阅读说明：本技术 用于治疗糖尿病的肽及方法 (For treating the peptide and method of diabetes ) 是由 卢克·范德·埃尔斯特 文森特·卡利尔 让-玛丽·圣-雷米 于 2018-03-06 设计创作，主要内容包括：本发明涉及肽诸如HCPYCSLQPLALEGSLQKRG及它们在治疗1型糖尿病和产生溶细胞性CD4+T细胞中的用途。(The present invention relates to peptide such as HCPYCSLQPLALEGSLQKRG and they treatment type 1 diabetes and generate cytolytic CD4+T cell in purposes.)

1. a kind of isolated immunogenic peptide, by amino acid sequence HCPYCSLQPLALEGSL QKRG [SEQ ID NO:26] group At.

2. peptide according to claim 1 is used as drug.

3. peptide according to claim 1, for treating or preventing type 1 diabetes.

4. a kind of pharmaceutical composition includes peptide described in claim 1 and pharmaceutically acceptable carrier.

5. it is a kind of for generate be directed to present insulin epitope APC cytolytic CD4+T cell mass in-vitro method, including Step:

Peripheral blood cells are provided；

Contact the cell with peptide described in claim 1 in vitro；With

The cell is expanded in the presence of IL-2.

6. for the cytolytic CD4 for the APC for presenting insulin epitope obtained by a kind of method as described in claim 5 + T cell group.

7. for the cytolytic CD4 for the APC for presenting insulin epitope obtained by a kind of method as described in claim 5 + T cell group is used as drug.

8. for the cytolytic CD4 for the APC for presenting insulin epitope obtained by a kind of method as described in claim 5 + T cell group, for treating or preventing type 1 diabetes.

9. isolated immunogenic peptide according to any one of claim 1 to 3 or medicine according to claim 4 Compositions, for treating or preventing type 1 diabetes, or the symptom for mitigating type 1 diabetes.

It include tetrapeptide array Cxx [CST] [SEQ ID NO:1] or [CST] 10. a kind of length is the peptide of 12 to 50 amino acid XxC [SEQ ID NO:2] and the sequence LALEGSLQK [SEQ ID NO:3] separated by 0 to 4 amino acid with the tetrapeptide.

11. peptide according to claim 10, comprising sequence C xx [CST] SLQPLALEGSLQK [SEQ ID NO:4] or [CST]xxCSLQPLALEGSLQK[SEQ ID NO:5]。

12. peptide described in 0 or 11 according to claim 1 includes sequence C xxCSLQPLALEGSLQK [SEQ ID NO:6].

13. peptide according to any one of claims 10 to 12 includes sequence HCxx [CST] SLQPLALEGSLQK [SEQ ID NO:7] or H [CST] xxCSLQPLALEGSLQK [SEQ ID NO:8].

14. peptide described in any one of 0 to 13 according to claim 1 includes sequence HCxxCSLQPLALEGSLQK [SEQ ID NO:9]。

15. peptide described in any one of 0 to 14 according to claim 1, comprising sequence C xx [CST] [SEQ ID NO:1] or [CST] xxC [SEQ ID NO:2] redox motif sequence and sequence SLQPLALEGSLQKRG [SEQ ID NO:20].

Background technique

Several strategies have been described to prevent the unwanted immune response for antigen.WO2008/017517 Describe a kind of new strategy of the use comprising giving the MHC II class antigen of antigen protein and the peptide of oxidoreducing enzyme motif.These CD4+T cell transformation is the cell type with cell dissolution characteristic, referred to as cytolytic CD4+T cell (cytolytic by peptide CD4+T cell, molten cell CD4+T cell).These cells can be thin to kill those antigen presentations by triggering Apoptosis Born of the same parents (APC), the antigen presenting cell present the antigen of the derivative peptide.WO2008/017517 demonstrates allergy and itself exempts from This concept of epidemic disease (such as type-1 diabetes mellitus).Herein, insulin can serve as autoantigen.

WO2009101207 and Carlier et al. (2012) Plos one 7,10e45366 is further retouched in more detail Antigentic specificity cytolytic cells are stated.

WO2009101206 describes the MCH II class table with oxidoreducing enzyme motif and soluble alloantigen The purposes of the peptide of position, to prevent immune response (such as the needle in diabetic for this antigen when for alternative medicine To the unwanted immune response of insulin injection).

WO2016059236 discloses the peptide further modified, wherein there are additional group near oxidoreducing enzyme motif Propylhomoserin.

Design be directed to type-1 diabetes mellitus peptide when, it may be considered that several factors, for example, autoantigen type (insulin, GAD 65 ...), the specific domain of autoantigen and epitope, oxidoreducing enzyme motif, oxidoreducing enzyme motif and epitope sequences Between length and amino acid sequence.

Summary of the invention

The present invention provides the new peptides for being derived from insulin, are used to treat type 1 diabetes.

Compared with the peptide of the prior art, peptide of the invention has an advantage in that the cytolytic CD4+T for having used these peptides to generate There is cell increased IFN-γ and sFasL to produce.It is additionally considered that the generation of the granzyme B in the CD4+T cell increases.

The expression increase of these markers shows that compared with prior art peptide, peptide of the invention has bigger production The ability of raw cytolytic CD4+T cell.

One aspect of the present invention is related to the peptide that length is 12 to 50 amino acid, and it includes tetrapeptide array Cxx [CST] [SEQ ID NO:1] or [CST] xxC [SEQ ID NO:2] (i.e. redox motif) and with 0 to 5, the tetrapeptide interval ammonia Base acid, preferably by the sequence LALEGSLQK [SEQ ID NO:3] (i.e. epitope) of 0 to 4 amino acid (being spaced connector).

The embodiment of these peptides includes sequence C xx [CST] SLQPLALEGSLQK [SEQ ID NO:4] or [CST] xxCSLQPLALEGSLQK[SEQ ID NO:5]。

The other embodiment of these peptides includes sequence C xxCSLQPLALEGSLQK [SEQ ID NO:6].

The other embodiment of these peptides includes sequence HCxx [CST] SLQPLALEGSLQK [SEQ ID NO:7] or H [CST]xxCSLQPLALEGSLQK[SEQ ID NO:8]。

The other embodiment of these peptides includes sequence HCxxCSLQPLALEGSLQK [SEQ ID NO:9].

The other embodiment of these peptides includes Cxx [CST] [SEQ ID NO:1] or [CST] xxC [SEQ ID NO:2] Redox motif sequence and sequence SLQPLALEGSLQKRG [SEQ ID NO:20].

The specific embodiment of these peptides is made of following sequence:

Cxx[CST]SLQPLALEGSLQK[SEQ ID NO:4]、

[CST]xxCSLQPLALEGSLQK[SEQ ID NO:5]、

CxxCSLQPLALEGSLQK[SEQ ID NO:6]、

HCxx[CST]SLQPLALEGSLQK[SEQ ID NO:7]、

H [CST] xxCSLQPLALEGSLQK [SEQ ID NO:8] or

HCxxCSLQPLALEGSLQK[SEQ ID NO:9]。

Other specific embodiments of these peptides are made of following sequence:

Cxx[CST]SLQPLALEGSLQKRG[SEQ ID NO:10]、

[CST]xxCSLQPLALEGSLQKRG[SEQ ID NO:11]、

CxxCSLQPLALEGSLQKRG[SEQ ID NO:12]、

HCxx[CST]SLQPLALEGSLQKRG[SEQ ID NO:13]、

H [CST] xxCSLQPLALEGSLQKRG [SEQ ID NO:14] or

HCxxCSLQPLALEGSLQKRG[SEQ ID NO:15]。

In the specific embodiment of these sequences, Cxx [CST] [SEQ ID NO:1] be CPY [CST] [SEQ ID NO: 16], [CST] xxC [SEQ ID NO:2] is [CST] PYC [SEQ ID NO:17], more specific CxxC [SEQ ID NO:18] It is CPYC [SEQ ID NO:19].

One specific embodiment is peptide HCPYCSLQPLALEGSLQKRG [SEQ ID NO:26].

