CN1535313A - Protein C or activated protein C-like molecules - Google Patents

Abstract

The present invention relates to novel conjugates between polypeptide variants of protein C and a non-polypeptide moiety, such as PEG or sugar moieties. In particular, the present invention provides novel protein C conjugates having an increased resistance to inactivation by e.g. human plasma and alpha1-antitrypsin. Consequently, such conjugates have an increased in vivo half-life. Preferred examples include protein C conjugates, wherein at least one additional in vivo N-glycosylation site has been introduced. The conjugates of the invention are useful for treating a variety of diseases, including septic shock.

Description

PROTEIN C or activatory PROTEIN C-sample molecule

Technical field

The present invention relates to the polypeptide variants of PROTEIN C and the new conjugate of non-polypeptide fraction, the instrument (means) and the method that prepare described conjugate, the pharmaceutical composition that comprises described conjugate, and the purposes of described conjugate in treatment are especially treated various blood coagulation diseases.The invention still further relates to the polypeptide portion of described conjugate.

Background technology

Blood coagulation is by multiple blood constitutent, or the factor, between the process that causes fibrin clot to form gradually formed of complex interactions.Usually the blood constitutent that participates in blood coagulation " cascade reaction " is protoenzyme or proenzyme,, does not have the albumen of enzymic activity that is, and the effect of activator can make it change activity form into.Adjusting to blood coagulation mainly realizes (Esmon, J Biol Chem 1989 by activatory PROTEIN C (APC) by protein cleavage effect deactivation prothrombinase and VIIIa; 264; 4743-4746).

PROTEIN C is a kind of serine protease, and it is with the form circulation of the proenzyme in the blood plasma, and half life is about 7 hours, and blood plasma level is generally 3-5mg/l.It produces in the liver in vivo with the form of 461 amino acid whose strand precursor polypeptide.This polypeptide experiences multiple posttranslational modification, comprises a) 42 amino acid whose signal peptides of cutting; B) cutting Methionin and arginine residues (156 and 157) produces the proenzyme (a kind ofly have 155 amino acid whose light chains, have 262 amino acid whose heavy chains and link to each other with a kind of via disulfide linkage) of double-stranded non-activity; C) it is carboxylated 9 glutaminic acid residues of light chain to be carried out vitamin K-dependence, thereby obtains 9 Gla residues at the N-of light chain end; With d) (site is in light chain, and three in heavy chain) links to each other with carbohydrate in four sites.At last, the dodecapeptide (activation peptide, 158-169 position) by removing heavy chain N-end makes above-mentioned double-stranded proenzyme activation, produces activated form PROTEIN C (APC).

PROTEIN C activates by being positioned at the limited protein cleavage with zymoplasm zymoplasm regulin (thrombomodulin) formation complex body the endotheliocyte surface of internal cavity.As mentioned above, activation causes terminal 12 the amino acid whose little peptides (being called the activation peptide) that discharge from the N-of heavy chain.The half life of APC in blood plasma, be about 15 minutes.

Under the situation that its cofactor Protein S is arranged, APC makes prothrombinase and VIIIa inactivation by the protein cleavage effect, thereby reduces generation (Esmon, the Thromb Haemost 1993 of zymoplasm; 70; 29-35).Protein S with another kind of plasma proteins, the conjugated protein form reverse circulation that combines of C4b-.Only the free Protein S can be used as the cofactor of APC.Because C4b-is conjugated protein to be a kind of acute phase reactant, this proteic blood plasma level has big-difference very and therefore influences the anticoagulating active of protein C system in a lot of diseases.

The assignment of genes gene mapping of coding human protein C in karyomit(e) 2q13-q14 (Patracchini etc., HumGenet 1989; 81; 191-192), it strides 11kb, comprises a coding region (exon II-IX) and a 5 ' non-translational region that holds (encompassing) exon I.Demonstrate higher homology with other vitamin K-dependence blood coagulating protein such as plasma thromboplastin component and X by exon II-IX encoded protein structural domain.Encode former peptide and comprise 38 amino acid whose sequences of 9 Glu residues of exon II coded signal peptide, exon III.Former peptide comprise can with calcium ion bonded carboxylase binding site, this enzyme is converted into di-carboxylic acid (Gla) with the Glu residue, and this is the combination of phosphatide and the necessary step of anticoagulating active of PROTEIN C (Cheung etc., Arch Biochem Biophys 1989; 274; 574-581).Exon IV, V and VI encode respectively one section short catenation sequence and two kinds of EGF-spline structure territories.Exon VII coding contains the structural domain and the dipeptides 156-157 of 12 amino acid whose activation peptides, and described activation peptide can be released after PROTEIN C is by activated by thrombin, after described dipeptides is cut, produces the albumen of sophisticated double chain form.Exon VIII and IX encoding serine proteolytic enzyme structural domain.

By Foster etc., PNAS.USA 1986 for the complete amino acid sequence of human protein C; 82; The 4673-4677 report, it comprises signal peptide, former peptide, light chain, heavy chain, and activation peptide.

PROTEIN C combines with endothelial cell protein acceptor (EPCR).APC and combining of EPCR make the APC can not deactivation prothrombinase and VIIIa, and PROTEIN C then obviously strengthens the active rate of zymoplasm-zymoplasm regulin complex body to PROTEIN C with combining of EPCR.These interactional physiological significances it be not immediately clear.But obviously, PROTEIN C and EPCR combine with the strictness of phosphatide dependent/non-dependent mode depend on the existing of Gla structural domain (Esmon etc., Haematologica 1999; 84; 363-368).

APC is subjected to the inhibition of protein C inhibitor and α-1-antitrypsin and α-2-macroglobulin in blood plasma.

According to Mather etc., EMBO J1996; 15; The report of 6822-6831, the experimental three-dimensional structure of human APC has been determined to the level of resolution of 2.8 .They have reported the X-line structure of the APC that does not contain the Gla structural domain.This structure comprise covalently bound inhibitor (the D-Phe-Pro-Arg chloromethyl ketone, PPACK).

PROTEIN C is now separated from the thrombogen enriched material by the monoclonal antibody affinity chromatography preparation.In addition, PROTEIN C also can be recombinated by the expression of mammalian cell and be produced or obtain by modified protein C.

APC can be used for treating heredity and acquired PROTEIN C defective, someone advises APC is used for following patient as antithrombotics: the lupus of some forms, after outbreak (stroke) or the myocardial infarction, phlebothrombosis, disseminated inravascular coagulation (DIC), septic shock, embolism (emboli) is transplanted as bone marrow transplantation burn as pulmonary infarction, gestation, great surgical operation/wound and adult respiratory distress syndrome (ARDS).

Reorganization APC is by Eli Lilly and Co preparation, treat pyemic III clinical trial phase (Bernard etc., N Engl J Med (2001), 344, pp.699-709) finish in the near future.The severe sepsis patient in 96 hours with the dosage infusion administration of 24 μ g/kg/h.

But,, must reach and keep required treatment or prophylactic effect with higher relatively dosage and frequent administration because the half life of APC is short.The result is be difficult to dosage is fully adjusted, and the needs of intravenously administrable high level APC will bring problem and spend higher continually.

Have the long molecule that circulates half life and will reduce essential administration number of times and better therapeutic APC level effectively is provided, treat effect simultaneously and also strengthen.

The circulation half life of APC, can be passed through, and for example, reduces kidney and remove, and reduces the protein cleavage degraded or reduce and suppress and be increased.This can pass through, for example APC and non-polypeptide fraction coupling are realized, described non-polypeptide fraction such as PEG or carbohydrate, they can cause that described proteic kidney is removed minimizing and/or effective blocks protein cracking performance enzyme or inhibitor and contact with above-mentioned proteic physiology.In addition, this can realize that also described sudden change causes the protein C molecule retentive activity but blocked inhibitor and this proteic combination by protein C molecule is suddenlyd change.

The description of PEGization wild-type APC is referring to JP 8-92294.

WO 91/09960 discloses a kind of hybrid protein, and it partly comprises modification at the heavy chain of PROTEIN C.

WO 01/59084 has described the PROTEIN C variant, and it comprises that D167F+D172K replaces and in the position 10,11,12,32,194,195,228,149,254,302 or 316 also comprise at least one replacement.Disclosed variant it is said and has the enhanced anticoagulating active among the WO 01/59084.

WO 98/44000 has extensively described the PROTEIN C variant with enhanced acid amides lytic activity.

EP 0 323 149 has described the zymogen forms of PROTEIN C, and it has following sudden change: D167F/G/Y/W in heavy chain.These variants it is said to the susceptibility increase of thrombin activation.

WO 00/66754 reported position 194,195,228,249, and 254,302 or 316 natural residue replaces the half life that causes APC in the human blood to be increased than wild-type APC.Disclosed variant is not included in the scope of the present invention among the WO00/66754.

WO 99/63070 has described the terminal truncation type PROTEIN C of C-.

EP 0 946 715 has reported chimeric protein c polypeptide, and wherein the Gla structural domain of PROTEIN C is replaced by the Gla structural domain from other vitamin k dependent polypeptides (as proconvertin, factor X and thrombogen).

WO 99/20767 and WO 00/66753 disclose the vitamin K-dependence polypeptide variants, and they comprise modification in the Gla structural domain.

US 5,453, and 373 disclose the derivative of human protein C, and their glycosylation pattern and region of activation all change, as N313Q and N329Q.US 5,453, and disclosed variant is not included in this in 373

In the scope of invention.

US 5,460, and 953 disclose the dna sequence dna of the PROTEIN C of coding zymogen forms, their modified one or more Natively glycosylated sites of having removed.More specifically, US 5,460, and 953 disclose variant N97Q, N248Q, N313Q and N329Q.US 5,460, and disclosed variant is not included in the scope of the present invention in 953.Disclosed variant all is not included in the scope of the present invention in the above-mentioned prior art document.

US 5,270, and 178 relate to specific proteins C variant, and wherein I 171 is deleted, and Asp is replaced by Asn.

US 5,041, and 376 relate to the method for identifying and sheltering proteic functional site of transhipment type or epi-position, have wherein introduced extra N-connecting-type glycosylation site.

US 5,766, and 921 relate to the resistance of human plasma or the deactivation of α l-antitrypsin enhanced PROTEIN C variant, comprise the replacement from corresponding ox type heavy chain in its heavy chain.

WO 01/57193 has reported the PROTEIN C variant that comprises two sudden changes, and one of them sudden change occurs in position 10,11,32 or 33, and another sudden change occurs in position 194,195,228,249,254,392 or 316.

WO 01/36462 relates to the PROTEIN C variant, and it 12 comprises replacement in the position, and chooses in the position 10 and/or 11 wantonly and also have and replace.

WO 00/26354 relates to the method for the glycosylated protein variant that weakens of preparation allergenicity.

WO 00/26230 relates to the method for the protein variant of screening reduced immunogenicity.

People's wild-type protein C comprises its precursor forms, dna sequence dna and corresponding aminoacid sequence be disclosed in US 4,775,624 and US 4,968,626 in.

Disclosed any variant all is not included in the scope of the present invention in above-mentioned patent/patent application.

The invention summary

The present invention relates to the polypeptide variants of PROTEIN C and the new conjugate of non-polypeptide fraction, prepare the tool and method of described conjugate, comprise the pharmaceutical composition of described conjugate, and the purposes of described conjugate in treatment, various blood coagulation diseases especially treated.The invention still further relates to the polypeptide portion of described conjugate.

Correspondingly, first aspect present invention relates to the conjugate that contains at least one non-polypeptide fraction that links to each other with the protein c polypeptide covalency, described protein c polypeptide comprises the aminoacid sequence that is different from parent's protein c polypeptide, and its difference has been to introduce and/or remove at least one and has contained the amino-acid residue of the group that links to each other with described non-polypeptide fraction.

The present invention relates to the variant of parent's protein c polypeptide on the other hand, and the position of group comprises replacement to described variant: D172, D189, S190, K191, K192 being selected from down, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, R312, T315, F316, V334, S336, N337, M338, I348, L349, D351, R352, E357, E382, G383, L386, L387 and H388, prerequisite is, described replacement is not to be selected from: T254S, T254A, T254H, T254K, T254R, T254N, T254D, T254E, T254G, T254Q, Y302S, Y302A, Y302T, Y302H, Y302K, Y302R, Y302N, Y302D, Y302E, Y302G, Y302Q, F316S, F316A, F316T, F316H, F316K, F316R, F316N, F316D, F316E, F316G and F316Q.

