO2 red fluorescent protein positive is cell and transitional period in the G0/G1 phase Double positive cells.It is dyed by Ki67 (a kind of albumen that can distinguish G0 and G1, do not expressed in the G0 phase), inventor's discovery The cell of mKO2 very high (hi) is in the G0 phase, not the expression (as shown in Figure 1 b) of Ki67.Nevertheless, these mKO2hi's Cell still has GL7 and the FAS expression (as illustrated in figure 1 c) of peer-level.By sorting, inventor is contaminated using rupture of membranes The method of color has carried out one of the very important transcription factor of GC B cell BCL6, and (root is it was found that B cell without BCL6 molecule GC can not be formed) dyeing, find this crowd of mKO2hiCell low expression BCL6, while the high expression CD38 (mark of memory B cell Will object), and CD38 molecule is that GC B cell does not express (as shown in Figure 1 d).This crowd of mKO2hiCD38+Cell height expression And localized molecules CCR7, the CD62L of GC feature are not met, also similar simultaneously or higher levels of express is consistent with GC feature Activation markers molecule, such as CD44, CD80, CD73 (as shown in fig. le).Inventor also obtains using sheep red blood cell (SRBC) is immune Similar result, it was demonstrated that this group of cells may be that group of cells for jumping out the cell cycle for leaving GC to be looked for of inventor.
In order to exclude mKO2hiCell is just to enter GC environment, and inventor utilizes NIP dyeing (NIP is used to mark NP) Examine these cells to the specificity of antigen, as a result, it has been found that they have the NIP dye level similar with other GC B cells (result is as shown in Figure 1 f).Inventor further utilizes NIP to detect the BCR sequence of different GC B cells, is dashed forward with analyzing its high frequency Become and finds mKO2 with antigen affinity, inventorhiCell possesses and mKO2loCell, the similar mutation rate of entirety GC B cell, But there is very high antigen affinity simultaneously (result is as shown in Figure 1 g).This means that these cells live through the sieve in GC Select process, it is more likely that be the cell of GC to be left.
Next, inventor has carried out positioning analysis to this group of cells using immunohistochemistry, by surface molecular CXCR4 with CD86 total dyeing (the two molecules known be used to distinguish area pellucida and dark space) (Victora GD, Schwickert TA, Fooksman DR,Kamphorst AO,Meyer-Hermann M,et al.2010.Germinalcenter dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent Reporter.Cell143:592-605), find overwhelming majority mKO2hiCell be all located at area pellucida, this and common GC B are thin Born of the same parents are distributed different (result is as shown in figure 1h).In conjunction with GC localized molecules EFNB1 and non-GC molecule CD38, inventor can be immune MKO2 is searched out in fluorescent staininghiCell.Inventors have found that these cells are more likely to be distributed in the fringe region (knot of GC Fruit is as shown in Fig. 1 i~1k), which imply that they possibly leave GC.In in vitro culture experiment, inventor has found mKO2hiCarefully Born of the same parents are easy to lose the characteristic molecular GL7 and FAS of GC, the further indication difference (knot of they and common GC B cell Fruit is as shown in figure 11).
This group of cells are defined as CD19 by inventor+GL7+Fas+CD38+Bcl6-mKO2hiCell obtains mouse in non-Fucci Under background, inventor uses the CD19 of G0 phase+GL7+Fas+CD38+Bcl6-To define this group of cells.
Embodiment 2CD19+GL7+Fas+CD38+Bcl6-mKO2hiIt is the precursor of memory B cell
In the present embodiment, inventor passes through experimental verification CD19+GL7+Fas+CD38+Bcl6-mKO2hiIt (writes a Chinese character in simplified form into mKO2hiCD38+) cell has and be divided into the potential of memory B cell.