In the above-described embodiment, redox motif is located at the N-terminal side of epitope.

In another group of embodiment, peptide has redox motif in the C-terminal side of epitope.

Another aspect of the present invention relates to any peptides disclosed above to be used as drug, in particular for treating or preventing 1 Patients with type Ⅰ DM or symptom for mitigating type 1 diabetes.

On the other hand it is related to pharmaceutical composition, it includes any peptide disclosed above and pharmaceutically acceptable loads Body.

On the other hand it is related to a kind of for generating the cytolytic CD4+T cell mass for being directed to the APC for presenting insulin epitope In-vitro method, comprising the steps of:

Peripheral blood cells are provided；

Contact the cell with any immunogenic peptide disclosed above in vitro；With

The cell is expanded in the presence of IL-2.

On the other hand it is related to presenting the cytolytic CD4+T cell of APC for insulin as obtained by the above method Cell mass be used as drug.

On the other hand it is related to the cell mass as obtained by the above method for treating or preventing type 1 diabetes.

Detailed description of the invention

Fig. 1: with the release of sFasL in the cell line of p17-003 or p17-001 generation.

By p17-003 or p17-001 specific cell (IL5 positive cell；Black histograms) with load have peptide p17-003 Or the antigen presenting cell of p17-001 stimulates 24 hours (peptide depending on the generation for them) again, and it is small to co-culture 24 When after collect supernatant.IL-5 negative cells are for compareing PBMC groups (open histograms).As a result it indicates to be corrected according to cell number SFasL concentration the +/- SD of average value.

Fig. 2: the response the figure illustrates cell line to the stimulation of p17-003 or p17-001 in terms of cell factor generation.

Specific embodiment

Will be in conjunction with the particular implementation description present invention, but the invention is not restricted to the described embodiment, but only by weighing Benefit requires limitation.Any reference marker in claim is not necessarily to be construed as limitation range.There is provided following term or definition only It is used to help understand the present invention.Unless being specifically defined herein, otherwise all terms used herein have and field of the present invention The identical meanings that technical staff understands.Definition provided herein should not be interpreted as having to be managed than those of ordinary skill in the art The small range of the range of solution.

As clear to the skilled person, unless otherwise stated, all methods not being described in detail specifically, step Suddenly, technology and operation can with and execute in a way known.For example, referring again to standard manual, mentioned above General background technique and other bibliography cited therein.

As used herein, unless the context clearly determines otherwise, singular ' one ', ' one kind ' and ' being somebody's turn to do ' include single Several and plural object.When with aspect used herein, claim or embodiment used in connection with, term " any " refers to Any single (i.e. any one) and all combinations of the aspect, claim or the embodiment referred to.

Term as used herein is ' comprising (comprising) ', ' comprising (comprises) ' and ' comprising (comprised It of) ' is ' including (including) ', ' including (includes) ' or ' containing (containing) ', ' containing (contains) ' Synonym, and be inclusiveness or open, however not excluded that other unlisted members, element or method and step.It is described Term also covers embodiment " substantially by ... form " and " Consists of ".

It includes all digital and scores in respective range and cited for including by the numberical range that endpoint is stated Endpoint.

When refer to parameter, amount, when away from etc. measurable magnitudes when, the term as used herein ' about ' means to cover designated value +/- 10% or smaller, preferably +/- 5% or smaller, more preferably +/- 1% or smaller, more preferably +/- 0.1% or smaller variation, As long as these variations are suitable for carrying out in disclosed invention.It should be understood that value sheet cited in modifier ' about ' Body also specifically and is preferably disclosed.

As it is used herein, term " being used for " used in " for treating the composition of disease " should also disclose accordingly Treatment method and preparation be used to manufacture the corresponding uses of drug for treating disease.

Term " peptide " as used herein refers to the amino acid sequence of 12 to 200 amino acid comprising being keyed by peptide Molecule, but it may include non-amino acid structure.

Peptide according to the present invention can contain any conventional 20 kinds of amino acid or its modified forms, or can be containing logical Cross chemical peptide synthesis or by chemistry or the non-naturally occurring amino acid of enzymatically modifying incorporation.

Term " antigen " as used herein refers to the structure of macromolecular, usually protein (being with or without polysaccharide), or Person is made of the protein compositions comprising one or more haptens and comprising t cell epitope.

Term " antigen protein " as used herein refers to the protein comprising one or more t cell epitopes.As herein Autoantigen or autoantigen protein used, which refers to, is present in intracorporal mankind or animal albumen, in same human body or animal Cause immune response in vivo.

Term " epitope " refers to one or several parts (it can be with constrained conformation epitope) of antigen protein, by antibody or Its part (Fab', Fab2' etc.) or the receptor-specific of cell surface for being present in B or T cell lymphocyte are identified and are tied It closes, and it can induce immune response by the combination.

In the context of the present invention, term " t cell epitope " refers to dominant, subdominance or secondary T cell table Position, i.e. a part of antigen protein, are identified and are combined by the receptor-specific of the cell surface of T lymphocyte.Epitope is dominant , subdominance or it is secondary, depending on for the epitope cause immune response.In all possible protein T cell In epitope, the dominant T cell that depends on identifies these epitopes and can activate their frequency.

T cell epitope is to be made of the epitope of MHC II class molecular recognition the sequence of +/- 9 amino acid, is suitble to The groove of MHC II molecule.In the peptide sequence for representing t cell epitope, the amino acid number in epitope is P1 to P9, epitope Amino acid N-terminal number is P-1, P-2 etc., and amino acid C-terminal number P+l, P+2 etc. of epitope.By MHC II class molecule The peptide of non-MHC I class molecular recognition is referred to as the restricted t cell epitope of MHC II class.

Term " MHC " refers to " major histocompatibility antigen ".In the mankind, mhc gene is referred to as HLA, and (" mankind are white thin Extracellular antigen ") gene.Although the convention not followed always, some documents refer to HLA protein molecule using HLA, and And the gene of coding HLA protein is referred to using MHC.Therefore, term " MHC " and " HLA " are equivalent as used herein 's.HLA system in human body has its equivalent, i.e. H2 system in mouse.Studying the most in-depth HLA gene is 9 kinds so-called Classical mhc gene: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLAs DQB1, HLA-DRA And HLA-DRB1.In the mankind, MHC points are three regions: I, II and Group III.A, B and C gene belongs to MHC I class, and 6 D Gene belongs to II class.MHC I class molecule is made of the single polymorphic chain containing 3 structural domains (α 1,2 and 3), with cell surface β2-microglobulin it is associated.II class molecule is made of 2 polymorphic chains, and every polymorphic chain contains 2 chains (1 He of α 1 and 2 and β 2)。

I class MHC molecule is almost expressed on all karyocytes.

The peptide fragment presented under the background of I class MHC molecule by CD8+T lymphocyte (cytolytic T cell or CTL it) identifies.It is cytolytic effect object that CD8+T lymphocyte is often mature, and cleavable has the cell of stimulator antigen. II class MHC molecule is mainly expressed on the lymphocyte of activation and antigen presenting cell.(T-helper is thin for CD4+T lymphocyte Born of the same parents or Th) it is activated by the identification of the unique peptide fragments presented by II class MHC molecule, the II class MHC molecule is generally found On antigen presenting cell such as macrophage or dendritic cells.CD4+T lymphopoiesis and secrete cytokines such as IL-2, IFN- γ and IL-4 supports antibody-mediated and cell-mediated response.

Functional HLA is characterized in that the deep engagement groove that endogenous and external potentially antigenic property peptide combine.This is recessed Slot is further characterized in that specific shape and physicochemical properties.HLA I class binding site is closed, because peptide end is consolidated It is scheduled on the end of groove.They further relate to the hydrogen bond network with conservative HLA residue.In view of these limitations, the length limit of binding peptide In 8,9 or 10 residues.However, it has been proved that the peptide of up to 12 amino acid residues can also combine HLA I class.Different HLA The structure of compound relatively confirms general binding pattern, and wherein peptide, or can be with using the conformation of the extension of opposite linear Make center residue to protrude groove.