The present invention relates to the polypeptide portion of conjugate of the present invention on the other hand.

A further aspect of the present invention relates to the nucleotide sequence of polypeptide portion in the code book invention conjugate, the nucleotide sequence of code book invention polypeptide variants, comprise the expression vector of nucleotide sequence of the present invention, and the host cell that comprises nucleotide sequence of the present invention or expression vector of the present invention.

A further aspect of the present invention relates to pharmaceutical composition that contains conjugate of the present invention or variant and the method for preparing and use conjugate of the present invention and variant.

Detailed Description Of The Invention

Definition

In the application and full text of the present invention, use following definition:

Term " conjugate " (or interchangeable be " link coupled polypeptide ") expression is by one or more polypeptide and the covalently bound heterozygosis that forms of one or more non-polypeptide fraction (as polymer molecule, lipophilic compound, sugar component or organic derivating agent) (referring to compound or chimeric) molecule.Preferably, conjugate is solvable under related concentrations and condition, and is promptly solvable in physiological liquid such as blood.The example of coupling polypeptide of the present invention comprises glycosylated polypeptides and/or PEGization polypeptide.

Term " covalently bound " expression polypeptide and non-polypeptide fraction are directly covalently bound each other, and be perhaps by one or more components that interleave, as bridge, spacerarm or connection component, covalently bound indirectly.

Term " non-coupling polypeptide " is the polypeptide portion of conjugate.

Term used herein " non-polypeptide fraction " expression be different from the peptide polymer that is formed by connecting by peptide bond by amino acid monomer, can with the linking group link coupled molecule of polypeptide of the present invention.The preferred embodiment of this quasi-molecule comprises polymer molecule, sugar component, lipophilic compound or organic derivating agent.When being used for conjugate of the present invention in this article, be interpreted as non-polypeptide fraction is connected to conjugate by the polypeptide linking group polypeptide portion.As above explanation, non-polypeptide fraction may directly covalently be connected to linking group, or by one or more components that interleave, as bridge, spacerarm or joint component, covalently bound indirectly to linking group.

The term " polymer " molecule " be that none is amino-acid residue for wherein said monomer by the covalently bound molecule that forms of two or more monomers, but not being human albumin or another kind, described polymkeric substance do not enrich plasma proteins.Term " polymer " " can with the term " polymer " molecule " exchange.

Term " sugar component " is meant the glycan molecule that contains that contains one or more monosaccharide residue, and it can be by in the body or external glycosylation and is connected (with the polypeptide conjugate of generation glycosylated polypeptides form) with polypeptide.Term " body in glycosylation " be meant by, for example N-connects and is connected glycosylation with O-, and occurs any sugar component linking group of (occurring during promptly translating post-treatment in the glycosylation cell of the described polypeptide of expression) in vivo.Actual oligosaccharide structure depends on relevant glycosylation biology to a great extent.Term " external glycosylation " is meant the synthetic property glycosylation of external generation, is usually directed to sugar component is linked to each other with the linking group of polypeptide, can select linking agent for use.Will be explained below with external glycosylation in the body.

" N-glycosylation site " has sequence N-X-S/T/C, and wherein X is any amino acid except proline(Pro), and N is a l-asparagine, and S/T/C is Serine, Threonine or halfcystine, preferred Serine or Threonine, most preferably Threonine." O-glycosylation site " is Serine or threonine residues-OH base.

In term " linking group " the expression polypeptide can with non-polypeptide fraction such as polymer molecule, sugar component, lipophilic compound or organic derivating agent link coupled functional group, particularly its amino-acid residue or sugar component.Useful linking group and the non-polypeptide fraction that matches thereof see the following form.

Linking group	Amino acid	The example of non-polypeptide fraction	Coupling method/-activatory PEG	Reference
					-NH 2	N-terminal,	Polymkeric substance, for example	mPEG-SPA	Shearwater?Inc.Delgado?et?al.，

	Lys，His， Arg	PEG, band amide group or imido grpup	Tresylated mPEG	Critical?reviews?in?Therapeutic Drug?Carrier?Systems?9(3，4)： 249-304(1992)
					-COOH	C-terminal, Asp, Glu	Polymkeric substance, for example PEG is with ester or amide group oligosaccharide compositions	The external coupling of mPEG-Hz	Shearwater?Inc.
-SH	Cys	Polymkeric substance, PEG for example, band disulfide linkage, maleimide or vinyl sulfone(Remzaol group oligosaccharide compositions	The external coupling of PEG-vinyl sulfone(Remzaol PEG-maleimide	Shearwater?Inc.Delgado?et?al.， Critical?reviews?in?Therapeutic Drng?Carrier?Systems?9(3，4)： 249-304(1992)
					-OH	Ser，Thr， -OH，Lys	Oligosaccharide compositions has ester, ether, carbamate, the PEG of carbonate	The glycosylation that O-connects in the body
-CONH 2	Asn is as the part of N glycosylation site	Oligosaccharide compositions polymkeric substance, for example PEG	N glycosylation in the body
					Aromatic residue	Phe，Tyr， Trp	Oligosaccharide compositions	External coupling
-CONH 2	Gln	Oligosaccharide compositions	External coupling	Yan?and?Wold，Biochemistry， 1984，Jul?31；23(16)：3759-65
					Aldehyde ketone	The oligosaccharides of oxidation	Polymkeric substance, PEG for example, PEG-hydrazides	PEGization	Andresz?et?al.，1978， Makromol.Chem.179：301， WO92/16555，WO02/23114
Guanidine	Arg	Oligosaccharide compositions	External coupling	Lundblad?and?Noyes，Chemical Reagents?for?Protein Modification，CRC?Press?Inc.， Florida，USA
					Imidazole ring	His	Oligosaccharide compositions	External coupling	Identical with guanidine

For N-glycosylation in the body, term " linking group " is used to represent to constitute the amino-acid residue of N-glycosylation site in unconventional mode.Although the asparagine residue of N-glycosylation site is the residue that is connected with sugar component,, otherwise can not realize described connection unless there is other amino-acid residue in this N-glycosylation site during glycosylation.

Therefore, when non-polypeptide fraction is a sugar component, and coupling is when realizing by the N-glycosylation, term " amino-acid residue that contains the group that is connected with non-polypeptide fraction " changes coupling with the aminoacid sequence of desired polypeptides, the one or more amino-acid residues that are interpreted as constituting the N-glycosylation site are changed, thereby make functional N-glycosylation site be introduced into aminoacid sequence or remove from described sequence.

The name of amino acid whose name and atom (as, CA, CB, CD, CG, SG, NZ, N, O, C etc.) use by albumen database (PDB) definition (www.pdb.org), these titles are according to IUPAC nomenclature name (IUPAC Nomenclature and Symbolism for Amino Acids andPeptides (residue names, atom names etc.), Eur.J.Biochem.138,9-37 (1984), and, they are corrected in 1 (1985) at Eur.J.Biochem.152.

Term " amino-acid residue " expression is included in down the amino-acid residue in the group: L-Ala (Ala or A), halfcystine (Cys or C), aspartic acid (Asp or D), L-glutamic acid (Glu or E), stupid L-Ala (Phe or F), glycine (Gly or G), Histidine (His or H), Isoleucine (Ile or I), Methionin (Lys or K), leucine (Leu or L), methionine(Met) (Met or M), l-asparagine (Asn or N), proline(Pro) (Pro or P), glutamine (Gln or Q), arginine (Arg or R), Serine (Ser or S), Threonine (Thr or T), Xie Ansuan (Val or V), tryptophane (Trp or W) and tyrosine (Tyr or Y) residue.

Be used for identifying that the term of amino acid position/replacement illustrates as follows: the K174 of given aminoacid sequence represents shown in SEQ ID NO.2 or 4 that the 174th is occupied by lysine residue in the aminoacid sequence.K174S represents that the 174th lysine residue is replaced by serine residue.Alternative replacement available "/" is represented, represents that as K174S/T the 174th Methionin is replaced by Serine or threonine residues.Multiple replacement represent that as D172N+K174S the 172nd asparagicacid residue replaced by asparagine residue, and the 174th lysine residue is replaced by serine residue with "+" expression.The following expression of the insertion of additional amino acid: after K174, insert an alanine residue, be expressed as K174KA.The disappearance of amino-acid residue is represented with asterisk.For example, the disappearance of the 174th Methionin is expressed as K174 ^*Except as otherwise noted, herein the sequence number of amino-acid residue corresponding to the aminoacid sequence shown in SEQ ID NO:2 or 4.

Term " is different from " when with specific sudden change coupling, is meant to remove that specific amino acid difference is unusual to allow to exist other difference.For example, comprise the amino-acid residue of non-polypeptide fraction linking group except removing and/or introducing, protein c polypeptide may also comprise introducing and/or irrelevant other replacement, insertion or the disappearance of removal with these amino-acid residues.Therefore, be intended to remove and/or introduce the amino acid change of non-polypeptide fraction linking group except disclosed herein, should understand the aminoacid sequence of polypeptide conjugate of the present invention, can comprise when needed not necessarily and must or remove relevant other change, i.e. other replacement, insertion or disappearance with the introducing of connection site.For example, these changes may comprise one or more amino-acid residues of removing N-and/or C-end, or at N-and/or the one or more extra amino-acid residues of the terminal interpolation of C-, as adding a methionine residues at the N-end, and " conserved amino acid replacement ", that is, replacement is to have the amino acid of similar characteristics (as, p1 amino acid, acidic amino acid, polare Aminosaeren, basic aminoacids, hydrophobic amino acid and die aromatischen Aminosaeuren) between carry out.

The example of the conservative replacement among the present invention specifically can be selected from the listed group of following table.

??1	L-Ala (A) glycine (G) Serine (S) Threonine (T)
		??2	Aspartic acid (D) L-glutamic acid (E)
??3	L-asparagine (N) glutamine (Q)
		??4	Arginine (R) Histidine (H) Methionin (K)
??5	Isoleucine (I) leucine (L) methionine(Met) (M) Xie Ansuan (V)
		??6	Phenylalanine (F) tyrosine (Y) tryptophane (W)

Term " precursor protein C " is meant the PROTEIN C form by dna encoding herein, promptly, it comprises signal peptide (residue-42 is to-1), light chain (residue 1-155), Lys-Arg dipeptides (residue 156-157) and heavy chain (158-419), comprise activation peptide (residue 158-169), see SEQ ID NO:2.

Term " double-stranded proenzyme PROTEIN C " is meant excretory inactive protein C form, and it comprises light chain (residue 1-155) and heavy chain (158-419), comprising activation peptide (158-169), sees SEQ ID NO:4.

Term " strand zymogen protein C " is meant the PROTEIN C form of non-activity, comprises light chain (residue 1-155), and heavy chain (158-419) comprises bioactive peptide (158-169), and Lys-Arg dipeptides (residue 156-157), sees SEQ ID NO:4.

Term " zymogen protein C " is used in reference to the zymogen protein C of strand and double chain form.

Term " activatory PROTEIN C ", " activatory human protein C ", " APC " or " people APC " is used in reference to the activatory proenzyme, and they comprise light chain (residue 1-155) and the heavy chain (not containing the activation peptide) of SEQ ID NO:4.A kind of aminoacid sequence in back, that is, the aminoacid sequence of activatory PROTEIN C is referred to herein as " the APC part shown in the SEQ ID NO:4 in the aminoacid sequence " sometimes.

Term " PROTEIN C " comprises all aforesaid PROTEIN C forms, i.e. " precursor protein C " form, " zymogen protein C " form (single stranded form and double chain form) and " activatory PROTEIN C form ".

Term " parent " is meant and will carries out improved molecule according to the present invention.Parent's polypeptide of modifying by the present invention can be any protein c polypeptide, and therefore can be derived from any source, for example non-human mammal is originated, but the PROTEIN C that preferred parent's polypeptide is the people (promptly, people's precursor protein C, people's zymogen protein C or people's deutero-PROTEIN C) or its fragment or variant.