In view of CD19+GL7+Fas+CD38+Bcl6-mKO2hiTransition state GC cell subsets is in the G0 phase of cell cycle, So they centainly derive from the mKO2 for being in the G1 phase in GC cellloCD38-GL7+FAS+Cell, and be probably ready for It is differentiated to form memory B cell.In order to further detect the speciality and its development pathway of this group of transition state GC B cells, inventor's benefit The gene expression profile of the subgroup and its corresponding G1 phase GC cell is analyzed with RNA deep sequencing.Due to this group of transition state cells Proportion is minimum (account for about GC 5%), and reported unicellular RNA sequencing approach is optimized in inventor, keeps it suitable The about 100-200 lymphocyte population for be analyzed.Pass through the base of illumina high-flux sequence and DESeq2 software Because of Differential expression analysis, detect 460 genes in mKO2hiCD38+And mKO2loCD38-Expression is had differences between cell (padj < 0.01, as a result as shown in Figure 2 a).These difference expression genes can be divided into following a few classes according to gene function: (1) adjusting Control the gene of cell cycle, survival and apoptosis；(2) gene of cellular localization is influenced；(3) cell surface marker molecule can be with CD38 helps the gene that the transition state cell subsets is effectively identified in the mouse of non-UBP-2A-fucci background together, such as Ly6D (as shown in Figure 2 c)；(4) gene and (5) various transcription factors (as shown in Figure 2 b) of immune response are participated in.Wherein very Ironically, mKO2hiCD38+Cell has significantly raised such as S1pr1, S1pr4, Gpr183, Ccr7 and Sell (L- ) etc. selectin chemokine receptors and cell adhesion act on relevant molecule (and chemokine receptors and cell adhesion Relevant molecule is to determine that cell positions and the molecule of motion conditions in vivo), it means that these transition state cells are wanted The trend of the region GC limitation is left, also home position observation is intended in GC boundary to the cell on tissue sections with inventor for this The phenomenon that being distributed (as shown in Fig. 1 i-1k) is consistent.
It is well known that transcription factor regulated and control network is the key factor for determining cell phenotype and destiny.From sequencing data Inventor has found mKO2hiCD38+GC cell lowered the central transcription factor Bcl-6 (consistent with coloration result) of GC B cell And the helpful transcription factor Bach2 for maintaining GC status function that is reported before, and the transcription factor of inducing plasma cell differentiation There is no in mKO2 by BLIMP1hiCD38+GC cell detection to there is up-regulation.On the other hand, inventor has found the transition state cell Transcription factor Klf2 and Zbtb32 (result is as shown in Figure 2 b) have been raised, and the two transcription factors are all once reported in memory B High expression in cell.Transcription factor Bcl-6 and Bach2 are lowered in the B cell of antigentic specificity, significantly improve GC B cell Ratio, and be overexpressed up-regulation transcription factor KLF2 and ZBTB32 then reduce GC reaction (result is as shown in Figure 2 d), further Demonstrate mKO2hiCD38+GC B cell had been switched on it is a set of with maintain GC stage incompatible transcriptional control program, into one Step implies that it will leave centrum germinativum.
In order to further verify mKO2hiCD38+Destiny after leaving centrum germinativum, actually or memory B cell slurry it is thin Born of the same parents, inventor have similarly carried out 100 cell RNAs sequencings to memory B cell and thick liquid cell (positioned at spleen and marrow), and And it is compared analysis simultaneously with the data of above two groups of GC B cells.Bioinformatics principal component analysis be (principal component analysis Basic thought is to reduce the dimension of variable using orthogonal transformation, and (such as inventor is sequenced to obtain each sample a genes up to ten thousand Expression quantity, dimension is too high to be unfavorable for analyzing), but retain in former variable that (make a difference most important information maximum again simultaneously Part), to facilitate the cluster situation analyzed between different samples.) show that the thick liquid cell of spleen and marrow can be very good Cluster together, and differs greatly with other cell populations on first principal component.And when to pay close attention to other three classes thin by inventor When born of the same parents, it can be found that mKO2hiCD38+GC B cell fall in mKO2loCD38-GC B cell and memory B cell between, imply It was probably on the stage broken up toward memory B cell, rather than thick liquid cell (result is as shown in Figure 2 e).