With HLA I class binding site on the contrary, II class site at both ends is open.This allows peptide from actual bond area Extend, so that " suspension " is at both ends.Therefore, II class HLA can be 9 in conjunction with the peptide ligand of variable-length, the length range A to more than 25 amino acid residues.Similar to HLA I class, the affinity of II class ligand is determined by " constant " and " variable " component It is fixed.Constant portion is produced by the hydrogen bond network formed between the main chain of conserved residues and binding peptide in HLA II class groove again It is raw.However, the hydrogen bonding pattern is not limited to N- the and C- terminal residue of peptide, and it is distributed across on entire chain.The latter is important, because The conformation of complex peptides is limited to the binding pattern of Strict linear for it.This is typical for all II class allografts.By In certain positions of the polymorphism in II class binding site, determine that the second component of the binding affinity of peptide is variable.It is different Allograft different complementary pockets is formed in groove, thus solve peptide hypotype dependence selection or specificity.Weight It wants, to the limitation of the amino acid residue kept in II class pocket usually than I class " softer ".It is of the same race in different HLA II classes In abnormal shape, the cross reactivity of peptide wants much higher.It is suitble to +/- 9 of the MHC II class t cell epitope in MHC II molecule groove It is P1 to P9 that the sequence of amino acid (i.e. 8,9 or 10), which is usually numbered,.The end the plus Amino Acid N- number of the epitope is P-1, P-2 Amino acid C-terminal number Deng, the epitope is P+1, P+2 etc..

The epitope with reference to used in the context of the invention and refer in terms used herein " homologue ", and it is naturally occurring Epitope have at least 50%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% amino acid sequence The molecule of identity thus maintains the ability of the cell surface receptor of the epitope-binding antibodies or B and/or T cell.Table The specific homologue of position corresponds at most three, more particularly at most 2, is most particularly repaired in an amino acid The natural epitopes of decorations.

With reference to peptide of the invention, terminology used in this article " derivative " refers to containing at least peptide activity part (i.e. oxygen Change reduction motif and the MHC II class epitope of cytolytic CD4+T cell activity can be caused) and in addition to this also comprising can With the molecule of the complementary portion with different purposes (such as stabilized peptide or the pharmacokinetics or pharmacodynamic profiles of change peptide).

The term " sequence identity " of two sequences used herein is related to when by described two sequence alignments, has phase With the positional number of nucleotide or amino acid divided by the nucleotide or amino acid number in shorter sequence.Particularly, sequence identity is 70% to 80%, 81% to 85%, 86% to 90%, 91% to 95%, 96% to 100% or 100%.

Terms used herein " the Encoding polynucleotides of peptide (or nucleic acid) " and " polynucleotides (or nucleic acid) of encoded peptide " Refer to nucleotide sequence, when expressing in environment appropriate, causes to generate relevant peptide sequence or derivatives thereof or homologous Object.Such polynucleotides or nucleic acid include the normal sequence for encoding the peptide, and can be expressed with required active peptide The derivative and segment of these nucleic acid.The nucleic acid for encoding peptide according to the present invention or its segment is that coding is originated from mammal or right It should be in the peptide of mammal or the sequence of its segment (most particularly mankind's peptide fragment).

Term " immunologic derangement " or " immunological diseases " refer to a kind of disease, and wherein the reaction of immune system is to cause or maintain Dysfunction or the reason of non-physiological status in organism.Immunologic derangement especially includes anaphylactia and autoimmune disease Disease.

Term " autoimmune disease " or " autoimmune disorders " refer to since organism fails the group of its own Organism caused by " self " is identified as (down to inframolecular level) at part to exempt from for the exception of its own cell and tissue Disease caused by epidemic disease response.This group of disease can be divided into two classes, organ specific disease and systemic disease." anaphylactogen " is defined as A kind of substance, usually macromolecular or protein compositions, can (spy answers in susceptible (especially heredity disposition) individual Property) induce in patient and generate lgE antibody.It is mentioned in Liebers et al. (1996) Clin.Exp.Allergy 26,494-516 Similar definition is gone out.

Term " type 1 diabetes " (T1D) or " 1 type of diabetes " (also referred to as " type 1 diabetes " or " immune-mediated glycosuria Disease " is formerly referred to as " juvenile onset patients with type Ⅰ DM " or " insulin-dependent diabetes mellitus ") it is autoimmune disorders, usually Develop in Childhood susceptible individual.On the basis of T1D, pathogenesis is that most of produce is destroyed by autoimmune mechanism The pancreatic beta cell of raw insulin.In brief, organism loses the immune tolerance of the pancreatic beta cell to responsible insulin production, And immune response is induced, the mainly cell-mediated generation to autoantibody is related, leads to self destroying for β cell.

Term " therapeutically effective amount " refers to the amount of peptide of the invention or derivatives thereof, generates required treatment in patients Or preventive effect.It is reduce disease or disorder to a certain extent one or more for example, when referring to disease or disorder The amount of symptom, more specifically, it is partially or completely by physiology that is relevant to disease or disorder or leading to disease or disorder Or biochemical parameter restores to normal.In general, therapeutically effective amount is the amount of peptide of the invention or derivatives thereof, normal life will lead to Manage the improvement or recovery of situation.For example, it is daily amount peptide/kg when for treating the mammal influenced by immunologic derangement The weight of the mammal.Alternatively, in the case where being applied by gene therapy, adjust the amount of naked DNA or viral vectors with Ensure that part generates the relevant dose of peptide of the invention, its derivative or homologue.

When referring to peptide, term " natural " relate to the fact that the sequence and naturally occurring protein (wild type Or mutant) segment it is identical.In contrast, term " manually " refers to the sequence being not present in nature.By limited Modification, such as change/missing/insertion one or more amino acid in naturally occurring sequence, or pass through addition/removal day So there are the amino acid of the end N- or C- of sequence, artificial sequence is obtained from native sequences.

Amino acid is drawn herein with their full name, their trigram abbreviation or their letter abbreviations With.

The motif of amino acid sequence is described according to Prosite format herein.Motif is for describing sequence specific part Certain sequence variants.Symbol X is used to receive the position of any amino acid.For given position, by square brackets (' [] ') between list acceptable amino acid to indicate substitute.Such as: [CST] represents the amino for being selected from Cys, Ser or Thr Acid.Amino acid as an alternative is excluded by the way that they are listed instruction between braces (' { } ').Such as: { AM } is represented Any amino acid in addition to Ala and Met.Different elements in motif are separated from each other alternately through hyphen (-).It can lead to It crosses behind the element and places numerical value between bracket or numberical range to indicate duplicate identical element in motif.For example, X (2) Corresponding to X-X or XX；X (2,5) corresponds to 2,3,4 or 5 X amino acid, and A (3) corresponds to A-A-A or AAA.

To distinguish amino acid X, amino acid is known as external amino acid X (with lower stroke single in above-mentioned sequence those of between H and C Line), it is known as internal amino acid X those of in redox motif (with double underline in above-mentioned sequence).

X indicates any amino acid, especially l-amino acid, more particularly one of 20 kinds of naturally occurring l-amino acids.

The peptide of modified peptide motif sequence comprising t cell epitope and with reduction activation can be generated is in for antigen The antigentic specificity cytolytic CD4+T cell mass of delivery cell.

Therefore, from broadest sense, the present invention relates to peptide, it includes have the potentiality that trigger an immune response extremely The t cell epitope of a few antigen (itself or non-self), and there is the modified thio of reduction activation to peptide disulfide bond Reductase sequence motifs.T cell epitope and modified redox motif sequence can be directly adjacent to each other or appoint in peptide Selection of land is separated by one or more amino acid (so-called joint sequence).Optionally, peptide additionally include inner body targeting sequence and/ Or additional " flank " sequence.