Fragment is the part of total length people's PROTEIN C sequence, for example the terminal truncation type of its C-end or N-.The segmental object lesson of parent's protein c polypeptide comprises the people's of 1-15 amino-acid residue of C-terminal brachymemma and/or 1-3 amino-acid residue of the terminal brachymemma of N-albumen.

As mentioned above, parent's protein c polypeptide also can comprise the variant of people's PROTEIN C.The object lesson of human protein C variant comprises, for example at the terminal variant that adds methionine residues of N-, and contains the variant that one or more above-mentioned conserved amino acid replaces.Other example of variant comprises, has one or more amino acid to be substituted in the Gla of PROTEIN C structural domain or PROTEIN C variant that whole PROTEIN C Gla structural domain has wherein been replaced by another kind of Gla structural domain (as the Gla structural domain of Protein S).

The variant of term (parent's polypeptide) is meant a peptide species, and it has one or more amino-acid residue different with its parent's polypeptide, 1-15 amino-acid residue is arranged usually (as 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15 amino-acid residues), for example, 1-10 amino-acid residue or 1-5 amino-acid residue difference.

Term " sudden change " and " replacement " are used interchangeably at this.

The continuous fragment of the two or more nucleic acid molecules of term " nucleotide sequence " expression.Nucleotide sequence can be genome, cDNA, RNA, semi-synthetic or synthetic source or its arbitrary combination.

Term " polymerase chain reaction " or " PCR " are often referred to the method for the required nucleotide sequence of amplification in vitro, as described in United States Patent (USP) 4683195.PCR method relates generally to use and can carry out the primer extension building-up reactions with the Oligonucleolide primers of the preferential hybridization of template nucleic acid, and makes this reaction repeat many wheels.

" cell ", " host cell ", " clone " and " cell culture " are used interchangeably in this article, and all these terms all should be understood to comprise the cell growth or cultivate the offspring who is produced.

" conversion " and " transfection " is used interchangeably, and refers to DNA is introduced the process of cell.

" can be operatively connected " and refer to that two or more nucleotide sequences are covalently bound in the conformation that is relative to each other by modes such as enzymatic connections, make sequence exercise normal function.For example, participate in the preceding albumen of polypeptide excretory if the nucleotide sequence of coding presequence or secretion leader sequence is expressed as, the nucleotides sequence that then can be operationally connected to this polypeptide lists; Promotor or enhanser then can be operatively connected with this sequence if influence transcribing of encoding sequence; Ribosome bind site then can be operatively connected with encoding sequence if be in the position that promotes translation.Usually, " can be operatively connected " means connected nucleotide sequence is adjacency, the secretion leader sequence situation under, be adjacency and also under read state.Connect and occur in restriction site easily.If there is no this type of site is used synthetic oligonucleotide adapter or joint so, and the recombinant DNA method of the standard of use.

Term " introducing " mainly refers to replace existing amino-acid residue, also refers to insert other amino-acid residue.

Term " is removed " and is mainly referred to replace the amino-acid residue that will be removed with other amino-acid residue, also refers to the amino-acid residue that deletion (not being substituted) will be removed.

Term " function gonosome in half life " uses its common implication, promptly polypeptide or conjugate in vivo/still had for 50% bioactive time in the target organ, perhaps the activity of polypeptide or conjugate is time of 50% of initial activity.As in the function gonosome half life assay method alternative approach, can measure " serum half life ", that is, 50% polypeptide or conjugate molecule before being eliminated in blood plasma or blood flow the round-robin time.The mensuration of serum half life is usually simple than the mensuration of half life in the function gonosome, and the size of serum half life usually well in the functions gonosome half life size.Other replaceable term of serum half life comprises " blood plasma half life ", " circulation half life ", " serum removing ", " plasma clearance " and " removing half life ".Polypeptide or conjugate be by one or more effect in reticuloendothelial system (RES), kidney, spleen or the liver, by tissue factor, SEC acceptor or other receptor-mediated scavenging(action), or removes by special or non-specific proteolyzing.Remove and generally depend on the carbohydrate chain of size (for the cutoff value of glomerular filtration), electric charge, connection, and whether have this proteic cell receptor.The function that will keep is selected from anticoagulating active, acid amides lytic activity or receptor-binding activity usually.Half life and serum half life, can be measured by any appropriate method known in the art in the function gonosome.

The term " increase " that relates to half life in the function gonosome or blood plasma half life is used to represent the mensuration of corresponding half life under comparable conditions of conjugate or polypeptide, and (as APC) has statistics to increase significantly with respect to the reference molecule.Usually, when removing, protein cleavage degraded and/or inhibition to polypeptide reduce, half life or serum half life increase in the function gonosome.Therefore preferred conjugate is following conjugate, when they are activity form, compares half life or serum half life increase in the function gonosome with people's APC.Particularly preferred conjugate is following conjugate, the Billy of the serum half life of their serum half life (or function gonosome in half life) and people APC (or in the function gonosome half life) is at least 1.25, more preferably at least 1.50, as at least 1.75, for example at least 2, also more preferably at least 3, as at least 4, for example at least 5, most preferably at least 6, as at least 7, for example at least 8, at least 9 or at least 10.

The relevant purge mechanism of polypeptide of the present invention or conjugate comprises one or more reticuloendothelial cell system (RES), kidney, spleen or liver, receptor-mediated degraded, perhaps specificity or nonspecific proteins cracking.Term " kidney removing " uses its common implication, promptly occurs in the removing of kidney, for example, finishes by glomerular filtration, tubular excretion or the degraded in renal tubular cell.Kidney is removed the physical property that depends on conjugate, comprises molecular weight, size (for the cutoff value of glomerular filtration), symmetry, shape/rigidity and electric charge.The cutoff value that kidney is removed is commonly considered as the about 67kDa of molecular weight.Kidney is removed and generally can be measured by any suitable test, as the in vivo test of having set up.For example, can with mark (as, radio-labeled or fluorescent mark) the polypeptide conjugate give the patient, and measure marker activity in collected patient's urine and measure kidney and remove.The minimizing that kidney is removed can through and the reference molecule, compare and determine as APC.

Term " activity ", " APC activity " or " activity of activatory PROTEIN C " is meant that the activity form of conjugate of the present invention has kept the fundamental characteristics of APC.

This paper embodiment 9 has described a kind of suitable external APC activity test (being called " APC acid amides breaking test ").Therefore, more specifically, conjugate of the present invention records active at least 10% of the people APC that is with this paper embodiment 9 described " APC acid amides breaking test " when activity form, then can classify as to have " APC activity ".Preferably, the activity of described conjugate is people APC active at least 20%, as at least 30%, more preferably the activity of described conjugate is people APC active at least 40%, as at least 50%, also more preferably the activity of described conjugate is that people APC is active at least 60%, as at least 70%, most preferably the activity of described conjugate is that people APC is active at least 80%, as at least 90%.In an especially preferred embodiment, when detecting with this paper embodiment 9 described " APC acid amides breaking tests ", described conjugate has the activity basic identical or higher with people APC.Should be understood that conjugate of the present invention and wild-type people APC should under equal conditions test, promptly when testing as described in this paper embodiment 9, these two kinds of proteic concentration should be identical.

Perhaps, " APC activity " can carry out analyzed in vitro in this paper embodiment 10 described " ACP thrombotest ".More specifically, conjugate of the present invention if record at least 5% of anticoagulating active behaviour APC with this paper embodiment 10 described " APC thrombotest ", then can classify as and has " APC activity " when activity form.Preferably, at least 10% of the anticoagulating active behaviour APC of described conjugate, as at least 20%, for example at least 30%, more preferably at least 40% of the anticoagulating active behaviour APC of described conjugate, as at least 50%, also more preferably at least 60% of the anticoagulating active behaviour APC of described conjugate, as at least 70%, most preferably at least 80% of the anticoagulating active behaviour APC of described conjugate, as at least 90%.In an especially preferred embodiment, when detecting with this paper embodiment 10 described " APC thrombotests ", described conjugate has the anticoagulating active basic identical or higher with people APC.Example between typical PC active zone as, the 5-75% of the anticoagulating active of people APC is as 10-50%, as 10-40%.Should be understood that conjugate of the present invention and wild-type people APC should under equal conditions test, promptly when testing as described in this paper embodiment 10, these two kinds of proteic concentration should be identical.

Term " increases the resistance of α-1-antitrypsin deactivation " and " to the resistance increase of the deactivation of human plasma " is meant mainly that respectively conjugate of the present invention is subjected to the degree of the inhibition of α-1-antitrypsin or human plasma to be lower than people APC.Can select effective and preferred conjugate in order to ensure those skilled in the art in its early development, the inventor has developed suitable tentative experiment, and those skilled in the art can implement described test easily, thus the performance of its conjugate of entry evaluation.Therefore, " α-1-antitrypsin inactivation test " (this paper embodiment 11), " human plasma inactivation test I " (this paper embodiment 12) and " human plasma inactivation test II " (this paper embodiment 13) can be used for the potentiality of the selected conjugate of entry evaluation.By using above-mentioned any or all tests, can assess the adaptability of the deactivation of selected conjugate opposing α-1-antitrypsin and/or human plasma, ultimate principle is, if a kind of conjugate is subjected to the strongly inhibited of α-1-antitrypsin and/or human plasma, needn't carry out other test more usually.

Therefore, the particularly preferred conjugate of this paper is, when being its activity form, and when with the inhibitor concentration of 16.6 μ M when this paper embodiment 11 described " α-1-antitrypsin inactivation test " detects, have at least 20% residual activity.Preferred described conjugate has at least 30% residual activity, as at least 40%, and more preferably at least 50%, as at least 60%, also more preferably at least 70%, as at least 75%, most preferably at least 80%, as at least 85%.

Perhaps, or except above-mentioned test, also available " human plasma inactivation test I " checks the adaptability of selected conjugate.Therefore, the particularly preferred conjugate of this paper is, when being its activity form, and when detecting with this paper embodiment 12 described " human plasma inactivation test I ", has at least 20% residual activity.Preferred described conjugate has at least 30% residual activity, as at least 40%, and more preferably at least 50%, as at least 60%, also more preferably at least 70%, as at least 75%.

Perhaps, or except above-mentioned test, also available " human plasma inactivation test II " checks the adaptability of selected conjugate.Therefore, the particularly preferred conjugate of this paper is, when detecting with this paper embodiment 13 described " human plasma inactivation test II ", the ratio of the external half life of its activity form and the external half life of people APC is at least 1.25, and preferably at least 1.5, as at least 2, more preferably at least 3, as at least 4, also more preferably at least 5, as at least 6, most preferably at least 7, as at least 8, especially at least 9, as at least 10.

Term " immunogenicity that weakens " is meant conjugate of the present invention than the contrast molecule of measuring under suitable condition, for example wild-type people APC or wild-type human protein C, the lower immunne response that generation can be measured.Described immunne response can be that cellullar immunologic response or antibody-mediated replying are (to immunogenic further definition referring to, Roitt:Essential Immunology (the 8th edition, Blackwell)) for example.Usually, the antibody response reduction can be indicated reduced immunogenicity.Reduced immunogenicity can utilize any proper method known in the art, and the interior method of for example external or body is measured.

The method of calculation of describing in detail in term " at least 25% side chain is exposed to the surface " and " at least 50% side chain is exposed to the surface " reference example 1 wait and limit.

Conjugate of the present invention

Conjugate of the present invention is the result that the exploitation PROTEIN C is improved the overall New Policy of molecule.More specifically, by removing and/or introduce the amino-acid residue of the linking group that contains described non-polypeptide fraction, might change polypeptide specifically, this molecule is easier to and selected non-polypeptide fraction coupling, make coupling pattern optimizing (as, guarantee non-polypeptide fraction with the optimal dose optimum distribution on protein C molecule surface and guarantee only to exist the link coupled linking group in the molecule), thereby obtain new coupling molecule, it is compared with existing protein C molecule, have or do not have the APC activity, have one or more improved characteristics in addition.For example, when the amino-acid residue sum of the linking group that contains selected non-polypeptide fraction was increased to or is reduced to an optimum level, the change of the shape of molecule that the renal clearance of this conjugate causes because of coupling usually, size and/or electric charge significantly reduced.In addition, we find, might design being connected of linking group in the polypeptide portion of non-polypeptide fraction and described conjugate, cause the deactivation of human plasma or special inhibitor (as α-1-antitrypsin) significantly to weaken (seeing below).

