CN101006096A - Synthetic chemokines, methods of manufacture, and uses - Google Patents

Abstract

The invention relates to synthetic chemokines, methods of manufacture and uses thereof. The synthetic chemokines of the invention include one or more amino acid deletions at the C-terminus relative to the corresponding wild type chemokine, which also can include one or more covalently attached polymers, as well one or more additional amino acid changes or chemical modifications. The invention also provides synthetic chemokines in a substantially purified oligomeric form, such as, in a monomer or dimer form. Further provided are methods for synthesizing the synthetic chemokines of the invention, pharmaceutical formulations and kits thereof, and their use as research tools and medicaments.

Description

Synthetic chemokines, Preparation Method And The Use

Technical field

The present invention relates to chemokine and derivative thereof, Preparation Method And The Use.

The cross reference of related application

The application requires in the right of priority of the U.S. Patent application sequence number (SN) 60/557,400 of application on March 30th, 2004, and described application all is attached to herein by reference.

Background

Chemokine is the small protein matter that participates in white corpuscle transportation and various other biological procedureses.Most of chemokines are located and exacerbate inflammation by the cell activation of inducing chemotaxis and being present in the dissimilar inflammatory cell of inflammation part usually.Some chemokine has the characteristic except that chemotaxis when inflammation, blood vessel generation and tumor growth, for example induce killer cell propagation and activation, regulates the growth of hemopoietic progenitor cell type, and the transportation hemopoietic progenitor cell passes in and out marrow.(referring to for example Baggiolini etc., Ann.Rev.Immunology (1997) 75:675-705; Zlotnik etc., Critical Rev.Immunology (1999) 19 (1): 1-4; Wang etc., J.Immunological Methods (1998) 220 (1-2): 1-17; Moser etc., Intl.Rev.Immunology (1998) 16 (3-4): 323-344).

The aminoacid sequence of many chemokines, 26S Proteasome Structure and Function are known.The molecular weight of chemokine is about 8-10kDa, demonstrates the sequence homology of about 20-50% on protein level each other.These protein are also shared the common tertiary structure.All chemokines all have the conservative cysteine residues that many participations form intramolecular disulfide bond, and this conservative cysteine residues is used to identify chemokine and it is classified.For example, preceding two cysteine residues are called " C-X-C " chemokine (being also referred to as " α " chemokine) by an isolated chemokine of other amino acid in the sequence.Preceding two adjacent chemokines of cysteine residues are called " CC " chemokine (being also referred to as " β " chemokine) in the sequence.The difference of " C " chemokine and other chemokine is to lack a cysteine residues (being also referred to as " γ " chemokine).The C chemokine shows with some CC chemokine member similarity, but lacks first and the 3rd cysteine residues with CC and CXC chemokine feature.Preceding two cysteine residues are called " CXXXC " chemokine by a group member of three isolated chemokines of other amino acid and (are also referred to as " CX in the sequence ₃C " or " δ " chemokine).Also exist the chemokine subgroup.For example, the known CC chemokine that contains two extra conservative cysteine residues, term " C6-β " chemokine is used for this subgroup sometimes.Most of chemokines of Jian Dinging all are the members of CC chemokine and CXC chemokine class up to now.

The biological activity of chemokine is by receptor-mediated.These acceptors comprise chemokine specific receptors and the acceptor with overlapping ligand specificity, and this receptoroid is under the jurisdiction of the different chemokine combinations of CC chemokine or CXC chemokine class with some.For example, CXC chemokine SDF-1 α has specificity to the CXCR4 acceptor, and CC chemokine RANTES and CCR1 acceptor, CCR3 acceptor and CCR5 receptors bind.Another example is a chemokine eosinophilic granulocyte chemotactic protein (Eotaxin), and it is the part of CCR3 acceptor and CKR3 acceptor.(referring to for example Cyster, J.G., Science (1999) 286:2098-2102; Ponath etc., J.Experimental Medicine (1996) 183 (6): 2437-2448; Ponath etc., J.Clinical Investigation (1996) 97 (3): 604-12; Yamada etc., Biochem.Biophys.Res.Communications (1997) 231 (2): 365-368.

Chemokine relates to important disease approach, for example asthma, rhinallergosis, atopic dermatitis, cancer, virus disease, congee sample spot/atherosclerosis, rheumatoid arthritis and organ-graft refection.Yet, many chemokines and relate to the natural characteristics that their promote or increase the weight of white corpuscle inflammatory reaction and infection as the FAQs of the potential use aspect of therapeutical agent.For this reason, chemokine is carried out a large amount of modifications, attempted to produce the antagonist of corresponding Chemokine Receptors.Classical representative example is the RANTES situation.Under certain conditions, but wild-type RANTES exacerbate inflammation and HIV infect (Gordon etc., J.Virol. (1999) 73:684-694; Czaplewski etc., J.Biol.Chem. (1999) 274:16077-16084).By contrast, 26 (E26A) and the replacement on 66 (E66S) at the RANTES polypeptide chain, this molecule become its non-inflammatory form and improves the ability of it and it receptor competition HIV that (Appay etc., J.Biol.Chem. (1999) 274 (39): 27505-27512).In addition, it is terminal modified RANTES have been carried out N-, generation can be blocked the antagonist that HIV-1 infects, described modification comprises the brachymemma [RANTES 9-68] of N-end, add methionine(Met) (" Met-RANTES "), amino oxygen base pentane (" AOP-RANTES ") or nonanoyl (" NNY-RANTES ") (Arenzana-Seisdedos etc., Nature (1996) 383:400; Mack etc., J.Exp.Med. (1998) 187:1215-1224; Proudfoot etc., J.Biol.Chem. (1996) 271:2599-2603; Wells etc., WO 96/17935; Simmons etc., Science (1997) 276:276-279; Offord etc., WO 99/11666; Mosier etc., J.Virology (1999) 73 (5): 3544-3550).

Many wild-type chemokines as another thorny feature of potential therapeutical agent are: they have accumulative trend under high density, and mixing property is in conjunction with (Promiscuous binding) and variant activatable Chemokine Receptors (Murphy etc., Pharmacological Rev. (2000) 51 (1): 145-176), Rollins, BJ., Blood (1997) 90 (3): 909-928; Wells etc., Inflammation Res. (1999) 48:353-362)).Congregation may be thorny for process for preparation, also can increase the weight of in some cases illness (Czaplewski etc., J.Biol.Chem. (1999) 274 (23): 16077-16084; Czaplewski etc., " Engineering; Biology; andClinical Development of hMIP-1 α; " (1999) be stated from: Chemokines in Disease:Biology and Clinical Research, CA.Herbert writes, Humana Press Inc., Totwa, NJ; Trkola etc., J.Virol. (1999) 73 (8): 6370-6379; Appay etc., J.Biol.Chem. (1999) 274 (39): 27505-27512; Hunter etc., Blood (1995) 86 (12): 4400-4408; Lord etc., Blood (1995) 85 (12): 3412-3415; Lord etc., Brit.J.Cancer (1996) 74:1017-1022).Mixing property may be unwanted in conjunction with being the characteristics of chemokine under some treatment situation.Yet, chemokine still be hopeful very much as therapeutical agent (Murphy etc., Pharmacological Rev. (2000) 51 (1): 145-176), Rollins, BJ., Blood (1997) 90 (3): 909-928; Wells etc., Inflammation Res. (1999) 48:353-362).

International disclose No. 00/53223, WO openly representative in the chemokine document of new chemokine in a large amount of report property, according to it, can prepare antagonist and also can be connected with PEG or other water-soluble polymers.United States Patent (USP) the 6th, 168, disclose in No. 784 chemokine analogue NNY-Rantes and suggestion with the PEG chain at terminal modified this analogue of C-.Recently, even the more potent form (Offord etc. of various chemokines have been prepared, WO02/04499), comprise polymer-modified form, wherein polymkeric substance is connected on the position corresponding to C-end, glycosylation site, coaggregation sites and/or GAG binding site (Koechendoerfer etc., WO 02/04015).Wilkin etc. (Curr.Opinion Biotech. (1999) 9:412-426) have summarized proteinic chemosynthesis.

Although these methods have been improved the target chemokine as potential therapeutical agent in some cases, but one of challenge that preparation is suitable for medicinal chemokine is both to have improved effect, improve simultaneously the other medicines characteristic again, for example pharmacokinetic properties, acceptor specificity and gathering reduce.Equally, also wish to seek general policies and the method for preparing effective chemokine receptor anagonists, and seek they are used for preventing and/or treating the medicine of disease in preparation purposes.The present invention is just in order to satisfy the demand of these aspects and others.

Summary of the invention

The present invention relates to synthetic chemokines, Preparation Method And The Use.Synthetic chemokines of the present invention comprises the chemokine polypeptides chain with N-end and C-end, wherein this chemokine polypeptides chain comprises (i) aminoacid sequence and halfcystine pattern and the (ii) end of the C-with respect to wild-type chemokine brachymemma corresponding to the wild-type chemokine.The present invention also provides synthetic chemokines, described chemokine has C-end brachymemma and one or more other modification with respect to corresponding wild-type chemokine, described chemokine comprises the synthetic chemokines with one or more amino acid changes, and the synthetic chemokines of modifying with one or more covalently bound polymkeric substance and/or chemical adducts.The present invention also provides such synthetic chemokines: described synthetic chemokines has terminal modified and terminal modified and one or more other modifications of C-of N-with respect to corresponding wild-type chemokine, the for example one or more amino acid changes of described modification, and the form of modifying with one or more covalently bound polymkeric substance and/or chemical adducts.The composition of the synthetic chemokines of the present invention of the single oligomer form (for example monomer or dimer) that comprises basic purifying also is provided.The invention still further relates to the preparation method of synthetic chemokines of the present invention, relate to the pharmaceutical composition that comprises synthetic chemokines of the present invention, relate to the method for the treatment of mammiferous Chemokine Receptors-disease states mediated with synthetic chemokines of the present invention and medicine box.

The accompanying drawing summary

Fig. 1: the representative fast chromatogram of imitating liquid chromatography (LC) (FPLC) of synthetic NK chemokine.The FPLC antique catalog and the peak that show the corresponding dimer flow point of NK3 (Figure 1A) and NK4 (Figure 1B).

Fig. 2: the representative C4 high performance liquid chromatography (C4-HPLC) and size exclusion HPLC (HPLC-SEC) antique catalog of synthetic NK chemokine, and the chromatogram of corresponding electrospray ionization mass spectrum (ES-MS).Show that the purifying dimer of synthetic chemokines NK3 (Fig. 2 A) and NK4 (Fig. 2 B) merges C4-HPLC, SEC-HPLC and the ES-MS antique catalog of thing.

Fig. 3: the representative sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) of the synthetic NK chemokine of purifying, and circular dichroism (CD) spectrum.Each reduction and the dimeric SDS-PAGE gel of non-reducing purifying of diagram NK3 (Fig. 3 A) and NK4 (Fig. 3 C), and the dimeric CD spectrum of the purifying of NK3 (Fig. 3 B) and NK4 (Fig. 3 D).In the SDS-PAGE gel: Fig. 3 A, the 1st road is molecular weight (MW) standard substance, the 2nd road (reduction) and the 3rd road (non-reduced) is that the crude protein of NK3 merges thing; The 4th road (reduction) and the 5th road (non-reduced) are the monomer flow points of NK3; The 6th road (reduction) and the 7th road (non-reduced) are the dimer flow points of NK3; The 8th road (reduction) and the 9th road (non-reduced) are the aggregate flow points of NK3; The 10th road (blank).Fig. 3 C, the 1st road is the MW standard substance, the 2nd road (reduction) and the 3rd road (non-reduced) is that the crude protein of NK4 merges thing; The 4th road (reduction) and the 5th road (non-reduced) are the monomer flow points of NK4; The 6th road (reduction) and the 7th road (non-reduced) are the dimer flow points of NK4; The 8th road (reduction) and the 9th road (non-reduced) are the aggregate flow points of NK4; The 10th road (blank).

Fig. 4: the representative size exclusion chromatography-multi-angle determination of light scattering of the synthetic NK chemokine of purifying (Size Exclusion Chromatography-Multiangle Light ScatteringDetection, SEC-MALS) chromatogram.The SEC-MALS antique catalog of the purifying oligomerization aggregate (may be eight aggressiveness) (Fig. 4 C) of the NK3 dimer (Fig. 4 A) of diagram purifying, the NK4 dimer (Fig. 4 B) of purifying and NK contrast.

Fig. 5: show that one is combined into the single transfectant calcium of the people CCR5 flux mensuration figure of NK chemokine and contrast.

Fig. 6: show that one is combined into the single transfectant calcium flux mensuration figure (Fig. 6 A, Fig. 6 B) of people CCR3 of NK chemokine and contrast.

Fig. 7: show that one is combined into the single transfectant calcium flux mensuration figure (Fig. 7 A, Fig. 7 B) of people CCR1 of NK chemokine and contrast.

Fig. 8: relatively the plasma concentration (ng/ml) of synthetic NK chemokine and time in the rat body (minute) representative pharmacokinetics (PK) distribution plan.Show by intravenously (IV) (Fig. 8 A) or NK3, the NK10 of subcutaneous (SC) (Fig. 8 B) route of administration and the PK distribution plan of NK11.

The description of specific embodiments

The present invention relates to synthetic chemokines, Preparation Method And The Use.Specifically, the present invention relates to synthetic chemokines, especially the terminal modified chemokine molecule of C-.When measuring by suitable chemokine bioassay method, new biological activity synthetic chemokines of the present invention is regulated the activity of naturally occurring Chemokine Receptors.Described molecule can work by one or more characteristics of their institute's bonded Chemokine Receptors of antagonism and (for example suppress virus infection, cause receptor down-regulated, cause the acceptor internalization), thereby " antagonism " acceptor recirculation normal circulation process of getting back to cell surface.Under the situation of other biological respinse, described molecule can play the effect of receptor stimulant, for example induces the calcium flux, starts chemotaxis etc.Therefore, biological activity synthetic chemokines of the present invention can play the effect (comprising partial antagonism) of antagonist, but also can play the effect (comprising partial agonist) or the two the combination of agonist.Preferably show the molecule of at least a antagonistic properties, (for example blocking-up or part are blocked (1) virus infection to one or more biological characteristicses that described antagonistic properties meaning is their institute's bonded Chemokine Receptors of antagonism, (2) ability chemotaxis, (3) acceptor circulation etc.).Described molecule can work by the acceptor of combination rather than activation chemokine, perhaps can mediate their effect by alternate manner.It is shocking, found that the brachymemma of chemokine C-end residue has many useful unforeseeable characteristics, comprise retentive activity (for example above-mentioned activity) and other characteristic, comprise and reduce the aggregation tendency of chemokine when concentration increases.

I. synthetic chemokines

A. synthetic chemokines of the present invention

The preferred synthetic chemokines molecule of the present invention comprises the chemokine polypeptides chain with N-end and C-end, wherein said chemokine polypeptides chain comprises (i) aminoacid sequence and halfcystine pattern and the (ii) end of the C-with respect to wild-type chemokine brachymemma corresponding to the wild-type chemokine.The N-end comprises the amino acid of chemokine polypeptides chain, and described amino acid is the N end that forms the halfcystine of first disulfide linkage in the chemokine polypeptides chain.The C-end comprises the amino acid of chemokine polypeptides chain, and described amino acid is the C end that forms the halfcystine of last disulfide linkage in the chemokine polypeptides chain.

The brachymemma of C-end is the C-end of the distinctive core helical region of chemokine preferably." core helical region " is meant the C end of the halfcystine that is arranged in last disulfide linkage of chemokine polypeptides chain formation and can forms the chemokine polypeptides chain residue of alpha-helix.This comprises the C-that dangles end residue and the adjacent residues thereof that has free α-carboxylicesters.The important second structure characteristic of all chemokines is antiparallels, and it constitutes the βZhe Die bottom, and C-end alpha-helix is arranged on it.Therefore C-end alpha-helix is the consistent features of chemokine, this feature is identified easily, for example by homology modeling and comparison, for example compare by primary sequence and/or three-dimensional structure with known chemokine, perhaps by molecular replacement and energy minimization algorithm predicts structure (referring to for example Cytokine Reference, the 1st volume, Ligands, Acompendium of cytokines and other mediators of host defense, J.J.Oppendheim and M.Feldmann write, Acedemic Press, 2001)).

" chemokine polypeptides chain " is meant and the basic homologous polypeptide chain of naturally occurring wild-type chemokine polypeptides chain.Chemokine polypeptides chain, n terminal amino acid sequence, C-terminal amino acid sequence and form first and the halfcystine of last disulfide linkage constitutes the basis of synthetic chemokines of the present invention, can be according to the corresponding aminoacid sequence of naturally occurring chemokine, and, easily derive synthetic chemokines of the present invention by carrying out homology modeling (for example the aminoacid sequence with known C, CC, CXC and CXXXC chemokine compares) with similar other chemokine." n terminal amino acid sequence " is meant the aminoacid sequence of chemokine polypeptides chain, and it is adjacent and be its N end to form the halfcystine of first disulfide linkage in described aminoacid sequence and the naturally occurring chemokine polypeptides chain." C-terminal amino acid sequence " is meant the aminoacid sequence of chemokine polypeptides chain, and it is adjacent and be its C end to form the halfcystine of last disulfide linkage in described aminoacid sequence and the naturally occurring chemokine polypeptides chain.

Table 1: the universal architecture motif and the classification of chemokine ^*

Classification	The N-end	Cys	The N-ring	Cys	The variable region	Cys	The C-end
								CC CXC CX 3C XC	r1 r1 r1 r1	CC CXC CXXXC XC	r2；r3； r4 r2；r3； r4 r2；r3； r4 r2；r3； r4	C C C X	r2；r3； r4 r2；r3； r4 r2；r3； r4 r2；r3； r4	C C C C	r2；r3；r4； r5；r6 r2；r3；r4； r5；r6 r2；r3；r4； r5；r6 r2；r3；r4； r5；r6

^*Keyword: N-end=N-petiolarea; C-end=C-petiolarea; Cys=halfcystine pattern, wherein C is a halfcystine, X is a variable amino acid; N-ring=ring district; R1=pharmacophore district; R2=receptor-specific district; The r3=GAG land; R4=oligomerization district; R5=core helical region; R6=C-holds tail.

Be the example of known naturally occurring " wild-type " chemokine below, wherein many are because used different titles to be described, so occur repeatedly: 6Ckine, 9E3, ATAC, ABCD-1, ACT-2, ALP, AMAC-1, AMCF-1, AMCF-2, AIF, ANAP, Angie, β-R1, β-thromboglobulin (β-Thromboglobulin), BCA-1, BLC, the blr-1 part, BRAK, C10, CCF18, Ck-β-6, Ck-β-8, Ck-β-8-1, Ck-β-10, Ck-β-11, cCAF, CEF-4, CINC, C7, CKA-3, CRG-2, CRG-10, CTAP-3, DC-CK1, ELC, eosinophilic granulocyte chemotactic protein (Eotaxin), eosinophilic granulocyte chemotactic protein-2, Exodus-1, Exodus-2, ECIP-1, ENA-78, EDNAP, ENAP, FIC, FDNCF, FINAP, CX ₃C chemokine (Fractalkine), G26, GDCF, GOS-19-1, GOS-19-2, GOS-19-3, GCF, GCP-2, the GCP-2-sample, GRO1, GRO2, GRO3, GRO-α, GRO-β, GRO-γ, H400, HC-11, HC-14, HC-21, HCC-1, HCC-2, HCC-3, HCC-4 H174, in the heparin and albumen, Humig, I-309, ILINCK, I-TAC, Ifi10, IL8, IP-9, IP-10, IRH, JE, KC, lymphocyte chemotactic protein (lymphotactin), L2G25B, LAG-1, LARC, LCC-1, LD78-α, LD78-β, LD78-γ, LDCF, LEC, Lkn-1, LMC, LAI, LCF, LA-PF4, LDGF, LDNAP, LIF, LIX, LUCT, Lungkine, LYNAP, the Manchester inhibitor, MARC, MCAF, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MDC, MIP-1-α, MIP-1-β, MIP-1-δ, MIP-1-γ, MIP-3, MIP-3-α, MIP-3-β, MIP-4, MIP-5, Monotactin-1, MPIF-1, MPIF-2, MRP-1, MRP-2, M119, MDNAP, MDNCF, megakaryocyte stimulating factor, MGSA, Mig, MIP-2, mob-1, MOC, MONAP, NC28, NCC-1, NCC-2, NCC-3, NCC-4, N51, NAF, NAP-1, NAP-2, NAP-3, NAP-4, NAP S, NCF, NCP, neural chemokine, oncostatin A, P16, P500, PARC, pAT464, pAT744, PBP, the PBP sample, PBSF, PF4, the PF4 sample, PF4-ALT, PF4V1, PLF, PPBP, RANTES, SCM-1-α, SCI, SCYA26, SLC, SMC-CF, ST38, STCP-1, SDF-1-α, SDF-1-β, TARC, TCA-3, TCA-4, TDCF, TECK, TSG-8, TY5, TCF, TLSF-α, TLSF-β, TPAR-1, TSG-1.

The non-limiting purpose for illustrative, some example of wild-type chemokine polypeptides chain listed above and corresponding N-thereof end, N-ring and C-terminal amino acid sequence see Table 1 and table 2.In table 2, adopt standard single-letter amino acid code.Be appreciated that, other chemokine polypeptides chain is known, can derive from many different sourcess, comprise the enterable database of the public, for example Genome Database (Johns Hopkins University, Maryland USA), Protein Data Bank (Brookhaven National Laboratory ﹠amp; RutgersUniversity, New Jersey USA), Entrez (National Institutes of Health, Maryland USA), NRL 3D (Pittsburgh Supercomputing Center, CamegieMellon University, Pennsylvania USA), CATH (University CollegeLondon, London, UK), NIH Gopher Server (NIH, Maryland USA), ProLink (Boston University, Massachusetts USA), The Nucleic AcidDatabase (Rutgers University, New Jersey USA), Genebank (NationalLibrary of Medicine, Maryland USA), Expasy (Swiss Institute ofBioinformatics, Geneva Switzerland) etc.In addition, can by homology and pattern match, identify new chemokine according to standard technique known in the art (comprising database and the related tool that reaches this purpose) from different genes and protein sequencing program etc.