Peptide of the invention includes: having the MHC II class T of the antigen (itself or non-self) of the potentiality triggered an immune response Cell epitope and modified redox motif.The ability of motif sequence reduction sulfydryl in peptide can be measured to measure it also Former activity, such as in insulin solubility measurement, wherein the solubility of insulin is changed in reduction, or with fluorescence mark The substrate of note such as insulin.One example of this measurement is using fluorescent peptide and is described in Tomazzolli et al. (2006)Anal.Biochem.350,105-112.When two peptides with FITC label are covalently attached each other by disulphide bridges When, they become self-quenching.After being restored by peptide according to the present invention, the single peptide of reduction issues fluorescence again.

Modified redox motif can be located at the amino terminal side of t cell epitope or the carboxyl end of t cell epitope End.

Encounter the peptide fragment with reduction activation in thioreductase, the thioreductase be small disulphide also Protoenzyme, including glutaredoxin, kernel toxin, thioredoxin and other thiol/disulfide oxidoreducing enzyme (Holmgren (2000)Antioxid.Redox Signal.2,811-820；Jacquot et al.(2002)Biochem.Pharm.64, 1065-1069).They be it is multi-functional, be prevalent in many prokaryotes and eucaryote.They pass through conserved active Redox-active cysteines in structural domain consensus sequence play reduction activation, institute to the disulfide bond on protein (such as enzyme) State conserved active structural domain consensus sequence are as follows: CXXC [SEQ ID NO:18], CXXS [SEQ ID NO:23], CXXT [SEQ ID NO:24]、SXXC[SEQ ID NO:21]、TXXC[SEQ ID NO:22](Fomenko et al.(2003)Biochemistry 42,11214-11225；Fomenko et al. (2002) Prot.Science 11,2285-2296), wherein X represents any ammonia Base acid.Such structural domain exists in biggish protein, as protein disulfide isomerase (PDI) and phosphoinositide are special Property phospholipase C.

The reduction motif of 4 amino-acid oxidases known to such as Fomenko and WO2008/017517 includes position 1 and/or 4 Cysteine；Therefore the motif is CXX [CST] [SEQ ID NO:1] or [CST] XXC [SEQ ID NO:2].This tetrapeptide Sequence will be referred to as " motif ".Motif in peptide can be any substitute CXXC [SEQ ID NO:18], SXXC [SEQ ID NO:21], TXXC [SEQ ID NO:22], CXXS [SEQ ID NO:23] or CXXT [SEQ ID NO:24].Particularly, peptide contains There is sequence motifs CXXC [SEQ ID NO:18].

As explained in further detail, peptide of the invention can be prepared by chemical synthesis, allow to mix non-natural ammonia Base acid.Therefore, " C " in the redox motif of above-mentioned redox modification represents cysteine or with thiol group Another amino acid, such as trolovol, homocysteine or other natural or non-natural aminos with thiol functionalities Acid.In order to which with reduction activation, the cysteine being present in modified redox motif should not be used as two sulphur of cystine A part of bridge occurs.However, the redox motif of redox modification may include modified cysteine, such as first Base cysteine is converted into the cysteine with free sulfhydryl groups in vivo.X can be appointing in 20 kinds of natural amino acids What is a kind of, including S, C or T or can be unnatural amino acid.In specific embodiment, X is the amino with small side chain Acid, such as Gly, Ala, Ser or Thr.In further specific embodiment, X is not the amino acid with bulky side chains, example Such as Trp.In further specific embodiment, X is not cysteine.In further specific embodiment, through modifying Redox motif at least one X be His.In other further specific embodiments, modified oxidation is also At least one X in former base sequence is Pro.

Peptide can further include modification to increase stability or dissolubility, such as the modification end N- NH₂Group or C-terminal COOH group (such as COOH is modified into CONH₂Group).

In the peptide of the invention comprising modified redox motif, the motif is positioned such that when epitope is suitble to When MHC groove, the motif is retained in the outside of MHC engagement groove.By modified redox motif close in peptide Epitope sequences place [in other words, the joint sequence between motif and epitope be zero amino acid], or by the inclusion of 5 or The connector of the amino acid sequence of less amino acid is separated with t cell epitope.More particularly, the connector includes 1,2,3,4 or 5 A amino acid.Specific embodiment is that have 0,1 or 2 amino between epitope sequences and modified redox motif sequence The peptide of sour connector.In those modified redox motif sequences peptide adjacent with epitope sequences, compared with epitope sequences, This is designated as position P-4 to P-1 or P+1 to P+4.In addition to peptide linker, other organic compounds also are used as connector with by peptide Each section be connected to each other (for example, modified redox motif sequence is connect with t cell epitope sequence).

Peptide of the invention can be further in the N or C of the sequence comprising t cell epitope and modified redox motif End includes additional short amino acid sequence.This amino acid sequence is commonly referred to as ' flanking sequence ' herein.Flanking sequence It can be located between epitope and inner body targeting sequence and/or between modified redox motif and inner body targeting sequence.In In certain peptides not comprising inner body targeting sequence, short amino acid sequence can reside in modified redox motif in peptide And/or the N and/or C-terminal of epitope sequences.More particularly, flanking sequence is the sequence of 1 to 7 amino acid, most particularly 2 The sequence of amino acid.

Modified redox motif can be located at the N-terminal of epitope.

In some embodiments of the present invention, it provides comprising an epitope sequences and modified redox motif The peptide of sequence.In further specific embodiment, modified redox motif occurs multiple (1,2,3,4 in peptide Or even more repeatedly), such as the weight for the modified redox motif that can be spaced each other one or more amino acid It is multiple, or as the repetition being directly adjacent to each other.Alternatively, the N and C-terminal in t cell epitope sequence are provided which one or more Modified redox motif.

It include containing the duplicate peptide of t cell epitope sequence for other variants that peptide of the invention is imagined, wherein each epitope Sequence is before or after modified redox motif (for example, the weight of " modified redox motif-epitope " Multiple or " the modified modified redox motif of redox motif-epitope-" repetition).Herein, through modifying Redox motif may have identical sequence, but this is not enforceable.It is worth noting that, containing comprising itself The repetitive sequence of the peptide of the epitope of modified redox motif will also generate comprising ' epitope ' and ' modified oxidation be also Former base sequence ' sequence.In these peptides, modified redox motif rises outside the second epitope sequences in an epitope sequences To the effect of modified redox motif.

In general, peptide of the invention only includes a t cell epitope.As described below, the t cell epitope in protein sequence can By one of functional examination and/or silica prediction measurement or a variety of to identify.Amino in t cell epitope sequence Acid group is according to their Position Numbers in MHC protein binding groove.Existing t cell epitope is by 8 to 25 amino acid groups in peptide At being more particularly made of 8 to 16 amino acid, but most particularly by 8,9,10,11,12,13,14,15 or 16 amino acid Composition.

In embodiment particularly, t cell epitope is made of the sequence of 9 amino acid.Further specific real It applies in mode, t cell epitope is the epitope [the restricted t cell epitope of MHC II class] by MHC-II class molecular presentation to T cell. Usual t cell epitope sequence refers to the octapeptide for being suitable for MHC II albumen crack or more specifically nonapeptide sequence.

The t cell epitope of peptide of the invention can correspond to the natural epitopes sequence of protein or can be its modification version This is similar to natural T-cell epitope sequence on condition that modified t cell epitope retains its ability being incorporated in the crack MHC Column.Modified t cell epitope can have binding affinity identical with natural epitopes to MHC protein, but can also have There is the affinity of reduction.Particularly, the binding affinity of modified peptides is lower than original peptide is not less than 10 times, is more particularly not less than 5 times.Peptide of the invention has stabilization to protein complex.Therefore, the stabilization of peptide-MHC compound, which compensates for, repairs Adoring epitope reduces the affinity of MHC molecule.