No matter the amino-acid residue that comprises non-polypeptide fraction linking group is to be removed or to introduce, and it selects the character based on selected non-polypeptide fraction, and, in most cases, based on the used method of coupling between polypeptide and the non-polypeptide fraction.For example, when non-polypeptide fraction is a polymer molecule, divide the period of the day from 11 p.m. to 1 a.m as polyoxyethylene glycol or polyalkylene oxide deutero-, the amino-acid residue that comprises linking group can be selected from: Methionin, halfcystine, aspartic acid, L-glutamic acid, Histidine, or tyrosine, preferred halfcystine and Methionin, preferred especially Methionin.When non-polypeptide fraction was sugar component, linking group can be, glycosylation site in the body for example, preferred N-glycosylation site.

In case the linking group of non-polypeptide fraction is introduced in the protein c polypeptide of the present invention or when being removed from described polypeptide, carry out the common following selection in modified polypeptides position:

Described optimum seeking site is positioned at the surface of protein c polypeptide, is more preferably the position that is occupied by those amino-acid residues that have (more preferably more than 50%) side chain more than 25% to be exposed to the surface.Described position determines according to the analysis to the three-dimensional structure of people's wild-type APC molecule, and method therefor is as described in this paper method part.In addition, in the inhuman APC polypeptide (comprising its variant) that contains with wild-type human protein C homologous aminoacid sequence, can arrange and determine easily by corresponding sequence or three-dimensional structure are suitably contrasted with the source position.

In order to determine the optimum distribution of linking group, calculate the distance between the amino-acid residue on this polypeptide surface based on the three-dimensional structure of described polypeptide.More specifically, to the distance between the CB of the amino-acid residue that comprises this linking group, or a kind of amino acid whose functional group (NZ of Methionin, the CG of aspartic acid, the CD of L-glutamic acid, the SG of halfcystine) and the distance that comprises between the CB of another kind of amino-acid residue of linking group determine.For glycine, use CA but not CB.In the polypeptide portion of conjugate of the present invention, any described distance is preferably greater than 8 , is preferably greater than 10 especially, to avoid or to reduce the allos coupling.

As described in the follow-up chapters and sections of this paper, the sum (comparing with parent's protein C molecule) according to the present invention with the amino-acid residue that changes is no more than 15 usually.The definite number and the type of the amino-acid residue that is introduced into depend on that for example, required coupling characteristic and degree are (for example, the identity of non-polypeptide fraction, need or have how many non-polypeptide fraction and described polypeptide coupling, need to implement still avoid described polypeptide coupling, or the like).Preferably, the polypeptide portion of conjugate of the present invention or polypeptide of the present invention comprise following aminoacid sequence, it has 1-15 amino-acid residue different with aminoacid sequence shown in the SEQ ID NO:4, and as 1-8 or 2-8 amino-acid residue difference, for example 1-5 is individual or 2-5 amino-acid residue difference.Therefore, the polypeptide portion of conjugate of the present invention or polypeptide of the present invention comprise with aminoacid sequence shown in the SEQ ID NO:4 1,2, and 3,4,5,6,7,8,9,10,11,12,13,14 or 15 different aminoacid sequences of amino acid.

Preferably, conjugate of the present invention is compared with wild-type people APC, and have one or multinomial following improved characteristic: half life increases in the function gonosome, serum half life increase, resistance to inhibitor increases, and kidney is removed and reduced, and reduced immunogenicity and/or bioavailability increase.Conjugate of the present invention has multiple advantage with respect to existing APC product, comprises inject time longer (longer duration betweeninjections), the albumen of administration still less, and side effect is littler.In addition, anticoagulating active weakens to have to be beneficial in the anti-inflammatory effect of keeping the APC conjugate and reduces risk of bleeding.This is even more important in conjugate has the situation of blood plasma half life of prolongation.Compare with the anticoagulating active that allows more effective with safe treatment, above-mentioned new features should be able to strengthen anti-inflammatory effect.

Usually, the molecular weight of conjugate of the present invention, reduces but less molecular weight can make kidney remove preferably at least about 70kDa at least about 67kDa.The glycosylation site of having found polymer molecule (as PEG) or having been introduced is effective especially to the molecular weight of adjusting conjugate.

Conjugate of the present invention comprises the non-polypeptide fraction of sufficient amount or type, to improve above-mentioned one or more desired characteristic of protein c polypeptide.Usually, conjugate of the present invention comprises 1-10 first non-polypeptide fraction, the especially 1-8 or 1-5 first non--polypeptide fraction.

Conjugate of the present invention also can comprise the second different non-polypeptide fraction of at least one-polypeptide fraction non-with first.For example, conjugate of the present invention can comprise 1-10 second non--polypeptide fraction, especially 1-8 or 1-5 second non--polypeptide fraction.For example, when first non--polypeptide fraction was sugar component (the especially sugar component that connects in the body), second non--polypeptide fraction can be a PEG type polymkeric substance.The sugar component that connects in the described body can link to each other with glycosylation site in the natural body of described polypeptide, or links to each other with the site of introducing.

In a highly preferred embodiment, of the present invention non--polypeptide fraction is introduced into the avtive spot district of PROTEIN C, ultimate principle is, in this specific region of protein C molecule, introduce one or more non-polypeptide fraction and will weaken combining of inhibitor (as α-1-antitrypsin) and APC, but the while still keeps substantial APC activity.This causes described conjugate to have half life than wild-type people APC significant prolongation, because the liver acceptor is avoided the elimination effect of inhibitor/APC complex body or is to have reduced at least.The amino-acid residue in the avtive spot district that is positioned at PROTEIN C is selected and can be described in detail as this paper embodiment 2.

Term used herein " avtive spot district " in embodiment 2, has shown the real amino acid residue of forming the avtive spot district with reference to the qualification of this paper embodiment 2.

In the particularly preferred embodiment of the present invention, the linking group of non-polypeptide fraction is introduced into the position that is occupied by following amino-acid residue in the avtive spot district, and described amino-acid residue has at least 25% side chain to be exposed to surface (seeing this paper embodiment 3), that is, the linking group of non-polypeptide fraction is introduced into the position that is selected from down group: D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, N248, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, K311, R312, N313, R314, T315, F316, V334, S336, N337, M338, I348, L349, D351, R352, E357, E382, G383, L386, L387 and H388 (getting rid of H211 and C384).The preferred linking group of being introduced is the linking group of sugar component, particularly N-glycosylation site (referring to the chapters and sections that are entitled as " with of the present invention conjugate of sugar component as non--polypeptide fraction ") in the body.

With of the present invention conjugate of sugar component as non-polypeptide fraction

As indicated above, a preferred embodiment of the present invention relates to the conjugate of having introduced the covalently bound glycosylation site of at least one and protein c polypeptide (especially N-glycosylation site in the body), described protein c polypeptide comprises the aminoacid sequence that is different from parent's protein c polypeptide, especially be different from aminoacid sequence or its variant of SEQ IDNO:4, difference is the glycosylation site of at least one introducing.

Preferred described glycosylation site is introduced into the position that is occupied by following amino-acid residue, and described residue has the side chain of at least 25% (as at least 50%) to be exposed to the surface.This amino acid residue is described next definite according to this paper embodiment 1.When should be understood that the introducing coupling when N-glycosylation site in term " side chain of at least 25% (or at least 50%) is exposed to the surface " and the body, the surface of amino acid side chain can be near characteristic in the position that in fact its represent with sugar component is connected.Under many circumstances, must be with respect in fact for asparagine residue that sugar component links to each other, introducing Serine or threonine residues on for+2 position (certainly, unless this position is occupied by Serine or threonine residues), these positions of having introduced Serine or threonine residues can be covered, and promptly it has the side chain less than 25% to be exposed to the surface.

In order to weaken inhibitor, combine with APC's as α-1-antitrypsin, preferably described glycosylation site is introduced the avtive spot district (such as this paper embodiment 2 qualifications) in be exposed to the position (being limited) that those surperficial amino-acid residues occupy by the side chain that has at least 25% as this paper embodiment 3, promptly preferably N-glycosylation site in the body is introduced: D172N+K174S, D172N+K174T, D189N+K191S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, K192N+L194S, K192N+L194T, K193N+A195S, K193N+A195T, D214N, D214N+S216T, E215N+K217S, E215N+K217T, S216N+K218S, S216N+K218T, K217N+L219S, K217N+L219T, K218N+L220S, K218N+L220T, L220N+R222S, L220N+R222T, V243N+V245S, V243N+V245T, V245N+P247S, V245N+P247T, S250N, S250N+S252T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, T254N+N256S, T254N+N256T, D255N+D257S, D255N+D257T, L296N, L296N+T298S, Y302N, Y302N+S304T, H303N, H303N+S305T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T, E309N+K311S, E309N+K311T, A310N+R312S, A310N+R312T, R312N+R314S, R312N+R314T, T315N+V317S, T315N+V317T, F316N+L318S, F316N+L318T, V334N, V334N+S336T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, I348N+G350S, I348N+G350T, L349N+D351S, L349N+D351T, D351N+Q353S, D351N+Q353T, R352N+D354S, R352N+D354T, E357N+D359S, E357N+D359T, G383N+G385S, G383N+G385T, L386N+H388S, L386N+H388T, L387N+N389S, L387N+N389T, H388N+Y390S or H388N+Y390T.

More preferably N-glycosylation site in the body is introduced: S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, D189N+K191S, D189N+K191T, D214N, D214N+S216T, K217N+L219S, K217N+L219T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S305N+E307S, S305N+E307T, E307N+E309S, E307N+E309T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S or L386N+H388T.

Also more preferably N-glycosylation site in the body is introduced: D189N+K191T, K191N+K193T, D214N, K251N, S252N, T253N+D255T, Y302N, S305N+E307T, S336N+M338T, V339T, M338N, or G3833N+G385T; Most preferably N-glycosylation site in the body is introduced: D189N+K191T, K191N+K193T, D214N, T253N+D255T, S305N+E307T, S336N+M338T, M338N, G383N+G385T or L386N+H388T.In an especially preferred embodiment, the N-glycosylation site is selected from D189N+K191T, D214N or L386+H388T in the body of being introduced.

As mentioned above, the resistance of the deactivation of α-1-antitrypsin and/or human plasma is increased, can measure by " α-1-antitrypsin inactivation test " as herein described, " human plasma inactivation test I " or " human plasma inactivation test II ".

Conjugate of the present invention can comprise glycosylation site in the single body (except beyond 97,248,313 and 329 the glycosylation site Already in).But for proteolytic enzyme cutting site of effectively sheltering parent's polypeptide surface and/or the combination that effectively weakens inhibitor, the polypeptide portion of wishing described conjugate usually comprises glycosylation site in the more than one body, especially glycosylation site in 2-5 (extra) body, as 2,3, glycosylation sites in 4 or 5 (extra) bodies preferably replace by listed one or more above and introduce described glycosylation site.

In addition, aminoacid sequence with N-glycosylation site modified polypeptides at least one above-mentioned body may differently with parent's polypeptide be, introduced at least one " conjugate of the present invention; wherein non-polypeptide fraction links to each other with cysteine residues " defined cysteine residues of chapters and sections as mentioned, or introduced at least one " conjugate of the present invention, wherein non-polypeptide fraction links to each other with non-cysteine residues " defined non-cysteine residues of chapters and sections as mentioned.

And the polypeptide portion of conjugate of the present invention also can comprise extra known sudden change with benefit.For example, except above-mentioned glycosylation site, comprise replacement: L194 on the position that the polypeptide portion of described conjugate also can be organized under being selected from, A195, L228, Y249 or their combination, especially L194S, L194S+T245S or L194A+T254S (seeing WO 00/66754).Preferred extra other example that replaces comprises, near known position of easily being degraded by the protein cleavage effect or its, replaces or introduces one or more glycosylation site.The H10 (seeing WO 98/48822) that one of them known position of easily being degraded by the protein cleavage effect is wild-type people APC.

Should be understood that in order to prepare the conjugate of this aspect of the present invention, described polypeptide must in the glycosylation host cell, express or receptor outside glycosylation, described cell should be able to be connected sugar component on the glycosylation site.The example of glycosylation host cell can vide infra and be entitled as the chapters and sections of " with the coupling of sugar component ".