Table 2: the aminoacid sequence of exemplary human chemokine

Rantes

SPYSSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV

FVTRKNRQVC ANPEKKWVRE YINSLEMS(SEQ ID NO：3)

MIP-1-β

APMGSDPPTA CCFSYTLRKL PRHFVIDYFE TTSLCSQPAV

VFQTKKGRQV CANPSESWVQ EYVDDLELN(SEQ ID NO：4)

vMIP-II

GDTLGASWHR PDKCCLGYQK RPLPQVLLSS WYPTSQLCSK

PGVIFLTKRG RQVCADKSKD WVKKLMQQLP VTAR(SEQ ID NO：5)

SCM-1

GSEVSDKRTC VSLTTQRLPV SRIKTYTITE GSLRAVIFIT

KRGLKVCADP QATWVRDVVR SMDRKSNTRN NMIQTKPTGT

QQSTNTAVTL TG(SEQ ID NO：6)

Eotaxin

GPASVPTTCC FNLANRKIPL QRLESYRRIT SGKCPQKAVI

FKTKLAKDIC ADPKKKWVQD SMKYLDQKSP TPKP(SEQ ID NO：7)

1309

KSMQVPFSRC CFSFAEQEIP LRAILCYRNT SSICSNEGLI

FKLKRGKEAC ALDTVGWVQR HRKMLRHCPS KRK(SEQ ID NO：8)

MCP-1

QPDAINAPVT CCYNFTNRKI SVQRLASYRR ITSSKCPKEA

VIFKTIVAKE ICADPKQKWV QDSMDHLDKQ TQTPKT(SEQ IDNO：9)

MCP-3

QPVGINTSTT CCYRFINKKI PKQRLESYRR TTSSHCPREA

VIFKTKLDKE ICADPTQKWV QDFMKHLDKK TQTPKL(SEQ IDNO：10)

mMCP-5

GPDAVSTPVT CCYNVVKQKI HVRKLKSYRR ITSSQCPREA

VIFRTILDKE ICADPKEKWV KNSINHLDKT SQTFILEPSC LG(SEQ ID NO：11)

MIP-1α(CCL3)

SLAADTPTAC CFSYTSRQIP QNFIADYFET SSQCSKPGVI

FLTKRSRQVC ADPSEEWVQK YVSDLELSA(SEQ ID NO：12)

MIP-3α

ASNFDCCLGY TDRILHPKFI VGFTRQLANE GCDINAIIFH

TKKKLSVCAN PKQTWVKYIV RLLSKKVKNM(SEQ ID NO：13)

MIP-3β

GTN DAEDCCL SVTQKPIPGY IVRNFHYLLI KDGCRVPAVV

FTTLRGRQLC APPDQPWVER IIQRLQRTSA KMKRRSS(SEQ ID No：14)

MIP-5(CCL15)

QFINDAETEL MMSKLPLENP VVLNSFHFAA DCCTSYISQS

IPCSLMKSYF ETSSECSKPG VIFLTKKGRQ VCAKPSGPGV

QDCMKKLKPY SI(SEQ ID NO：15)

MPIF-1

RVTKDAETEF MMSKLPLENP VLLDRFHATS ADCCISYTPR

SIPCSLLESY FETNSECSKP GVIFLTKKGR RFCANPSDKQ

VQVCMRMLKL DTRIKTRKN(SEQ ID NO：16)

LEC

QPKVPEWVNT PSTCCLKYYE KVLPRRLVVG YRKALNCHLP

AIIFVTKRNR EVCTNPNDDW VQEYIKDPNL PLLPTRNLST

VKIITAKNGQ PQLLNSQ(SEQ ID NO：17)

HCC

TKTESSSRGP YHPSECCFTY TTYKIPRQRI MDYYETNSQC

SKPGIVFITK RGHSVCTNPS DKWVQDYIKD MKEN(SEQ ID NO：18)

SLC

SDGGAQDCCL KYSQRKIPAK VVRSYRKQEP SLGCSI PAIL

FLPRKRSQAE LCADPKELWV QQLMQHLDKT PSPQKPAQGC

RKDRGASKTG KKGKGSKGCK RTERSQTPKG P(SEQ ID NO：19)

MDC

GPYGANMEDS VCCRDYVRYR LPLRVVKHFY WTSDSCPRPG

VVLLTFRDKEICADPRVPWV KMILNKLSQ(SEQ ID NO：20)

TARC

ARGTNVGREC CLEYFKGAIP LRKLKTWYQT SEDCSRDAIV

FVTVQGRAIC SDPNNKRVKN AVKYLQSLER S(SEQ ID NO：21)

TECK

QGVFEDCCLA YHYPIGWAVL RRAWTYRIQE VSGSCNLPAA

IFYLPKRHRK VCGNPKSREV QRAMKLLDAR NKVFAKLHHN

MQTFQAGPHA VKKLSSGNSK LSSSKFSNPI SSSKRNVSLL

ISANSGL(SEQ ID NO：22)

SDF1α

KPVSLSYRCP CRFFESHVAR ANVKHLKILN TPACALQIVA

RLKNNNRQVC IDPKLKWIQE YLEKALN(SEQ ID NO：23)

IP-10

VPLSRTVRCT CISISNQPVN PRSLEKLEII PASQFCPRVE

IIATMKKKGE KRCLNPESKA IKNLLKAVSK EMSKRSP(SEQ IDNo：24)

IL-8

AVLPRSAKEL RCQCIKTYSK PFHPKFIKEL RVIESGPHCA

NTEIIVKLSD GRELCLDPKE NWVQRVVEKF LKRAENS(SEQ IDNO：25)

MIG

TPVVRKGRCS CISTNQGTIH LQSLKDLKQF APSPSCEKIE

IIATLKNGVQ TCLNPDSADV KELIKKWEKQ VSQKKKQKNG

KKHQKKKVLK VRKSQRSRQK KTT(SEQ ID NO：26)

GCP-2

GPVSAVLTEL RCTCLRVTLR VNPKTIGKLQ VFPAGPQCSK

VEVVASLKNG KQVCLDPEAP FLKKVIQKIL DSGNKKN(SEQ IDNO：27)

GROα

ASVATELRCQ CLQTLQGIHP KNIQSVNVKS PGPHCAQTEV

IATLKNGRKA CLNPASPIVK KIIEKMLNSD KSN(SEQ ID NO：28)

GROβ

APLATELRCQ CLQTLQGIHL KNIQSVKVKS PGPHCAQTEV

IATLKNGQKA CLNPASPMVK KIIEKMLKNG KSN(SEQ ID NO：29)

GROγ

ASVVTELRCQ CLQTLQGIHL KNIQSVNVRS PGPHCAQTEV

IATLKNGKKA CLNPAS PMVQ KIIEKILNKG STN(SEQ ID NO：30)

FK

QHHGVTKCNI TCSKMTSKIP VALLIHYQQN QASCGKRAII

LETRQHRLFC ADPKEQWVKD AMQHLDRQAA ALTRNG(SEQ IDNO：31)

Especially valuable is the synthetic chemokines with the brachymemma of C-end, and described chemokine has been eliminated one or more amino-acid residues of coaggregation sites with respect to corresponding wild-type chemokine." coaggregation sites " is meant the residue that causes the protein monomers self-association.Most of chemokines all have the potentiality that form homodimer, manyly also can form the tetramer, even bigger polymer (Cytokine Reference, the 1st volume, Ligands, Acompendium of cytokines and other mediators of host defnse, J.J.Oppendheim and M.Feldmann write, Acedemic Press, 2001).Usually under high density, can form in the chemokine of dimer and multimeric complexes, can find coaggregation sites (the Cytokine Reference of chemokine, the 1st volume, Ligands, Acompendium of cytokines and other mediators of host defense, J.J.Oppendheim and M.Feldmann write, Acedemic Press, 2001).For example, Rantes and MIP1 β are the Classical examples that forms the chemokine of aggregate by monomer self-association under the high density, and chemokines such as IL-8, SDF1 α and vMIP then do not have such trend on significance degree.

One of discovery of the present invention is: be positioned at the C-end, participate in the one or more residues of accumulative normally polar residues or charged residue usually, these residues can be eliminated by brachymemma, do not lose the biological activity of wanting simultaneously again.Illustrative purposes can be passed through aforesaid different technologies for example, identifies coaggregation sites in a large amount of chemokines.For example, wild-type Rantes has two at least and participates in the accumulative residues: 26 and 66 s' L-glutamic acid (is Glu26 and Glu66; Or E26 and E66), wherein E66 is positioned at the C-end of this molecule.As another example, chemokine MIP1 α has two at least and participates in the accumulative residue: D26 and E66.Equally, MIP1 β all has two main coaggregation siteses on D27 and E67 position.In another example, MCP-1 and eosinophilic granulocyte chemotactic protein have the accumulative of participation residue: residue P8 and the D68 of MCP-1; The D66 of eosinophilic granulocyte chemotactic protein.In addition, at least one participation accumulative residue is positioned at the C-end.Compare these chemokines and other chemokine, further disclose coaggregation sites and be usually located at C-and hold core helical region, for example E66 of Rantes and M67, the E66 of MIP1 α to C-end, the E67 of MIP1 β, the D66 of the D68 of MCP-1 or eosinophilic granulocyte chemotactic protein.

Therefore, in a preferred embodiment, synthetic chemokines of the present invention has the brachymemma of C-end, and this brachymemma is the C-end core helical region of corresponding wild-type chemokine.As mentioned above, making us interested residue in the chemokine oligomerization especially is polar residues or charged residue, and described residue is abundant at chemokine C-petiolarea content.Therefore, preferred synthetic chemokines of the present invention comprises the brachymemma of C-end, this brachymemma comprises the one or more disappearances with amino-acid residue of polarity or electrically charged side chain with respect to described wild-type chemokine, for example arginine, Methionin, aspartic acid and L-glutamic acid.Synthetic chemokines more preferably of the present invention comprises the brachymemma of the C-end core helical region of corresponding wild-type chemokine, and wherein the brachymemma of C-end comprises the one or more disappearances with amino-acid residue of polarity or electrically charged side chain with respect to described wild-type chemokine.

An advantage of described C-end brachymemma is: the gained synthetic chemokines that can prepare pure substantially single oligomer form.Having been found that the brachymemma of one or more amino-acid residues of corresponding wild-type chemokine C-end coaggregation sites, is enough for eliminating gathering basically.Described molecule has significantly improved purifying and handling property, especially under the situation that purified product concentration increases.Therefore, the invention still further relates to the synthetic chemokines with the brachymemma of C-end, described chemokine is the oligomer state of being made up of monomer or dimer basically.

In a preferred embodiment, the composition that comprises the synthetic chemokines with the brachymemma of C-end is provided, wherein said synthetic chemokines is the oligomer state of being made up of monomer or dimer basically, and the maximum non-aggregate concentration of wherein said synthetic chemokines is greater than corresponding wild-type chemokine.For example, the common concentration range of synthetic chemokines of the present invention for example at the most approximately every milliliter 20 milligrams (mg) (20mg/ml) keep the oligomer state form by monomer or dimer basically simultaneously.More preferably the concentration range of synthetic chemokines of the present invention is about 0.5mg/ml to 15mg/ml, and most preferably from about 1mg/ml is to about 10mg/ml.Certainly, measure the actual concentrations scope of the given synthetic chemokines of the present invention under the certain condition easily, for example,, easily determine to keep the maximum tolerated concentration of single substantially oligomer form by handling process for preparation.

For other chemokine, can identify coaggregation sites by various well-known methods, comprise C-end coaggregation sites, for example use various techniques known in the art to carry out homology modeling, L-Ala scanning and the active compound that increases concentration in the solution is compared and monitor gathering, self-association.(referring to for example Czaplewski etc., J.Biol.Chem. (1999) 274 (23): 16077-16084; Czaplewski etc., " Engineering; Biology; andClinical Development of hMIP-1 α; " (1999) be stated from: Chemokines in Disease:Biology and Clinical Research, CA.Herbert writes, Humana Press Inc., Totwa, NJ; Trkola etc., J.Virol. (1999) 73 (8): 6370-6379; Appay etc., J.Biol.Chem. (1999) 274 (39): 27505-27512; Hunter etc., Blood (1995) 86 (12): 4400-4408; Lord etc., Blood (1995) 85 (12): 3412-3415; Lord etc., Brit.J.Cancer (1996) 74:1017-1022).As mentioned above, the coaggregation sites of many chemokines is known, the technology that coaggregation sites is inferred in evaluation also is well-known (in addition referring to CytokineReference, the 1st volume, Ligands, A compendium of cytokines and othermediators of host defense, J.J.Oppendheim and M.Feldmann write, Acedemic Press, 2001).Therefore, can by screening or combination separately, identify the coaggregation sites of chemokine according to disclosed information, homology modeling.Therefore the example that this paper provided is an illustrative example of the present invention, rather than limitation of the present invention.

Synthesis of biologically active chemokine of the present invention also preferably comprises one or more modifications, described modification is given the inhibition of wanting or is promoted the active characteristic of corresponding wild-type chemokine, promptly measure, for example be used for the mensuration of receptors bind, competition, calcium flux, map kinase, chemotaxis, downward modulation, virus infection etc. by suitable Chemokine Receptors.It is terminal modified and/or C-is terminal modified and/or the modification in connecting the zone of N-petiolarea and C-petiolarea, for example covalently bound polymkeric substance that preferred described molecule has N-.More preferably described synthetic chemokines comprises that one or more N-are terminal modified and C-is terminal modified, most preferably in N-end, C-end and the modification in connection N-end and C-petiolarea.

Specifically, synthetic chemokines of the present invention can comprise the chemokine polypeptides chain with C-end brachymemma and one or more other amino-acid residues, and this amino-acid residue is different from the amino-acid residue of corresponding position in the wild-type chemokine.This comprises N-end capping group, C-end capping group and interior finishing.For example, the C-end can be by the amino acid end-blocking of formula-NH-CH (R)-C (O)-Z, wherein R be with the wild-type chemokine in the identical or different amino acid side chain of amino acid side chain of corresponding position, Z is that C-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.) or polymkeric substance connect residue.For example, preferred C-end capping group has formula-NH-CH (R)-C (O)-NH ₂But, have the amino advantage of C-end that the stabilizing local hydrogen bond is provided, and therefore make whole protein stabilization.Other example of capping group comprise dyestuff, fluorescent mark, small-molecule drug, purifying or jointing (for example Histidine polymkeric substance (His-tag)), with sero-abluminous fusions, antibody or antibody fragment fusion constructs, aliphatic chain, water-soluble polymers etc.Other example comprises end capped brachymemma C-end, this end-blocking is to come end capped with being less than complete amino acid complement, this amino acid complement is from the C-terminal amino acid sequence that is removed with respect to corresponding wild-type chemokine, and the result produces for example synthetic chemokines that has C-terminal amino acid sequence frameshit or one or more residue disappearance with respect to corresponding wild-type chemokine.Equally, provide and have brachymemma of C-end and N-end capping group or have N-end capping group simultaneously and the synthetic chemokines of C-end capping group.Therefore the end-blocking type can be the target synthetic chemokines provides advantages characteristic.

In a preferred embodiment, synthetic chemokines of the present invention can comprise the chemokine polypeptides chain with other amino-acid residue that the brachymemma of C-end and the chemical adducts of one or more quilt modify.What is particularly worth mentioning is that chemical adducts comprises small molecules and polymkeric substance etc., for example dyestuff, medicine, lipid, carbohydrate, nucleic acid, detectable label, metal chelator, toxin, aliphatic chain and polymkeric substance, especially water-soluble polymers.

In a valuable especially embodiment, provide the synthetic chemokines that comprises the small molecules adducts.Described small molecules adducts preferably is connected with the N-end and/or the C-end of target synthetic chemokines.Described small molecules adducts comprises the group that can form covalent linkage with the N-end or the C-end of amino-acid residue or derivative amino residue.This connection can be undertaken by any amount of covalent linkage, includes but not limited to acid amides, amino, ester, thioesters, selenium ester, ether, thioether, selenide, oxime, Schiff's base (non-reduced type or reduced form) etc.Described group can help to protect N-end and/or C-end not by the proteolysis enzyme liberating, and/or helps protection stability.They also help to improve synthetic and handling property (being included in the solubleness in the aqueous solution) and help the specific administration approach of pharmaceutical preparation and be used to have the patient of needs.Also can use chemical adducts as short capping group.Example includes but not limited to capping group and other amino acid capping groups such as acyl group, acid amides; and methylglycine (trimethyl-glycine), N-methylsarcosine and other irregular amino acid analogue; and acids (for example succsinic acid or other class acidoid), this depends on the end-use of expection.

In a preferred embodiment, synthetic chemokines of the present invention comprises chemokine polypeptides, and described polypeptide has the brachymemma of (i) C-end; The (ii) one or more chemical adducts that is connected with chemokine polypeptides chain amino acid residue.Described molecule preferably has chemical adducts, comprises with N-end, C-to hold the aliphatic chain that is connected or connects simultaneously the two.More preferably described synthetic chemokines also comprises one or more amino acid derivative of one or more (iii) N-ends; The (iv) polymkeric substance that is connected with the chemokine polypeptides chain.

In one embodiment, the chemokine polypeptides chain of the synthetic chemokines that C-of the present invention is terminal modified also is included in the chemokine that its N-end and/or C-end are modified by aliphatic chain or many rings (preferred hydrophobicity aliphatic chain), for example referring to Koechendoerfer etc., WO 02/04015 and Offord etc., WO 02/04499.In brief, suitable hydrophobicity aliphatic chain includes but not limited to that length is the hydrophobicity aliphatic chains of 5 carbon (C5) to 22 carbon (C22).This chain can be unsaturated and/or not branched, perhaps can have saturated and/or branch in various degree.The hydrophobicity aliphatic chain has general formula C _n(R _m)-, be C wherein _nBe the number of carbon, R _mBe the substituent number that is selected from hydrogen, alkyl, acyl group, aryl or its combination, n and m can be identical or different.

The hydrophobicity aliphatic chain is connected with the chemokine polypeptides chain by any suitable covalent linkage.The example of suitable covalent linkage includes but not limited to: acid amides, ketone, aldehyde, ester, ether, thioether, thioesters, thiozolidine, oxime, oxizolidine, Schiff's base and schiff's base type key (for example hydrazides).The chemistry that is suitable for bonding system is well-known, and (referring to for example Rose etc., WO 94/25071 to can be used for this purpose; " Chemistry of Protein Conjugation and Cross-Linking ", S.S.Wong writes, CRC Press, Inc. (1993); Perspectives in BioconjugateChemistry, Claude F.Modres writes, ACS (1993)).Can be different fully with the bonding unit that hydrocarbon chain and chemokine polypeptides chain link together, precondition is that the total length of N-petiolarea and filling space and naturally occurring chemokine are approximate.Have been found that the C-petiolarea is more flexible on the one hand at this, so total length can be different with the N-petiolarea largely with the filling space.

In a preferred embodiment, the hydrophobicity aliphatic chain that is connected with the N-end is that length is the hydrocarbon chains of 5 carbon (C5) to 10 carbon (C10), and the hydrophobicity aliphatic chain that is connected with the C-end is that length is the lipids of 12 carbon (C12) to 20 carbon (C20).The example of C5-C10 hydrocarbon chain includes but not limited to :-C ₅H ₁₁,-C ₅H ₉,-C ₅H ₇,-C ₅H ₅,-C ₅H ₃,-C ₆H ₁₃,-C ₆H ₁₁,-C ₆H ₉,-C ₆H ₇,-C ₆H ₅,-C ₆H ₃,-C ₇H ₁₅,-C ₇H ₁₃,-C ₇H ₁₁,-C ₇H ₉,-C ₇H ₇,-C ₇H ₅,-C ₇H ₃,-C ₈H ₁₇,-C ₈H ₁₅,-C ₈H ₁₃,-C ₈H ₁₁,-C ₈H ₉,-C ₈H ₇,-C ₈H ₅,-C ₈H ₃,-C ₉H ₁₉, C ₉H ₁₇,-C ₉H ₁₅,-C ₉H ₁₃,-C ₉H ₁₁,-C ₉H ₉,-C ₉H ₇,-C ₉H ₅,-C ₉H ₃,-C ₁₀H ₂₁,-C ₁₀H ₁₉, C ₁₀H ₁₇,-C ₁₀H ₁₅,-C ₁₀H ₁₃,-C ₁₀H ₁₁,-C ₁₀H ₉,-C ₁₀H ₇,-C ₁₀H ₅With-C ₁₀H ₃Suitable lipid includes but not limited to lipid and many ring steroidal deutero-lipids of fatty acid derived.Lipid acid includes but not limited to saturated and unsaturated fatty acids.The example of saturated fatty acid is lauric acid (C12), tetradecanoic acid (C14), palmitinic acid (C16), stearic acid (C18) and eicosanoic acid (C20).The example of unsaturated fatty acids comprises that oleic acid (C18), linolic acid (C18), linolenic acid (C18), eleostearic acid (eleosteric acid) are (C18) and arachidonic acid (C20).Many lopps include but not limited to: aldosterone, Dihydrocholesterol, cholesterol, cholic acid, stercorin, corticosterone, cortisone, dehydrocholesterol, desmosterol, digitogenin, ergosterol, estradiol, hydroxycorticosterone (hydoxycorticosterone), lathosterol, prednisone, pregnenolone, Progesterone, testosterone, zymosterol etc.In general, lipid acid is connected with the chemokine polypeptides chain by acid constituents usually, therefore obtains the acyl group connection portion, though also can adopt other key.

In another preferred embodiment; the hydrophobicity aliphatic chain that comprises in the biological activity synthetic chemokines of the present invention comprises the saturated or unsaturated acyl group chain of C5-C20, for example nonanoyl, nonene acyl group, amino oxygen base pentane, lauroyl, myristoyl, cetylate, lauryl, palmitoyl, eicosane acyl group, oleoyl or courage acyl group (cholyl).For example, N-end substituting group can be nonanoyl (nonaoyl) or amino oxygen base pentane, and C-end substituting group can be saturated or unsaturated fatty acids, preferred C12-C20 lipid acid, or encircle the steroidal lipid, for example cholesterol more.

As mentioned above, synthetic chemokines of the present invention can comprise other amino acid or add other parts on the polypeptide chain to." amino acid " or " amino-acid residue " be meant the amino acid that comprises 20 kinds of genetic codings, at nature the rare or amino acid that exists of common amino acid and any non-natural not, for example irregular amino acid; When under peptide, polypeptide or proteinic situation, be meant amino-acid residue sometimes." amino acid derivative " is meant the derivative of amino acid or amino acid sample chemical entities.

Specifically, synthetic chemokines of the present invention can comprise one or more amino acid derivative, for example referring to Koechendoerfer etc., WO 02/04015 and Offord etc., WO02/04499.The preferred amino acids derivative has formula-(N-C _nR-CO)-, C wherein _nBe 1-22 carbon, R is hydrogen, alkyl or aryl, and wherein N and C _n, N and R or C _nCan constitute ring texture with R.In addition, N, C _nCan have one or more hydrogen with R separately in its reduction form, this depends on amino acid derivative.Moieties can be that replace or unsubstituted, and it can be straight chain, side chain or ring-type, and can comprise one or more heteroatomss.Aryl can be that replace or unsubstituted, and comprises one or more heteroatomss.Amino acid derivative can be from the beginning prepared or commercial source can be derived from (referring to for example Calbiochem-Novabiochem AG, Switzerland; Advanced Chemtech, Louisville, KY, USA; Lancaster Sythesis, Inc., Windham, NH, USA; Bachem California, Inc., Torrance, CA, USA; Genzyme Corp., Cambridge, MA, USA).The example of described amino acid derivative includes but not limited to aminoisobutyric acid (Aib), oxyproline (Hyp), 1,2,3,4-tetrahydroisoquinoline-3-COOH (Tic), indoline-2-formic acid (indol), (P (4 for 4-two fluoro-proline(Pro), 4DiF)), L-thiazolidine-4-formic acid (Thz), the high proline(Pro) of L-(HoP), 3,4-dehydrogenation-proline(Pro) (Δ Pro), 3, (F (3 for the 4-dopa, 4-DiOH)), pBzl,-3, (F (3 for the 4-dopa, 4-DiOH, pBzl)), benzophenone (p-Bz), cyclohexyl-L-Ala (Cha), 3-(2-naphthyl)-L-Ala (β Nal), cyclohexyl-glycine (Chg) and phenylglycocoll (Phg).

In another embodiment, synthetic bioactive protein of the present invention can contain " irregular " amino-acid residue.Term used herein " irregular " amino-acid residue is meant the amino acid of can't help the RNA coding and not assembling at rrna.At this on the one hand, the present invention allows selectivity and handiness widely in design and/or structure synthetic bioactive protein.Can be used for the amino acid whose example that does not assemble at rrna of the present invention comprises: amino acid (R.Simon etc., Proc.Natl.Acad.Sci.U.S.A. (1992) 89:9367-71 that D-amino acid, beta-amino acids, vacation-L-glutamic acid, γ-An Jidingsuan, ornithine, homocysteine, N-replace; WO91/19735 (Bartlett etc.), United States Patent (USP) the 5th, 646, No. 285 (Baindur)), alpha-amino group methylene ethoxyacetic acid (structure thing such as amino acid-Gly dipeptides) and alpha-amino group alcohol acid etc.Can adopt the structure thing such as reducing amide of structure things such as containing thioamides, vinylogous amide, diazanyl, methylene radical oxygen base, thio-methylene, phosphonamide, oxyamide, hydroxy ethylene, reducing amide, replacement and the peptide analogs of beta-sulfonamido amine.

In a preferred embodiment, the present invention also provides the synthetic chemokines that comprises chemokine polypeptides, and described polypeptide has the brachymemma of (i) C-end; The (ii) polymkeric substance that is connected with the chemokine polypeptides chain.It is terminal modified and/or C-is terminal modified that more preferably synthetic chemokines also comprises one or more above-mentioned N-.

As described in WO 02/04014, preferred synthetic chemokines of the present invention has polymkeric substance: C-end site, coaggregation sites, glycosylation site and GAG binding site on one or more residues that are selected from following site." C-holds the site " is meant that chemokine C-holds to the chemokine polypeptides chain residue of C-end alpha-helix." GAG binding site " is meant and is used to encode GAG bonded residue; Normally having the residue (for example Methionin and arginine) of primary amine or secondary amine, is Histidine sometimes, and this Histidine constitutes positive charge bunch at protein surface." glycosylation site " is meant the enzyme bonded residue of coding sugar (oligosaccharides) chain, and for example N-connects the glycosylation site that is connected with O-.Preferred glycosylation site is the site at chemokine C-petiolarea.The glycosylation site that connects of N-most preferably.Glycosylation site can be natural site or the site that adds target protein through through engineering approaches.

Residue in these sites can be used for connecting, and precondition is that they have the side chain (amino acid side chain that promptly has functional group, for example Methionin, aspartic acid, L-glutamic acid, halfcystine, Histidine etc.) that is suitable for the polymkeric substance connection.The different aminoacids displacement that perhaps, can be had the side chain that is suitable for the polymkeric substance connection at the residue in these sites.In addition, the amino acid whose side chain of genetic coding can be used for polymkeric substance through chemically modified and connect, and perhaps can use the alpha-non-natural amino acid that has suitable side chains functional group.The synthetic combination that is connected with chemistry of peptide is adopted in preferred method of attachment.Hold being connected of site for polymkeric substance with C-, be necessary terminal joint or the spacer of adding of C-, for example C-end capping group to the chemokine polypeptides chain of C-end brachymemma.Can be by functional side be provided, be used for C-end coupling with water-soluble polymers and target chemokine, finish this step, thereby influence near original C-end group group function this position or its as small as possible.