Sequence comprising t cell epitope and reducing compound in peptide, which can be further connected to, promotes peptide intake to evening Amino acid sequence (or another organic compound) in phase inner body to process and to present in MHC II class determinant.In advanced stage Body targets the signal as present in albuminous cell matter tail portion and mediates, and corresponds to the peptide motif sufficiently identified.Late endosomal Targeting sequence allows MHC-II class molecule to handle and effectively present the t cell epitope of antigenic source.Such inner body targets sequence packet It is contained in such as gp75 albumen (Vijayasaradhi et al. (1995) J.Cell.Biol.130,807-820), mankind CD3 γ Albumen, HLA-BM 11 (Copier et al. (1996) J.Immunol.157,1017-1027), DEC205 receptor cytoplasm In tail portion (Mahnke et al. (2000) J.Cell Biol.151,673-683).Its of the peptide of sorting signals is played to inner body Its example is disclosed in the summary of Bonifacio and Traub (2003) Annu.Rev.Biochem.72,395-447.Alternatively, institute Stating sequence can be the sequence of subdominance or secondary t cell epitope from protein, promote the intake in late endosomal and The t cell response for antigen is not overcome.Late endosomal targets the amino terminal or carboxyl that sequence can be located at antigenic source peptide End effectively to take in and to process, and can also pass through the peptide sequence idol of such as at most 10 amino acid of flanking sequence Connection.When being used to target purpose using secondary t cell epitope, the latter is usually located at the amino terminal of antigenic source peptide.

Therefore, present invention contemplates the peptide of antigen protein and its purposes in initiation specific immune response.These peptides It can correspond to protein fragments, the protein fragments are in its sequence comprising by most 10, preferably 7 or less amino The t cell epitope and reducing compound that acid separates.Alternatively, and for most of antigen proteins, peptide of the invention pass through by The redox motif that reducing compound, reduction more particularly as described herein are modified is coupled to antigen in the end N- or the end C- The t cell epitope (directly adjacent or have at most 10, the connector of more particularly at most 7 amino acid) of albumen and produces It is raw.In addition, the t cell epitope sequence that can modify protein compared with naturally occurring sequence and/or modified oxidation are also Former base sequence, and/or (or modification) one or more flanking sequences and/or targeting sequence can be introduced.Therefore, depending on whether can To find feature of the invention in the sequence of target antigen albumen, peptide of the invention may include ' artificial ' or ' naturally occurring ' Sequence.

The length of peptide of the invention can be with significant changes.The length of peptide can be from 13 or 14 amino acid (i.e. by 8-9 ammonia 5 epitope of base acid, the modified redox motif with histidine adjacent thereto amino acid compositions) variation is up to 20,25,30,40 or 50 amino acid.For example, peptide may include the side of the inner body targeting sequence of 40 amino acid, about 2 amino acid Wing sequence, the motif of 5 amino acid as described herein, the connector of 4 amino acid and 9 amino acid t cell epitope peptide.

Therefore, in specific embodiment, complete peptide by 13 amino acid be up to 20,25,30,40,50,75 or 100 amino acid compositions.More particularly, the case where reducing compound is modified redox motif as described herein Under, comprising optionally by the epitope of connector connection and modified redox motif, (referred to herein as ' epitope-is through modifying Redox motif ' sequence) but not include the length of (artificial or natural) sequence of inner body targeting sequence be vital. ' the modified redox motif of epitope-' is more particularly with the length of 13,14,15,16,17,18 or 19 amino acid.This The peptide of kind 13 or 14 to 19 amino acid is optionally with inner body targeting signal coupling, and the size of the inner body targeting signal is less It is important.

As described above, in certain embodiments, peptide of the invention includes to be connect with t cell epitope sequence such as this paper institute The redox motif for the reduction modification stated.

In further specific embodiment, peptide of the invention is the peptide comprising t cell epitope, which exists The amino acid sequence with redox property is not included in its native sequences.

However, in an alternative embodiment, t cell epitope may include any amino for ensuring epitope in conjunction with the crack MHC Acid sequence.When the target epitope of antigen protein includes modified redox motif as described herein in its epitope sequences When, immunogenic peptide according to the present invention includes modified redox motif sequence as described herein and/or and epitope The end sequence N- or C- coupling another reduction sequence sequence so that (be embedded in existing warp in the epitope in crack The redox motif of modification is opposite) attached by modified redox motif may insure reduction activation.

Therefore, it t cell epitope and motif direct neighbor or is separated from each other, but is not overlapped.In order to assess " direct neighbor " or The design of " separated ", it is determined that be suitble to 8 in the crack MHC or nine amino acid sequence, and determined this octapeptide or nonapeptide with The distance between redox motif tetrapeptide or modified redox motif pentapeptide (including histidine).

In general, peptide of the invention is not natural (therefore protein fragments of not instead of original sample), in addition to t cell epitope Artificial peptide also containing modified redox motif as described herein, thus the modified redox motif is logical It crosses and is directly separated by the connector that at most seven, most particularly at most four or at most 2 amino acid form with the t cell epitope It opens.

It has been shown that peptide (or composition comprising this peptide) according to the present invention is applied and (injected) to mammal When, which causes the activation of T cell, identifies the t cell epitope of antigenic source, and by the reduction of surface receptor to T cell Additional signal is provided.This super optimal activation causes T cell to obtain the cell dissolution spy for the cell for presenting t cell epitope Property, and to the rejection characteristic of onlooker's T cell.In this way, the t cell epitope containing antigenic source and in the epitope It is dynamic to can be used for lactation for the peptide of the present invention containing modified redox motif or the composition comprising the peptide outside The direct immunization of object (including the mankind).Therefore, the present invention provides peptides of the invention or derivatives thereof, are used as drug.Therefore, originally Invention provides treatment method, and it includes one or more peptides according to the present invention are applied to patient in need.

The present invention provides a kind of methods, in this way, can be caused by being immunized with small peptide with cell dissolution The T cells with antigenic specificity of characteristic.It has been found that sequence and (ii) containing the coding t cell epitope of (i) from antigen have The consensus sequence of redox property and optionally further also comprising promote peptide intake late endosomal in so as to effective MHC- The peptide for the sequence that II class presents causes suppressor T lymphocyte.

The immunogenic properties of peptide of the invention are particularly interesting in treating and preventing immune response.

Peptide described herein is used as drug, and more specifically to manufacture is for preventing or treating mammal (especially people Class) immunologic derangement drug.

The present invention, which is described, needs this treatment by using peptide of the invention, its homologue or derivatives for treatment or prevention Or prevention mammal immunologic derangement method, this method include step: to immunologic derangement or have be immunized it is disorderly Peptide of the invention, its homologue or the derivative of the mammal application therapeutically effective amount of random risk, to mitigate immune The symptom of disorder.Contemplate the treatment to human and animal such as pet and farm-animals.In one embodiment, to be treated Mammal is the mankind.Above-mentioned immunologic derangement is selected from anaphylactia and autoimmune disease in certain embodiments.

Peptide of the invention or the pharmaceutical composition comprising such peptide as defined herein preferably pass through it is subcutaneous or intramuscular to Medicine.Preferably, the peptide or the pharmaceutical composition comprising such peptide subcutaneously (SC) can be injected at the exterior portion of upper arm in elbow Between portion and shoulder in intermediate region.When needing individually injection two or more times, they can be applied simultaneously in two-arm With.

Peptide according to the present invention is applied comprising its pharmaceutical composition with treatment effective dose.It is exemplary but non-limiting Dosage be 50 to 1500 μ g, preferably 100 to 1200 μ g.More specific dosage can between 50 to 250 μ g, Between 250 to 450 μ g or between 850 to 1300 μ g, this depends on the situation and the severity of disease of patient.Dosage can Comprising applying single dose or concurrently or consecutively application 2,3,4,5 or more dosage.Illustrative non-limiting dosage regimen It is as follows:

Low dosage scheme is applied 50 μ g peptides comprising SC, is injected in two times, every time 25 μ g (100 μ L every time), subsequent three 25 μ g peptides of continuous injection, are each injected, every time 12.5 μ g (50 μ L every time) in two times.

Middle dosage scheme is applied 150 μ g peptides comprising SC, is injected in two times, every time 75 μ g (300 μ L every time), subsequent three 75 μ g peptide of continuous administration, is each injected, every time 37.5 μ g (150 μ L every time) in two times.