Conjugate of the present invention, wherein non-polypeptide fraction links to each other with cysteine residues

Another preferred embodiment of the present invention relates to the conjugate that comprises the covalently bound non-polypeptide fraction (especially polymer molecule) of at least one and protein c polypeptide, described protein c polypeptide comprises the aminoacid sequence that is different from parent's protein c polypeptide, especially the aminoacid sequence or its variant that are different from SEQ ID NO:4, difference are to introduce and/or remove (the preferred introducing) at least one cysteine residues.Therefore, in a preferred embodiment of the present invention, described non-polypeptide fraction has halfcystine as linking group.Preferably described halfcystine linking group is introduced the position that is occupied by following amino-acid residue, described amino-acid residue has the side chain of at least 25% (as at least 50%) to be exposed to the surface.This amino acid residue such as this paper embodiment 1 qualification.In the described position, those positions that in parent's polypeptide, occupy most preferably by T or S residue (preferred S residue).Correspondingly, the conjugate that has halfcystine-modification be with cysteine residues introduce at least one be selected from down the group position in those: S3, S11, S12, T37, S42, S61, T68, S75, S77, S82, S99, S119, S153, S190, S216, S252, T253, T268, S270, S281, S304, S305, T315, S332, S336, S340, S367, or S416, described position is S3 more preferably, S11, S12, S42, S61, S75, S77, S82, S99, S119, S153, S190, S216, S252, S270, S281, S304, S305, S332, S336, S340, S367 or S416.

With aforesaid similar manner (referring to the chapters and sections that above are entitled as " being the conjugate of the present invention of non-polypeptide fraction with sugar component "), preferably cysteine residues is introduced the avtive spot district (such as this paper embodiment 2 qualification) in be exposed to the position (being limited) that occupies of those amino-acid residues on surface by the side chain that has at least 25% as this paper embodiment 3, that is, preferably described cysteine residues is introduced and is selected from down the position of organizing: D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, R312, T315, F316, V334, S336, V339, M338, I348, L349, D351, R352, E357, G383, E385, L386, L387 or H388.More preferably described cysteine residues is introduced D189, S190, K191, D214, K217, K251, S252, T253, Y302, S305, E307, S336, V339, M338, G383 or L386.

The polypeptide portion of conjugate described in this embodiment comprises 1-10 the cysteine residues of introducing usually, especially 1-5 or 1-3 the cysteine residues of introducing, for example cysteine residues of 1,2 or 3 introducing.

Non--the polypeptide fraction of this conjugate on the one hand of the present invention can be, when using given coupling method, have cysteine residues as any molecule of linking group (as polymeric constituent, lipophilic group or organic derivating agent), preferred described non--polypeptide fraction is a polymer molecule, any molecule described in " with the coupling of polymer molecule " chapters and sections.Preferred described polymer molecule is selected from linearity or ramose polyoxyethylene glycol or polyalkylene oxide.Most preferably, described polymer molecule is PEG, as VS-PEG.Coupling between described polypeptide and the polymkeric substance can realize by any suitable method, as being to describe in the chapters and sections of title with " with the coupling of polymer molecule ", for example uses single stage method related in the above-mentioned chapters and sections or multistep processes.When but polypeptide only comprises a link coupled cysteine residues, preferably with have at least about 10kDa at least about 15kDa molecular weight (12kDa according to appointment, about 15kDa, or about 20kDa) the direct coupling of the first non-polypeptide fraction is perhaps by the indirect coupling of low-molecular weight polymer (seeing WO99/55377).When conjugate comprised the two or more first non-polypeptide fraction, these non-polypeptide fraction molecular weight separately was generally about 5kDa, about 10kDa, or about 12kDa.

Conjugate in this embodiment can comprise at least one second non-polypeptide fraction, as 1-10, and 1-8,1-5 or 1-3 described component.When first non--polypeptide fraction is polyalkylene oxide or PEG derived polymers, the preferred sugar component of second non--polypeptide fraction, the especially component that connects in the body.Sugar component can appear at one or more Natively glycosylated site of parent's polypeptide, or appears at the glycosylation site of introducing.The glycosylation site of suitable introducing, especially the N-glycosylation site is described in the chapters and sections that are entitled as " being the conjugate of the present invention of non-polypeptide fraction with sugar component ".

And the polypeptide portion of conjugate of the present invention also can comprise extra known sudden change with benefit.For example, except the cysteine residues of above-mentioned introducing, the polypeptide portion of described conjugate also can comprise replacement in the position of group under being selected from: L194, A195, L228, Y249 or their combination, especially L194S, L194S+T245S or L194A+T254S (seeing WO 00/66754).Preferred extra other example that replaces comprises, near known position of easily being degraded by the protein cleavage effect or its, replaces or introduces one or more cysteine residues.The H10 (seeing WO 98/48822) that one of them known position of easily being degraded by the protein cleavage effect is wild-type people APC.

Conjugate of the present invention, wherein non-polypeptide fraction is connected with non-halfcystine component

According to content disclosed herein, present technique personnel are appreciated that the method at glycosylation site and cysteine residues that use is above given an example, and the amino-acid residue that comprises other linking group can be introduced parent's polypeptide by replacing.For example, one or more amino-acid residue (L-glutamic acid or aspartic acid), tyrosine, Serine or the Methionin that comprise acidic-group can be introduced above-mentioned position (referring to the chapters and sections that are entitled as " being the conjugate of the present invention of non-polypeptide fraction with sugar component " and " conjugate of the present invention, wherein non-polypeptide fraction is connected with cysteine residues ").

Polypeptide variants of the present invention

The general on the other hand neomorph that relates to parent's protein c polypeptide of the present invention.Described neomorph is the important intermediate compound of preparation conjugate of the present invention.In addition, and the embodiment that is provided from content and this paper of following discloses as can be seen, and described variant itself has interesting characteristic.

Therefore, the present invention aspect the most widely relates to the neomorph of parent's protein c polypeptide, and described variant is formed the polypeptide portion of conjugate of the present invention, more particularly APC part.As shown in the Examples, introduced one or more glycosylation site in some variants, they are not utilized, but they have interesting characteristic, especially the inhibiting resistance of α-1-antitrypsin is increased and the resistance of human plasma deactivation is increased.These variants the avtive spot district (such as this paper embodiment 2 qualification) in comprise at least one replacement, especially, they comprise aminoacid replacement by the position (being limited as this paper embodiment 3) that occupies of those amino-acid residues that the side chain that has at least 25% is exposed to the surface in the avtive spot district.Therefore, the position of group comprises replacement to the preferred variant in this aspect of the present invention: D172, D189, S190, K191, K192 being selected from down, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, R312, T315, F316, V334, S336, N337, M338, I348, L349, D351, R352, E357, E382, G383, L386, L387 or H388, condition is, described replacement is not to be selected from: T254S, T254A, T254H, T254K, T254R, T254N, T254D, T254E, T254G, T254Q, Y302S, Y302A, Y302T, Y302H, Y302K, Y302R, Y302N, Y302D, Y302E, Y302G, Y302Q, F316S, F316A, F316T, F316H, F316K, F316R, F316N, F316D, F316E, F316G or F316Q.

Listed above, the position that occupies of those amino-acid residues that be positioned at the avtive spot district, is exposed to the surface by the side chain that has at least 25% shows that these a large amount of class positions are occupied by charged amino-acid residue.To PROTEIN C, especially the three-dimensional structural analysis of aforementioned region is found, the charged residue of at least a portion is interact with each other.For example, it is believed that K251 and D214 form salt bridge.And, be also shown in the electronegative amino-acid residue (D214, E215 and E357) of cluster.Under the condition that is not subjected to any concrete theory, charged amino-acid residue in the aforementioned region perhaps is the such amino-acid residue of a part at least, for catch and/or bound substrates/inhibitor very important.Therefore, the particularly preferred aminoacid replacement of the present invention comprises, be exposed to the locational charged amino-acid residue that those surperficial amino-acid residues occupy by the side chain that has at least 25% in the avtive spot district, by uncharged amino-acid residue, especially not charged but have amino-acid residue (Gly, the Ser of polar side chain, Thr, Cys, Tyr, Asn or Gln) replace; And be exposed to the locational charged amino-acid residue that those surperficial amino-acid residues occupy by the side chain that has at least 25% in the avtive spot district, replaced by the amino-acid residue of oppositely charged.

Make charged target amino acid residue become oppositely charged, the specific examples that this amino acid replaces comprises: D172K, D172R, D189K, D189R, K191D, K191E, K192D, K192E, K193D, K193E, D214K, D214R, E215K, E215R, K217D, K217E, K218D, K218E, K251D, K251E, D255K, D255R, R306D, R306E, E307K, E307R, K308D, K308E, E309K, E309R, R312D, R312E, D351K, D351R, R352D, R352E, E357K, E357R, E382K and E382R, as D214K, D214R, E215K, E215R, K251D, K251E, E357K and E357R, e.g.D214K, D214R, K251D and K251E.

Make charged target amino acid residue be had the aminoacid replacement of polar side chain, the specific examples that this amino acid replaces comprises: D172G/S/T/C/Y/N/Q, D189G/S/T/C/Y/N/Q, K191G/S/T/C/Y/N/Q, K192G/S/T/C/Y/N/Q, K193G/S/T/C/Y/N/Q, D214G/S/T/C/Y/N/Q, E215G/S/T/C/Y/N/Q, K217G/S/T/C/Y/N/Q, K218G/S/T/C/Y/N/Q, K251G/S/T/C/Y/N/Q, D255G/S/T/C/Y/N/Q, R306G/S/T/C/Y/N/Q, E307G/S/T/C/Y/N/Q, K308G/S/T/C/Y/N/Q, E309G/S/T/C/Y/N/Q, R312G/S/T/C/Y/N/Q, D351G/S/T/C/Y/N/Q, R352G/S/T/C/Y/N/Q, E357G/S/T/C/Y/N/Q and E382G/S/T/C/Y/N/Q are as D214G/S/T/C/Y/N/Q, E215G/S/T/C/Y/N/Q, K251G/S/T/C/Y/N/Q and E357G/S/T/C/Y/N/Q, D214Q for example, E215Q, K251Q and E357Q, especially K251Q.Another kind of interesting replacement may be K251N+T253A.

Other specific examples of interesting replacement comprise in the chapters and sections that are entitled as " being the conjugate of the present invention of non-polypeptide fraction with sugar component " and " conjugate of the present invention; wherein non-polypeptide fraction is connected with cysteine residues " disclosed those, especially be selected from down the replacement of group: K251N, S252N, Y302N or S190+K192T, be preferably selected from K251N or S252N, most preferably K251N.

Be appreciated that, as long as it is suitable, details relevant with conjugate of the present invention and specific descriptions (as, the activation of PROTEIN C, the quantity that replaces, the resistance increase of the explanation of conjugate at the deactivation of α-1-antitrypsin and human plasma used in the preparation of conjugate) also be same or analogous for variant of the present invention.Therefore, as long as suitable, statement and the detailed description relevant with conjugate of the present invention after carrying out necessary correction, can be applied to PROTEIN C variant disclosed herein.

The non-polypeptide fraction of conjugate of the present invention

As mentioned above, the non-polypeptide fraction of conjugate of the present invention is preferably selected from polymer molecule, lipophilic compound, sugar component (by glycosylated mode in the body) and organic derivating agent.The polypeptide portion that all these materials all can be conjugate provides desired characteristic, particularly increases in the function gonosome half life and/or increases the blood plasma half life.The polypeptide portion of conjugate can be only and one type non-polypeptide fraction coupling, but also can with two or more dissimilar non-polypeptide fraction couplings, for example, with polymer molecule and sugar component coupling, with lipophilic group and sugar component coupling, with organic derivating agent and sugar component coupling, with lipophilic group and polymer molecule coupling etc.Can simultaneously or carry out in order with two or more dissimilar non-polypeptide fraction couplings.

The method for preparing conjugate of the present invention

In following chapters and sections " with the coupling of lipophilic compound ", the coupling with the non-polypeptide fraction of all kinds is described in " with the coupling of polymer molecule ", " with the coupling of sugar component " and " with the coupling of organic derivating agent ".Usually, polypeptide conjugate of the present invention is prepared as follows: helping cultivating proper host cell under the condition of described expression of polypeptides, obtain described polypeptide, wherein a) described polypeptide comprises at least one N-or O-glycosylation site, and described host cell is to carry out glycosylated eukaryotic cell in the body, and/or b) described polypeptide is in the non-polypeptide fraction of external coupling.