As described in WO 02/04014, can finish the precursor synthetic chemokines and be connected and keep the external biological activity with polymkeric substance between the subject polymer in one or more sites.The external biological activity is the good standard of Function of Evaluation, the standard test that is used for each chemokine of this purpose is well-known (referring to for example Cytokine Reference, the 1st volume, Ligands, Acompendium of cytokines and other mediators of host defense, JJ.Oppendheim and M.Feldmann write, Acedemic Press, 2001; CytokineReference, the 2nd volume, Receptors, A compendium of cytokines and othermediators of host defense, J.J.Oppendheim and M.Feldmann write, Acedemic Press, 2001).As mentioned above, preferred connection site is selected from the precursor chemokine residue corresponding to C-end site, coaggregation sites, glycosylation site and GAG binding site.

Polymkeric substance can be connected with the chemokine polypeptides chain by one or more spacers or joint (if present), can comprise Biostatic or Biodegradable polymeric chain or unit.For example, the polymkeric substance that has a repeat key has stability in various degree under physiological condition, and this depends on the unstable of key.Can be according to known lower molecular weight analogue hydrolysis rate, according to the hydrolysis relative rate of the polymkeric substance that has described key under physiological condition, it is classified, and for example stability is from high to low successively: polycarbonate (O-C (O)-O-)＞polyester (C (O)-O-)＞polyurethane(s) (NH-C (O)-O-)＞poe (O-C ((OR) (R '))-O-)＞polymeric amide (C (O)-NH-).Equally, the bonding system that water-soluble polymers is connected with target molecule can be Biostatic or biodegradable, and for example stability is from high to low successively: carbonic ether (O-C (O)-O-)＞ester (C (O)-O-)＞carbamate (NH-C (O)-O-)＞ortho ester (O-C ((OR) (R '))-O-)＞acid amides (C (O)-NH-).These keys are provided by way of example, but are not in order to limit the polymer chain that is used for water-soluble polymers of the present invention or the key type of bonding system.

Polymer-modified synthetic chemokines of the present invention preferably has biocompatible polymkeric substance, and promptly they are nontoxic to biosystem, and many these base polymers are all known.Described polymkeric substance can be a hydrophobicity or hydrophilic, biodegradable, abiotic degradable or its combination.These polymkeric substance include but not limited to natural polymer (for example collagen, gelatin, Mierocrystalline cellulose, hyaluronic acid, polysaccharide and polyamino acid) and synthetic polymer (for example polyester, poe, polyanhydride etc.).The example of the non-degradation polymer of hydrophobicity comprises polydimethylsiloxane, urethane, tetrafluoroethylene, polyethylene, polyvinyl chloride and polymethylmethacrylate.The example of the non-degradation polymer of wetting ability comprises poly-(methacrylic acid 2-hydroxyl ethyl ester), polyvinyl alcohol, poly-(N-V-Pyrol RC), polyalkylene, polyacrylamide and multipolymer thereof.

Preferred polymkeric substance is water miscible." water-soluble polymers " is meant the polymkeric substance that energy is water-soluble, and the atom and molecule amount is greater than about 1,000 dalton (Da).The preferred effective fluid power of this polymkeric substance is learned molecular weight greater than 5,000Da, more preferably from about 10,000-500,000Da, most preferably from about 10,000-300,000Da." effective fluid power molecular weight " is meant the effective water-solvate size of the polymer chain through measuring based on the size exclusion chromatography method (SEC) of water.When water-soluble polymers contains have the polyalkylene oxide repeating unit polymer chain of (for example oxyethane repeating unit), the atom and molecule amount of preferred each chain is between about 200Da and about 80, between the 000Da, preferably between about 1,500Da and about 42 is between the 000Da, most preferably between 2,000Da is to about 40, between the 000Da.Except as otherwise noted, otherwise molecular weight is meant the atom and molecule amount, can be the mean value of targeted species.

Water-soluble copolymer component can have the molecular weight and the polymkeric substance subunit of wide region.These subunits can comprise biological polymer, synthetic polymer or its combination.The example of described water-soluble polymers comprises: dextran and glucan derivative (comprise T 500, amino crosslinked dextrin of P-and carboxyl methyl dextrin), Mierocrystalline cellulose and derivatived cellulose (comprising methylcellulose gum and carboxymethyl cellulose), starch and dextrin and starch derivative and hydroylacte, polyalkylene glycol and derivative thereof (comprise polyoxyethylene glycol, methoxy poly (ethylene glycol), the polyoxyethylene glycol homopolymer, the polypropylene glycol homopolymer, the multipolymer of ethylene glycol and propylene glycol), wherein said homopolymer and multipolymer are unsubstituted or are at one end replaced by following group: alkyl, heparin and heparin fragment, polyvinyl alcohol and polyvinyl ethyl ether, polyvinylpyrrolidone, aspartyl-phenylalanine methyl ester and polyoxyethylene polyvalent alcohol, and dextran and glucan derivative, dextrin and dextrin derivative.In the various derivatives that are appreciated that the water-soluble polymers of specifically enumerating are also included within.

Above-mentioned water-soluble polymers is well-known, especially based on the polymkeric substance of polyalkylene oxide, for example polyoxyethylene glycol " PEG " is (referring to for example " Poly (ethylene glycol) Chemistry:Biotechnical and Biomedical Applications ", J.M.Harris writes, PlenumPress, New York, NY (1992); " Poly (ethylene glycol) Chemistry andBiological Applicatiohs ", J.M.Harris and S.Zalipsky write, ACS (1997); And international patent application: WO 90/13540, WO 92/00748, and WO 92/16555, WO94/04193, WO 94/14758, and WO 94/17039, WO 94/18247, and WO 94/28937, and WO 95/11924, WO 96/00080, and WO 96/23794, and WO 98/07713, WO98/41562, WO 98/48837, and WO 99/30727, WO 99/32134, WO 99/33483, and WO 99/53951, and WO 01/26692, WO 95/13312, WO 96/21469, WO97/03106, WO 99/45964 and U.S. Patent number 4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).

Preferred water-soluble polymers comprises the oxyethane as the continuous repeating unit of following formula :-(CH ₂-CH ₂-O)-.Preferably containing the polymers of ethylene oxide example is polyoxyethylene glycol (" PEG ") and daiamid epoxy ethane, for example is described in respectively hereinafter and WO 00/12587.In brief, polyalkylene oxide and daiamid epoxy alkane can be classical polyoxyethylene glycol (PEG) forms, or mix other polymkeric substance for example in the polymeric amide.For example, preferred polymkeric substance based on polymeric amide is that molecular weight is greater than about 1,000 daltonian formula-[C (O)-X-C (O)-NH-Y-NH] _n-or-[NH-Y-NH-C (O)-X-C (O)] _n-polymeric amide, wherein X and Y are identical or different divalent radicals, they can be side chain or straight chain, and n is 2-100, the discrete integer of 2-50 more preferably, and wherein one of X and Y or both comprise biocompatible, be the water-soluble straight or branched repeating unit of nonantigenic basically.For polymeric amide, most preferred water-soluble repeating unit comprises formula-(CH ₂-CH ₂-O)-or-(CH ₂-CH ₂-O)-oxyethane.The quantity of described water-soluble repeating unit can be obviously different, but more preferably element number is 2-500,2-400,2-300,2-200,2-100, most preferably is 2-50.The example of more preferred is that wherein one of X and Y or both are selected from :-((CH ₂) _N1-(CH ₂-CH ₂-O) _N2-(CH ₂) _N1-)-or-((CH ₂) _N1-(O-CH ₂-CH ₂) _N2-(CH ₂) _N1-), wherein n1 is 1-6,1-5,1-4,1-3 most preferably, and wherein n2 is 2-50,2-25,2-15,2-10,2-8, most preferably is 2-5.The example of highly preferred embodiment is that wherein X is-(CH ₂-CH ₂)-, and wherein Y be-(CH ₂-(CH ₂-CH ₂-O) ₃-CH ₂-CH ₂-CH ₂)-or-(CH ₂-CH ₂-CH ₂-(O-CH ₂-CH ₂) ₃-CH ₂)-.

For example, the chain based on PEG is amphipathic, non-immunogenic and insensitive to the proteolytic ferment cutting.Preparation has reduced immunogenicity and antigenicity with PEG or the material modified based on the chain of PEG.Referring to for example Abuchowski etc., J.Biol.Chem. (1977) 252 (11): 3578-3581; Tsutsumi etc., Jpn.J.Cancer Res. (1994) 85:9-12; Poly (ethylene glycol) Chemistry and Biological Applications, ACSSymposium Series 680, J.M.Harris and S.Zalipsky write, AmericanChemical Society, 1997; Poly (ethylene glycol) Chemistry, Biotechnicaland Biomedical Applications, Topics in Applied Chemistry, J.M.Harris writes, Plenum Press, New York, NY, 1992).PEG also is used to increase the molecular size of the material that it connects, thereby increases its biological halflife.These advantageous feature of the material that PEG modifies make them very useful on various therapeutic are used.Therefore, the present invention also comprises the pharmacokinetics of improving polypeptide of the present invention, promptly by on such site polypeptide being modified or " PEGization ", can allow protein to keep its inherent biological activity.

Be grafted on the protein with the PEG chain or based on the PEG chain, this is known.Referring to for example WO 00/12587.In addition referring to for example Zalipsky, United States Patent (USP) the 5th, 122, No. 614, this patent has been put down in writing PEG has been transformed on its N-succinimdyl carbonate derivative.Also known with active group modification PEG chain, so that to the protein grafting.Referring to for example Harris, United States Patent (USP) the 5th, 739, No. 208, this patent has been put down in writing the derivative by sulfone part activatory PEG, is used for selectivity link molecule and lip-deep mercaptan (thiol) part; And Harris etc., United States Patent (USP) the 5th, 672, No. 662, this patent disclosure have an active ester of the PEG acid of single propionic acid or butyric acid part.There is detailed summary: Zalipsky in this field in following document, Bioconjugate Chemistry (1995) 6:150-165.Except using PEG, Wright (EP 0 605 963 A2) has also introduced connection reagent, and described reagent contains the water-soluble polymers that can form the hydrazone key with proteinic aldehyde radical.Above-mentioned all reference all are attached to herein by reference.

Being appreciated that provides synthetic chemokines, and described chemokine can be from these assemblies one or its combination of (character and the selection that are polymkeric substance connection site, polymkeric substance are used for polymer-modified precursor chemokine).The polymkeric substance connection site also can be overlapping, and perhaps on same site, for example coaggregation sites and GAG site are adjacent or are positioned at same site.In addition, provide and have two above polymkeric substance and connect thereon synthetic chemokines.For example, the present invention also comprises the biological activity synthetic chemokines, this chemokine comprises chemokine polypeptides chain and the water-soluble polymers that is connected with second site in first site thereon, and wherein first site is selected from the GAG site, and second site is selected from coaggregation sites and C-end site.Of the present invention this especially allows people to increase molecular weight and water-soluble (therefore improving other desired characteristic that circulation half life and water-soluble polymers are provided) on the one hand, eliminate simultaneously unwanted characteristic again, for example assemble, particularly the GAG bond type on the polymkeric substance connection site.

In a preferred embodiment, provide the synthetic chemokines with the brachymemma of C-end and water-soluble polymers, described polymkeric substance is connected on the chemokine polypeptides chain amino acid position with respect to the GAG binding site of corresponding wild-type chemokine.For illustrative purposes, wild-type Rantes has two main GAG binding sites at least, and described site comprises corresponding to the amino acid that is selected from following Rantes residue: Lys44, Lys45, Arg47, Lys55, Lys56 and Arg59 (being K44, K45, R47, K55, K56 and R59).Therefore, when synthetic chemokines is Rantes analogue and water-soluble polymers when being connected on its GAG binding site, the GAG binding site comprises corresponding to the amino acid that is selected from following Rantes residue: K44, K45, R47, K55, K56 and R59.In a preferred embodiment, water-soluble polymers is connected corresponding on the position that is selected from following Rantes residue: K44, K45, R47, wherein more preferably K45 position.In this embodiment, being connected 45 polymkeric substance has and keeps basically again when reducing synthetic Rantes chemokine analogue with the CCR1 receptors bind and the bonded benefit of CCR5.

As another example, when synthetic chemokines is the analogue of MIP1 α and water-soluble polymers when being connected on its GAG binding site, the GAG binding site comprises corresponding to the amino acid that is selected from following MIP1 α residue: R17, R45 and R47.Equally, when synthetic chemokines is the analogue of MIP1 β and water-soluble polymers when being connected on its GAG binding site, described coaggregation sites can comprise corresponding to the amino acid that is selected from the MIP1 β residue of upper/lower positions: R18, R45 and R46.In these two examples, the site that preferably is used for the polymkeric substance connection is at the R45 of MIP1-α and the R46 of MIP1 β.For Rantes, design these and modify to be partial to that the chemokine analogue is combined with CCR5.

In another example, when synthetic chemokines is the analogue of SDF1-α and water-soluble polymers when being connected on its GAG binding site, the GAG binding site comprises the amino acid corresponding to following SDF1-α residue: K24, H25 and K27.In addition, can develop the residue adjacent with these positions, being used for polymkeric substance connects, to reach substantially the same result, for example N22 or N30 or N33, especially therefore N33 comprises the biological activity synthetic chemokines of the present invention with the water-soluble polymers that is connected on its GAG binding site.

In another example, when synthetic chemokines is the analogue of IL-8 and water-soluble polymers when being connected on its GAG binding site, the GAG site comprises corresponding to the amino acid that is selected from following IL-8 residue: K20, R60, K64, K67 and R68.The polymkeric substance connection site that is preferred for the IL-8 synthetic analogues is corresponding to its K64 position.Another example is MCP-1, and when synthetic chemokines is the analogue of MCP-1 and water-soluble polymers when being connected on its GAG site, the GAG site comprises corresponding to the amino acid that is selected from following MCP-1 residue: K58 and H66, wherein preferred K58.

Be appreciated that the GAG binding site easily identifies, and be polymer-modified preferred sites, can select these sites, be used for screening required bioactive method and connect according to qualifications by routine.The bioassay method that is suitable for this purpose is well-known, referring to following document: Cytokine Reference, the 1st volume, Ligands, A compendium ofcytokines and other mediators of host defense, J.J.Oppendheim and M.Feldmann write, Acedemic Press, 2001; In addition referring to for example Wells etc., Inflamm.Res. (1999) 48:353-3362; Lalani etc., J.Virol. (1997) 71:4356-4363; Rot, A., Eur J Immunol (1993) 23:303-306; Witt etc., Curr Biol (1994) 4:394-400; Hoogewerf etc., Biochem (1997) 36:13570-13578; Marquezini etc., Cardiology (1995) 86:143-146; Wasty etc., Diabetologia (1993) 36:316-322.Kuschert etc. (Biochem. (1999) 38:12959-12968), (J.Biol.Chem. (2001) 276 (14): 10620-10626) reported GAG combination and chemokine for Koppman etc. (J.Immunol. (1999) 163:2120-2127) and Proudfoot etc.

Specifically, can identify the GAG binding site of chemokine according to disclosed information, homology modeling, by screening or its combination.In addition, side chain is positioned at molecular surface because at least one participates in GAG bonded residue, and away from N-end pharmacophore district, so these sites are suitable for connecting water-soluble polymers usually.For example, it is common heparin binding motif that BBXB and BBBXXB aminoacid sequence motif have demonstrated concerning some protein (comprising some chemokines), and wherein B represents alkaline residue.(referring to for example Cardin etc., Arteriosclerosis (1989) 9:21-32; Hileman etc., Bioessays (1998) 20 (2): 156-167; Proudfoot etc., J.Biol.Chem. (2001) 276 (14): 10620-10626).Yet the GAG binding site is not limited to BBXB or BBBXXB motif.For example, the GAG binding site among the Rantes ( ⁴⁴RKNR ⁴⁷(SEQ ID NO:32) and ⁵⁵KKWVR ⁵⁹(SEQ IDNO:33)), SDF-1 ( ²⁴KHLK ²⁷(SEQ ID NO:34)), MIP-1 α ( ⁴⁵KRSR ⁴⁸(SEQID NO:35)) and MIP-1 β ( ⁴⁵KRSK ⁴⁸(SEQ ID NO:36)) have the BBXB motif, and the main GAG of IL-8 is spatially separated in conjunction with residue (Lys20, Lys64 and Arg68) and MCP-1 (Lys59 and Arg66), but all constitutes alkaline electric charge bunch at protein surface.Therefore the alkaline electric charge of these parts has been explained its heparin binding characteristic.Also can pass through the L-Ala scanning of alkaline residue (for example Lys, His and Arg), NMR and in conjunction with in measuring active compound is compared at the GAG/ heparin, and be suitable for this purpose NMR research, identify that (referring to for example Proudfoot etc., J.Biol.Chem. (2001) 276 (14): 10620-10626 in the GAG site; Trkola etc., J.Virol. (1999) 73 (8): 6370-6379; Appay etc., J.Biol.Chem. (1999) 274 (39): 27505-27512; Hunter etc., Blood (1995) 86 (12): 4400-4408; Lord etc., Blood (1995) 85 (12): 3412-3415; Lord etc., Brit.J.Cancer (1996) 74:1017-1022).As mentioned above, the GAG binding site of many chemokines is known, the technology that is used to identify the GAG site of inferring also is well-known (in addition referring to Cytokine Reference, the 1st volume, Ligands, A compendium ofcytokines and other mediators of host defense, JJ.Oppendheim and M.Feldmann write, Acedemic Press, 2001).

Except having the synthetic chemokines that one or more N-are terminal modified, C-is terminal modified and/or polymer-modified, synthetic chemokines of the present invention also can be included in one or more aminoacid replacement, insertion or the disappearance in the polypeptide chain, for example referring to WO 02/04015.In a preferred embodiment, can change on the ring at the N-of chemokine polypeptides chain, to increase its specificity/selectivity the target acceptor.Like this, the N-of biological activity synthetic chemokines of the present invention ring can seal specific receptors, influences other possible coreceptor simultaneously as small as possible." N-ring " is meant 20-26 aminoacid sequence district, and this district is contiguous to define the N-petiolarea of the specific chemokines polypeptide chain of the first conservative halfcystine pattern/or its C-end (referring to table 1 and table 2).For example, when holding from chemokine polypeptides chain N-when the C-extreme direction is read, the N-ring of CC chemokine is the amino acid district, this district is positioned between the first and second conservative cysteine amino acids and the contiguous first and second conservative cysteine amino acids/or the C-end of the first and second conservative cysteine amino acids, and the contiguous the 3rd conservative cysteine amino acids/or the N-end of the 3rd conservative cysteine amino acids.

For the chemokine polypeptides chain, the aminoacid sequence of this component and the basic homology of corresponding naturally occurring wild type molecule.Term used herein " basic homology " comprises the aminoacid sequence that has at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% (preferred 95-99%) sequence homology with given sequence.This term can include but not limited to such aminoacid sequence: described sequence has disappearance, insertion or the replacement of 1-20,1-10 or 1-5 single amino acid with respect to given sequence, and precondition is that the gained polypeptide can combine with at least a Chemokine Receptors of corresponding wild-type chemokine.

For example, this area is well-known, and some amino acid can be by other aminoacid replacement, but does not change the polypeptide characteristic basically, includes but not limited to that amino acid is conservative replace.Such possibility comprises within the scope of the present invention.Also it should be noted, often can carry out amino acid whose disappearance or insertion, but do not change the polypeptide characteristic basically.The present invention includes such disappearance or insertion (its can be for example length be corresponding naturally occurring chemokine specificity chemokine polypeptides sequence at the most 10%, 20% or 50%).

Specifically, can modify substantially chemokine, comprise mixing and mate different chemokine polypeptides sections, to produce other diversity, for example among the WO 99/11655 record module " exchange " synthetic method (molular ' cross-over ' synthesis aproach), described reference all is attached to herein by reference.Recombination method also can be used for producing the variation of main chain.For example, orthogenesis technology (for example phage display or modular shuffling etc.) can be used for producing the chemokine that receptor-specific increases.In the treatment of HIV and prevention, described the employing phage display, the ability of chemokine derivative or analogue binding chemotactic factor receptor has been measured (United States Patent (USP) 6,214,540; DeVico etc.).

Another method of modifying chemokine is a phage display.For example, display technique of bacteriophage has been used for detecting or identifying part, receptor protein inhibitor or the promotor (United States Patent (USP) the 6th of CXC Chemokine Receptors 3 (CXCR3), 140, No. 064, Loetscher etc.), it is characterized in that selective binding can inducing cell one or more chemokines (United States Patent (USP) the 6th, 184 of reaction, No. 358, Loetscher etc.).The application of phage display has been described in molecule marker and selection (United States Patent (USP) the 6th, 180, No. 336, Osbourn etc.), the mark of specificity antigen binding molecules and subsequent purification (referring to for example WO92/01047) and mensuration peptide combinations, be used for prevention and treatment HIV infection and immunological disease (United States Patent (USP) the 6th, 090, No. 388, Wang, with Offord etc., WO 02/04499).

B. exemplary synthetic chemokines of the present invention

The most preferred synthetic chemokines albumen of the present invention is Rantes derivative (perhaps be called Nonakine, or " NK ").Preferred Nonakine of the present invention sees Table 3.

Table 3: exemplary synthetic Rantes derivative ^*

J-X ¹X ²X ³SSDX ⁷X ⁸PC CFAX ¹⁴IAX ¹⁷PLP RAHIKX ²⁶YFYT SGKCSNPAVVFVTX ⁴⁴X ⁴⁵NX ⁴⁷QVC ANPEKKWVRE YINSX ⁶⁵X ⁶⁶X ⁶⁷X ⁶⁸-Z(SEQ ID NO：1)

^*J is a N-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), and it can exist or not exist; X ¹, X ², X ³, X ⁷, X ⁸, X ¹⁴, X ¹⁷, X ²⁶, X ⁴⁴, X ⁴⁵, X ⁴⁷, X ⁶⁵, X ⁶⁷, X ⁶⁷And X ⁶⁸Connect residue for amino acid, aminoacid replacement residue, aminoacid deletion residue or the polymkeric substance that is selected from corresponding position among the wild-type Rantes independently of one another; Z is that C-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.) or polymkeric substance connect residue, and can exist or not exist.

According to table 3 and SEQ ID NO:1, preferred Nonakine has one or more following modifications:

J=N-holds capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), and it can exist or not exist.

X ¹=Serine, amino acid derivative (for example glyoxylyl) or N-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), it can exist or not exist.

X ²=proline(Pro), amino acid derivative (for example Thioproline) or N-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), it can exist or not exist.Work as J-X ¹In any one or a plurality of when not existing, can comprise until X ¹N-end disappearance, X wherein ²Can be proline(Pro), amino acid derivative (for example Cyclohexylglycine) or N-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), it can exist or not exist.

X ³=tyrosine, amino acid derivative (for example Cyclohexylglycine) or N-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), it can exist or not exist.Work as J-X ²In any one or a plurality of when not existing, can comprise until X ²N-end disappearance, X wherein ³Can be tyrosine, amino acid derivative (for example Cyclohexylglycine) or N-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), it can exist or not exist.

X ⁷=Threonine, amino acid derivative (for example N-methylthreonine), it can exist or not exist.Work as J-X ⁶In any one or a plurality of when not existing, can comprise until the N-end disappearance of 6 aspartic acid, wherein X ⁷Can be Threonine, amino acid derivative (for example N-methylthreonine) or N-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), it can exist or not exist.

X ⁸=Threonine, amino acid derivative (for example N-methylthreonine) or N-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), it can exist or not exist.Work as J-X ⁷In any one or a plurality of when not existing, can comprise until X ⁷N-end disappearance, X wherein ⁸Can be Threonine, amino acid derivative (for example N-methylthreonine) or N-end capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), it can exist or not exist.

X ¹⁴=tyrosine, L-Ala or amino acid derivative (for example Cyclohexylglycine).

X ¹⁷=arginine, L-Ala, amino acid derivative (for example amino-butyric acid (Abu)); or polymkeric substance connects residue; described residue comprise have can covalently bound side chain amino acid or amino acid analogue; or combine with water-soluble polymers (for example have protection or do not protect the side chain of functional group's (for example amino oxygen base, levulinic acid, pyruvic acid, amino, mercaptan, selenol, thioesters, selenium ester), or the side chain that is connected with polymkeric substance by covalent linkage (for example oxime, hydrazone, acid amides, thioesters, selenium ester, thioether, selenide,  azoles alkane, thiazolidine) etc.).

X ²⁶=L-glutamic acid, L-Ala or amino acid derivative (for example Abu).

X ⁴⁴=arginine, L-Ala, amino acid derivative (for example Abu), or as X ¹⁷Polymkeric substance connect residue.

X ⁴⁵=Methionin, L-Ala, amino acid derivative (for example Abu), or as X ¹⁷Polymkeric substance connect residue.

X ⁴⁷=arginine, L-Ala, amino acid derivative (for example Abu), or as X ¹⁷Polymkeric substance connect residue.