High dose scheme is applied 450 μ g peptides comprising SC, is injected in two times, every time 225 μ g (900 μ L every time), subsequent three A 225 μ g peptide of continuous administration, is each injected, every time 112.5 μ g (450 μ L every time) in two times.

For all above-mentioned peptides, it is contemplated to other variants, wherein there are one or two between histidine and cysteine A amino acid X.In general, these external amino acid X are not His, Cys, Ser or Thr.

Peptide of the invention can be also used for in-vitro diagnosis method, for the restricted CD4+T cell of II class in test sample. In the method, make the complex contacts of sample Yu MHC II class molecule and peptide according to the present invention.By measurement compound with The combination of cells in sample detects CD4+T cell, and wherein CD4+T cell is deposited in the combination instruction sample of compound and cell In.

Compound can be the fusion protein of peptide and MHC II class molecule.

Alternatively, the MHC molecule in compound is the tetramer.The compound can be used as shla molecule offer or can To be connected on carrier.

Therefore, in certain embodiments, treatment and prevention method of the invention includes that application is immune as described herein Originality peptide, wherein peptide includes the t cell epitope of antigen protein, in disease to be treated (for example, those of as described above) It works.In further specific embodiment, the epitope used is dominant epitope.

Peptide according to the present invention is prepared by synthetic peptide, wherein t cell epitope and modified redox motif are by 0 It is separated to 5 amino acid.In some embodiments, it can be obtained by introducing 1,2 or 3 mutation outside epitope sequences Modified redox motif, to keep sequence background present in protein.In general, referring to as native sequences a part Nonapeptide, the amino acid in P-2 and P-l and P+10 and P+11 is retained in peptide sequence.These flanking residues usually it is stable with The combination of MHC II class.In other embodiments, the sequence N-terminal or C-terminal of epitope and resisting containing t cell epitope sequence The sequence of former albumen is unrelated.

Therefore, based on the above method of designed peptide, by chemical peptide synthesis, recombinant expression method or in more specifically situation The proteolysis or Chemical fragmentation of lower protein generates peptide.

The peptide generated in the above-mentioned methods can be tested in method in vitro and in vivo with the presence or absence of t cell epitope, and Their reduction activation is tested in can measuring in vitro.It is controlled as last quality, during these peptides can measure in vitro It is tested, to verify whether peptide can produce CD4+T cell, dissolves antigen presenting cell by apoptotic pathways, it should Antigen presenting cell present antigen contain in the peptide with modified redox motif there is also epitope sequences.

Recombinant DNA technology can be used to generate in bacterium, yeast, insect cell, plant cell or mammalian cell Peptide of the invention.In view of the limited length of peptide, they can be prepared by chemical peptide synthesis, wherein by by different amino Acid is coupled each other to prepare peptide.Chemical synthesis is particularly suitable for being included in such as D- amino acid, having non-naturally occurring side chain Amino acid or the side chain with modification natural amino acid, such as the cysteine of methylation.

Chemical peptide symthesis method is fully described, and peptide can be from such as Applied Biosystems and other public affairs Si Deng company orders.

Peptide synthesis can be used as Solid phase peptide synthesis (SPPS) or progress opposite with solution phase peptide synthesis.The foremost side SPPS Method is t-Boc and Fmoc solid state chemistry:

During peptide synthesis, a variety of blocking groups are used.For example, protecting hydroxyl and carboxyl functional group by tert-butyl, lead to T-Boc radical protection lysine and tryptophan are crossed, trityl as protecting group asparagine, glutamine, cysteine and group are passed through Propylhomoserin, and pass through pdf radical protection arginine.If appropriate, this blocking group can be stayed on peptide in post synthesis.It uses Peptide, can be connected to each other to form longer peptide by connection strategy (the chemo-selective coupling of two unprotected peptide fragments), Such as Kent (Schnelzer&Kent (1992) Int.J.Pept.Protein Res.40,180-193) initially described and example It is summarized such as Tam et al. (2001) Biopolymers 60,194-205, this provides beyond SPPS range and realizes albumen The great potential of matter synthesis.Many protein that size is 100-300 residue have been successfully synthesized by this method.Due to The huge advance of SPPS, synthetic peptide is in research fields such as biochemistry, pharmacology, Neurobiology, zymetology and molecular biology Continue to play increasingly important role.

Alternatively, it can be synthesized in suitable expression vector by using the nucleic acid molecules for encoding peptide of the invention Peptide, the expression vector include coding nucleotide sequence.Use the password of automatic dna synthesizer and well-known genetic code Son-amino acid relationship can easily prepare this DNA molecular.It is this using oligonucleotide probe and conventional hybridization method DNA molecular can also be used as genomic DNA or cDNA is obtained.Such DNA molecular can be incorporated into expression vector (including plasmid) In, it is suitable for expressing in the suitable hosts such as such as bacterium (such as Escherichia coli), yeast cells, zooblast or plant cell DNA and generate polypeptide.

Physics and the chemical property (such as solubility, stability) of target peptide are checked to determine the peptide if appropriate for for controlling It treats in composition.In general, this is optimized by adjusting the sequence of peptide.Optionally, this field public affairs can be used in post synthesis The technology known comes modified peptides (chemical modification, such as add/lack functional group).

Think t cell epitope itself by conjunction with the appropriate HLA molecule on Antigen Presenting Cell surface and stimulating related T Cell subsets and the earliest events for triggering t helper cell level.These events cause T cell to be proliferated, lymphokine secretion, part Inflammatory reaction, recruitment of the additional immuno cell to the position, and activation B cell cascade reaction, cause to generate antibody.These antibody A kind of isotype lgE in the development of allergic symptom have basic importance, and its generate in t helper cell level Cascade of events early stage is influenced by the property for the lymphokine secreted.T cell epitope is the primary element of T cell receptor identification Or minimum unit, wherein epitope includes amino acid residue necessary to Receptor recognition, ammonia of these amino acid residues in protein It is continuous in base acid sequence.

However, when application has the peptide of t cell epitope and redox motif, it is believed that following event can occur:

Antigen (i) specificity T is led to by the homologous interaction of the antigenic source peptide with MHC-II class molecular presentation The activation of cell；

It restores enzyme sequence and restores T cell surface protein, such as CD4 molecule, the second structural domain contains limited disulphide bridges.This By signal transduction to T cell.Increase in a series of related consequences with oxidative pathway, important event is the increase that calcium current enters Transposition with from NF-kB transcription factor to nucleus.The latter causes the transcription of IFN-γ and granzyme to increase, so that cell is by luring Guided cell Apoptosis mechanism obtains cell dissolution characteristic；Cytolytic properties are mutual by being related to granzyme B secretion and Fas-FasL The mechanism of effect influences to present the cell of peptide.Since killing functions of immunocytes is obtained by apoptosis pathway, cytolytic Cell is the term than cytotoxic cell more suitable for these cells.The destruction of antigen presentation target cell is prevented to positioned at phase The activation of the other T cells of epitope specifically on synantigen, or to the irrelevant antigen for by same antigen being in delivery cell processing Activation；Another consequence of t cell activation is to inhibit the activation of onlooker's T cell by cell-cell contact dependent mechanism. In this case, if cytolytic T lymphocyte and onlooker's T cell are very close, i.e., the table in same antigen presenting cell It is activated on face, then the T cell of the antigenic activation presented by different antigen presenting cells is also suppressed.

Experimental data disclosed in PCT application WO2008/017517 and the present inventor's publication by above-mentioned reference, card The real mechanism of action of above-mentioned hypothesis.

The present invention provides the methods for generating antigentic specificity cytolytic CD4+T cell in vivo or in vitro, and solely The method is stood on, based on feature representation data separation cytolytic CD4+T cell and other cell masses (such as Foxp3+Tregs) Method.

The present invention describes the vivo approaches for generating antigentic specificity CD4+T cell.One specific embodiment It is related to the method for producing or separating CD4+T cell, animal (including mankind) is immunized with the peptide of invention as described herein, Then CD4+T cell is separated from immune animal.The present invention is described for generating the molten cell of antigentic specificity for being directed to APC The in-vitro method of property CD4+T cell.The present invention provides generate the antigentic specificity cytolytic CD4+T cell for being directed to APC Method.