In order to functionally verify the precursor of transition state GC B cell really memory B cell, inventor passes through stream Formula sub-elects the CD38 of NP specificity out of proteantigen NP-KLH peritoneal immunity Mice Body+GL7+FAS+CD19+Bcl6-'s GC B cell (about 2x104) donor source as B cell, while in the Mice Body crossed of protein isolate antigen OVA peritoneal immunity Donor source of the CD4 positive T cell as T cell, by their adoptive transfers simultaneously a to half lethal dose irradiate by In body Mice Body, peritoneal immunity is carried out to Recipient mice followed by proteantigen NP-OVA.Inventors have found that CD38+GL7+ FAS+CD19+Bcl6-GC B cell almost have and the internal really identical secondary immune response ability of memory B cell, energy Germinal center reaction is quickly reentered at 7 days after secondary immunity and generates thick liquid cell.And for another specific antigen MD4B cell or polyclonal B progenitor cells, and same NP special CD38-GL7+FAS+GC B cell it is all not similar The ability of quick second set response (shown in result figure 2f-2g).
To sum up, inventor is it is concluded that have mKO2hiCD38+GL7+FAS+CD19+Bcl6-The GC transition of feature State B cell is the precursor that memory B cell is formed in centrum germinativum, is defined as GC-MP cell (GC-derived memory B cell precursors).Simultaneously for ease of use, inventor is by mKO2loCD38-GL7+FAS+Be in G1 The GC B cell of phase is known as GC-MPP cell, because wherein containing the precursor of GC-MP.
3 IL-9 of embodiment (IL-9) promotes the development of GC-MP cell and the differentiation to memory B cell
In order to identify the extracellular factor for promoting GC-MP development and differentiation, inventor further utilizes Bioinformatic methods point Sequencing data is analysed, it is desirable to obtain certain clues and hint.Inventor is enriched with analysis (GSEA) by gene set and compares GC-MP The signal path that difference expression gene is enriched between cell and GC-MPP cell, wherein JAK-STAT signal path causes invention People's great interest (result is as shown in Figure 5 a), implying has special character with GC-MPP on JAK-STAT access in GC-MP cell, Or the access promotes GC-MPP and forms GC-MP or the access to the function of GC-MP itself and subsequent differentiation with important Effect.On the other hand, due to being not directly related between the mRNA level in-site of transcription factor and the activity of transcription factor, for GC- Key transcription factor in MP constructs the special transcription factor-target of B cell using the sequencing data and algorithm of previous report Gene action network, then analyzed by the differential expression of transcription factor targets gene transcribed in GC-MP and GC-MPP because The active variation of son.Wherein the activity of STAT1 transcription factor rises the most significant in GC-MP cell, has prompted STAT1 transcription The development of factor pair GC-MP and function play an important role (result is as shown in Figure 5 b).Activate the main letter of JAK-STAT access Number source, that is, cell factor, and be the cell factor that can activate STAT1.Coincidentally, inventor is in Differential expression analysis It was found that IL-9 receptor (IL-9R) significantly high expression (result is as shown in Figure 2 b) on GC-MP, and before this IL-9 access by Report can activate STAT1 access (and up-regulation L-selectin).Due to no commercialized for the anti-of mouse IL-9R Body, in order to detect protein expression level of the IL-9R on GC-MP cell membrane, inventor by using cell and IL-9 incubation again The experimental method that the antibody of IL-9 is dyed finds have in the film surface of almost all of GC B cell and non-GC B cell The expression of a small amount of IL-9R, but expression quantity will significantly high (result such as Fig. 3 a, shown in 3b) on GC-MP.