X ⁶⁵=leucine or amino acid derivative (for example leu-acid amides).Work as X ⁶⁶When-Z does not exist, can be leucine, amino acid derivative or Z.

X ⁶⁶=L-glutamic acid, L-Ala or amino acid derivative (for example glu-acid amides), and can exist or not exist.Work as X ⁶⁷When-Z does not exist, can be L-glutamic acid, L-Ala, amino acid derivative or Z.

X ⁶⁷=methionine(Met), Serine, L-Ala, amino acid derivative (for example met-acid amides, nor-leucine), or as X ¹⁷Polymkeric substance connect residue, and can exist or not exist.Work as X ⁶⁸When-Z does not exist, can be methionine(Met), Serine, L-Ala, amino acid derivative, polymkeric substance connection residue or Z.

X ⁶⁸=Serine, L-Ala, amino acid derivative (for example ser-acid amides, amino-butyric acid (Abu)), or as X ¹⁷Polymkeric substance connect residue, and can exist or not exist.

Z=C-holds capping group (for example one or more other amino acid, polymkeric substance, chemical adducts etc.), or as X ¹⁷Polymkeric substance connect residue, and can exist or not exist.

In a preferred embodiment, the aminoacid sequence of synthetic chemokines of the present invention is SEQ ID NO:1 and has the biological activity of at least a corresponding Rantes acceptor of downward modulation.Synthetic chemokines with aminoacid sequence SEQ ID NO:1 is preferably included in one or more following locational at least one aforesaid modification: J, 1,2,3,4,7,8,14,17,26,44,45,47,66,67,68 and Z that are selected from.Synthetic chemokines with aminoacid sequence SEQ ID NO:1 also can be included in one or more other locational modifications.The synthetic chemokines that more preferably has an aminoacid sequence SEQ ID NO:1 comprises one or more modifications at 44,45,47, and in one or more modifications at 26 and 66.Even more preferably such combination: it comprises one or more modification and one or more modification and one or more modifications on J, 1,2 and 3 on 26 and 66 at 44,45,47.

In another preferred embodiment, the bioactive synthetic chemokines that has aminoacid sequence SEQ ID NO:1 and have an at least a corresponding Rantes acceptor of downward modulation comprises the brachymemma of C-end, and described brachymemma comprises one or more aminoacid deletion that are selected from upper/lower positions: 66,67 and 68.Have aminoacid sequence SEQ ID NO:1 and comprise one or more synthetic chemokines of C-end brachymemma that are selected from 66,67 and 68 aminoacid deletion, also preferably comprise at least one and be selected from one or more aforesaid modifications: J, 1,2,3,4,7,8,14,17,26,44,45,47 and Z with upper/lower positions.More preferably have aminoacid sequence SEQ ID NO:1 and comprise one or more synthetic chemokines that are selected from the modification of 66,67 and 68 the C-end brachymemma of aminoacid deletion and the one or more positions in J, 1,2,3,44,45,47.Even more preferably such combination: it comprises that containing one or more C-that are selected from 66,67 and 68 aminoacid deletion holds brachymemmas, and in one or more J, 1,2 and 3 modification, and the modification of the one or more positions in 44,45,47.Most preferred synthetic chemokines with aminoacid sequence SEQ ID NO:1 and the brachymemma of C-end comprises such C-end brachymemma: described brachymemma comprises that C-holds to the aminoacid deletion of terminal 65 of core helical region, promptly lacks 66,67 and 68 amino acid.

The most preferred Nonakine of the present invention sees Table 4, and has a detailed description in an embodiment.

Table 4: with respect to the PSC-RANTES shown in the SEQ ID NO:2, have aminoacid sequence and modification that locus specificity changes, as follows.

B ¹X ²Z ³SSDT ⁷TPC CFAY ¹⁴IAR ¹⁷PLP RAHIKE ²⁶YFYT SGKCSNPAVVFVTR ⁴⁴K ⁴⁵NR ⁴⁷QVC ANPEKKWVRE YINSLE ⁶⁶M ⁶⁷S ⁶⁸(SEQID NO：2)

B ¹=nonanoyl; X ²=Thioproline; Z ³=Cyclohexylglycine

Compound	Backbone modifications with respect to SEQ ID NO:2
													7	14	17	26	44	45	47	66	67	68	69	70
	NK	T	Y	R	E	R	K	R	E	K plp	S	K pal													△
NK1													T	A	A	A	A	A	A	S	M	S	K plp	L
	NK2	T	A	A	E	A	K plp	A	E	M	S	△													△
NK3													T	Y	R	E	R	K plp	R	△	M	S	△	△
	NK4	T	Y	R	E	R	K plp	R	△	△	△	△													△
NK5													NmeT	A	A	A	R	K plp	R	△	M	S	△	△
	NK6	T	A	K plp	A	A	A	A	S	M	S	△													△
NK7													NmeT	A	A	A	A	A	A	S	M	S	K plp	L
	NK8	T	A	A	A	A	K plp	A	△	M	S	△													△
NK9													T	Y	R	E	R	K plp	R	△	M	K pal	△	△
	NK10	T	Y	R	E	R	K PEG	R	△	M	S	△													△
NK11													T	Y	R	E	R	K PEG	R	△	M	S	△	△
	NK12	T	Y	R	E	R	K plp	R	△	M	S	△													△
NK13													T	Y	R	E	R	K PEG	R	△	△	△	△	△

Keyword: plp=precise length polyamide polymer; The PEG=polyoxyethylene glycol; Pal=cetylate derivative; △=disappearance or do not exist.All amino acid are all represented with one-letter code.The key that connects Methionin and plp is by the oxime that levulinie acid-the amino oxygen base forms, and the key of connection Methionin and PEG is the oxime that is formed by amino oxygen base-aldehyde.

II. synthetic chemokines of the present invention is synthetic

Can pass through the whole bag of tricks, prepare synthetic chemokines of the present invention, described method comprises recombinant dna expression (partly or entirely synthetic), chemistry complete synthesis (all synthetic) and unites semi-synthetic (all synthetic) of using recombinant dna expression and chemosynthesis.Hold the synthetic chemokines of brachymemma for the C-of the present invention that produces by recombinant dna expression fully, described molecule lacks the modification (for example the chemokine polypeptides chain contains the one or more amino acid that do not assemble at rrna) of non-genetic coding usually.

Therefore, preferred synthetic method comprises the chemosynthesis of at least one aspect, for example, the chemistry of amino acid whose progressively chain assembling (solid-phase peptide is synthetic) and/or peptide fragment connects (for example natural chemistry connects) and/or chain assembling back (post chain assembly) method (connections of for example chemical adducts, polymkeric substance etc.).Peptide synthetic preferably according to nineteen sixty for " the Merrifield "-chemistry of early development solid-phase peptide synthetic schemes progressively, adopt standard automatic peptide synthesizer to carry out.The peptide Connection Step can adopt solid phase or liquid phase connection strategy.Chemistry connects the formation that comprises the selectivity covalent linkage between first chemical composition and second chemical composition.Be present in the chemo-selective that unique functional group that can react to each other on first and second components can be used for providing ligation.For example, peptide is connected with the chemistry of polypeptide and comprises and have compatible, unique C-terminal amino acid residue that can react to each other and the peptide of n terminal amino acid residue or the chemo-selective reaction of polypeptide fragment.

In one embodiment, all amino-acid residues of synthetic bioactive protein can link together by peptide bond (being amido linkage).Perhaps, two amino-acid residues (or the C-of two polypeptide end residue and N-end residue) can (Schn  lzer be connected to each other by non-amido linkage (for example the connection that is connected, forms  azoles alkane of thioester bond, oxime key, thioether bond, directed disulfide linkage, thiazolidine key (thiozolidine bond), formation hydrazone etc.), M. and Kent, S.B.H., Science (1992) 256:221-225; Rose, K., J.Amer.Chem Soc. (1994) 116:30-33; Englebretsen, D.R. etc., Tetrahedron Lett.36:8871-8874; Baca, M. etc., J.Amer.Chem Soc. (1995) 117:1881-1887; Liu, CF. etc., J.Amer.Chem Soc. (1994) 116:4149-4153; Liu, C.F. etc., J.Amer.Chem Soc. (1996) 118:307-312; Dawson, P.E. etc. (1994) Science 266:776-779; Gaertner etc., Bioconj.Chem. (1994) 5 (4): 333-338; Zhang etc., Proc.Natl.Acad.Sci. (1998) 95 (16): 9184-9189; Tam etc., WO 95/00846; United States Patent (USP) the 5th, 589, No. 356) or by other method (Yan that is connected to each other, L.Z. and Dawson, P.E., " Synthesis ofPeptides and Proteins without Cysteine Residues by Native ChemicalLigation Combined with Desulfurization; " J.Am.Chem.Soc.2001,123,526-533, described document is attached to herein by reference; Gieselnan etc., Org.Lett.20013 (9): 1331-1334; Saxon, E. etc., " Traceless " Staudinger Ligation forthe Chemoselective Synthesis of Amide Bonds.Org.Lett.2000,2,2141-2143.Therefore the present invention allow to develop various peptide bond modifications, surrogate and etc. the structure thing replace, be used to prepare biological activity protein.

When connection relates to the participation of the polypeptide with N-end cysteine residues, preferably adopt natural chemical connection process (Dawson etc., Science (1994) 266:776-779; Kent etc., WO 96/34878; Kent etc., WO 98/28434)).This method has proved the effective ways that produce natural amido linkage at connecting portion.Natural chemistry connects second peptide or the reaction of the chemo-selective between the polypeptide that comprises first peptide or the polypeptide fragment with C-end α-carboxyl thioesters part and have N-end cysteine residues.The sulfydryl permutoid reaction obtains the intermediate of initial thioesters-connection, and the spontaneous rearrangement of this intermediate obtains natural amido linkage at connecting portion, the sulfydryl of the cysteine side chain of regenerating simultaneously.In many examples, the sequence of natural protein comprises the cysteine residues of correct position, makes can synthesize the polypeptide fragment with N-end cysteine residues and be used for the native chemical ligation.In other example, can carry out peptide and synthesize, so that cysteine residues is incorporated in the polypeptide, reach this purpose.

Polymkeric substance is well-known with being connected of polypeptide, can select to connect chemistry by the corresponding active group that exists on the chemokine polypeptides chain, and perhaps preferred natural higher locus specificity comes being connected of controlling polymers and polypeptide more or less.Generally speaking, the polymkeric substance connection can be connected N-end or C-end, naturally occurring amino acid side chain, replace natural the existence on the amino acid whose amino acid derivative, perhaps by being connected another part on the target position that is suitable for this purpose.It is well-known being suitable for the chemistry that polymkeric substance is connected with protein.Referring to for example WO 02/04014; ROSE; Perspectives in Bioconjugate Chemistry, Claude F.Meares writes, American Chemical Society, 1993; Abuchowski etc., J.Biol.Chem. (1977) 252 (11): 3578-3581; Tsutsumi etc., Jpn.J.CancerRes. (1994) 85:9-12; Poly (ethylene glycol) Chemistry and BiologicalApplications, ACS Symposium Series 680, J.M.Harris and S.Zalipsky write, American Chemical Society, 1997; Poly (ethylene glycol) Chemistry, Biotechnical and Biomedical Applications, Topics in Applied Chemistry, J.M.Harris writes, Plenum Press, New York, NY, 1992).

In a preferred embodiment, the invention provides the preparation method of synthetic chemokines of the oligomer form of basic purifying.This method comprises: the synthetic proteic protein library of synthetic chemokines (protein pool) that contains, described synthetic chemokines albumen comprises the chemokine polypeptides chain with N-end and C-end, wherein said chemokine polypeptides chain comprises (i) aminoacid sequence and halfcystine pattern and the (ii) end of the C-with respect to wild-type chemokine brachymemma corresponding to the wild-type chemokine; Proteic one or more oligomer forms of the described synthetic chemokines of purifying from described protein library then, thereby the synthetic chemokines of the single substantially oligomer form of preparation.

The preparation method of biological activity synthetic chemokines of the present invention also is provided.This method comprises that (i) synthesizes the wild-type analogue, described analogue comprises the polypeptide chain that has with the basic homologous aminoacid sequence of wild-type chemokine, and wherein said polypeptide chain comprises C-end brachymemma and is selected from the part modification of aliphatic chain, amino acid derivative and polymkeric substance at its one or more N-ends, N-ring and C-end; (ii) screen Chemokine Receptors bonded chemokine analogue with corresponding wild-type chemokine.

Preferably, finish the synthetic of synthetic chemokines of the present invention by the combination of chemosynthesis (it is synthetic promptly not have rrna) or biology synthetic (being that rrna is synthetic) and chemosynthesis.For chemosynthesis, can intactly prepare synthetic chemokines of the present invention by the following method: progressively (for example solid phase or liquid phase peptide are synthetic for chain assembling or fragment condensation technology, use Fmoc and tBoc method), the chemistry of the peptide fragment of complete preparation connects the perhaps combination of chain assembling and biological preparation by the chain assembling.Assembling of described progressively chain or fragment condensation and interconnection technique be well-known in the art (referring to for example Kent, S.B.H., Ann.Rev.Biochem. (1988) 57:957-989; Dawson etc., Methods Enzymol. (1997) 287:34-45; Muir etc., Methods Enzymol. (1997) 289:266-298; Wilken etc., Current Opinion InBiotechnology (1998) 9:412-426; Ingenito etc., J.Amer.Chem.Soc. (1999) 121 (49): 11369-11374; Muir etc., Chemistry ﹠amp; Biology (1999) 6:R247-R256).

Connect for chemistry, first peptide fragment with N-end functional group is connected with second peptide fragment with C-end functional group, C-end functional group and N-end functional group reactions form covalent linkage betwixt.According to selected functional group, ligation produces the product with natural amido linkage or non-natural covalent linkage at connecting portion.Usually adopt progressively chain assembling or fragment condensation, preparation is used for first or second peptide fragment that chemistry connects.Specifically, when biological activity synthetic chemokines of the present invention is when preparing by the connection peptides fragment, prepared fragment contains the suitable chemo-selective active group that dangles, and described active group is suitable active group to the expection chemo-selective reactive chemistry that is used to connect.These chemistry include but not limited to that native chemical connects (Dawson etc., Science (1994) 266:776-779; Kent etc., WO 96/34878), extend general chemistry and connect (Kent etc., WO 98/28434), the chemistry that forms oxime connects (Rose etc., J.Amer.Chem.Soc. (1994) 116:30-33), form connection (the Schn  lzer etc. of thioesters, Science (1992) 256:221-225), the connection of formation thioether (Englebretsen etc., Tet.Letts. (1995) 36 (48): 8871-8874), form the connection (Gaertner etc. of hydrazone, Bioconj..Chem. (1994) 5 (4): 333-338) with the connection that is connected and forms  azoles alkane that forms thiazolidine (Zhang etc., Proc.Natl.Acad.Sci. (1998) 95 (16): 9184-9189; Tam etc., WO 95/00846).

Select the given reaction conditions that connects chemistry, to keep the interaction of required connection component.For example, can change the composition of ratio, water content and each peptide of water-soluble, the peptide of pH and temperature, peptide and component, to optimize ligation.Add or be not added in specificity and the speed that the reagent that makes the peptide solubilising in varying degrees also can be used for controlling required ligation.By comparing with one or more inside and/or external control, can easily determine reaction conditions to estimate required chemo-selective reaction product.

Preferred chemical synthesis process adopts native chemical to connect, and described method is disclosed in Kent etc., and WO 96/34878; Have N-proteinic preparation method terminally chemically modified and/or that C-is terminally chemically modified and be disclosed in Offord etc., WO 99/11666, and described document is attached to herein by reference.Generally speaking, second peptide of the N-end halfcystine of oxidation sulfydryl side chain reacts with having not to make first peptide that contains C-end thioesters.In the presence of the mercaptan (preferred benzyl sulfhydrate, thiophenol, 2-nitro thiophenol, 2-thiobenzoic acid, 2-sulfo-pyridine etc.) of catalytic amount, make the not oxidation sulfydryl side chain and the condensation of C-end thioesters of N-end halfcystine.First and second peptides connect by the beta-amino thioester bond, produce the peptide prod that comprises first and second peptides that connect with amido linkage through resetting, thereby produce the intermediate peptide.

For the combination by chemistry and biological preparation prepares the synthetic chemokines that contains non-genetic coding amino-acid residue, can prepare a kind of peptide fragment by chemosynthesis, other then use recombinant methods, and then, obtain full length product with chemical the connection these fragments being linked together.For example, intein (intein) expression system can be used for developing the induction type oneself nicking activity of ' intein ' protein-montage element, to produce C-end thioesters peptide fragment.Specifically, intein mercaptan for example DTT, beta-mercaptoethanol or halfcystine in the presence of, the oneself cutting of experience specificity produces the peptide fragment that has C-end thioesters.(referring to for example Muir etc., Chemistry ﹠amp; Biology (1999) 6:R247-R256; Chong etc., Gene (1997) 192:277-281; Chong etc., Nucl.Acids Res. (1998) 26:5109-5115; Evans etc., Protein Science (1998) 7:2256-2264; Cotton etc., Chemistry ﹠amp; Biology (1999) 6 (9): 247-256).Then, this peptide fragment that has C-end thioesters can be used for connecting second peptide that has N-end thioesters-active function groups, for example has the peptide fragment of N-end halfcystine, is used for native chemical and connects.

At chain between erecting stage, chain assembling back or its Assemble Duration, can mix polymkeric substance, aliphatic chain, amino acid derivative etc.For example, for chain mixing between erecting stage,, mix amino acid derivative and/or be connected with the amino acid of aliphatic chain progressively or in fragment condensation and/or the connection chain assembling process.These amino acid can be peptide in a step-wise fashion adds peptide chain in the growth between synthesis phase on, add on the peptide fragment after the assembling, so that connect, perhaps in some cases, can be by cutting from polymer support, provide the N-that dangles terminal modified or C-is terminal modified, wherein cleaved products produces required hydrophobicity aliphatic chain.For chain assembling back, mix amino acid or derivatives thereof (to protect or not protect form) between erecting stage at chain with active function groups, be used to connect required hydrophobic part with its unprotected activity form then, promptly in the conjugation reaction after peptide is synthetic.Can perhaps after polypeptide chain is folding, finish in the connection after finishing the chain assembling on the linear peptide chain of sex change.In a preferred embodiment, between synthesis phase, on the target amino acid position, add amino acid derivative, and after peptide is synthetic, add N-end, C-end and/or N-end/C-end hydrophobicity aliphatic chain by conjugation reaction at peptide.In a large number in conjunction with in the chemistry any all can use (referring to for example Plaue, S etc., Biologicals. (1990) 18 (3): 147-57; Wade, J.D. etc., Australas Biotechnol. (1993) 3 (6): 332-6; Doscher, M.S., Methods Enzymol. (1977) 47:578-617; Hancock, D.C. etc., Mol Biotechnol. (1995) 4 (1): 73-86; Albericio, F. etc., Methods Enzymol. (1997) 289:313-36), and can use the connection chemistry, this depends on required covalent attachment.According to this area standard technique, can finish the folding of biological activity synthetic chemokines of the present invention.Referring to for example WO 99/11655; WO 99/11666; Dawson etc., Methods Enzymol. (1997) 287:34-45).

Antagonistic activity for screening synthetic chemokines compound by based on external or intravital mensuration, detects these compounds, and the feature of described mensuration is a directly or indirectly receptors bind corresponding with it of chemokine ligand.The example of Chemokine Receptors and corresponding wild-type chemokine thereof comprises CXXXCR1 (CX ₃The C chemokine); XCR1 (SCM-1); CXCR2 (GRO, LIX, MIP-2); CXCR3 (MIG, IP-10); CXCR4 (SDF-1); CXCR5 (BLC); CCR1 (MIP-1 α, RANTES, MCP-3); CCR2 (MCP-1, MCP-3, MCP-5); CCR3 (eosinophilic granulocyte chemotactic protein, RNATES, MIP-1 α); CCR4 (MDC, TARC); CCR5 (RANTES, MIP-1 α, MIP-1 β; CCR6 (MIP-3 α); CCR7 (SLC, MIP-3 β); CCR8 (TCA-3); And CCR9 (TECK).The interior measuring method of external and body that is used for these systems is well-known and easy acquisition, perhaps can from the beginning create.Referring to No. the 5th, 652,133, United States Patent (USP) for example; United States Patent (USP) the 5th, 834, No. 419; WO 97/44054; WO 00/04926; With WO 00/0492.For example express natural, the conversion and/or the transgenic cell line of one or more Chemokine Receptors, when being exposed to biological activity synthetic chemokines of the present invention, be generally used for monitoring the inhibiting effect of chemokine inductive chemotaxis or these incidents.Also can use animal model, for example be used to monitor the reaction profile type when using biological activity synthetic chemokines of the present invention to handle, perhaps be used for the pharmacokinetics and the pharmacodynamics characteristic of characterizing compounds.Utilize the coating cell fusion mediated of target cell system and coating clone to measure in order to characterize the inhibitor of The compounds of this invention, can to use, screen the ability that biological activity synthetic chemokines prevention HIV of the present invention infects as virus infection.Certainly, in order to reach this purpose, also can use acellular virus infection to measure.

As an example, generally speaking, in order to estimate chemotactic antagonistic action, can use peripheral blood leucocyte, for example according to the scheme of having set up that is used for purifying monocyte, T lymphocyte and neutrophilic granulocyte, isolating peripheral blood leucocyte from normal donor.Can make up one group of test cell of expressing C, CC, CXXXC and CXC Chemokine Receptors, and after each compound of the present invention who is exposed to serial dilution, it be estimated.Can use natural chemokine in contrast.For example, one group with the expression cassette cells transfected promptly applicable to this purpose, the described expression cassette various Chemokine Receptors of encoding.(described cell can derive from different commercial source and/or academic source, or can prepare according to standard scheme for example can to use transformant expression CXCR4/ fusion/LESTR, CCR3, CCR5, CXC4; Referring to for example Risau etc., Nature387:671-674 (1997); Angiololo etc., Annals NY Acad.Sci. (1996) 795:158-167; Friedlander etc., Science (1995) 870:1500-1502), screen the antagonist of RANTES, SDF-1 α or chemokines such as SDF-1 β and MIP.The result can be expressed as chemotaxis index (" CI "), represents the increase multiple of cell migration of stimulator inductive and the cell migration of control media inductive, and carries out significance,statistical and detect.

Also can carry out for example receptors bind mensuration, estimate competitive inhibition and acceptor recirculation effect (referring to Signoret, N. etc., " Endocytosis and recycling of the HIV coreceptorCCR5, " J Cell Biol.2000 151 (6): 1281-94; Signoret, N. etc., " Analysis ofchemokine receptor endocytosis and recycling, " Methods Mol Biol.2000; 138:197-207; Pelcheh-Matthews, A. etc., " Chemokine receptortrafficking and viral replication, " Immunol ReV.1999 Apr:168:33-49; Daugherty, B.L. etc., " Radiolabeled chemokine binding assays, " MethodsMol Biol.2000; 138:129-34; Mack, M. etc., " Downmodulation andrecycling of chemokine receptor, " Methods Mol Biol.2000; 138:191-5; All documents all are attached to herein by reference).This method is well-known, and according to standard scheme, this method in the presence of the natural chemokine that unlabelled concentration increases, is used the biological activity synthetic chemokines of the present invention of tape label usually.Certainly, mark can be on one or both parts.In such is measured, can compare with natural white corpuscle or one group of acceptor-transfectional cell of expressing the target Chemokine Receptors, analyze binding data, for example use computer program, for example LIGAND (P.Munson, DMsion of Computer Researchand Technology, NIH, Bethesda, MD), and carry out the analysis of Scatchard graphic representation, adopt " a bit (one site) " and " 2 points (two site) " model.With the combining competition speed and can try to achieve as follows of unmarked part: % inhibition=1-(in the combination in the presence of the unmarked chemokine /) * 100 in the combination that only has in the presence of the medium.

For the ability of SCREENED COMPOUND prevention or alleviation virus infection and disease, can come SCREENED COMPOUND at one group of cell of the suitable acceptor of stably express that is exposed to different virus strain and contrast.For example, can use the U87/CD4 cell of expressing CCR3, CCR5, CXC4 or CXCR4 acceptor, screen the infection of having a liking for M (M-tropic), having a liking for T (T-tropic) and two preferendum HIV strains.With respect to chemokine concentration and contrast concentration, the percentage that can approach to infect to the inhibition of virus infection.Referring to for example McKnight etc., Virology (1994) 207:8-18; Mosier etc., Science (1993) 260:689-692; Simmons etc., Science (1997) 276:276-279; Wu etc., J.Exp.Med. (1997) 185:168-169; Trkola etc., Nature (1996) 384:184-186.It is another example that is used to screen receptor-binding antagonists that the calcium mobilization measures (Calcium mobilizationassay), for example identify the antagonist of natural chemokine, described chemokine has chemotaxis (Jose etc., J.Exp.Med.179:881-887 (1994)) to neutrophilic granulocyte and eosinophilic granulocyte.As another example, can pass through chick chorioallantoic membrane (CAM) and measure, estimate angiogenic activity (Oikawa etc., Cancer Lett. (1991) 59:57-66 of The compounds of this invention.