In one embodiment, such method is provided, it includes: separation peripheral blood cells pass through basis in vitro Immunogenic peptide of the invention stimulates cell mass, and especially in the presence of IL-2, expands stimulated cell mass.According to this hair Bright method has the advantages that generate a large amount of CD4+T cells, and it is (logical to can produce the CD4+T cell special to antigen protein It crosses and uses the peptide comprising antigentic specificity epitope).

In alternative embodiments, CD4+T cell can be generated in vivo, i.e., it is described herein by being injected to subject Immunogenic peptide, and collect the cytolytic CD4+T cell that generates in vivo.

The obtainable antigentic specificity cytolytic CD4+T cell for APC is particularly useful by means of the present invention Immunotherapy, prevention allergic reaction and treatment autoimmune disease are configured in applying to mammal.It contemplates of the same race different The use of body and autogenous cell.

Cytolytic CD4+T cell mass is obtained as described below.

Antigentic specificity cytolytic CD4+T cell as described herein can be used as drug, more specifically to adoptive Cell therapy more specifically to treats the recurrence of acute allergic reaction and autoimmune disease (such as multiple sclerosis). Isolated cytolytic CD4+T cell or cell mass, more particularly antigentic specificity cytolytic CD4+T cell mass, for making Make the drug for preventing or treating immunologic derangement.Disclose by using separation or generation cytolytic CD4+T cell The method treated.

As explained in WO2008/017517, the expression characteristic based on cell can will be directed to the molten cell of APC Property CD4+T cell is distinguished with natural Treg cell.More specifically, with natural Treg cell faciation ratio, cytolytic CD4+T is thin Born of the same parents group shows one or more following characteristics:

The expression of rear surface marker is activated to increase, the surface marker includes CD103, CTLA-4, Fasl and ICOS；

Centre expression CD25；

CD4, ICOS, CTLA-4, GITR are expressed, and low expression or is not expressed CD127 (IL7-R), CD27 is not expressed；

Transcription Factor T-bet and egr-2 (Krox-20) are expressed, but does not express transcription inhibitory factor Foxp3；

High yield IFN-γ and do not produce or only produce micro IL-10, IL-4, IL-5, IL-13 or TGF-β.

In addition, cytolytic T lymphocyte expresses CD45RO and/or CD45RA, CCR7, CD27 are not expressed and presents high level Granzyme B and other granzymes and FasL.

Peptide of the invention can cause specific T-cells and send out onlooker's T cell after being administered to live animal (usually people) Wave inhibitory activity.

In a particular embodiment, cytolytic cells group of the invention is characterized in that expression FasL and/or interference Plain γ.In a particular embodiment, cytolytic cells group of the invention is further characterized in that expression granzyme B.

The mechanism also implies and experimental result shows peptide of the invention, although the specific T-cells comprising certain antigen Epitope, but can be used for preventing or treating the disorder as caused by the immune response for other t cell epitopes of same antigen, or In some cases, or even for treating the disorder as caused by the immune response for other t cell epitopes of other not synantigens, If other not synantigens can be by the MHC II class molecule near the T cell by peptide activation of the invention via identical mechanism It presents.

The separation cell mass of cell type as characterized above is disclosed, other than being antigentic specificity, i.e., also It is able to suppress antigen-specific immune response.

The present invention provides the pharmaceutical compositions comprising one or more peptides according to the present invention, also include pharmaceutically may be used The carrier of receiving.As described above, the invention further relates to the compositions for being used as drug, or it is related to controlling by using the composition The method for treating mammalian immune disorder, and it is related to the composition for manufacturing the drug for preventing or treating immunologic derangement Purposes.It is suitable for treating or preventing immunologic derangement (especially air borne and food-borne mistake for example, pharmaceutical composition can be It is quick) and anaphylactogen disease vaccine.As the example of pharmaceutical composition described further herein, peptide according to the present invention It is adsorbed on and is suitable for being applied on the adjuvant of mammal, such as aluminium hydroxide (alum).In general, by subcutaneous route, interval 2 In week, 3 times 50 μ g of injection are adsorbed on the peptide on alum.It will be apparent to one skilled in the art that other administration routes are It is possible including oral, intranasal or intramuscular.Moreover, frequency injection and injection volume can change according to the patient's condition to be treated.This Outside, other adjuvants in addition to alum can be used, precondition is that they promote in the presentation of MHC-II class and T cell activation Peptide presents.Therefore, although active constituent can be administered alone, they are provided usually as pharmaceutical formulation.Animal doctor of the invention It include at least one active constituent as described above and one or more pharmaceutically acceptable carriers with the formula with people. The present invention relates to pharmaceutical compositions, and it includes the one or more peptides according to the present invention mixed with pharmaceutically acceptable carrier As active constituent.Pharmaceutical composition of the invention should include the active constituent of therapeutically effective amount, as follows about treatment or pre- Shown in anti-method.Optionally, composition also includes other therapeutic components.Suitable other therapeutic components and they common dose Amount, depending on the classification belonging to them, be well known to those skilled in the art, and can from for treat immune disorders its It is selected in its known drug.

The term as used herein " pharmaceutically acceptable carrier " refers to any material prepared with active constituent or substance, Promoting active constituent to apply or travel to position to be treated, such as by dissolution, dispersion or the diffusion composition, And/or to assist its storage, transport or handle without damaging its validity.They include any and all solvents, dispersion Jie Matter, coating material, antibacterial and antifungal agent (such as phenol, sorbic acid, methaform), isotonic agent (such as sugar or sodium chloride) etc..It can To include supplementary element with the acting duration of immunogenic peptide in control composition.Pharmaceutically acceptable carrier can be Solid or liquid or the gas for being compressed to form liquid, i.e., composition of the invention can be suitable as concentrate, lotion, molten Liquid, particle, pulvis, spray, aerosol, suspension, ointment, emulsifiable paste, tablet, pill or powder.For pharmaceutical composition and Its suitable pharmaceutical carrier being formulated is well known to those skilled in the art, and in the present invention to their selection without spy It does not limit.They can also include additive, such as wetting agent, dispersing agent, sticker, adhesive, emulsifier, solvent, coating Material, antibacterial agent and antifungal agent (such as phenol, sorbic acid, methaform), isotonic agent (such as sugar or sodium chloride) etc., precondition It is that they meet pharmacy practice, i.e., will not causes the carrier and additive of permanent lesion to mammal.Drug of the invention Composition can be prepared in any known fashion, such as by by active constituent and selected in the rapid program of one or more steps Carrier material and (under appropriate circumstances) other additives such as surfactant are uniformly mixed, coat and/or are ground together.It Can also be prepared by being micronized, such as to obtain microspheres form usually with about 1 to 10 μ m diameter, i.e., for manufacturing For controlling or the microcapsules of sustained release activity ingredient.