Whether can influence the development of GC-MP cell mass in order to verify IL-9 and be differentiated to form memory B cell, inventor passes through IL-9 IL-9 blocking antibody (anti-IL-9) is injected to the mouse peritoneal after immune, observes it after 24 hours to hair tonic The rapid contribution of central reaction.Go out mouse GC after PBS control processing, IL-9 processing and anti-IL-9 are handled by airflow classification GC-MP and GC-MPP cell and 100 cell RNA sequencings of progress in B cell, GSEA is analysis shows that IL-9 processing is compared at PBS Comprehend the marker gene made in GC-MPP cell enrichment GC-MP, consistently, PBS processing is handled compared to anti-IL-9, can energy The marker gene being enriched in GC-MP in GC-MPP cell, i.e. hint IL-9 signal may promote GC-MPP to change to GC-MP. In addition, IL-9 processing can also make GC-MP cell and memory B cell be more nearly (result figure 3c, 3d, 3e institute on express spectra Show).Inventor equally on tissue sections home position observation to originally just the relatively close boundary GC GC-MP IL-9 handle after meeting Closer to the boundary GC, and the effect of anti-IL-9 is then opposite (result such as Fig. 3 f, shown in 3g), implies that IL-9 signal can be with shadow The gene expression in relation to cellular localization is rung, B cell is pushed to leave GC environment.And, the result of flow cytometer showed consistent with this Also show the ratio regular meeting decline of GC-MP after IL-9 is handled, it is likely to which leaving the differentiation of GC state becomes memory B cell, on the contrary Anti-IL-9 processing will increase the ratio of GC-MP, i.e., in the case where lacking IL-9 signal, more cells can be trapped in GC shape State and can not leaving continues to break up (result is as illustrated in figure 3h).To sum up, the signal of IL-9 can persistently instruct GC B cell to enter The state of GC-MP pushes the GC-MP formed to leave GC state and continue to break up.
In order to verify the function of IL-9, inventor blocks in mouse the 8th day injection IL-9 or IL-9 that NP-KLH is immunized Antibody is observed behind two days and three days.It was found that the processing of IL-9 can be obviously improved the ratio of memory B cell, and blocking antibody The ratio (result figure 4a, shown in 4b) of memory B cell can then be significantly inhibited.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, modifies, replacement and variant.

Claims (9)

1. a kind of memory B cell precursor, which is characterized in that the memory B cell precursor is with marker CD19+ GL7+Fas+CD38+Bcl6-B cell, the memory B cell precursor be G0 phase cell.
2. memory B cell precursor according to claim 1, which is characterized in that be directed to marker CD38, the memory B cell precursor cell has the expression opposite with centrum germinativum.
3. a kind of method for obtaining the described in any item memory B cell precursors of claim 1~2, which is characterized in that packet It includes:
Cell is sorted using fluidic cell separating method based on CD19, GL7, Fas, Bcl6 and CD38.
4. a kind of described in any item memory B cell precursors of the promotion claim 1~2 of non-treatment purpose are thin to memory B The method of dysuria with lower abdominal colic characterized by comprising contact the memory B cell precursor with IL-9.
5. a kind of described in any item memory B cell precursors of the inhibition claim 1~2 of non-treatment purpose are thin to memory B The method of dysuria with lower abdominal colic characterized by comprising contact the memory B cell precursor with IL-9 antagonist.
6. the purposes of reagent in medicine preparation, the drug is for promoting the described in any item memory B of claim 1~2 thin Born of the same parents' precursor is converted to memory B cell, and the reagent is IL-9.
7. the purposes of reagent in medicine preparation, the drug is for inhibiting the described in any item memory B of claim 1~2 thin Born of the same parents' precursor is converted to memory B cell, and the reagent is IL-9 antagonist.
8. a kind of method of the acquisition memory B cell of non-treatment purpose characterized by comprising
Contact the described in any item memory B cell precursors of claim 1~2 with IL-9.
9. the purposes of reagent in medicine preparation, the drug is for adjusting memory B cell precursor/memory in centrum germinativum B cell ratio, the reagent are selected from least one of following:
(1)IL-9；
(2) IL-9 antagonist.
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