Should be understood that design should make the quantity of gained molecule at the non-polypeptide fraction that is connected, the size of this quasi-molecule and form (as linearity or branch), and the connection site each side the best on described polypeptide to link coupled.The molecular weight of the non-polypeptide fraction that uses can, select as the effect that reaches based on hope.For example, if the link coupled main purpose is to obtain to have the high-molecular weight conjugate (removing as reducing kidney), want the coupling non-polypeptide fraction of few high molecular of trying one's best usually, with the molecular weight that obtains to expect.If it is higher to wish to shelter degree, can use capacity the non-polypeptide fraction of lower molecular weight (as molecular weight from about 300Da to about 5kDa, as molecular weight from 300Da to 2kDa).

Coupling with polymer molecule

With polypeptide link coupled polymer molecule can be any suitable polymers molecule, as natural or synthetic homopolymer or heteropolymer, the about 300-100 of the common scope of molecular weight, 000Da, 500-20 according to appointment, 000Da, more preferably from about 500-15,000Da, even more preferably from about 2-12kDa, 3-10kDa according to appointment.When term " about " used herein related to a certain molecular weight, " pact " just represented about molecular-weight average, and reflected certain molecular weight distribution is promptly arranged such fact usually in certain polymer formulations.

The example of homopolymer comprise polyalcohols (that is, poly--OH), polyamine (that is poly--NH, ₂) and poly carboxylic acid (that is, gather-COOH).Heteropolymer is the polymkeric substance that contains different coupling group (as hydroxyl and amino).

The example of suitable polymers molecule comprises and is selected from following polymer molecule: polyalkylene oxide (PAO), comprise polyalkylene glycol (PAG), as polyoxyethylene glycol (PEG) and polypropylene glycol (PPG), ramose PEG, polyvinyl alcohol (PVA), polycarbonate, polyvinylpyrrolidone, polyethylene is maleic anhydride altogether, polystyrene is maleic anhydride altogether, dextran comprises Sensor Chip CM 5, or any other is suitable for reducing immunogenicity and/or increases half life in the function gonosome and/or the biological polymer of serum half life.Another example of polymer molecule is human albumin or another abundant plasma proteins.In general, polyalkylene glycol-derived polymers is biocompatible, nontoxic, no antigen, non-immunogenicity, has various water-soluble characteristics and be easy to secrete from the biology of living.

PEG is preferred polymer molecule because its with, for example polysaccharide such as dextran are compared the crosslinked reactive group of energy that only has minute quantity.Interested especially is single function PEG, as mono methoxy polyethylene glycol (mPEG) because its coupling chemofacies to simple (only reactive group can be used for polysaccharide on the linking group coupling).Therefore, crosslinked danger is weakened, and the gained polypeptide conjugate more reaction of homogeneous and polymer molecule and polypeptide is easier to control.

For polymer molecule is connected with polypeptid covalence, the hydroxyl terminal groups of polymer molecule is necessary for activated form, and (example comprises primary amine group promptly to have reactive functional group, hydrazides (HZ), sulfydryl (thiol), succinate (SUC), succinimido succinate (SS), succinimido succinic diamide (SSA), succinyl phosphorons amino propyl acid ester (SPA), succinimido butyric ester (SBA), succinimido carboxymethylation thing (SCM), benzotriazole carbonic ether (BTC), N-hydroxy-succinamide (NHS), aldehyde, nitrophenyl carbonate (NPC) and tresylate (TRES)).Suitably the activatory polymer molecule has commodity, for example can derive from Shearwater Polymer, Inc., Huntsville, AL, USA or derive from PolyMASC Pharmaceuticals plc, UK.

Perhaps, polymer molecule can for example, activate described in WO 90/13540 by ordinary method known in the art.Use the specific examples of activated form linearity or branched polymer molecule to be described in Shearwater Polymers among the present invention, Inc.1997 and 2000 catalogues (FunctionalizedBiocompatible Polymers for Research and pharmaceuticals, Polvethylene Glycoland Derivatives is incorporated herein for reference).

The specific examples of activatory PEG polymkeric substance comprises (SPA-PEG for example, SSPA-PEG, SBA-PEG, SS-PEG with lower linear PEG:NHS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG and MAL-PEG comprise its mPEG form; With ramose PEG such as PEG2-NHS, comprise its mPEG form, and US5,932,462 and US 5,643,575 (all being incorporated herein by reference) those disclosed.In addition, be incorporated herein and disclose useful polymer molecule and/or PEGization chemical process: US 5,824,778 in the following publication for reference, US 5,476,653, and WO 97/32607, EP 229,108, and EP 402,378, US4,902,502, US 5,281,698, US 5,122,614, US 5,219, and 564, WO 92/16555, WO94/04193, and WO 94/14758, and WO 94/17039, WO 94/18247, and WO 94/28024, WO95/00162, and WO 95/11924, WO 95/13090, and WO 95/33490, and WO 96/00080, WO97/18832, WO 98/41562, and WO 98/48837, and WO 99/32134, and WO 99/32139, WO99/32140, WO 96/40791, and WO 98/32466, and WO 95/06058, EP 439 508, WO97/03106, and WO 96/21469, and WO 95/13312, EP 921 131, and US 5,736,625, WO98/05363, EP 809 996, US 5,629, and 384, WO 96/41813, and WO 96/07670, US5,473,034, US 5,516,673, EP 605 963, US 5,382, and 657, EP 510 356, and EP 400 472, EP 183 503 and EP 154 316.

The coupling of polypeptide and activatory polymer molecule can be by using any ordinary method, for example, undertaken by following reference described (also having described the proper method of activated polymer molecule in the described reference): R.F.Taylor, (1991), " Protein immobilisation.Fundamental andapplications ", Marcel Dekker, N.Y.; S.S.Wong, (1992), " Chemistry of ProteinConjugation and Crosslinking ", CRC Press, Boca Raton; G.T.Hermanson etc., (1993), " Immobilized Affinity Ligand Techniques ", Academic Press, N.Y.).The technician knows, the functional group that activation method that will use and/or coupling chemistry depend on the linking group (the above example that provided) of described polypeptide and polymkeric substance (for example, be amine, hydroxyl, carboxyl, aldehyde, sulfydryl, succinimide, maleimide, vinysulfone or halogenated acetic acid salt).PEGization can relate to polypeptide on all available linking groups (that is, being exposed to the linking group on polypeptide surface) coupling, or can with one or more specific linking groups as, the direct coupling of N-terminal amido (as US5,985,265 is described).And, coupling can one the step in finish or with the multistep mode finish (as, WO 99/55377 is described).

Should be understood that PEG turns into need being designed to, make the gained molecule number, these bulks of molecule and the form of the PEG molecule that connects (as, linearity or branch), and connection Ei site each side the best of peptide molecule.For example, can select the molecular weight of employed polymkeric substance according to the effect that need reach.

Only with protein on during a linking group (as the N-terminal amido) coupling, advantageously linearity or ramose polymer molecule have high molecular, preferably about 10-25kDa, 15-25kDa according to appointment, for example about 20kDa.

In general, polymkeric substance link coupled condition is to make available polymkeric substance linking group as much as possible and reacted polymer molecule.This can realize for the suitable molar excess of described polypeptide by making described polymer phase.Usually, the mol ratio of activatory polymer molecule and polypeptide reaches as high as about 1000-1, as the highest about 200-1, or the highest about 100-1.In some cases, for obtaining optimum response, described mol ratio also may be lower slightly, as the highest about 50-1, and 10-1,5-1,2-1 or 1-1.

The invention still further relates to via joint and make polymer molecule and polypeptide coupling.Suitable joint is known to the skilled.Preferred examples is cyanuryl chloride (Abuchowski etc., (1977), J.Biol.Chem., 252,3578-3581; US4179337; People such as Shafer (1986), J.Polym.Sci.Polym.Chem.Ed., 24,375-378).

After the coupling,, as by primary amine is joined in the reaction mixture, seal residual activated form polymer molecule, and remove the polymer molecule of the inactivation that is produced by suitable method by methods known in the art.

Should understand, based on various conditions (as amino acid sequence of polypeptide, the character of the activated form PEG compound that uses and specific PEGization condition, the mol ratio that comprises PEG and polypeptide), may obtain PEGization in various degree, the common higher mole ratio of the PEGization of higher degree from PEG and polypeptide.Yet, derive from the PEGization polypeptide of any given PEGization process, comprise the stochastic distribution of the slightly different polypeptide conjugate of PEGization degree usually.

Coupling with sugar component

Be glycosylation in the body of realizing protein C molecule (comprising one or more glycosylation sites), the nucleotide sequence of this polypeptide of coding must be inserted among the glycosylated eukaryotic expression host.Expression host cell can be selected from fungi (filamentous fungus or yeast), insect or zooblast, or is selected from transgenic plant cells.In one embodiment, host cell is that mammalian cell is (as COS cell, Chinese hamster ovary celI, bhk cell or HEK cell, as the HEK293 cell), perhaps insect cell (as the SF9 cell), perhaps yeast cell such as yeast saccharomyces cerevisiae (Saccharomyces Cerevisiae) or finish red saccharomyces pastorianus (Pichia pastoris), or any host cell that hereinafter further describes.

Sugar component (as dextran) with as described in the amino-acid residue of polypeptide also can use WO87/05330 and Aplin etc., CRC Crit Rev.Biochem., pp.259-306,1981 described methods at external covalent coupling.The external coupling of sugar component or PEG and protein bound type and peptide mating type Gln-residue also can be undertaken by transglutaminase (TGases).Transglutaminase with a kind of mode catalysis donor transamination of so-called crosslinking reaction to albumen-and peptide-bonded Gln residue on.Donor amino can be albumen-or peptide-bonded, as the ε amino in the lysine residue, or also can be the part of little or big organic molecule.In transglutaminase-catalyzed crosslinked as the little organic molecule such as the putrescine (1, the 4-diaminobutane) of amino donor.In transglutaminase-catalyzed crosslinked as the big organic molecule of amino donor as contain amino-PEG (Sato etc., 1996, Biochemistry 35,13072-13080).

Usually, transglutaminase is the enzyme of high special, be not all Gln residues that are exposed to protein surface all can be used for transglutaminase-catalyzed contain the crosslinked of amino material.On the contrary, having only minority Gln residue is the natural substrate of transglutaminase, but still unknowable as the definite parameter of the transglutaminase substrate that is fit to for control Gln residue.Therefore, in order to make the crosslinking reaction sensitivity of albumen to transglutaminase-catalyzed, frequent prerequisite is at one section aminoacid sequence of position increase easily, the known substrate that can be used as transglutaminase well of described aminoacid sequence.Known several aminoacid sequence can be the substrate of transglutaminase, or comprises the substrate of transglutaminase, as Substance P, and elafin, Fibrinogen, fibronectin, α ₂-plasmin inhibitor, alpha-casein and beta-casein.

Coupling with organic derivating agent

The covalent modification of described polypeptide can be by polypeptide one or more linking groups and organic derivating agent react and carry out.Suitable derivating agent and method are well known in the art.For example, the most normal and alpha-halogen acetic ester (with corresponding amine) of cysteinyl residue as Mono Chloro Acetic Acid or chlor(o)acetamide reaction, produces carboxymethyl or carboxy and amide groups methyl-derivatives.The cysteinyl residue also can be derived with following substance reaction: bromine trifluoroacetone, α-bromo-β-(4-imidazolyl) propionic acid, chloracetyl phosphoric acid ester, N-alkyl maleimide, 3-nitro-2-pyridine disulphide, methyl 2-pyridine disulphide, pCMBA salt, 2-chloromercuri-4-nitrophenol or chloro-7-nitro benzo-2-Evil-1,3-diazole.The histidyl-residue is by deriving in the pH5.5-7.0 reaction with the diethyl pyrocarbonate, because this reagent is specific to the histidyl-side chain relatively.The PBPB thing is also effective; This reaction is preferably reacted in the 0.1M of pH6.0 cacodylic acid sodium.Lysyl and n terminal residue can react with succinyl oxide or other carboxylic acid anhydride.Derive with these reagent and to have the effect of the charge reversal that makes the lysyl residue.Be suitable for making containing alpha-amino other reagent of residue deutero-and comprising the inferior acid amides (methylpicolinimidate) of imino esters such as picoline, phosphoric acid Vitamin B6, Vitamin B6, chloro borohydride, trinitro-benzene-sulfonic acid, adjacent methyl-isourea, 2 the catalytic reaction with glyoxylate of 4-diacetylmethane and transaminase.The arginyl residue is by modifying with the reaction of one or more conventional reagent, described reagent comprise the phenyl oxalic dialdehyde, 2,3-dimethyl diketone, 1,2-cyclohexanedione and triketohydrindene hydrate.The requirement of deriving of arginine residues is reflected under the alkaline condition to be carried out, because the pKa of guanidine functional group is higher.