III. medicine, morbid state and treatment

Biological activity synthetic chemokines of the present invention serves many purposes, and comprises as research tool, diagnostic reagent and therapeutical agent.Specifically, have been found that biological activity synthetic chemokines of the present invention has the valuable pharmacological characteristic, and show and can block effectively that the inflammatory reaction relevant with corresponding wild type molecule---these inflammatory reactions give rise to diseases, comprise asthma, rhinallergosis, atopic dermatitis, congee sample spot/atherosclerosis, cancer, organ-graft refection and rheumatoid arthritis.Therefore, they can be used for treating asthma, rhinallergosis, atopic dermatitis, congee sample spot/atherosclerosis, organ-graft refection and rheumatoid arthritis.For example, some biological activity synthetic chemokines of the present invention (for example RANTES and SDF-1 α or SDF-1 beta antagonists) also shows and suppresses the HIV-1 infection, and antagonist (for example vMIP-II analogue) also can be used for same purpose.Therefore, RANTES of the present invention or SDF-1 α or SDF-1 beta antagonists and vMIP-II analogue can be used for suppressing the intravital HIV-1 of Mammals.Measure the potentiality that compound is used for anti-HIV-1 by the described method of following examples.By the well-known method of those skilled in the art, measure the potentiality that compound is used for anti-inflammatory response.In addition, be appreciated that biological activity synthetic chemokines of the present invention can use separately, or the use that combines with one another, and unite use that these medicines play synergy in the given disease of treatment with other non-chemotactic factor substance.

The non-limiting purpose for illustrative is specific examples and their relevant biological characteristicses of some wild-type chemokine molecules, with the common purposes of explanation preparation biological activity synthetic chemokines of the present invention below.For example, SCM-1 is the C-chemokine that spleen is expressed.It is in fact relevant with the CXC chemokine with the CC chemokine, its main difference be it in these protein, only have in 4 conservative halfcystines second and the 4th halfcystine (Yoshida etc., FEBS Letters (1995) 360 (2): 155-159); J.Biol.Chem. such as Yoshida (1998) 273 (26): 16551-16554).The mankind, two height homologous SCM-1 albumen are arranged, SCM-1 α and SCM-1 β, their difference is two aminoacid replacement.Find that SCM-1 and lymphocyte chemotactic protein (lymphotactin, a kind of mouse lymphotactin specificity chemokine) have about 60% identity.Therefore, SCM-1 and lymphocyte chemotactic protein are represented the people's prototype and the mouse prototype of C-chemokine or γ-chemokine.These two kinds of SCM-1 molecules through engineeredization with the mouse L1.2 cell of expressing orphan receptor GPR5 in specificity induce migration; In various tissues, GPR5 mainly expresses in placenta, and in spleen and thymus gland faint expression is arranged.Therefore, the antagonist of SCM-1 can be used for blocking the normal function of GPR4.

As another example, CX ₃The soluble form of C chemokine (Fractalkine, a kind of 76 amino acid whose CXXXC-chemokines) is potent chemoattractant to T cell and monocyte, but concerning neutrophilic granulocyte is not.After being subjected to TNF or IL1 stimulation, CX ₃The C chemokine significantly increases.CX ₃People's acceptor of C chemokine is called CX ₃CR1.This receptor mediates CX simultaneously ₃The adhesion of C chemokine and shift function.This people's acceptor is expressed in neutrophilic granulocyte, monocyte, T-lymphocyte and several solid organ (comprising brain).This receptor has shown with CD4 and has worked as the coreceptor of envelope protein, and described envelope protein is the main isolate from HIV-1.A cell-cytogamy is measured and is proved CX ₃Effective and the specificity inhibition fusion of C chemokine.(referring to for example Bazan etc., Nature (1997) 385 (6617): 640-644; Combadiere etc., J.Biol.Chem. (1998) 273 (37): 23799-23804; Rossi etc., Genomics (1998) 47 (2): 163-170; Faure etc., Science (2000) 287:2274-2277).Therefore, it is evident that CX ₃The antagonist of C chemokine can be used for treating the various arthritis diseases (for example sacroiliitis) that relate to TNF or IL1 approach, and can be used as HIV infection blocker.

Eosinophilic granulocyte chemotactic protein (Eotaxin) is an other example.This protein length is 74 amino acid, because of its characteristic halfcystine pattern is categorized as the CC-chemokine.Have been found that in the guinea pig bronchial alveolar wass it can be used as the model of alterative inflammation, and relate to the asthma relative disease.In fact the eosinophilic granulocyte chemotactic protein induces the remarkable accumulation of skin eosinophilic granulocyte when 1-2pM dosage, and the accumulation of not remarkably influenced neutrophilic granulocyte.The eosinophilic granulocyte chemotactic protein is external potent stimulant to cavy and people's eosinophilic granulocyte.This factor table reveal with the cavy eosinophilic granulocyte on RANTES share binding site.The eosinophilic granulocyte chemotactic protein is induced the reaction of calcium flux in normal people's eosinophilic granulocyte, but does not induce the reaction of calcium flux in neutrophilic granulocyte or monocyte.With other CC-chemokine pre-treatment eosinophilic granulocyte, can not be to this reaction desensitization.In basophilic granulocyte, compared with RANTES, the eosinophilic granulocyte chemotactic protein can be induced higher levels of chemotactic response, but only has fringing effect concerning the generation of the release of histamine or leukotriene C.It also works to the chemotaxis of B cell lymphoma cell.The principal recipient of eosinophilic granulocyte chemotactic protein is CCR3.(referring to for example Bartels etc., Biochem.Biophys.Res.Comm. (1996) 225 (3): 1045-51); Jose etc., J.Exp.Med. (1994) 179:881-887); Ponath etc., J.Clin.Investigation (1996) 97 (3): 604-612); Ponath etc., J.Exp.Med (1996) 183 (6): 2437-2448); Yamada etc., Biochem.Biophys.Res.Comm. (1997) 231 (2): 365-368).Therefore, the antagonist of eosinophilic granulocyte chemotactic protein can be used as the potent conditioning agent of asthma and the relevant allergic disease of other eosinophilic granulocyte.

RANTES is another example of target chemokine, and the antagonist of this target chemokine is valuable especially.It is the CC-chemokine that participates in from inflammation, organ transplantation to numerous diseases such as HIV infection.The synthetic of RANTES induced by TNF-α and IL1-α, but is not subjected to inducing of TGF-β, IFN-γ and IL6.In culture, circulation T cell and T cell clone produce RANTES, but present any T-clone of measuring all can not produce RANTES.Behind the T LS, can suppress the expression of RANTES.RANTES has chemotaxis to T cell, people's eosinophilic granulocyte and basophilic granulocyte, and leukocyte recruitment is being played active effect aspect the inflammation site.RANTES also activates eosinophilic granulocyte, so that it discharges for example cationic protein of eosinophilic granulocyte.It has changed the density of eosinophilic granulocyte and its density is descended, and this is considered to show the state of general cell activation and relates to common disease, for example asthma and rhinallergosis.RANTES also is specific, activated dose of the potent eosinophilic granulocyte of oxidative metabolism.RANTES has increased the adhesion of monocyte to endotheliocyte.It optionally supports to express monocyte and the lymphocytic migration of T-of cell surface marker CD4 and UCHL1.These cells are considered to have the helper cell of the pre-stimulation of memory T cell function.RANTES has activated people's basophilic granulocyte and has caused the release of histamine from the basophilic granulocyte donor of some selection.In others, RANTES also can suppress to discharge histamine by some cytokine (comprising that one of the most potent histamine inductor is MCAF) institute's inductive basophilic granulocyte.

Recently, show that RANTES suppresses other biological activity except that chemotaxis.It can induce the propagation and the activation of the killer cell that is called as CHAK (C-C-chemokine activation killer cell), and this class cell and IL2 institute activatory cell are similar.RANTES is expressed and participates in ongoing inflammatory process in rheumatoid arthritis by people's synovioblast.Be high-affinity receptor (about 700 the binding site/cells that identify RANTES on the THP-1 person monocytic cell's property leukemia cell; Kd=700pM), this receptor reacts with RANTES in chemotaxis and calcium mobilization's mensuration.With MCAF (monocyte chemotactic and activation factor) or MIP-1-α (macrophage inflammatory protein) preincubate, can significantly suppress the chemotactic response of THP-1 cell to RANTES.RANTES is to finish by MCAF and MIP-1-α with monocytic the combination.The acceptor of RANTES is CCR1, CCR3 and CCR5.The clinical application of RANTES antagonist and significance are many-sided.For example, natural RANTES antibody can significantly suppress the relevant cellular infiltration of experimental glomerular mesangium productive nephritis.In addition, in experience and people's kidney allograft thing of kidney organ's transplant rejection dependency cellular rejection, it seems and highly to express natural RANTES (Pattison etc., Lancet (1994) 343 (8891): 209-11 (1994).Chemically modified form (amino oxygen base pentane-RANTES or the AOP-RANTES of RANTES; With n-nonanoyl-RANTES or NNY-RANTES) demonstrated effect as chemokine CCR-5 receptor antagonist, and have the ability that HIV-1 infects that suppresses.Therefore, the RANTES analogue that antagonist N-of the present invention is terminal modified, C-is terminal modified and N-end/C-is terminal modified can be used as for example antiphlogiston of following disease of treatment: asthma, rhinallergosis, atopic dermatitis, organ transplantation, congee sample spot/atherosclerosis and rheumatoid arthritis.

Chemokine SDF-1 α and SDF-1 beta antagonists are other examples, are under the jurisdiction of the CXC class of chemokine.The difference of SDF-1 β is that the C-end has 4 other amino acid.These chemokines are compared the identity that has greater than 92% with its non-human counterpart.SDF-1 is wide expression in the cell except that hemocyte.SDF-1 in lymphocyte and monocyte, but does not act on neutrophilic granulocyte at interaction in vitro, and SDF-1 is the high efficiency decoy to monocyte in vivo.It also induces actin polymerization in the born of the same parents in lymphocyte.SDF-1 can play the effect of chemoattractant in the external and body simultaneously for people's hemopoietic progenitor cell, produce the progenitor cell of mixed type and primary type more.SDF-1 it seems the formation that also participates in the compartment space diaphragm.When responding the combination of SDF-1 and IL-3, the chemotaxis of CD34+ cell increases.SDF has demonstrated the also instantaneous rising of inducing cell matter calcium in these cells.The principal recipient of SDF-1 is CXCR4, and it also plays the function of the main T-lymphocyte coreceptor of HIV1.Referring to for example Aiuti etc., J.Exp.Med.1997) 185 (1): 111-120 (1997); Bleul etc., J.Exp.Med. (1996) 184 (3): 1101-1109 (1996); Bleul etc., Nature (1996) 382 (6594): 829-833; D ' Apuzzo etc., European J.Immunol. (1997) 27 (7): 1788-1793; Nagasawa etc., Nature (1996) 382:635-638); Oberlin etc., Nature (1996) 382 (6594): 833-835.So for example, SDF-1 alpha-2 antagonists of the present invention or SDF-1 beta antagonists can be used as for example antiphlogiston of following disease of treatment: asthma, rhinallergosis, atopic dermatitis, congee sample spot/atherosclerosis and rheumatoid arthritis.In addition, raising and/or activating for short inflammatory cell, SDF-1 alpha-2 antagonists of the present invention or SDF-1 beta antagonists can be used for separately or unite with other compound (RANTES antagonist analogue for example of the present invention) being used to block the intravital SDF-1 of Mammals, RANTES, MIP-1 α and/or MIP-1 β effect, and perhaps treatment or blocking-up HIV-1 infect.

Therefore, another aspect of the present invention relates to pharmaceutical composition and is used for having the mammiferous methods of treatment of needs, described method to comprise to treat the compound that comprises one or more chemokines of the present invention or its pharmacy acceptable salt of significant quantity." pharmacy acceptable salt " is meant the biological effectiveness of reservation polypeptide of the present invention and the salt of characteristic, and described salt does not have the characteristic of undesired biological nature or others.Salt can be acid salt or subsalt.Acid salt is derived by mineral acid and organic acid, described mineral acid is hydrochloric acid, Hydrogen bromide, sulfuric acid (obtaining vitriol and hydrosulfate), nitric acid, phosphoric acid etc. for example, and described organic acid is acetate, propionic acid, oxyacetic acid, pyruvic acid, oxalic acid, oxysuccinic acid, propanedioic acid, succsinic acid, toxilic acid, fumaric acid, tartrate, citric acid, phenylformic acid, styracin, amygdalic acid, methylsulfonic acid, ethyl sulfonic acid, Whitfield's ointment, tosic acid etc. for example.Base addition salt can be derived by mineral alkali, comprises sodium salt, sylvite, lithium salts, ammonium salt, calcium salt, magnesium salts etc.Salt derived from organic bases comprises by following organic bases deutero-salt: primary amine, secondary amine, tertiary amine, comprise the replacement amine and the cyclic amine of naturally occurring replacement amine, comprise Isopropylamine, Trimethylamine 99, diethylamine, triethylamine, tripropyl amine, thanomin, 2-dimethylaminoethanol, Tutofusin tris, Methionin, arginine, Histidine, caffeine, PROCAINE HCL, PHARMA GRADE, sea crust amine, choline, trimethyl-glycine, quadrol, glycosamine, N-alkylated glucamine, Theobromine, purine, piperazine, piperidines, N-ethylpiperidine etc.Preferred organic bases is Isopropylamine, diethylamine, thanomin, piperidines, Tutofusin tris and choline.

Term used herein " treatment " comprises any treatment to mammalian diseases, particularly human diseases, comprising: (i) to disease-susceptible humans but be not diagnosed as prophylactic generation among the patient of this disease; (ii) suppress disease, promptly stop its development; Or (iii) alleviate disease, promptly cause disappearing of disease.

Term used herein " by the mammiferous morbid state that prevents or alleviate with biological activity synthetic chemokines treatment of the present invention ", comprise all morbid states of art-recognized biological activity synthetic chemokines treatment available of the present invention, and found that available concrete The compounds of this invention treats the morbid state that prevents or alleviate.These morbid states include but not limited to asthma, rhinallergosis, atopic dermatitis, virus disease, congee sample spot/atherosclerosis, rheumatoid arthritis and organ-graft refection.

Term used herein " treatment significant quantity " is meant when the Mammals that needs is arranged, be enough to reach curative effect (as above definition, for example as antiphlogiston, antasthmatic, immunosuppressive drug or anti-autoimmune disorder medicine) thus suppress the amount of the biological activity synthetic chemokines of the present invention of mammiferous virus infection.The consumption that constitutes " treatment significant quantity " is different and different because of chemokine derivative, illness or disease and severity and subject Mammals, its body weight, age etc., can be considered existing knowledge and specification sheets thereof and is conventionally determined this consumption by those of ordinary skills.Term used herein " q.s. " is meant the add-on that is enough to reach described effect (for example making solution reach volume required (for example 100ml)).

Give chemokine of the present invention and pharmacy acceptable salt (being activeconstituents) thereof with the treatment significant quantity, described treatment significant quantity is exactly when the Mammals that needs is arranged, and is enough to reach the amount of aforesaid curative effect.Give biological activity synthetic chemokines of the present invention as herein described, any accepted administering mode of medicine that can be by having similar applications carries out.Term used herein " biological activity synthetic chemokines of the present invention ", " pharmacy acceptable salt of polypeptide of the present invention " and " activeconstituents " are used interchangeably.

The level of biological activity synthetic chemokines of the present invention in preparation can change in the used entire area of those skilled in the art, contains the vehicle of 0.01% (weight) to the biological activity synthetic chemokines of the present invention of about 99.99% (weight) and about 0.01% (weight) to 99.99% (weight) of having an appointment in for example total preparation.More generally be that the level of biological activity synthetic chemokines of the present invention is about 0.5% (weight) to about 80% (weight).

Although the human dosage level of biological activity synthetic chemokines of the present invention can also be optimized, common daily dosage portion is about 0.05-25mg/kg body weight/day, most preferably from about the 0.01-10mg/kg body weight/day.Therefore, for the people who gives a 70kg, dosage range will be about 0.07mg to 3.5g every day, preferred every day about 3.5mg to 1.75g, most preferably every day about 0.7mg to 0.7g.The dosage of antagonist depends on the mode of the character of a morbid state, disease that patient and institute prevent or alleviate or severity, administration and timetable and the doctor's that prescribes judgement certainly.Such use optimization is within those of ordinary skills' technical scope.

Can be by any acceptable system or topical routes, for example by parenteral, oral (especially for baby preparation), intravenously, nasal cavity, segmental bronchus suck (being aerosol formulation), through skin or local approach, with solid, semisolid or liquid form or aerosol dosage forms, for example tablet, pill, capsule, pulvis, liquid preparation, solution, emulsion, injection, suspensoid, suppository, aerosol etc.Also can give the slowly-releasing or the controlled release form of biological activity synthetic chemokines of the present invention, comprise slow release type injection, osmotic pump, pill, through skin (comprising electromigration) patch etc., so that postpone to give described polypeptide with set rate, preferably to be suitable for the unit dosage that single gives exact dosage desired.Composition can comprise conventional pharmaceutical carrier or vehicle and biological activity synthetic chemokines of the present invention, also can comprise other medicinal reagent, pharmacy reagent, carrier, auxiliary material etc. in addition.Carrier can be selected from various oil, comprises the oil in oil, animal oil, vegetables oil or synthetic source, for example peanut oil, soybean oil, mineral oil, sesame wet goods.Water, salt solution, G/W and ethylene glycol all are preferred liquid vehicles, in particular for injection solution.Suitable pharmaceutical carrier comprises starch, Mierocrystalline cellulose, talcum powder, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, whiting, silica gel, Magnesium Stearate, sodium stearate, glyceryl monostearate, sodium-chlor, skim-milk, glycerine, propylene glycol, water, ethanol etc.Pharmaceutical carrier that other is suitable and preparation thereof are referring to " Remington ' sPharmaceutical Sciences ", and E.W.Martin writes.

If necessary, institute's administered agents composition also can contain a small amount of nontoxic auxiliary material, for example wetting agent or emulsifying agent, pH buffer reagent etc., and for example sodium acetate, mono laurate sorb are smooth, Emulphor FM etc.

Although may need more activeconstituentss, can adopt oral administration, give biological activity synthetic chemokines of the present invention with conventional per daily dose scheme, described scheme can be adjusted according to needed prevention or the painful degree of alleviation.For such oral administration, can constitute pharmaceutically acceptable non-toxic composite by mixing any usual excipients, described vehicle is the N.F,USP MANNITOL, lactose, starch, povidone iodine, Magnesium Stearate, soluble saccharin, talcum powder, Mierocrystalline cellulose, croscarmellose sodium, glucose, gelatin, sucrose, magnesiumcarbonate etc. of pharmaceutical grade for example.This based composition is the form of solution, suspensoid, dispersible tablet, pill, capsule, pulvis, sustained release preparation etc.Oral preparations is specially adapted to treat gastrointestinal tract disease.For system's purpose, can adjust oral administration biaavailability by using the vehicle that can improve the picked-up of body round-robin, for example comprise the preparation of acetylated amino acids.Referring to No. the 5th, 629,020, No. the 5th, 935,601, United States Patent (USP) for example and United States Patent (USP).

Composition can be the form of capsule, pill or tablet, thus in the composition except that containing activeconstituents, also can contain thinner, for example lactose, sucrose, calcium phosphate etc.; Disintegrating agent, for example croscarmellose sodium, starch or derivatives thereof; Lubricant, for example Magnesium Stearate etc.; And tackiness agent, for example starch, polyvinylpyrrolidone, gum arabic, gelatin, Mierocrystalline cellulose and derivative thereof etc.

The liquid administration composition can make by for example biological activity synthetic chemokines of the present invention (about 0.5% to about 20%) and optional excipient substance being dissolved in, being scattered in the carrier, make solution or suspensoid thus, described carrier is water, salt solution, G/W, glycerine, ethylene glycol, ethanol, sanitas etc. for example.If necessary, institute's administered agents composition also can contain a small amount of nontoxic auxiliary material, for example wetting agent, suspension agent, emulsifying agent or solubilizing agent, pH buffer reagent etc., for example sodium acetate, Trisodium Citrate, cyclodextrin derivative, polyoxyethylene, the mono laurate sorb is smooth or the stearic acid sorb is smooth etc.The actual fabrication method of described formulation is well known by persons skilled in the art, or conspicuous; For example referring to Remington ' sPharmaceutical Sciences, Mack Publishing Company, Easton, PennsylVania.Composition that is given or preparation under any circumstance all can contain effective prevention or alleviate the activeconstituents of amount of the patient's that treats symptom.For the baby oral administration, preferred liquid preparation (for example syrup or suspensoid).

For the solid dosage that contains liquid, for example preferably will being dissolved in, the solution or the suspension of propylene carbonate, vegetables oil or triglyceride level wrap in the gelatine capsule.For liquid dosage form, the solution that is dissolved in the polyoxyethylene glycol for example can be with pharmaceutically acceptable liquid vehicle (for example water) dilution of q.s, so that administration according to quantity.

Perhaps, can be by activeconstituents being dissolved in or being scattered in vegetables oil, ethylene glycol, triglyceride level, the propylene glycol ester (for example propylene carbonate) etc., again these solution or suspension are wrapped in hard or the soft gelatin capsule shell, with preparation liquid or semisolid oral formulations.

When using biological activity synthetic chemokines of the present invention to treat above illness, preferably give activeconstituents as herein described through parenteral.The feature of parenteral admin is normally injected, can be through subcutaneous, intramuscular or intravenous injection, and can comprise intracutaneous or peritoneal injection and breastbone inner injection or infusion techniques.Injection can be prepared into conventionally form, for example liquor agent or suspensoid, be suitable for before injection, being mixed with the solution that is dissolved in liquid or the solid form of suspensoid, emulsion or based on the microsphere of biocompatible polymkeric substance (for example in liposome, polyethyleneglycol derivative, poly-(D, C) lactide etc.).Suitable vehicle is for example water, salt solution, glucose, glycerine, ethanol etc.In addition, if necessary, institute's administered agents composition also can contain a small amount of nontoxic auxiliary material, for example wetting agent or emulsifying agent, pH buffer reagent, solubilizing agent, protein carrier etc., for example sodium acetate, polyoxyethylene, mono laurate sorb are smooth, Emulphor FM, cyclodextrin, serum albumin etc.

Biological activity synthetic chemokines of the present invention can give through parenteral, and for example by described molecule being dissolved in the suitable solvent (for example water or salt solution) or being incorporated in the Liposomal formulation, redispersion is in acceptable transfusion.Can give the typical daily dosage portion of polypeptide of the present invention through an infusion, or give through a series of infusions of periodic intervals.For parenteral admin, the aqueous solution agent of the activeconstituents of specially suitable water-soluble form (for example water-soluble salt form) is arranged, perhaps moisture injection suspension, described preparation contains thickening material, for example Xylo-Mucine, sorbyl alcohol and/or dextran, and also contain stablizer if necessary.Activeconstituents, optional with vehicle, also can be made into lyophilized form, before parenteral admin, add suitable solvent and make solution.

Recently the method that is designed for parenteral admin adopts implantation slow release or sustained release system, thereby keeps the constant dosage level.Referring to No. the 5th, 041,292, No. the 3rd, 710,795, United States Patent (USP) for example, No. the 5th, 714,166, United States Patent (USP) and United States Patent (USP), described document is attached to herein by reference.

The percentage of the activeconstituents that contains in this class parenteral composition depends on its concrete property basically, and polypeptide active and patient's demand.Yet the activeconstituents percentage in the adoptable solution is 0.01% to 10%, also can be higher, if composition is solid and will be diluted to above percentile words subsequently.Preferred composition comprises the activeconstituents of 0.02-8% in solution.

Another method that gives biological activity synthetic chemokines of the present invention is utilized fast injection (injecting) and continuous infusion (instillation).When therapeutic treatment is that this is particularly preferred method when being used to prevent HIV-1 to infect.

Giving aerosol, is the effective ways that directly biological activity synthetic chemokines of the present invention given respiratory tract.Some advantages of this method are: 1) it has stoped the enzymatic degradation effect, gastrointestinal absorption is poor or because of the loss of the therapeutical agent due to the liver first-pass effect; 2) it gives activeconstituents, and described composition is because of its molecular size, electric charge or to the avidity at outer (extra-pulmonary) position of lung, and can't arrive target site in respiratory tract by other method; 3) it provides by alveolar and rapid absorption in body; With 4) it avoids other tract is exposed to activeconstituents, and this is very important exposing under the situation that may produce undesired side effect.Because these reasons, so give aerosol particularly advantageous for other disease for the treatment of asthma, local pulmonary infection and lung and respiratory tract or illness.