It is ready to use in the suitable surfactant of pharmaceutical composition of the present invention, also referred to as emulgent or emulsifier, is to have Nonionic, cation and/or the anionic materials of good emulsification, dispersion and/or wetting property.Suitable anion surface active Agent includes water-soluble soap and water-soluble synthetic surface activating agent.Suitable soap be higher fatty acids (C10-C22) alkali metal or Alkali salt, unsubstituted or substituted ammonium salt, such as oleic acid or stearic sodium salt or sylvite, or from coconut oil or butter In obtainable natural acid mixture sodium salt or sylvite.Synthetic surfactant includes the sodium salt or calcium of polyacrylic acid Salt；Fatty sulphonates and sulfate；Sulphonated benzimidazole derivative and alkylaryl sulfonates.Fatty sulphonates or sulfate are logical Normal form is alkali or alkaline earth metal salt, unsubstituted ammonium salt or the alkyl or acyl group base with 8 to 22 carbon atoms The ammonium salt that group replaces, for example, lignin sulfonic acid or dodecyl sodium sulfonate sodium salt or calcium salt or obtained from natural acid The alkali or alkaline earth metal salt (such as lauryl sodium sulfate) and rouge of the mixture of aliphatic alcohol sulfate, sulfuric acid or sulphonic acid ester The alkali or alkaline earth metal salt of fat alcohol/ethylene oxide adduct sulfonic acid.Suitable sulphonated benzimidazole derivative usually contains There are 8 to 22 carbon atoms.The example of alkylaryl sulfonates is dodecyl benzene sulfonic acid or dibutyl naphthalenesulfonic acid or naphthalene sulfonic acids/first Sodium, calcium or the alkanolamine (alcanolamine salt) of aldehyde condensation products.It also suitable is corresponding phosphate, such as phosphorus The salt and nonylphenol and ethylene oxide of acid esters and/or the adduct or phosphatide of propylene oxide.Conjunction for this purpose Suitable phosphatide is the cephalin or lecithin epoxy-type phosphatide of natural (being originated from animal or plant cell) or synthesis, such as phosphatide Acyl ethanol amine, phosphatidylserine, phosphatidyl glycerol, lysolecithin, cuorin, dioctanylphosphatidyl-choline, two palmityls Phosphatidyl choline and its mixture.

Suitable nonionic surfactant includes alkyl phenol, fatty alcohol, the rouge for containing at least 12 carbon atoms in molecule Fat acid, aliphatic amine or amide polyethoxylated and gather propenoxylated derivative, alkylated aromatic sulfonic acid ester and dialkyl sulfosuccinate Succinate, such as the polyglycol ether derivative of aliphatic series and alicyclic alcohol, saturation and unsaturated fatty acid and alkyl phenol, institute It states derivative and usually contains 3 to 10 glycol ether groups and 8 to 20 carbon atoms in (aliphatic series) hydrocarbon part, and in alkyl phenol Moieties contain 6 to 18 carbon atoms.Other suitable nonionic surfactants are polyethylene oxide and polypropylene glycol, alkane Contain second diamino-polypropylene glycol water-soluble addition object of 1 to 10 carbon atom in base chain, which contains 20 to 250 Ethylene glycol ether group and/or 10 to 100 propylene glycol ether groups.The usually every propylene glycol units of these compounds contain 1 to 5 second Diol units.The representative example of nonionic surfactant be nonyl phenol-polyethoxy ethanol, castor oil polyglycol ether, Polypropylene/polyethylene oxide adducts, tributyl phenoxypolyethoxy ethanols, polyethylene glycol and Octylphenoxypolyethoxy Ethyl alcohol.Polyethylene sorbitan (such as polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sugarcane The aliphatic ester of sugar and pentaerythrite is also suitable nonionic surfactant.Suitable cationic surfactant includes tool There are the quaternary ammonium salt of 4 alkyl optionally replaced by halogen, phenyl, substituted phenyl or hydroxyl, especially halide；For example, containing There is at least one C8C22 alkyl (such as cetyl, lauryl, palmityl, myristyl, oleyl etc.) as N- substituent group And contain the season of unsubstituted or halogenated low alkyl group, benzyl and/or hydroxy lower alkyl as further substituent group Ammonium salt.

The more detailed description of surfactant suitable for this purpose can be in such as " detergent and cream of McCutcheon Agent annual (McCutcheon's Detergents and Emulsifiers Annual) " (MC Publishing Crop.,Ridgewood,New Jersey,1981)、“Tensid-Taschenbucw',2d ed.(Hanser Verlag, Vienna, 1981) and " surfactant encyclopedia (Encyclopaedia of Surfactants), (Chemical Publishing Co., New York, 1981) it is found in.Peptide according to the present invention, its homologue or derivative (and its physiology Acceptable salt or pharmaceutical composition on are included in term " active constituent ") it can be by being suitable for disease to be treated Shape and any approach application for being suitable for compound to be administered (being herein protein and segment).Possible approach includes area Domain property, systemic, oral (solid form or sucking), rectum, nasal cavity, part (including eye, cheek and sublingual), vagina and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intra-arterial, intrathecal and Epidural cavity).Preferred administration method can be according to for example The situation of recipient or disease to be treated and change.As described herein, compatible with other ingredients of formula and connect to it In the sense that receptor is harmless, carrier is most preferably " acceptable ".Formula includes being suitble to oral, rectum, nose, part (including cheek With it is sublingual), vagina or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intra-arterial, intrathecal and Epidural cavity) application that A little formulas.

Formula suitable for parenteral administration includes aqueous and non-aqueous aseptic parenteral solution, can contain antioxidant, buffering Agent, bacteriostatic agent and solute keep formula and the blood of expected recipient isotonic；And aqueous or non-aqueous sterile suspensions, it can wrap Include suspending agent and thickener.Formula can reside in unit dose or multi-dose container, such as the ampoule and bottle of sealing, and And can be stored under the conditions of freeze-drying (freeze-drying), will be before use, it is only necessary to add sterile liquid carrier, such as infuse It penetrates and uses water.Extemporaneous injection solution and suspension can be prepared by the aseptic powdery, particle and tablet of aforementioned type.

Typical single dose unit formulation is (as described above or its is appropriate containing daily dosage or the daily sub-doses of unit Those of part) active constituent.It should be understood that other than the ingredient being above especially mentioned, formula of the invention can be with Other reagents including this field routine about the formulation types discussed, for example, it is orally administered those may include Flavoring agent.Peptide, its homologue or derivative according to the present invention can be used to provide controlled release drug formula (" controlled release formulation "), Containing one or more the compound of the present invention as active constituent, wherein the release of active constituent can be controlled and be adjusted Section is to allow lower frequency to be administered or improve the pharmacokinetics or toxicity profile of given the compounds of this invention.It can be according to normal Rule method is suitable for the controlled release formulation of oral administration to prepare, and wherein discrete unit includes one or more the compound of the present invention. It may include supplementary element with the acting duration of active constituent in control composition.It therefore, can be suitable poly- by selecting Object carrier is closed to realize controlled release composition, the polymer support be, for example, polyester, polyaminoacid, polyvinylpyrrolidone, second Alkene-vinyl acetate copolymer, methylcellulose, carboxymethyl cellulose, protamine sulfate etc..Can also by by activity at Divide and is incorporated into polymeric material (such as hydrogel, polylactic acid, hydroxymethyl cellulose, polymethyl methacrylate and other above-mentioned polymerizations Object) particle (such as microcapsules) in, come Drug controlled release rate and acting duration.These methods include colloid drug Transmission system such as liposome, microballoon, microemulsion, nanoparticle, Nano capsule etc..Depending on administration method, pharmaceutical composition can Protectiveness can be needed to be coated.Medicament forms suitable for injection include aseptic aqueous solution or dispersion liquid and the nothing for its extemporaneous preparation Bacterium powder.Therefore, typical carriers for this purpose include biocompatible aqueous buffer solution, ethyl alcohol, glycerol, propylene glycol, gather Ethylene glycol etc. and its mixture.In view of when a variety of active ingredients is applied in combination, they are not necessarily in mammal to be treated In simultaneously directly play its combination therapy effect, corresponding composition is also possible to the form of pharmaceutical reagent box or packaging, wherein Two kinds of ingredients, which are comprised in, to be separated but in adjacent repository or compartment.In the latter case, every kind of active constituent therefore can It is prepared by a manner of being suitable for the administration method different from other ingredients, such as one of which can be oral or extra-parenteral match The form of side, and another ampule form or aerosol form that can be for intravenous injection.

As in vitro and in vivo is confirmed, the cytolytic CD4+T cell obtained in the present invention is in MHC-II class dependence APC apoptosis is induced after alloactivation, influences dendritic cells and B cell, and (2) there is no the feelings of IL-10 and/or TGF-β Onlooker's T cell is inhibited by contact dependent mechanism under condition.As being discussed in detail in WO2008/017517, cytolytic CD4 + T cell can be distinguished with natural and adaptability Treg.

It will be illustrated by the following examples the present invention now, providing the embodiment does not have any restrictions intention.In addition, this All bibliography of text description are explicitly included in herein by reference.