In addition, these reagent can react with Methionin and arginine guanidine radicals.Carboxyl side group (aspartyl or glutamyl) carries out selective modification by reacting with carbodiimide (R-N=C=N-R '); wherein R is different alkyl with R '; as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-nitrogen-4,4-dimethyl amyl group) carbodiimide.And aspartyl and glutamyl are by being converted into asparagus fern acyl amide group (asparaginyl) and glutamy amide group (glutaminyl) with the ammonium ion reaction.

Coupling with lipophilic compound

Polypeptide and lipophilic compound can directly or pass through to use joint coupling each other.Lipophilic compound can be natural compounds such as saturated or unsaturated fatty acids, lipid acid diketone, terpenes, prostaglandin(PG), VITAMIN, carotenoid or steroid hormone, perhaps synthetic compound such as carbonic acid, alcohol, amine and have one or more alkyl-, aryl-, alkenyl-sulfonic acid or other polynary unsaturated compound.Can be by methods known in the art, as press Bodanszky at Peptide Synthesis, John Wiley, New York, 1976 and WO96/12505 described in method carry out coupling (optional connect) between polypeptide and the lipophilic compound by joint.

Coupling with labeling polypeptide

Polypeptide can be expressed as the fusion rotein with marker, promptly usually by 1-30, as the aminoacid sequence or the peptide section of 1-20 amino-acid residue formation.Except can be fast and easily carry out purifying, marker be to finish link coupled convenient tool between labeling polypeptide and the non-polypeptide fraction.Particularly, marker is used on titer plate or other carrier such as the paramagnetic beads and finishes coupling, and the polypeptide of mark can fix by marker like this., for example, the polypeptide link coupled advantage with mark on the titer plate is, the polypeptide of mark can directly be fixed on the titer plate (in principle without any purifying) and carry out coupling from culture broth.Therefore, can reduce the sum of operation steps (from expressing coupling).And marker can play the work of spacerarm molecule in order to guarantee the making fixed polypeptide be easier to coupling.The coupling that the applying marking polypeptide carries out can be the coupling with any non-polypeptide fraction disclosed herein, as with the coupling of polymer molecule such as PEG.

It is not crucial using which kind of concrete marker, as long as this marker can be expressed and can be fixed on the suitable surface or carrier substance with polypeptide.Many suitable markers can obtain by the commercial channel, for example, can purchase the Laboratories in Unizyme, Denmark.For example, described marker can be following arbitrary sequence:

His-His-His-His-His-His

Met-Lys-His-His-His-His-His-His

Met-Lys-His-His-Ala-His-His-Gln-His-His

Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln

Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-Gln

Or following arbitrary sequence:

EQKLI SEEDL (being described in Mol.Cell.Biol.5:3610-16,1985 C-end marker)

DYKDDDDK (C-or N-end marker)

YPYDVPDYA

The antibody of anti-above-mentioned marker can obtain by the commercial channel usually, for example purchases in ADI AvesLab and Research Diagnostics.

Available subsequently commercially available enzyme cuts off marker from polypeptide.

The method for preparing the polypeptide portion of polypeptide variants of the present invention or conjugate of the present invention

The polypeptide portion of polypeptide variants of the present invention or conjugate of the present invention (optional is the glycosylation form) can prepare by any suitable method known in the art.These methods comprise the nucleotide sequence that makes up this polypeptide of coding and express this sequence in suitable conversion or transfecting host.Preferred host cell is γ-carboxylated host cell, as mammalian cell.Yet, can produce polypeptide of the present invention by the combination of chemical synthesis process or chemical synthesis process or the combination of chemical synthesis process and recombinant DNA technology, but efficient is very low.

The nucleotide sequence of the polypeptide portion of code book invention polypeptide variants or conjugate of the present invention can be by separating or composite coding parent PROTEIN C, as the nucleotide sequence with PROTEIN C of aminoacid sequence shown in SEQ ID NO:2 and 4 makes up, and changes described nucleotide sequence then to introduce (promptly insert or replace) or to eliminate (promptly remove or replace) related amino acid residue.

Nucleotide sequence can be modified through site-directed mutagenesis easily by currently known methods.Perhaps, nucleotide sequence can prepare by chemosynthesis, for example by using oligonucleotide synthesizer, wherein comes design oligonucleotides based on required amino acid sequence of polypeptide, and preferred those codons of selecting the host cell preference of production recombinant polypeptide.For example, can synthesize the small molecules oligonucleotide of the each several part of the required polypeptide of a plurality of codings, assemble by PCR, ligation or ligase chain reaction (LCR) (Barany, PNAS 88:189-193,1991) then.Each oligonucleotide contains 5 usually ' or 3 ' overhang be used for complementary assembling.

Other has the nucleotide sequence modifying method to can be used for producing polypeptide variants, so that polypeptide variants is used for the high flow capacity screening, the disclosed method that relates to homology exchange among the US5093257 for example, and the method for related gene reorganization, be the reorganization between two or more homologous nucleotide sequences, cause producing and compare the new nucleotide sequence that has many Nucleotide to change with the nuclei originis nucleotide sequence.The one or many circulation that gene reorganization (being also referred to as DNA reorganization) relates to the random fragmentation of nucleotide sequence and ressembles selects coding to have the nucleotide sequence of the polypeptide of desired characteristic by screening thereupon.Reorganization can take place based on the nucleic acid of homology in order to make, and the relevant portion of described nucleotide sequence preferably has 50% identity at least, the identity as at least 60%, more preferably at least 70% identity, the identity as at least 80%.Reorganization can be carried out in external or body.

The example of suitable outer-gene reorganization method: Stemmer etc. are disclosed in the following document, (1994), Proc.Natl.Acad.Sci.USA; Vol.91, pp.10747-10751; Stemmer (1994), NaturemVol.370, pp.389-391; Smith (1994), Nature Vol.370, pp.324-325; Zhao etc., Nat.Biotechnol.1998.Mar; 16 (3): 258-61; Zhao H. and Arnold, FB, Nucleic AcidsResearch, 1997, Vol.25.No.6 pp.1307-1308; Shao etc., Nucleic Acids Research1998, Jan 15; 26 (2): pp.681-83; And WO95/17413.

Reorganization method in a kind of suitable body is disclosed among the WO97/07205.Other technology of carrying out nucleic acid mutagenesis by reorganization in external or the body exists, as open among WO97/20078 and the US5837458.Special shuffling technology comprises " gene cluster reorganization (Family shuffling) ", " synthetic reorganization " " computer (insliico) reorganization ".

Gene cluster reorganization is to make the gang's homologous gene from different plant species carry out the reorganization of one or many and the circulation of follow-up screening or selection.The gene cluster shuffling technology is disclosed in, as: Crameri etc., (1998), Nature, vol.391, pp.288-291; Christians etc., (1999), Nature Biotechnology, vol.17, pp.259-264; Chang etc. (1999), Nature Biotechnology, vol.17, pp.793-797; And Ness etc. (1999), Nature Biotechnology, vol.17 is among the 893-896.

Synthetic reorganization relates to according to the sequence alignment between the purpose homologous gene provides eclipsed synthetic oligonucleotide library.Make the reorganization of synthetic oligonucleotide, the gained recombinant nucleic acid sequence is screened, can be used for further reorganization circulation as needs.Synthetic reorganization can be referring to WO00/42561.

Computer (in silico) reorganization refers to utilize computer system to implement or simulation (modelled), therefore a kind of DNA reorganization method that partially or completely need not carry out physical operations to nucleic acid.Computer reorganization can be referring to WO00/42560.In case after the assembling (by synthetic, site-directed mutagenesis or other method), the nucleotide sequence of coded polypeptide is inserted in the recombinant vectors immediately, and in the target transformed host cell, expresses necessary regulating and controlling sequence with PROTEIN C and be operably connected.

Certainly, should understand the nucleotide sequence that not all carrier and expression regulation sequence can both be expressed polypeptide described herein equally well.Not all host can both be with the identical same good function of expression system performance.Yet those skilled in the art need not experimentize and just can make one's options to these carriers, expression regulation sequence and host.For example, when selecting carrier, must consider the host, because carrier must duplicate therein maybe and can be incorporated in the karyomit(e).Also should consider the expression of any other protein (as antibiotic marker) of carrier copy number, the ability of controlling copy number and vector encoded.When selecting expression regulation sequence, also should consider multiple factor.These factors comprise, as, the relative length of sequence, its controllability and with the consistency of the nucleotide sequence of coded polypeptide, to consider the potential secondary structure especially.Should consider they and the toxicity of the consistency of selected carrier, nucleotide sequence coded product, host's secretion characteristic, ability, host's fermentation and the difficulty or ease of cultivation demand and nucleotide sequence coded product purification that the host correctly folds polypeptide when selecting the host.Recombinant vectors can be the carrier of self-replicating, promptly as the outer entity of karyomit(e) exist, it duplicates a kind of carrier that is independent of chromosome duplication, for example plasmid.In addition, carrier can be when introducing host cell, the carrier that can be incorporated in the host cell gene group and duplicate with the karyomit(e) of its integration.

Carrier is expression vector preferably, and wherein the nucleotide sequence of code book invention polypeptide can be transcribed required extra fragments with nucleotide sequence and is operably connected.Carrier is derived by plasmid or viral DNA usually.The multiple suitable expression vector that is used for expressing at host cell described herein is commercially available or document description is arranged.The expression vector that can be used for eucaryon host comprises, as, contain carrier from the expression regulation sequence of SV40, bovine papilloma virus, adenovirus and cytomegalovirus.Concrete carrier such as pCDNA3.1 (+) Hyg (Invitrogen, Carlsbad, CA, USA) and pCI-neo (Stratagene, LaJola, CA, USA).The expression vector that can be used for yeast cell comprises 2 μ plasmid and derivatives thereof, and (US 4,931 for the POT1 carrier, 373), the pJSO37 carrier (is described in Okkels, Ann.New York Acad.Sci.782,202-207,1996) and pPICZ A, B or C (Invitrogen).The expression vector that can be used for insect cell comprises pVL941, pBG311 (Cate etc., ＂ Isolation of Bovine and Human Genes forMullerian Inhibiting Substance And Expression of the Human Gene In AnimalCells ＂, Cell, 45, pp.685-98 (1986), pBluebac 4.5 and pMelbac (all from Invitrogen).The expression vector that can be used for host bacterium comprises known bacterial plasmid, and Tathagata comprises pBR322 from the plasmid of intestinal bacteria, pET3a and pET12a are (from Novagen Inc., WI, USA), the plasmid of broad host range, as RP4, phage DNA, for example multiple derivative of lambda particles phage, for example NM989, with other DNA phage, as M13 and thread single stranded DNA phage.

Other carrier that uses among the present invention comprises increase those carriers of a plurality of copies of the nucleotide sequence that allows coded polypeptide.This carrier that increases is known in the art.For example, they comprise can be by DHFR amplification carrier (for example referring to Kaufman, U.S.Pat.No.4,470,461, Kaufmanand Sharp, ＂ Construction Of A Modular Dihydrofolate Reductase cDNA Gene:Analysis Of Signals Utilized For Efficient Expression ＂, Mol.Cell.Biol., 2, pp.1304-19 (1982)) and can pass through glutamine synthetase (" GS ") amplification carrier (for example referring to US5,122,464 and EP338,841).

Recombinant vectors can further comprise the dna sequence dna that described carrier is duplicated in target host cell.An example (when host cell is mammalian cell) of this type of sequence is the replication origin of SV40.When host cell was yeast cell, the proper sequence that carrier is duplicated was yeast plasmid 2 μ replicator REP 1-3 and replication origins.