3 class medicinal inhalation devices are arranged: nebulizer, metered-dose inhaler and Diskus.Atomisation unit produces high velocity air, makes chemokine derivative (being mixed with liquid form) be injected into vaporific and enters patient's respiratory tract.Metered-dose inhaler is equipped with preparation and pressurized gas usually, in case start, just can spray quantitative polypeptide with pressurized gas, thereby the reliable method of the amount of setting medicine is provided.Diskus gives polypeptide with free-pouring powder type, and when breathing by device, described dry powder can be dispersed in patient's the air-flow.In order to obtain free-pouring powder, the chemokine derivative is prepared with vehicle (for example lactose).Quantitative chemokine derivative is preserved with capsule form, when each the unlatching, given the patient.All aforesaid methods all can be used for administration of the present invention.

Pharmaceutical preparation based on liposome also is applicable to chemokine of the present invention.Referring to No. the 5th, 766,627, No. the 5th, 631,018, United States Patent (USP) for example, No. the 5th, 723,147, United States Patent (USP) and United States Patent (USP).The benefit that it is believed that liposome relates to because of tissue distribution due to the wrapping kmedicine by liposome and the favourable change on the pharmacokinetic parameter, and can be used for polypeptide of the present invention by those skilled in the art.Also can use for the controlled release liposome liquid pharmaceutical formulation of injecting or oral administration is used.

For being administered systemically by suppository, typical binders and carrier comprise for example polyoxyethylene glycol or triglyceride level, for example PEG 1000 (96%) and PEG 4000 (4%).This class suppository can be that about 0.5% (weight) to about 10% (weight), preferred about 1% (weight) to the mixture of about 2% (weight) constitutes by containing the activeconstituents scope.

The present invention also provides as the medicine box of studying reagent and being used for methods of treatment of the present invention.Described medicine box can comprise the synthetic chemokines that is contained in the compartment that separates separately or the container, can be according to as the required formulation of research reagent or be used for providing described chemokine through the required formulation of required approach (for example intravenously, subcutaneous etc.) administration.The container of medicine box can be for example sterile vials or prefilled syringe.The working instructions of synthetic chemokines also can be provided in the medicine box.Can change the component in the medicine box, so that the above specification sheets that is provided to be provided.For example, medicine box can comprise one or more other reagent, for example other curative or medicine.

As mentioned above and what will further specify in following specific embodiment is to have found that biological activity synthetic chemokines of the present invention can be used as naturally occurring chemokine antagonists.Specifically, find to render a service enhanced biological activity synthetic chemokines of the present invention and can be used as antagonist in analysis and treatment various disease states, described morbid state is asthma, rhinallergosis, atopic dermatitis, cancer therapy, organ-graft refection, virus disease, congee sample spot/atherosclerosis, rheumatoid arthritis and organ-graft refection for example.Biological activity synthetic chemokines of the present invention also can be used for designing the small molecules antagonist with the related acceptor that screens them.For example, The compounds of this invention is carried out the structure diversity transformation, promoted the more rational method of design, screening and the fine setting of better micromolecular compound, described micromolecular compound relates to the medicine of the disease of Chemokine Receptors natural radioactivity as treatment.

Abbreviation

Use following abbreviation herein:

The Abu=aminobutyric acid

Acm=acetylamino methyl sulfydryl-protecting group [promptly-CH ₂NHCOCH ₃]

The Aib=aminoisobutyric acid

AoA=amino oxygen base ethanoyl

Arg (Tos)=L-arginine (side chain N δ tosyl group-protection)

The absolute reticulocyte count of ART=

Asp (cHex)=L-aspartic acid (side links polyhexamethylene-protection)

The AUC=area under curve

The Boc=tert-butoxycarbonyl

The Bom=benzyloxymethyl

CD=circular dichroism

The CDI=carbonyl dimidazoles

The CHO=Chinese hamster ovary

CL=clearance rate (ml/hr/kg)

The Cmax=peak concentration

Cys (4MeBzl)=L-halfcystine (side chain (4-methyl) benzyl-protection)

Cys (Acm)=L-halfcystine (the side chain acetylamino methyl [promptly-CH ₂NHCOCH ₃]-protection)

DBU=diazabicyclo undecane

The DCM=methylene dichloride

The DIC=DIC

The DIEA=diisopropylethylamine

The DMF=dimethyl formamide

The Dmg=N-methylsarcosine

The DMSO=methyl-sulphoxide

The Dnp=dinitrophenyl

DPC=dodecylphosphoric acid choline

Dpr=L-1, the 2-diaminopropionic acid

ED50=reaches the required effective dose of 50% maximum efficiency

EDA=(4,7,10)-trioxa tridecane-1,13-diamines (being also referred to as TTD)

The ELISA=enzyme-linked immunoassay

The ES-MS=electrospray ionization mass spectrometry

The FBS=foetal calf serum

Glu (cHex)=L-L-glutamic acid (side links polyhexamethylene-protection)

HATU=phosphofluoric acid O-(7-azepine benzo triazol-1-yl)-1,1,3, the 3-tetramethyl-ammonium

His (Dnp)=L-Histidine (side chain Nlm dinitrophenyl-protection)

The HOBT=N-hydroxybenzotriazole

The HPLC=high pressure liquid chromatography (HPLC)

IMDM=IscoveShi improvement DulbeccoShi substratum

The IPA=Virahol

The Lev=levulinic acid

Lys (CIZ)=L-Methionin (side chain 2-chlorine benzyloxycarbonyl)-protection

MBHA=4-methyldiphenyl methylamine

The mcg=microgram

The MRT=mean residence time

Mtt=4-methyl trityl

The MTT=tetrazolium bromide

The NHS=N-N-Hydroxysuccinimide

-OCH ₂-Pam-resin=-O-CH ₂-Bz-CH ₂CONHCH ₂(AMBERLITE XAD-4)-resin

Pbo=4-(CH ₃S (O)-) benzyl

The PBS=phosphate buffered saline(PBS)

RSA=rat blood serum albumin

The SDS=sodium lauryl sulphate

The SDS-PAGE=SDS-polyacrylamide gel electrophoresis

Ser (Bzl)=L-Serine (side chain benzyl-protection)

The Succ=succinyl

TTD=(4,7,10)-trioxa tridecane-1,13-diamines (can exchange with EDA and use)

Embodiment

Following examples are provided, so that how to prepare and use whole disclosure of the present invention and explanation for those of ordinary skills provide, and must not be considered as the restriction of contriver to its invention scope, following experiment can not be considered as is whole and only experiments of being carried out.Though made effort,, still had some experimental errors and deviation to guarantee the accuracy of used numerical value (for example consumption, temperature etc.).Except as otherwise noted, otherwise part be weight part, molecular weight is a weight-average molecular weight, and temperature is a centigradetemperature, and pressure is normal atmosphere or near normal atmosphere.

Embodiment 1: general purpose material and method

Peptide synthetic: according to U.S. Patent Application Serial Number 09/141,833 is that No. the 6th, 168,784, present United States Patent (USP) is described, can be by the synthetic peptide for preparing the chemokine derivative of solid-phase peptide.On the improved 430A peptide synthesizer of user (Applied Biosystems), carry out solid phase synthesis, use in the original position and phosphofluoric acid 2-(1H-benzotriazole-1-yl)-1,1,1,3, the activation scheme of 3-tetramethyl-urea  is carried out progressively Boc chemistry chain extension (Schnolzer etc., Int.J.PeptideProtein Res. (1992) 40:180-193).Synthetic N-end peptide fragment on the resin that produces thioesters.Before the research position, connect residue (C holds to the extension of N end) and manual extension peptide, then with this resin cracking.Each synthesis cycle all is made up of following steps: to remove N α-Boc, DMF stream was washed 1 minute with pure TFA processing 1-2 minute, with 1.0mmol activated b oc-amino acid coupling in advance 10-20 minute, carried out second time DME stream again and washed in the presence of excessive DIEA.In the presence of excessive DIEA (3mmol), N α-Boc-amino acid (1.1mmol) is activated in advance and reaches 3 minutes with 1mmol HBTU (DMF of 0.5M).After each manual coupling step, with the residual unhindered amina (Sarin etc., Anal.Biochem. (1981) 117:147-157) of triketohydrindene hydrate evaluation of measuring.At standard-O-CH ₂The synthetic amino acid that comprises the C-end fragment on the-phenyl acetylamino methyl resin.After chain assembling is finished,, as scavenging agent, peptide is carried out deprotection and from resin it cut down with 5% p-cresol by handling 1 hour at 0 ℃ with anhydrous HF.In all cases, imidazoles side chain DNP protecting group all is retained on the His residue, because remove the step of DNP and be not suitable for C-end thioester group.Yet, in ligation, progressively remove DNP by mercaptan, obtain unprotected His.The amino side-chain that is used on the Lys residue that polymkeric substance connects is protected with Fmoc.After the peptide chain assembling, remove the Fmoc group with 20% piperidines/DMF.Levulinic acid or isopropylidene amino oxygen base-acetate (amino oxygen base-acid that its amino oxygen base is protected by acetone) are coupled on the amino of Lys.After the cracking, go out this two kinds of peptides, be dissolved in the acetonitrile solution and freeze-drying with ice-cold ether sedimentation.Use the RP-HPLC purified peptide, adopt C4-post (deriving from Vydacs), use buffer B (acetonitrile/0.081% trifluoroacetic acid)/buffer A (H of linear gradient ₂The O/0.1% trifluoroacetic acid), carry out ultraviolet detection at the 214nm place.Use electron spray mass spectrometry, (Micromass, Manchester England) come analytic sample to use Platform II instrument.

Native chemical connects: except as otherwise noted, otherwise according to standard scheme, adopt native chemical to connect and be used for connection peptides, to produce total length chemokine polypeptides chain (Dawson etc., Science (1994) 266:776-779); Wilken etc., Chem.Biol. (1999) 6:43-51; Camarero etc., Current Protocols in Protein Science (1999) 18.4.1-18.4.21).

Folding: except as otherwise noted, otherwise according to standard technique, at Cys-SH/ (Cys-S) ₂Existence under, finish folding (Wilken etc., Chem.Biol. (1999) 6:43-51) of polypeptide chain.

Chemical characterization: characterize by the material of several technology to purifying.For example, C4 reversed-phase HPLC, SEC-MALS (size exclusion-chromatography and multi-angle scattering of light), CD, SDS-PAGE and ESI-MS.MALDI is used for the PEG formed material.The mixture of about 50-100 μ l purifying is injected in the C4 reversed-phase HPLC post, this pillar has used 25% buffer A (water that contains 0.1%TFA) and 75% buffer B (acetonitrile that contains 0.08%TFA) to carry out balance, and material is used linear gradient (25-65% buffer A) wash-out 20 minutes.Collect the material that elutes then, check its structure with ESI-MS.The purity of material is determined with the integration of HPLC chromatography main peak and impurity peaks.Proteinic state of aggregation is measured with SEC-HPLC-MALS.MALS links to each other with the HP1100 HPLC that Superdex 200 SEC posts are housed with RI (refractive index) detector (deriving from Wyatt Technology Corporation).Running buffer is the 50mM sodium phosphate (pH7.4) that contains 0.5MNaCl.Calculate molecular weight with ASTRA software (providing) by Wyatt TechnologyCorporation.The state of aggregation of molecule is also studied by the SDS-PAGE gel, uses NUPAGE 4-2% gel, runs glue with the MOPS damping fluid.Use JASCO 600 instruments, obtain UV CD spectrum far away.Every curve all is the mean value of 4 scannings in the 190-260nm scope.

Embodiment 2:RANTES derived peptide and compound

Make up following RANTES derivative (NK).Be used for connecting the peptide make up various NK analogues and see the following form 4 (modify, remove fmoc again and connect) according to embodiment 1 by chemistry.Total length chemokine polypeptides chain sees the following form 5.

The peptide of table 4:NK analogue

G2071:B ¹X ²Z ³SSDTTPC CFAAIAAPLP RAHIKAYFYT SGK-thioesters

(SEQ ID NO：37)

G2088：CSNPAVV FVTAANAQVC ANPEKKWVRE YINSLSMSK ^(lev)L-COOH

(SEQ ID NO：38)

G2089:B ¹X ²Z ³SSDTTPC CFAAIAAPLP RAHIKEYFYT SGK-thioesters

(SEQ ID NO：39)

G2092：CSNPAVV FVTAK ^(lev)NAQVC ANPEKKWVRE YINSLEMS-COOH

(SEQ ID NO：40)

G2832:B ¹X ²Z ³SSDTTPC CFAYIARPLP RAH ^(Dnp)IKEYFYT SGK-thioesters

(SEQ ID No：41)

G2094：CSNPAVV FVTRK ^(lev)NRQVC ANPEKKWVRE YINSLMS-COOH

(SEQ ID NO：42)

G2095：CSNPAVV FVTRK ^(lev)NRQVC ANPEKKWVRE YINSL-CONH ₂

(SEQ ID NO：43)

G2133:B ¹X ²Z ³SSDT ^(Nme)TPC CFAAIAAPLP RAHIKAYFYT SGK-thioesters

(SEQ ID NO：44)

G2097：CSNPAVV FVTAK ^(lev)NAQVC ANPEKKWVRE YINSLMS-COOH

(SEQ ID NO：45)

G2136:B ¹X ²Z ³SSDTTPC CFAAIAK ^(lev)PLP RAHIKAYFYT SGK-thioesters

(SEQ ID NO：46)

G2135：CSNPAVV FVTAANAQVC ANPEKKWVRE YINSLSMS-COOH

(SEQ ID NO：47)

G2142：CSNPAVV FVTRK ^(lev)NRQVC ANPEKKWVRE YINSLMK ^(palm)-COOH

(SEQ ID NO：48)

G2143:CSNPAVV FVTRK ^{(AoA-acetone)}NRQVC ANPEKKWVRE YINSLMS-COOH

(SEQ ID NO：49)

G2163:CSNPAVV FVTRK ^{(AoA-acetone)}NRQVC ANPEKKWVRE YINSL-CONH ₂

(SEQ ID NO：50)

G1804：CSNPAVV FVTRKNRQVC ANPEKKWVRE YINSLEK ^(lev)SK ^(palm)L-COOH

(SEQ ID NO：51)

B wherein ¹=n-nonanoyl (NNY); X ²=L-Thioproline (Thz); Z ³=L-Cyclohexylglycine (Chg); K ^(lev)=Lys (N ε-levulinic acyl group); H ^(Dnp)=His-(N-im-2,4-dinitrophenyl); T ^(Nme)=N α (CH ₃) Thr; K ^(palm)=Lys (N ε-C (O)-(CH ₂) ₇-NH-C (O)-(CH ₂) ₁₄-CH ₃); And K ^{(AoA-acetone)}=Lys (N ε-C (O)-CH ₂-O-N=C (CH ₃) ₂).All amino acid all adopt one-letter code to represent.

Table 5: aminoacid sequence and modification with locus specificity variation, with respect to the PSC-RANTES shown in the SEQ ID NO:2, as follows.

B ¹X ²Z ³SSDT ⁷TPC CFAY ¹⁴IAR ¹⁷PLP RAHIKE ²⁶YFYT

SGKCSNPAVV FVTR ⁴⁴K ⁴⁵NR ⁴⁷QVC ANPEKKWVRE

YINSLE ⁶⁶M ⁶⁷S ⁶⁸(SEQ ID NO：2)

NK	Position with respect to SEQ ID NO:2 is modified												The N-end	The C-end
															7	14	17	26	44	45	47	66	67	68	69	70
	NK	T	Y	R	E	R	K	R	E	K plp	S	K palm															L	1832	1804
NK 1													T	A	A	A	A	A	A	S	M	S	K plp	L	2071	2088
	NK 2	T	A	A	E	A	K plp	A	E	M	S	△															△	2089	2092
NK 3													T	Y	R	E	R	K plp	R	△	M	S	△	△	1832	2094
	NK 4	T	Y	R	E	R	K plp	R	△	△	△	△															△	1832	2095
NK 5													T Nme	A	A	A	R	K plp	R	△	M	S	△	△	2133	2094
	NK 6	T	A	K plp	A	A	A	A	S	M	S	△															△	2136	2135
NK 7													T Nme	A	A	A	A	A	A	S	M	S	K plp	L	2133	2088
	NK 8	T	A	A	A	A	K plp	A	△	M	S	△															△	2071	2097
NK 9													T	Y	R	E	R	K plp	R	△	M	K palm	△	△	1832	2142
	NK 10	T	Y	R	E	R	K peg20	R	△	M	S	△															△	1832	2143
NK 11													T	Y	R	E	R	K peg5	R	△	M	S	△	△	1832	2143
	NK 12	T	Y	R	E	R	K xplp	R	△	M	S	△															△	1832	2143
NK 13													T	Y	R	E	R	K peq20	R	△	△	△	△	△	1832	2163

B wherein ¹=NNY; X ²=Thz; Z ³=Chg; T ^Nme=N α (CH ₃) Thr; K ^Plp(N ε-oxime-plp), wherein plp is precise length polyamide polymer GP 41 to=Lys; K ^Xplp(N ε-oxime-Xplp), wherein Xplp is precise length polyamide polymer GP43 to=Lys; K ^Peg5=Lys (N ε-oxime-5kDa mPEG), wherein 5kDa mPEG is a straight chain 5kDa mono methoxy polyethylene glycol; K ^Peg20=Lys (N ε-oxime-20kDa mPEG), wherein 20kDamPEG is a straight chain 20kDa mono methoxy polyethylene glycol; K ^Palm=Lys (N ε-C (O)-(CH ₂) ₇-NH-C (O)-(CH ₂) ₁₄-CH ₃); △=disappearance or do not exist.All amino acid are all represented with one-letter code.

Synthesizing of embodiment 3:RANTES derivative compound

NK analogue synthetic has following SEQ ID NO, the as described below structure in the table 5.

Sequence (NK):

B ¹X ²Z ³SSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV

FVTRKNRQVC ANPEKKWVRE YINSLEK ^(plp)SK ^(palm)L-COOH(SEQ IDNO：52)

Sequence (NK1):

B ¹X ²Z ³SSDTTPC CFAAIAAPLP RAHIKAYFYT SGKCSNPAVV

FVTAANAQVC ANPEKKWVRE YINSLSMSK ^(plp)L-COOH(SEQ ID NO：53)

Sequence (NK2):

B ¹X ²Z ³SSDTTPC CFAAIAAPLP RAHIKEYFYT SGKCSNPAVV

FVTAK ^(plp)NAQVC ANPEKKWVRE YINSLEMS-COOH(SEQ ID NO：54)

Sequence (NK3):

B ¹X ²Z ³SSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV

FVTRK ^(plp)NRQVC ANPEKKWVRE YINSLMS-COOH(SEQ ID NO：55)

Sequence (NK4):

B ¹X ²Z ³SSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV

FVTRK ^(plp)NRQVC ANPEKKWVRE YINSL-CONH ₂(SEQ ID NO：56)

Sequence (NK5):

B ¹X ²Z ³SSDT ^(Nme)TPC CFAAIAAPLP RAHIKAYFYT SGKCSNPAVV

FVTRK ^(plp)NRQVC ANPEKKWVRE YINSLMS-COOH(SEQ ID NO：57)

Sequence (NK6):

B ¹X ²Z ³SSDTTPC CFAAIAK ^(plp)PLP RAHIKAYFYT SGKCSNPAVV

FVTAANAQVC ANPEKKWVRE YINSLSMS-COOH(SEQ ID NO：58)

Sequence (NK7):

B ¹X ²Z ³SSDT ^(Nme)TPC CFAAIAAPLP RAHIKAYFYT SGKCSNPAVV

FVTAANAQVC ANPEKKWVRE YINSLSMSK ^(plp)L-COOH(SEQ ID NO：59)

Sequence (NK8):

B ¹X ²Z ³SSDTTPC CFAAIAAPLP RAHIKAYFYT SGKCSNPAVV

FVTAK ^(plp)NAQVC ANPEKKWVRE YINSLMS-COOH(SEQ ID NO：60)

Sequence (NK9):

B ¹X ²Z ³SSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV

FVTRK ^(plp)NRQVC ANPEKKWVRE YINSLMK ^(palm)-COOH(SEQ ID NO：61)

Sequence (NK10):

B ¹X ²Z ³SSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV

FVTRK ^(peg20)NRQVC ANPEKKWVRE YINSLMS-COOH(SEQ ID NO：62)

Sequence (NK11):

B ¹X ²Z ³SSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV

FVTRK ^(peg5)NRQVC ANPEKKWVRE YINSLMS-COOH(SEQ ID NO：63)

Sequence (NK12):

B ¹X ²Z ³SSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV

FVTRK ^(xplp)NRQVC ANPEKKWVRE YINSLMS-COOH(SEQ ID NO：64)

Sequence (NK13):

B ¹X ²Z ³SSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV

FVTRK ^(peg20)NRQVC ANPEKKWVRE YINSL-CONH ₂(SEQ ID NO：65)

B wherein ¹=n-nonanoyl; X ²=L-Thioproline (Thz); Z ³=L-Cyclohexylglycine (Chg); T ^(Nme)=L-N Alpha-Methyl-Threonine; K ^(plp)=Lys (N ε-[lev-AoA oxime]-{ GP41) is promptly by levulinic acyl group-amino oxygen base oxime and water-soluble side chain accurate polyamide polymer GP41 bonding and the Methionin of modifying on its side chain ε (epsilon) nitrogen (referring to WO02/04150) has bonding structure [ε nitrogen Lys]-C (O)-CH ₂-CH ₂-C (CH ₃)=N-O-CH ₂-C (O) NH-{GP41}; K ^(Xplp)=Lys (N ε-[AoA-PyT oxime]-{ GP43}) is promptly by amino oxygen base-pyruvoyl oxime and water-soluble side chain accurate polyamide polymer GP43 bonding and the Methionin of modifying on its side chain ε nitrogen (referring to WO 02/04150) has bonding structure [ε nitrogen Lys]-C (O)-CH ₂-O-N=C (CH ₃)-C (O) NH-{GP43}; K ^(peg5)=Lys (N ε-AoA-butyryl radicals oxime]-5kDa mPEG}), promptly the Methionin of modifying on its side chain ε nitrogen has straight chain 5kDa mono methoxy-butyryl radicals PEG and bonding structure [ε nitrogen Lys]-C (O)-CH ₂-O-N=CH-(CH ₂) ₃-{ PEG ^5kDa-O-CH ₃; K ^(Peg20)=Lys (N ε-[AoA-propionyl oxime]-and 20kDa mPEG}), promptly the Methionin of modifying on its side chain ε nitrogen has straight chain 20kDa mono methoxy-propionyl PEG and bonding structure [ε nitrogen Lys]-C (O)-CH ₂-O-N=CH-CH ₂-{ PEG ^20kDa-O-CH ₃; K ^(Palm)=Lys (N epsilon-amino octyl group cetylate (Lys (N ε-aminooctanylpalmitate)), promptly the Methionin of modifying on its side chain ε nitrogen has structure [ε nitrogen Lys]-C (O)-(CH ₂) ₇-NH-C (O)-(CH ₂) ₁₄-CH ₃

The connection of peptide: adopt the listed peptide combination of listed peptide of table 4 and table 5, connect analogue NK, NK1, NK2, NK3, NK4, NK5, NK6, NK7, NK8, NK9, NK10, NK11, NK12 and NK13.Connect for typical peptide, adopt the mol ratio of 1.5: 1 (peptide 1: peptide 2 and polymkeric substance) usually.Each component is dissolved in the 6M Guanidinium hydrochloride/200mM phosphoric acid of newly joining, and (pH7.0 wherein contains 5mg/ml L-methionine(Met) ^*) in, concentration is 1mM.Choose wantonly this solution is carried out supersound process with the dissolving peptide.

In suitable stink cupboard (so that getting rid of the stink of thiophenol), thiophenol is joined in the reactant, concentration is 0.5%.Reactant at room temperature stirred gently spend the night.Reaction process is checked with analysis mode HPLC, with protein C 4 posts, uses the gradient buffering liquid B of 5-65% in 30 minutes.Collect each peak, identify the product peak with mass spectrum.

After stirring is spent the night, in mixture, add the beta-mercaptoethanol of 1 part of reaction volume and the 6M Guanidinium hydrochloride/100mM tris (pH8.5) of 3 parts of reaction volumes, stirred 10 minutes.Again with Glacial acetic acid with the pH regulator of reactant to 4.0-4.5.TCEP is joined in the reactant, and its add-on is 0.25 times of combined wt of peptide, then with its dissolving and stirred 20 minutes.

Again with sample on the solution to using on the 20% buffer B equilibrated C4 HPLC post.Collect circulation liquid and washings, handle with 10% SYNTHETIC OPTICAL WHITNER.For 5 * 25cm Vydac C4 post, use following parameter: flow velocity: 50ml/min, flow point size: 12.5ml.Protein elutes in 80 minutes with the 25-45% buffer B.Flow point is analyzed with ES-MS again, merges suitable flow point.With the amalgamation liquid freeze-drying, be used for folding reaction next time again.

Polymkeric substance-modification:, before the protein main chain assembling or after the main chain assembling, add polymkeric substance in order to obtain polymer-modified chemokine.

A. polymkeric substance connectivity scenario #1: as described below, polymkeric substance is connected with oximate between the peptide by polymkeric substance, described peptide has the levulinic acyl group, is used for analogue NK1, NK2, NK3, NK4, NK5, NK6, NK7, NK8, NK9.(peptide that has the levulinic acyl group: bPLP) mol ratio was used for the oxime connection in 1: 1.5.Each component is dissolved in 50%ACN/H ₂Among the O, making PLP concentration is 60mg/ml.This 50 ACN/H ₂Do not add TFA in the O solution.Proved that the existence of TFA can reduce the yield of PLP-peptide prod.This solution is also carried out supersound process so that dissolving PLP and peptide.

In case the peptide dissolving is spent the night reactant 40 ℃ of mild stirring.Reaction process is checked with the analysis mode HPLC that has protein C 4 posts, in 40 ℃, with 1ml/min speed, uses the B gradient of 5-65% in 30 minutes, the gradient of enumerating below the employing (#NEW5-65.M).Collect each peak, identify the product peak with mass spectrum.

With the buffer A dilution of reactant, make ACN content be lower than 20% again with 2-2.5 times of reaction volume.With sample on the gained solution to on the 20% buffer B equilibrated C4 HPLC post.For 5 * 25cm Vydac C4 post, use following gradient: flow velocity 50ml/min, flow point size: 12.5ml.In case when the 214nm absorbancy begins to rise, just collect flow point.Collected first peak is unreacted PLP, is product then, is unreacted peptide at last.

Each flow point is analyzed with ES-MS, merges suitable flow point.With these amalgamation liquid freeze-drying, be used for next ligation again.Behind the purifying, be about 45% based on the typical PLP-peptide yield that limits peptide weight.

B. polymkeric substance connectivity scenario #2: connect and folding according to above native chemical, before removing the acetone protecting group part on the AoA-Lys residue and connecting polyoxyethylene glycol (PEG)-aldehyde, at first assemble NK 10,11,12 and 13 protein main chains.Again with the folding protein of C4 reversed-phase column purifying, with the H that contains 0.1%TFA ₂The O-ACN damping fluid, in 80 minutes with the linear gradient of 25-45%.Flow point checks that with ES-MS the flow point that will contain target material combines and freeze-drying.

Dry powder is dissolved in the 70%ACN/H that contains 0.1%TFA that newly joins ₂Among the O, concentration is 10-15mg/ml.Methoxy amine hydrochlorate is dissolved in the same damping fluid, and making protein concn is 2M.Isopyknic protein and methoxyl group amine aqueous solution were mixed and at room temperature stir 1 hour.Remove the partial acetone of amino oxygen base end again, confirm to react with ESI-MS and finish.Protein soln uses the damping fluid H that contains 0.1%TFA with C4 reversed-phase column purifying ₂O and acetonitrile.Come elute protein with stepwise gradient, with the amalgamation liquid freeze-drying.

Dry powder is dissolved in 50% acetonitrile that the contains 0.1%TFA-H that newly joins ₂In the O damping fluid, concentration is 10mg/ml.PEG5Kd-aldehyde and PEG20Kd-aldehyde are dissolved in the same damping fluid, and concentration is 20mg/ml.Peptide solution is mixed with PEG solution, and this PEG solution contains excessive 0.5 times PEG with respect to protein.Reactant was at room temperature stirred 1 hour.To Superdex 200 posts, this post has been used 50mM sodium phosphate (pH7.4 contains 0.5M NaCl) balance with sample on the protein soln.Again with degree gradient separations such as protein and PEG usefulness.To contain the proteinic flow point of PEGization and combine and be stored in-80 ℃.

Folding: as to adopt following proposal to fold.

A. folding scheme #1: the chemokine analogue first round is folding is undertaken by following proposal.For example, the molecular weight of NK 1 is as follows before and after folding: Zhe Die G2071-G2088-PLP=23 not, 777Da; G2071-G2088-PLP=23 after folding, 773Da.

For this scheme, measure and carry out required folding damping fluid (2M Guanidinium hydrochloride/100mM tris, consumption pH8.5) of the folding reaction of 0.5mg/ml G2071-G2088-PLP.Before beginning folding reaction, L-halfcystine and L-Gelucystine dihydrochloride are dissolved in the folding damping fluid of aequum, its concentration is as follows: 8mM L-halfcystine; 1mM L-Gelucystine dihydrochloride.L-halfcystine and the folding damping fluid of L-Gelucystine dihydrochloride used in the folding reaction are newly joined.

Mild stirring on one side, on one side the folding damping fluid of aequum is joined among the not folding G2071-G2088-bPLP.With 6N HCl or 6N NaOH with the pH regulator of reactant to 8.2-8.5 and at room temperature stirred 90 minutes.Usually before folding reaction in batches, should test reaction earlier, with the suitable time length of determining that reaction is required.The reaction times that prolongs shows assembles for example gathering increase of Nonakine (NK) alkali of analogue.

Again with Glacial acetic acid with pH regulator to 4.0-4.5 and the quencher reactant.After folding reaction quencher, should carry out purification step immediately.

With sample on the gained solution to using on the 20% buffer B equilibrated C4 HPLC post.For 5 * 25cm Vydac C4 post, use following gradient: flow velocity: 50ml/min, flow point size: 12.5ml.In case when the 214nm absorbancy begins to rise, just collect flow point.First peak is the G2071-G2088-PLP product that folds, and then is false folding or accumulative G2071-G2088-PLP peak.

Because the folding and mass discrepancy of a little between the unfolded protein not, merging flow point need analyze a small amount of amalgamation liquid by the analysis mode HPLC that has protein C 4 posts, adopts aforesaid method #NEW5-65.M.Can satisfy the minimum purity level of accepting to determine which part by comparing each % purity of amalgamation liquid in a small amount, thus definite suitable amalgamation liquid.With the amalgamation liquid freeze-drying,, perhaps carry out buffer-exchanged then, it is changed to suitable preparation damping fluid so that store.Behind the purifying, folding usually yield is about 40%.

B. folding scheme #2: following folding scheme is used to keep the NK analogue, except as otherwise noted.Total length wire polypeptide for connecting is dissolved in lyophilized powder in the 7.5M guanidine hydrochloride solution, and concentration is 0.2-2mg/ml.Add after halfcystine and the halfcystine, concentration is respectively 1mM and 0.1mM.Add the Tris damping fluid, final concentration is 0.1M, and pH is 8.5.At 4 ℃, protein soln was dialysed 16-20 hour to folding damping fluid (containing 0.6M chlorination guanidine, 100mM Tris damping fluid (pH8.5), 1mM halfcystine, 0.1mM Gelucystine).

By the weight shutoff value is 3500 jar device (stirred cell device) or any allied equipment, and folding protein soln is concentrated into 8-20mg/ml.Will be on spissated protein soln sample to use 50mM sodium phosphate (pH7.4 contains 0.5M sodium-chlor) Superdex 200 posts (26 * 60 inches) that balance is crossed (Amersham Bioscience, MA) on.With gradient such as degree such as grade, use level pad, target dimer or monomeric products and aggregate are separated.Flow point is by the reversed-phase HPLC inspection, with C4 post and acetonitrile damping fluid.Also available analyses type Superdex 200 post inspections of flow point, this column chromatography are carried out separating in the same damping fluid with the preparation type.The flow point that will contain target material combines, with liquid nitrogen freezing and be stored in-80 ℃.

Embodiment 4: chemical property

Example results is seen Fig. 1-4.In brief, as shown in Figure 1, the NK analogue usually earlier use the FPLC purifying, and collection is corresponding to the flow point at the oligomer peak single step purification of advancing of going forward side by side.The representative result of the FPLC purifying of diagram NK3 (Figure 1A) and NK4 (Figure 1B) dimer flow point.

As shown in Figure 2, the C4-HPLC of synthetic NK chemokine and HPLC-SEC antique catalog and corresponding ES-MS chromatogram.The purifying dimer of diagram synthetic chemokines NK3 (Fig. 2 A) and NK 4 (Fig. 2 B) merges C4-HPLC, SEC-HPLC and the ES-MS antique catalog of thing.

Fig. 3 illustrates the purity of each flow point of NK analogue of measuring with SDS-PAGE.Specifically, each reduction and the dimeric SDS-PAGE gel of non-reducing purifying of diagram NK 3 (Fig. 3 A) and NK 4 (Fig. 3 C), and the purifying dimer CD of NK3 (Fig. 3 B) and NK4 (Fig. 3 D) spectrum.In the SDS-PAGE gel: Fig. 3 A, the 1st road is molecular weight (MW) standard substance, the 2nd road (reduction) and the 3rd road (non-reduced) is that the crude protein of NK3 merges thing; The 4th road (reduction) and the 5th road (non-reduced) are the monomer flow points of NK3; The 6th road (reduction) and the 7th road (non-reduced) are the dimer flow points of NK 3; The 8th road (reduction) and the 9th road (non-reduced) are the aggregate flow points of NK3; The 10th road (blank).Fig. 3 C, the 1st road is the MW standard substance, the 2nd road (reduction) and the 3rd road (non-reduced) is that the crude protein of NK 4 merges thing; The 4th road (reduction) and the 5th road (non-reduced) are the monomer flow points of NK 4; The 6th road (reduction) and the 7th road (non-reduced) are the dimer flow points of NK 4; The 8th road (reduction) and the 9th road (non-reduced) are the aggregate flow points of NK 4; The 10th road (blank).Fig. 3 also shows the representative CD spectrum of the folding NK analogue of purifying, and this spectrum proof exists the secondary structure that has usually in the folding chemokine.

In Fig. 4, representational SEC-MALS data presentation is with respect to the NK compound that forms aggregate, the typical consequence of the NK compound of purifying from crude mixture.The SEC-MALS antique catalog of the purifying oligomer (may be eight aggressiveness) (Fig. 4 C) of the NK3 dimer (Fig. 4 A) of diagram purifying, the NK4 dimer (Fig. 4 B) of purifying and NK contrast.

More universal result from the oligomer content of the various NK analogues of C4-HPLC sees Table 6.

Table 6: chemical characterization

The NK analogue	The oligomer state	Relative purity %
			NK	Aggregate	N/A
NK1	Dimer, monomer, aggregate	99/63
			NK2	Dimer, monomer, aggregate	97
NK3	Dimer	98
			NK4	Dimer		100
NK5	Monomer, aggregate	96
			NK6	Monomer, dimer, aggregate	100
NK7	Monomer, aggregate	100
			NK8	Dimer, aggregate, monomer	100
NK9	Aggregate	N/A
			NK10	Dimer
	100
		NK11	Dimer		100
NK12	Dimer					97
		NK13	Dimer		100

^*Relative purity % through the C4-HPLC purifying

Embodiment 5: the receptor signal conduction of calcium flux

A.CCR 5 Ca ⁺⁺Flux list transfectant is measured. and in measuring,, duplicate with the agonist activity of 10 kinds of concentration determination NK trial targets and contrast based on the CCR5 calcium flux of cell.The dynamics research data presentation goes out 3 experimental concentration the highest and the minimum experimental concentration of every kind of compound.To the maximum RFU of every kind of trial target, draw the concentration-response curve.The rank order of rendeing a service is seen Fig. 5, and this figure has compared every kind of multiple difference at the thinner contrast that compound is shown when 10 μ M.Attention: the damping fluid contrast of identical test compound is deducted from the numerical value of this compound, and when observing, low pH (6.5 and 5.0) the induced fluorescence background of these samples increases.In addition, some compound is also observed biphasic reaction when maximum concentration (10 μ M).

General scheme is as follows.For cell cultures, at 37 ℃ of incubator/5%CO ₂Allow the CHO-K1 clone of CCR5 transfection in the MEM α that has replenished 10%FBS (hot deactivation) and 500 μ g/ml Totomycin, grow down.When cell covers with 80-90%, divide biography with its ratio in 1: 6.Before calcium is measured, according to the following steps, with the inoculation of CHO-K1 clone onboard: the culturing bottle that CCR5-CHO-K1 clone will be housed washs with HBSS/20mM Hepes, in each culturing bottle, add 0.05% trypsinase-0.05%EDTA-HBSS (37 ℃ of preheatings) again, place it in 37 ℃ of incubator/5%CO ₂Down, up to cell desorption (about 1 minute).After cell desorption from the culturing bottle gets off, their are inhaled beat several, unicellular to obtain.Again substratum is joined in the cell centrifugal 7 minutes in 1000xg.Remove substratum after centrifugal, cell precipitation is resuspended in the substratum.Carry out cell counting and check vigor with Trypan Blue.Inoculate 50,000 cells/well/100 μ L substratum again in 96 orifice plates, Jiang Geban was hatched 24 hours, began then to measure.

Before the analysis, test compound is diluted with 1X reagent damping fluid, reach the final mensuration concentration of 2X.(MDS Pharma Canada) and with temperature is set in 24 ℃ (room temperatures) to demarcate FLEXstation.Every hole adds 100 μ l sample-loading buffers, and assay plate was hatched 1 hour at 37 ℃.After hatching, assay plate is moved to FLEXstation, wherein add NK test compound and contrast, obtain data.In the FLEXstation unit, the every row adding 100 μ l compounds at plate add entire plate.In 120 seconds cycle, read a secondary data per two seconds.FLEXstation is set in 24 ℃ and demarcate the 485nm exciting light and 525nm emission light, and cutoff is 515nm.Obtain the calcium flux data then and analyze.

B.CCR1 ﹠amp; CCR3 calcium flux list transfectant is measured. and utilize recombinant cell lines, preparation NK analogue is used for screening, and described clone is used to measure functional response at the G albumen-coupled receptor (GPCR) of the calcium flux of CCR1 and CCR3 through optimization.In case the acceptor irriate also increases cellular calcium, photoprotein (photoprotein) produces CO simultaneously in conjunction with calcium ion and substrate coelenteron fluorescein (coelenterazine) that oxidation added ₂And flash.For the antagonist screening, on orifice plate, in reconstitution cell, add the NK analog compounds in advance.Enter the preincubate cycle, time length is variable, and it is porose to inject institute at the agonist of the fixed concentration of acceptor.Recording light emission in 20 seconds is with the effect of determination test NK analog compounds, because antagonist and the radiative ratio that is suppressed to.Measure the calcium flux between the NK analogue again.The results are shown in Figure 6 and Fig. 7.

Embodiment 6: HIV (human immunodeficiency virus)-resistant activity

In vitro human peripheral blood lymphocytes (PBMC) is measured, measure the biological activity that different N onakine analogue infects HIV.The results are shown in Table 7, wherein the given NK compound of specific oligomer state was hatched 1 hour with PBMC, add HIV-1 virus strain 92/US/712 (" adding virus preincubate before ") again, perhaps add (" adding simultaneously ") with virus in the same time that adds HIV-1 virus strain 92/US/712.

Table 7: the antiviral activity among the human PBMC

Compound	The oligomer state	Add virus preincubate before		Add simultaneously with virus
						IC 50(nM)	IC 90(nM)	IC 50(nM)	IC 90(nM)
		NK *	Aggregate	1.40	2.89					0.62	4.87
NK1	Aggregate					＞1000.0	＞1000.0	＞1000.0	＞1000.0
		NK1 **	Monomer	＞1000.0	＞1000.0					931.72	＞1000.0
NK1 **	Dimer					＞1000.0	＞1000.0	＞1000.0	＞1000.0
		NK2	Aggregate	273.52	＞1000.0					448.4	＞1000.0
NK3 *	Dimer					0.49	1.84	0.76	4.02
		NK4 *	Dimer	0.25	2.10					0.52	2.36
NK5	Monomer					21.91	＞1000.0	47.15	354.67
		NK10	Dimer	0.20	0.40					0.02 ***	0.04 ***
NK11	Dimer					0.20	0.40	0.8	1.0
		NK12	Dimer	＜0.1 ***	＜0.1 ***					0.4	6.0

^*The mean value of twice experiment

^*The merging thing of purifying from aggregate

^{* *}Trend

Embodiment 7: pharmacokinetics

In normal Sprague Dawley (SD) rat,, studied pharmacokinetics (PK) characteristic of NK 3, NK 10 and NK 11 by intravenously (IV) and subcutaneous (SC) injection.Animal is divided into 6 groups, gives NK 3, NK 10 and NK 11 by single IV or SC injection.The elisa assay drug level is used in blood sampling in 72 hours after the administration.All animals all are healthy in research process.In on the same group SD rat not, give analogue with different concns, the result gathers as follows, sees Table 8 and Fig. 8 for 1mg/kg dosage (protein concentration).

After the IV administration, all 3 kinds of compounds all show two-phase density loss (Fig. 8).By contrast, NK 10 shows the highest clearance rate (CL) value (367ml/hr/kg), and NK 11 (73ml/hr/kg) is similar with NK 3 (98ml/hr/kg).3 kinds survey in the compound, NK 10 also demonstrates the longest apparent t1/2 (T _1/2) (be that NK 10 is 18 hours, and NK 11 is 3.4 hours, NK 3 is 2.8 hours).Yet, on average retention time (MRT), do not observe significant difference (NK 10 is 2.8 hours, and NK 11 is 2.3 hours, and NK 3 is 1.0 hours).

After the SC injection, the peak serum-concentration (C of 3 kinds of compounds being studied _Max) all similar (table 6 and Fig. 8).The C of compound N K 3, NK 11 and NK 10 _MaxBe respectively 2.1 μ g/ml, 1.4 μ g/ml and 1.3 μ g/ml.Serum exposes (AUC) also similar (table 8).The biological sharp weekly of NK 3, NK 11 and NK 10 is respectively 21%, 10% and 47%.The higher bioavailability of NK 10 does not cause higher exposure (AUC), this be because it higher CL and other two kinds of compounds seemingly.

Table 8:PK parameter

Compound	By way of	C max pg/ml	T max hr	AUC hr *pg/ml	Cl/F ml/hr/kg	Vz/F ml/kg	T 1/2 hr	MRT hr	F ％
										NK
3 NK 3 NK 11 NK 11 NK 10 NK 10	IV SC IV SC IV SC	10320000 145772 12065000 105051 8318500 145232	0.08 8.00 0.25 2.00 0.08 2.00	10173245 2109813 13680612 1435796 2724921 1280707	98.3 474.0 73.1 696.5 367.0 780.8	392.9 2033.0 358.4 10265.7 9552.2 21101.1	2.8 3.0 3.4 10.2 18.0 18.7	1.0 7.0 2.3 8.7 2.8 7.1	21 10 47

To term, scope be equal to the statement of embodiment

Should be known in that the invention that this paper describes is not limited to described specific embodiments in the whole text, because embodiment certainly is different.Should be known in that also the used term of this paper only in order to describe specific embodiments, must not be considered as limitation of the present invention, because scope of the present invention only is subjected to the restriction of appended claims in the whole text.

Although numerical range is provided, be appreciated that each interval value, to 1/10th of lower limit unit, unless clearly explanation, the numerical value between the upper and lower bound of this scope are arranged in the text in addition, any other description value or the interval value in described scope all are included within the present invention.Upper and lower bound in these can be included in more independently is also included within the present invention, is subjected to any specific exclusive restriction in the described scope.Although described scope comprises one or two described restrictions, do not comprise that each scope of its restriction is also included within the present invention.

Except as otherwise noted, otherwise all scientific and technical terminologies used herein all have the identical meanings with those skilled in the art's common sense.Although method of describing in the whole text with this paper and materials similar or any method and the material that are equal to also can be used for practice of the present invention or checking, this paper has only described preferable methods and material.All mentioned in the whole text publications of this paper are attached to herein by reference, with open method and/or the material relevant with the publication of being quoted with explanation.

Except as otherwise noted, otherwise, comprise multiple Rantes derivative, and, then comprise one or more Rantes derivatives and equivalent well known by persons skilled in the art thereof or the like for " described Rantes derivative " for " Rantes derivative ".

The publication that this paper is discussed in the whole text only provides its disclosure before the application's the applying date.This paper shall not be construed as in the whole text and admits that the present invention is not prior to these publications according to existing invention.In addition, the date of publication that is provided may be different with actual date of publication, and this may need independent confirmation.

Sequence table

<110>Gryphon Therapeutics

Bradburne，James

Miranda，Leslie

Paliard，Xavier

＜120〉synthetic chemokines, Preparation Method And The Use

<130>3504.294

<150>US 60/557,400

<151>2004-03-30

<160>65

<170>PatentIn version 3.3

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＜213〉artificial sequence

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＜221〉other features

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＜223〉amino acid, aminoacid replacement residue, aminoacid deletion residue or the polymkeric substance that independently is selected from corresponding position among the wild-type Rantes separately connects residue

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<222>(7)..(8)

＜223〉amino acid, aminoacid replacement residue, aminoacid deletion residue or the polymkeric substance that independently is selected from corresponding position among the wild-type Rantes separately connects residue

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＜221〉other features

<222>(14)..(14)

＜223〉amino acid, aminoacid replacement residue, aminoacid deletion residue or the polymkeric substance that is selected from corresponding position among the wild-type Rantes connects residue

<220>

＜221〉other features

<222>(17)..(17)

＜223〉amino acid, aminoacid replacement residue, aminoacid deletion residue or the polymkeric substance that is selected from corresponding position among the wild-type Rantes connects residue

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＜221〉other features

<222>(26)..(26)

＜223〉amino acid, aminoacid replacement residue, aminoacid deletion residue or the polymkeric substance that is selected from corresponding position among the wild-type Rantes connects residue

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<222>(45)..(45)

＜223〉amino acid, aminoacid replacement residue, aminoacid deletion residue or the polymkeric substance that independently is selected from corresponding position among the wild-type Rantes separately connects residue

<220>

＜221〉other features

<222>(47)..(47)

＜223〉amino acid, aminoacid replacement residue, aminoacid deletion residue or the polymkeric substance that is selected from corresponding position among the wild-type Rantes connects residue

<220>

＜221〉other features

<222>(65)..(68)

＜223〉amino acid, aminoacid replacement residue, aminoacid deletion residue or the polymkeric substance that independently is selected from corresponding position among the wild-type Rantes separately connects residue

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Xaa Xaa Xaa Ser Ser Asp Xaa Xaa Pro Cys Cys Phe Ala Xaa Ile Ala

1 5 10 15

Xaa Pro Leu Pro Arg Ala His Ile Lys Xaa Tyr Phe Tyr Thr Ser Gly

20 25 30

Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Xaa Xaa Asn Xaa Gln

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Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser

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Xaa Xaa Xaa Xaa

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<222>(2)..(2)

＜223〉L-Cyclohexylglycine

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Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg

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Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln Val

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Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

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Glu Met Ser

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Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala

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Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly

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Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln

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Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser

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Leu Glu Met Ser

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Ala Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cys Phe Ser Tyr Thr

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Leu Arg Lys Leu Pro Arg His Phe Val Ile Asp Tyr Phe Glu Thr Thr

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Ser Leu Cys Ser Gln Pro Ala Val Val Phe Gln Thr Lys Lys Gly Arg

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Gln Val Cys Ala Asn Pro Ser Glu Ser Trp Val Gln Glu Tyr Val Asp

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Asp Leu Glu Leu Asn

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Gly Asp Thr Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu

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Pro Thr Ser Gln Leu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys

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Gly Ser Glu Val Ser Asp Lys Arg Thr Cys Val Ser Leu Thr Thr Gln

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Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr Thr Ile Thr Glu Gly Ser

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Leu Arg Ala Val Ile Phe Ile Thr Lys Arg Gly Leu Lys Val Cys Ala

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Asp Pro Gln Ala Thr Trp Val Arg Asp Val Val Arg Ser Met Asp Arg

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Gln Gln Ser Thr Asn Thr Ala Val Thr Leu Thr Gly

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Gly Pro Ala Ser Val Pro Thr Thr Cys Cys Phe Asn Leu Ala Asn Arg

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Lys Ile Pro Leu Gln Arg Leu Glu Ser Tyr Arg Arg Ile Thr Ser Gly

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Lys Cys Pro Gln Lys Ala Val Ile Phe Lys Thr Lys Leu Ala Lys Asp

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Leu Asp Gln Lys Ser Pro Thr Pro Lys Pro

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Lys Ser Met Gln Val Pro Phe Ser Arg Cys Cys Phe Ser Phe Ala Glu

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Gln Glu Ile Pro Leu Arg Ala Ile Leu Cys Tyr Arg Asn Thr Ser Ser

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Ile Cys Ser Asn Glu Gly Leu Ile Phe Lys Leu Lys Arg Gly Lys Glu

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Ala Cys Ala Leu Asp Thr Val Gly Trp Val Gln Arg His Arg Lys Met

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Leu Arg His Cys Pro Ser Lys Arg Lys

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Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

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Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

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Gln Pro Val Gly Ile Asn Thr Ser Thr Thr Cys Cys Tyr Arg Phe Ile

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Lys His Leu Asp Lys Lys Thr Gln Thr Pro Lys Leu

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Gly Pro Asp Ala Val Ser Thr Pro Val Thr Cys Cys Tyr Asn Val Val

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Ser Ser Gln Cys Pro Arg Glu Ala Val Ile Phe Arg Thr Ile Leu Asp

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Asn His Leu Asp Lys Thr Ser Gln Thr Phe Ile Leu Glu Pro Ser Cys

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Leu Gly

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Ser Leu Ala Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser Tyr Thr Ser

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Gln Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Arg Ser Arg Gln

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Leu Glu Leu Ser Ala

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<210>13

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Ala Ser Asn Phe Asp Cys Cys Leu Gly Tyr Thr Asp Arg Ile Leu His

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Pro Lys Phe Ile Val Gly Phe Thr Arg Gln Leu Ala Asn Glu Gly Cys

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Asp Ile Asn Ala Ile Ile Phe His Thr Lys Lys Lys Leu Ser Val Cys

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Ala Asn Pro Lys Gln Thr Trp Val Lys Tyr Ile Val Arg Leu Leu Ser

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Arg Lys Asn

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Val Lys Ile Ile Thr Ala Lys Asn Gly Gln Pro Gln Leu Leu Asn Ser

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Gln

<210>18

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<210>19

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<400>19

Ser Asp Gly Gly Ala Gln Asp Cys Cys Leu Lys Tyr Ser Gln Arg Lys

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Ile Pro Ala Lys Val Val Arg Ser Tyr Arg Lys Gln Glu Pro Ser Leu

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Gly Cys Ser Ile Pro Ala Ile Leu Phe Leu Pro Arg Lys Arg Ser Gln

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Ala Glu Leu Cys Ala Asp Pro Lys Glu Leu Trp Val Gln Gln Leu Met

50 55 60

Gln His Leu Asp Lys Thr Pro Ser Pro Gln Lys Pro Ala Gln Gly Cys

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Arg Lys Asp Arg Gly Ala Ser Lys Thr Gly Lys Lys Gly Lys Gly Ser

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Lys Gly Cys Lys Arg Thr Glu Arg Ser Gln Thr Pro Lys Gly Pro

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<210>20

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Gly Pro Tyr Gly Ala Asn Met Glu Asp Ser Val Cys Cys Arg Asp Tyr

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Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys His Phe Tyr Trp Thr

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Ser Asp Ser Cys Pro Arg Pro Gly Val Val Leu Leu Thr Phe Arg Asp

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Lys Glu Ile Cys Ala Asp Pro Arg Val Pro Trp Val Lys Met Ile Leu

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Asn Lys Leu Ser Gln

65

<210>21

<211>71

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Ala Arg Gly Thr Asn Val Gly Arg Glu Cys Cys Leu Glu Tyr Phe Lys

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Gly Ala Ile Pro Leu Arg Lys Leu Lys Thr Trp Tyr Gln Thr Ser Glu

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Asp Cys Ser Arg Asp Ala Ile Val Phe Val Thr Val Gln Gly Arg Ala

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Ile Cys Ser Asp Pro Asn Asn Lys Arg Val Lys Asn Ala Val Lys Tyr

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Leu Gln Ser Leu Glu Arg Ser

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<210>22

<211>127

<212>PRT

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<400>22

Gln Gly Val Phe Glu Asp Cys Cys Leu Ala Tyr His Tyr Pro Ile Gly

1 5 10 15

Trp Ala Val Leu Arg Arg Ala Trp Thr Tyr Arg Ile Gln Glu Val Ser

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Gly Ser Cys Asn Leu Pro Ala Ala Ile Phe Tyr Leu Pro Lys Arg His

35 40 45

Arg Lys Val Cys Gly Asn Pro Lys Ser Arg Glu Val Gln Arg Ala Met

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Lys Leu Leu Asp Ala Arg Asn Lys Val Phe Ala Lys Leu His His Asn

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Met Gln Thr Phe Gln Ala Gly Pro His Ala Val Lys Lys Leu Ser Ser

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Gly Asn Ser Lys Leu Ser Ser Ser Lys Phe Ser Asn Pro Ile Ser Ser

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Ser Lys Arg Asn Val Ser Leu Leu Ile Ser Ala Asn Ser Gly Leu

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<211>67

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<400>23

Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

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His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro

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Ala Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln

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Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys

50 55 60

Ala Leu Asn

65

<210>24

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Val Pro Leu Ser Arg Thr Val Arg Cys Thr Cys Ile Ser Ile Ser Asn

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Gln Pro Val Asn Pro Arg Ser Leu Glu Lys Leu Glu Ile Ile Pro Ala

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Ser Gln Phe Cys Pro Arg Val Glu Ile Ile Ala Thr Met Lys Lys Lys

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Gly Glu Lys Arg Cys Leu Ash Pro Glu Ser Lys Ala Ile Lys Asn Leu

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Leu Lys Ala Val Ser Lys Glu Met Ser Lys Arg Ser Pro

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Ala Val Leu Pro Arg Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys

1 5 10 15

Thr Tyr Ser Lys Pro Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val

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Ile Glu Ser Gly Pro His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu

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Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln

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Arg Val Val Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser

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<210>26

<211>103

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Thr Pro Val Val Arg Lys Gly Arg Cys Ser Cys Ile Ser Thr Asn Gln

1 5 10 15

Gly Thr Ile His Leu Gln Ser Leu Lys Asp Leu Lys Gln Phe Ala Pro

20 25 30

Ser Pro Ser Cys Glu Lys Ile Glu Ile Ile Ala Thr Leu Lys Asn Gly

35 40 45

Val Gln Thr Cys Leu Asn Pro Asp Ser Ala Asp Val Lys Glu Leu Ile

50 55 60

Lys Lys Trp Glu Lys Gln Val Ser Gln Lys Lys Lys Gln Lys Asn Gly

65 70 75 80

Lys Lys His Gln Lys Lys Lys Val Leu Lys Val Arg Lys Ser Gln Arg

85 90 95

Ser Arg Gln Lys Lys Thr Thr

100

<210>27

<211>77

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<400>27

Gly Pro Val Ser Ala Val Leu Thr Glu Leu Arg Cys Thr Cys Leu Arg

1 5 10 15

Val Thr Leu Arg Val Asn Pro Lys Thr Ile Gly Lys Leu Gln Val Phe

20 25 30

Pro Ala Gly Pro Gln Cys Ser Lys Val Glu Val Val Ala Ser Leu Lys

35 40 45

Asn Gly Lys Gln Val Cys Leu Asp Pro Glu Ala Pro Phe Leu Lys Lys

50 55 60

Val Ile Gln Lys Ile Leu Asp Ser Gly Asn Lys Lys Asn

65 70 75

<210>28

<211>73

<212>PRT

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<400>28

Ala Ser Val Ala Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu Gln

1 5 10 15

Gly Ile His Pro Lys Asn Ile Gln Ser Val Asn Val Lys Ser Pro Gly

20 25 30

Pro His Cys Ala Gln Thr Glu Val Ile Ala Thr Leu Lys Asn Gly Arg

35 40 45

Lys Ala Cys Leu Asn Pro Ala Ser Pro Ile Val Lys Lys Ile Ile Glu

50 55 60

Lys Met Leu Asn Ser Asp Lys Ser Asn

65 70

<210>29

<211>73

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<400>29

Ala Pro Leu Ala Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu Gln

1 5 10 15

Gly Ile His Leu Lys Asn Ile Gln Ser Val Lys Val Lys Ser Pro Gly

20 25 30

Pro His Cys Ala Gln Thr Glu Val Ile Ala Thr Leu Lys Asn Gly Gln

35 40 45

Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Lys Lys Ile Ile Glu

50 55 60

Lys Met Leu Lys Asn Gly Lys Ser Asn

65 70

<210>30

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Ala Ser Val Val Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu Gln

1 5 10 15

Gly Ile His Leu Lys Asn Ile Gln Ser Val Asn Val Arg Ser Pro Gly

20 25 30

Pro His Cys Ala Gln Thr Glu Val Ile Ala Thr Leu Lys Asn Gly Lys

35 40 45

Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Gln Lys Ile Ile Glu

50 55 60

Lys Ile Leu Asn Lys Gly Ser Thr Asn

65 70

<210>31

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<400>31

Gln His His Gly Val Thr Lys Cys Asn Ile Thr Cys Ser Lys Met Thr

1 5 10 15

Ser Lys Ile Pro Val Ala Leu Leu Ile His Tyr Gln Gln Asn Gln Ala

20 25 30

Ser Cys Gly Lys Arg Ala Ile Ile Leu Glu Thr Arg Gln His Arg Leu

35 40 45

Phe Cys Ala Asp Pro Lys Glu Gln Trp Val Lys Asp Ala Met Gln His

50 55 60

Leu Asp Arg Gln Ala Ala Ala Leu Thr Arg Asn Gly

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<210>32

<211>4

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Arg Lys Asn Arg

1

<210>33

<211>5

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Lys Lys Trp Val Arg

1 5

<210>34

<211>4

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Lys His Leu Lys

1

<210>35

<211>4

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Lys Arg Ser Arg

1

<210>36

<211>4

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Lys Arg Ser Lys

1

<210>37

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(32)..(32)

＜223〉K-thioesters

<400>37

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Ala Ile Ala Ala

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Ala Tyr Phe Tyr Thr Ser Gly Xaa

20 25 30

<210>38

<211>37

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(36)..(36)

＜223〉Lys (N ε-levulinic acyl group)

<400>38

Cys Ser Asn Pro Ala Val Val Phe Val Thr Ala Ala Asn Ala Gln Val

1 5 10 15

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

20 25 30

Ser Met Ser Xaa Leu

35

<910>39

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(32)..(32)

＜223〉K-thioesters

<400>39

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Ala Ile Ala Ala

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Xaa

20 25 30

<210>40

<211>35

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(12)..(12)

＜223〉Lys (N ε-levulinic acyl group)

<400>40

Cys Ser Asn Pro Ala Val Val Phe Val Thr Ala Xaa Asn Ala Gln Val

1 5 10 15

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

20 25 30

Glu Met Ser

35

<210>41

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(22)..(22)

＜223〉His-(N-im-2,4-dinitrophenyl)

<220>

＜221〉other features

<222>(32)..(32)

＜223〉K-thioesters

<400>41

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg

1 5 10 15

Pro Leu Pro Arg Ala Xaa Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Xaa

20 25 30

<210>42

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(12)..(12)

＜223〉Lys (N ε-levulinic acyl group)

<400>42

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

1 5 10 15

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

20 25 30

Met Ser

<210>43

<211>35

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(12)..(12)

＜223〉Lys (N ε-levulinic acyl group)

<220>

＜221〉other features

<222>(35)..(35)

<223>L-CONH ₂

<400>43

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Leu Glu Val Asn

1 5 10 15

Arg Gln Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile

20 25 30

Asn Ser Xaa

35

<210>44

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(6)..(6)

<223>Nα(CH ₃)Thr

<220>

＜221〉other features

<222>(32)..(32)

＜223〉K-thioesters

<400>44

Xaa Xaa Ser Ser Asp Xaa Thr Pro Cys Cys Phe Ala Ala Ile Ala Ala

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Ala Tyr Phe Tyr Thr Ser Gly Xaa

20 25 30

<210>45

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(12)..(12)

＜223〉Lys (N ε-levulinic acyl group)

<400>45

Cys Ser Asn Pro Ala Val Val Phe Val Thr Ala Xaa Asn Ala Gln Val

1 5 10 15

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

20 25 30

Met Ser

<210>46

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(16)..(16)

＜223〉Lys (N ε-levulinic acyl group)

<220>

＜221〉other features

<222>(32)..(32)

＜223〉K-thioesters

<400>46

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Ala Ile Ala Xaa

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Ala Tyr Phe Tyr Thr Ser Gly Xaa

20 25 30

<210>47

<211>35

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<400>47

Cys Ser Asn Pro Ala Val Val Phe Val Thr Ala Ala Asn Ala Gln Val

1 5 10 15

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

20 25 30

Ser Met Ser

35

<210>48

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(12)..(12)

＜223〉Lys (N ε-levulinic acyl group)

<220>

＜221〉other features

<222>(34)..(34)

<223>Lys(Nε-C(O)-(CH ₂) ₇-NH-C(O)-(CH ₂) ₁₄-CH ₃)

<400>48

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

1 5 10 15

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

20 25 30

Met Xaa

<210>49

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(12)..(12)

<223>Lys(Nε-C(O)-CH ₂-O-N＝C(CH ₃) ₂)

<400>49

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

1 5 10 15

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

20 25 30

Met Ser

<210>50

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(12)..(12)

<223>Lys(Nε)-C(O)-CH ₂-O-N＝C(CH ₃) ₂)

<220>

＜221〉other features

<222>(32)..(32)

<223>L-CONH ₂

<400>50

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

1 5 10 15

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Xaa

20 25 30

<210>51

<211>37

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes peptide derivant

<220>

＜221〉other features

<222>(34)..(34)

＜223〉Lys (N ε-levulinic acyl group)

<220>

＜221〉other features

<222>(36)..(36)

<223>Lys(Nε-C(O)-(CH ₂) ₇-NH-C(O)-(CH ₂) ₁₄-CH ₃)

<400>51

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln Val

1 5 10 15

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

20 25 30

Glu Xaa Ser Xaa Leu

35

<210>52

<211>69

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(66)..(66)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<220>

＜221〉other features

<222>(68)..(68)

＜223〉Lys (N epsilon-amino octyl group cetylate)

<400>52

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Glu Xaa Ser Xaa Leu

65

<210>53

<211>69

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(68)..(68)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<400>53

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Ala Ile Ala Ala

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Ala Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Ala Ala Asn Ala Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Ser Met Ser Xaa Leu

65

<210>54

<211>67

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(44)..(44)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble side chain accurate polyamide polymer GP41 bonding and the Methionin of on its side chain ε nitrogen, modifying

<220>

＜221〉other features

<222>(44)..(44)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<400>54

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Ala Ile Ala Ala

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Ala Xaa Asn Ala Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Glu Met Ser

65

<210>55

<211>66

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(44)..(44)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble side chain accurate polyamide polymer GP41 bonding and the Methionin of on its side chain ε nitrogen, modifying

<220>

＜221〉other features

<222>(44)..(44)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<400>55

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Met Ser

65

<210>56

<211>64

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(44)..(44)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<220>

＜221〉other features

<222>(64)..(64)

<223>L-CONH ₂

<400>56

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Xaa

50 55 60

<210>57

<211>66

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(6)..(6)

＜223〉L-N Alpha-Methyl-Threonine

<220>

＜221〉other features

<222>(44)..(44)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<400>57

Xaa Xaa Ser Ser Asp Xaa Thr Pro Cys Cys Phe Ala Ala Ile Ala Ala

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Ala Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Met Ser

65

<210>58

<211>67

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(16)..(16)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble side chain accurate polyamide polymer GP41 bonding and the Methionin of on its side chain ε nitrogen, modifying

<220>

＜221〉other features

<222>(16)..(16)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<400>58

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Ala Ile Ala Xaa

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Ala Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Ala Ala Asn Ala Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Ser Met Ser

65

<210>59

<211>69

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(6)..(6)

＜223〉L-N Alpha-Methyl-Threonine

<220>

＜221〉other features

<222>(68)..(68)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<400>59

Xaa Xaa Ser Ser Asp Xaa Thr Pro Cys Cys Phe Ala Ala Ile Ala Ala

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Ala Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Ala Ala Asn Ala Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Ser Met Ser Xaa Leu

65

<210>60

<211>66

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(44)..(44)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<400>60

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Ala Ile Ala Ala

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Ala Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Ala Xaa Asn Ala Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Met Ser

65

<210>61

<211>66

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(44)..(44)

＜223〉by levulinic acyl group-amino oxygen base oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<220>

＜221〉other features

<222>(66)..(66)

＜223〉Lys (N epsilon-amino octyl group cetylate)

<400>61

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr lle Ala Arg

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Met Xaa

65

<210>62

<211>66

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(44)..(44)

＜223〉Methionin of on its side chain ε nitrogen, modifying with straight chain 20kDa mono methoxy-propionyl PEG

<400>62

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Met Ser

65

<210>63

<211>66

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(44)..(44)

＜223〉Methionin of on its side chain ε nitrogen, modifying with straight chain 5kDa mono methoxy-butyryl radicals PEG

<400>63

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Met Ser

65

<210>64

<211>66

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>44)..(44)

＜223〉by amino oxygen base-pyruvoyl oxime and water-soluble branched polyamides polymer-bound and the Methionin of on its side chain ε nitrogen, modifying

<400>64

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Met Ser

65

<210>65

<211>64

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Rantes derivative

<220>

＜221〉other features

<222>(1)..(1)

＜223〉(n-nonanoyl)-L-Thioproline

<220>

＜221〉other features

<222>(2)..(2)

＜223〉L-Cyclohexylglycine

<220>

＜221〉other features

<222>(44)..(44)

＜223〉Methionin of on its side chain ε nitrogen, modifying with straight chain 20 kDa mono methoxy-propionyl PEG

<400>65

Xaa Xaa Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg

1 5 10 15

Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys

20 25 30

Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Xaa Asn Arg Gln Val

35 40 45

Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu

50 55 60

Claims (48)

1. synthetic chemokines, described chemokine comprises the chemokine polypeptides chain with N-end and C-end, described chemokine polypeptides chain comprises (i) aminoacid sequence and halfcystine pattern and the (ii) end of the C-with respect to described wild-type chemokine brachymemma corresponding to the wild-type chemokine.

2. the synthetic chemokines of claim 1, wherein said N-end comprises the amino acid of described chemokine polypeptides chain, and described amino acid is the N-end that forms the halfcystine of first disulfide linkage in the described chemokine polypeptides chain.

3. the synthetic chemokines of claim 1, wherein said C-end comprises the amino acid of described chemokine polypeptides chain, and described amino acid is the C-end that forms the halfcystine of last disulfide linkage in the described chemokine polypeptides chain.

4. the synthetic chemokines of claim 3, wherein said C-end comprises the core helical region, and described brachymemma is the C-end of described core helical region.

5. the synthetic chemokines of claim 1, wherein said brachymemma comprises the one or more disappearances with amino-acid residue of polarity or electrically charged side chain with respect to described wild-type chemokine.

6. the synthetic chemokines of claim 5, wherein said amino-acid residue with polarity or electrically charged side chain is selected from arginine, Methionin, aspartic acid and L-glutamic acid.

7. the synthetic chemokines of claim 5, wherein said synthetic chemokines is the oligomer state of being made up of monomer basically.

8. the synthetic chemokines of claim 5, wherein said synthetic chemokines is the oligomer state of being made up of dimer basically.

9. the synthetic chemokines of claim 1, wherein said chemokine polypeptides chain comprises one or more amino-acid residues, and this amino-acid residue is different from the amino-acid residue of corresponding position in the described wild-type chemokine.

10. the synthetic chemokines of claim 9, wherein said chemokine polypeptides chain is modified by one or more amino-acid residues at its C-end, and this amino-acid residue is different from the amino-acid residue of corresponding position in the described wild-type chemokine.

11. the synthetic chemokines of claim 11, wherein said C-end is by formula-NH-CH (R)-C (O)-NH ₂The amino acid end-blocking, wherein R is the amino acid side chain identical or different with the amino acid side chain of described wild-type chemokine.

12. the synthetic chemokines of claim 9, wherein said chemokine polypeptides chain is modified by one or more amino-acid residues at its N-end, and this amino-acid residue is different from the amino-acid residue of corresponding position in the described wild-type chemokine.

13. the synthetic chemokines of claim 12, wherein said chemokine polypeptides chain is modified by the hydrophobicity aliphatic chain at its N-end.

14. the synthetic chemokines of claim 13, wherein said N-end by formula J-X-NH-CH (R)-C (O)-the amino acid end-blocking, wherein R is the amino acid side chain identical or different with the amino acid side chain of described wild-type chemokine, and X is a joint, and J-is described hydrophobicity aliphatic chain.

15. the synthetic chemokines of claim 14, wherein X comprises amino acid derivative.

16. the synthetic chemokines of claim 14, wherein J comprises formula CH ₂-(CH ₂) _n-, n=0-20 wherein.

17. the synthetic chemokines of claim 1, wherein said chemokine polypeptides chain is by one or more polymkeric substance covalent modifications.

18. the synthetic chemokines of claim 17, wherein said polymkeric substance is a straight chain.

19. the synthetic chemokines of claim 17, wherein said polymkeric substance is a side chain.

20. the synthetic chemokines of claim 17, wherein said polymkeric substance comprises formula-CH ₂-CH ₂The oxyethane repeating unit of-O-.

21. the synthetic chemokines of claim 20, wherein said polymkeric substance comprises polyoxyethylene glycol.

22. the synthetic chemokines of claim 20, wherein said polymkeric substance comprises polymeric amide.

23. the synthetic chemokines of claim 17, wherein said polymkeric substance comprises lipid acid.

24. the synthetic chemokines of claim 1, wherein said chemokine polypeptides chain comprise and the basic homologous aminoacid sequence of the aminoacid sequence of described wild-type chemokine.

25. the synthetic chemokines of claim 24, wherein said wild-type chemokine is the CC chemokine.

26. the synthetic chemokines of claim 24, wherein said wild-type chemokine is the CXC chemokine.

27. the synthetic chemokines of claim 1, wherein said wild-type chemokine is selected from Rantes, MIP1 α, MIP1 β or MCP-1.

28. the synthetic chemokines of claim 1, wherein said chemokine polypeptides chain comprises the aminoacid sequence of SEQ ID NO:1.

29. a composition, described composition comprise the synthetic chemokines with the aminoacid sequence shown in the SEQ ID NO:1.

30. comprising, a composition, described composition be selected from following synthetic chemokines: NK1, NK2, NK3, NK4, NK5, NK6, NK7, NK8, NK9, NK10, NK11, NK12 and NK13.

31. the synthetic chemokines of claim 1, wherein said chemokine polypeptides chain prepares by chemosynthesis.

32. the synthetic chemokines of claim 31, wherein said chemosynthesis comprise that the chemo-selective of the non-overlapped peptide fragment of described chemokine polypeptides chain connects.

33. it is that native chemical connects that the synthetic chemokines of claim 32, wherein said chemo-selective connect.

34. a pharmaceutical composition, described composition comprise in the claim 1,29 and 30 each synthetic chemokines or its pharmacy acceptable salt.

35. the pharmaceutical composition of claim 34, described composition comprise one or more and are selected from following vehicle: buffer reagent, carrier proteins, amino acid, washing agent, lipid, water-soluble polymers and sanitas.

36. the pharmaceutical composition of claim 34, described composition comprise one or more other biologically active agents except that described synthetic chemokines.

37. method for the treatment of the mammalian diseases state, described morbid state is by being alleviated with chemokine receptor anagonists treatment, and described method comprises that the Mammals that needs described treatment treats in the claim 1,29 and 30 of significant quantity each synthetic chemokines.

38. the method for claim 37, wherein said synthetic chemokines causes described Chemokine Receptors to be reduced with combining of Chemokine Receptors.

39. suffering from, the method for claim 37, wherein said Mammals be selected from following disease: AIDS, psoriatic, multiple sclerosis, cancer, asthma, rhinallergosis, atopic dermatitis, congee sample spot, atherosclerosis or rheumatoid arthritis.

40. the method for claim 39, wherein said mammiferous disease can be selected from following treatment: antiviral therapy, curing psoriasis, multiple sclerosis treatment, cancer chemotherapeutic, treating asthma, treating allergic rhinitis, treatment of atopic dermatitis, the treatment of congee sample spot, treatment of atherosclerosis and rheumatoid arthritis treatment.

41. the method for claim 40, wherein said synthetic chemokines before described treatment, simultaneously or give afterwards.

42. the preparation method of the synthetic chemokines of the oligomer form of a basic purifying, described method comprises:

The synthetic proteic protein library of synthetic chemokines that contains, described protein comprises the chemokine polypeptides chain with N-end and C-end, described chemokine polypeptides chain comprises (i) aminoacid sequence and halfcystine pattern and the (ii) end of the C-with respect to described wild-type chemokine brachymemma corresponding to the wild-type chemokine; With

Proteic one or more oligomer forms of the described synthetic chemokines of purifying from described protein library are with the synthetic chemokines of the single substantially oligomer form of preparation.

43. the method for claim 42, wherein said single oligomer form is selected from monomer and dimer.

44. the method for claim 42, wherein said synthetic chemokines albumen is by one or more polymkeric substance covalent modifications.

45. the method for claim 42, the wherein said synthetic chemosynthesis that comprises.

46. the method for claim 45, wherein said chemosynthesis comprise that the chemo-selective of the non-overlapped peptide fragment of described chemokine polypeptides derivative connects.

47. it is that native chemical connects that the method for claim 46, wherein said chemo-selective connect.

48. a medicine box, described medicine box comprise in the claim 1,29 and 30 that is contained in the container each chemokine polypeptides derivative.

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