Carrier also can contain selectable mark, as replenish those genes of host cell defective with its product, as the gene (P.R.Russell of Tetrahydrofolate dehydrogenase of encoding (DHFR) or schizosaccharomyces pombe (Schizosaccharomycespombe) TPI, Gene 40,1985, pp.125-130), perhaps give gene to the resistance of medicine such as penbritin, kantlex, tsiklomitsin, paraxin, Xin Meisu, Totomycin or methotrexate.For yeast saccharomyces cerevisiae, selectable mark comprises ura3 and leu2.For filamentous fungus, selectable mark comprises amdS, pyrG, arcB, niaD and sC.

Term " regulating and controlling sequence " comprises in this article to the essential or favourable all the components of expression of polypeptides of the present invention.Each regulating and controlling sequence can be natural or external concerning the nucleotide sequence of coded polypeptide.These regulating and controlling sequences include but not limited to leader sequence, polyadenylation sequence, propeptide sequence, promotor, enhanser or upstream activation sequences, signal peptide sequence and transcription terminator.Regulating and controlling sequence comprises promotor at least.

The present invention can use various expression regulation sequences.These spendable expression regulation sequences comprise the expression regulation sequence that links to each other with the structure gene of aforementioned expression vector and any sequence and the various combination thereof of known regulation and control protokaryon or eukaryotic cell or its viral genetic expression.

The example of the suitable regulating and controlling sequence that guidance is transcribed in the mammalian cell comprises the early stage and late promoter of SV40 and adenovirus, major late promoter as adenovirus 2, MT-1 (metallothionein gene) promotor, human cytomegalic inclusion disease virus immediate early gene promotor (CMV), people's EF-1 α (EF-1 α) promotor, the minimum heat shock protein 70 promotor of fruit bat, Rous sarcoma virus (RSV) promotor, people's ubiquitin C (UbC) promotor, the human growth hormone terminator, SV40 or adenovirus Elb district's polyadenylation signal and Kozak consensus sequence (Kozak, M.J Mol Biol 1987 Aug 20; 196 (4): 947-50).

In order to improve the expression in mammalian cell, the synthetic intron can be inserted 5 ' non-translational region of the nucleotide sequence of coding said polypeptide.The example of synthetic intron be from plasmid pCI-Neo synthetic intron (available from Promega Corporation, WI, USA).

Instruct the example of the suitable regulating and controlling sequence of transcribing to comprise polyhedrin promotor, P10 promotor, autographa california (Autographa californica) polyhedrosis virus basic protein promoter, baculovirus immediate early gene 1 promotor and baculovirus 39K delayed early gene promotor and SV40 polyadenylation sequence in the insect cell.The example of the suitable regulating and controlling sequence that uses in yeast host cell comprises promotor, yeast triose-phosphate isomerase (TPI) promotor, the promotor from Yeast sugar glycolysis gene or alcohol dehydrogenase gene, ADH2-4c promotor and the induction type GAL promotor of yeast α mating system.The example of the suitable regulating and controlling sequence that uses in filamentous fungal host cell comprises ADH3 promotor and terminator, by gene deutero-promotor, TPI1 terminator and the ADH3 terminator of coding aspergillus oryzae TAKA amylase triose-phosphate isomerase or Sumizyme MP, aspergillus niger αDian Fenmei, aspergillus niger or Aspergillus nidulans (A.Nidulans) glucoamylase, Aspergillus nidulans acetamidase, Rhizomucor miehei (Rhizomucor miehei) aspartate protease or lipase.The example of the suitable regulating and controlling sequence that uses in bacterial host cell comprises the promotor of lac system, trp system, TAC or TRC system and the main promoter region of lambda particles phage.

Whether the existence of signal peptide is depended on, for example, is used for producing and will and whether needs secretion by the expression host cell of polypeptide expressed (the interior or outer polypeptide of born of the same parents of born of the same parents).In order to use in filamentous fungus, signal peptide can be derived easily by the gene of gene, coding Palatase or the proteolytic enzyme or the Humicola lanuginosa lipase of the amylase of coding Aspergillus bacterial strain or glucoamylase.Signal peptide is preferably derived by the gene of coding aspergillus oryzae TAKA amylase, the neutral α-Dian Fenmei of aspergillus niger, aspergillus niger acid-stable starch enzyme or aspergillus niger glucoamylase.In order in insect cell, to use, signal peptide can derive easily by insect genes (referring to WO90/05783), as lepidopteran Manduca sexta adipokinetic hormone precursor (US5023328), bee variety toxin (Invitrogen), ecdysteroids UDP glucanotransferase (glucosyltransferase, egt) (Murphy etc., Protein Expression andPurification 4,349-357 (1993)) or people's steapsin (hpl) (Methods in Enzymology 284, pp.262-272,1997).The preferred signals peptide that uses in the mammalian cell is hFVII's or mouse Ig κ light chain a signal peptide (Coloma, M (1992) J.Imm.Methods 152:89-104).In order in yeast cell, to use, the appropriate signal peptide is that the carboxypeptidase signal peptide of yeast saccharomyces cerevisiae α-factor signal peptide (referring to US4870008), modification is (referring to people such as L.A.Valls, Cell 48,1987, pp.887-897), yeast BAR1 signal peptide (referring to WO87/02670), yeast aspartate protease 3 (YAP3) signal peptide are (referring to people such as M.Egel-Mitani, Yeast 6,1990, pp.127-137) and synthetic leader sequence TA57 (WO98/32867).Colibacillary appropriate signals peptide is signal peptide ompA (EP581821).

The nucleotide sequence of proteins encoded C polypeptide variants of the present invention is no matter by site-directed mutagenesis, synthetic, PCR or other which kind of method preparation, the optional nucleotide sequence that comprises the coded signal peptide.Need secrete from express cell as polypeptide then needs signal peptide.The sort signal peptide if exist, should be the cell recognition that can be expressed described polypeptide.Signal peptide can with described homologous peptide (as, usually link to each other with human protein C) or allos (differing from human protein C as its origin), perhaps can with host cell homology or allos, promptly described signal peptide is that this host cell signal peptide of usually expressing or its are not the signal peptides that this host cell is expressed usually.Accordingly, described signal peptide can be a protokaryon, as derives from bacterium such as intestinal bacteria, or eucaryon, as derive from Mammals or insect or yeast cell.

Can use any appropriate host to produce the polypeptide portion of polypeptide of the present invention or conjugate of the present invention, comprise bacterium, fungi (comprising yeast), plant, insect, Mammals or other suitable zooblast or clone and transgenic animal or plant.The example of bacterial host cell comprises gram positive bacterium such as bacillus, as bacillus brevis or subtilis, Pseudomonas or streptomyces, or gram negative bacterium, as coli strain.Carrier is introduced bacterial host cell can be passed through, for example, protoplast transformation is (referring to as Chang and Cohen, 1979, MolecularGeneral Genetics 168:111-115), use experience attitude cell is (referring to as Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), electroporation is (referring to as Shigekawaand Dower, 1988, Biotechniques 6:742-751), or coupling (referring to as Koehler andThorne, 1987, Journal of Bacteriology 169:5771-5278) carry out.The example of suitable filamentous fungal host cell comprises the Aspergillus bacterial strain, as aspergillus oryzae, aspergillus niger or Aspergillus nidulans, and Fusarium or trichoderma.The fungal cell can transform by relating to following process: protoplastis formation, protoplast transformation and cell walls are regenerated by known mode own.The appropriate method that transforms the Aspergillus host cell has been described in EP238023 and US5679543.People such as Malardier 1989, the appropriate method that transforms Fusarium has been described among Gene 78:147-156 and the WO96/00787.The example of suitable yeast host cell comprises bacterial strain (as yeast saccharomyces cerevisiae), Schizosaccharomyces, genus kluyveromyces, Pichia (as pichia pastoris phaff or P.Methanolica), Hansenula (as multiple-shaped nuohan inferior yeast) or the Yarrowia of saccharomyces.Yeast can use Becker and Guarente, InAbelson, J.N.And Simon, M.I., editors, Guide to Yeast Genetics and MolecularBiology, Methods in Enzymology Volume 194, pp 182-187, Academic Press, Inc., New York; People such as Ito, 1983, Journal of Bacteriology 153:163; With people 1978 such as Hinnen, Proceedings of the National Academy of Sciences USA 75:1920; And by Clontech Laboratories, Inc, Palo Alto, CA, USA (Yeastmaker ^TMThe product manual of yeast conversion system test kit) method described in transforms.The example of suitable insect host cell comprises lepidopteran clone, as fall army worm (Sf9 or Sf21) or cabbage looper cell (HighFive) (US5077214).Can transform insect cell and produce heterologous polypeptide therein by the described method of Invitrogen.The example of suitable mammalian host cell comprises that Chinese hamster ovary (CHO) clone is (as, CHO-K1; ATCC CCL-61), grivet clone (COS) (as, COS1 (ATCCCRL-1650), COS7 (ATCC CRL-1651)); Mouse cell (as, NS/O), hamster children mouse kidney (BHK) clone (as, ATCC CRL-1632 or ATCC CCL-10) and people's cell (as, HEK293 (ATCC CRL-1573)), and the vegetable cell in the tissue culture.Other suitable clone is well known in the art and can be available from public preservation center, as American type culture collection, and Rockville, Maryland.And mammalian cell (as Chinese hamster ovary celI) can carry out modification by method as described in the US5047335 and express sialytransferase, as, 1, the 6-sialytransferase is so that improve the glycosylation of protein c polypeptide.

In order to increase secretion, make polypeptide of the present invention and endo-protease, PACE (paired basic aminoacids saccharase) (described in US5986079) especially, it is favourable producing together as Kex2 endo-protease (described in WO00/28065).

The method that foreign DNA is introduced mammalian host cell comprises the transfection of transfection, electroporation, the mediation of DEAE-dextran of calcium phosphate mediation, liposome-mediated transfection, virus vector and by LifeTechnologies Ltd, Paisley, the transfection method that the described use of UK Lipofectamin2000 carries out.These methods are well known in the art, for example at people such as Ausbel (eds.), and 1996, CurrentProtocols in Molecular Biology, John Wiley ﹠amp; Sons, New York is described in the USA.The cultivation of mammalian cell is undertaken by the method for setting up, for example referring to Animal Cell Biotechnology, Methods and Protocols, Nigel Jenkins compiles, and 1999, Human Press Inc, Totowa, NewJersey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997.

In production method of the present invention, with methods known in the art culturing cell in being suitable for producing the nutritional medium of polypeptide.For example, cell can be in the laboratory by shake-flask culture, small-scale or large scale fermentation (comprise continuously, in batch, fed-batch or solid state fermentation) or suitable culture medium with allow to express and/or separate under the condition of described polypeptide and carry out industrial fermentation.Cultivation uses methods known in the art to carry out in the suitable nutrient medium that contains carbon source and nitrogenous source and inorganic salt.Suitable medium is commercially available maybe can be prepared by disclosed composition (for example, described in the catalogue of American type culture collection).If the polypeptide secretion is in nutritional medium, this polypeptide can directly reclaim from substratum.If polypeptide is not secreted, can from cell lysate, reclaim.

Can reclaim the gained polypeptide by methods known in the art.For example, can include but not limited to that centrifugal, filtration, ultrafiltration, extraction or precipitation reclaim polypeptide from nutritional medium by ordinary method.

Can by the whole bag of tricks known in the art include but not limited to chromatography (as, ion-exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method (as, the isoelectrofocusing of preparation property), dissolubility difference (as, ammonium sulfate precipitation) or extract (referring to, as Protein Purification, J.-C.Janson andLars Ryden, editors, VCH Publishers, New York, 1989) come purified polypeptide.

Pharmaceutical composition and purposes

The present invention relates to a kind of pharmaceutical composition on the other hand, and it comprises conjugate of the present invention or variant of the present invention, and a kind of pharmaceutically useful carrier or vehicle.In context, " pharmaceutically acceptable " means and do not cause in the patient of administration and anyly do not want or the carrier or the vehicle of deleterious effects.This class pharmaceutically acceptable carrier and vehicle be well known in the art (referring to, as Remington ' s PharmaceuticalSciences, 18th edition, A.R.Gennaro, Ed., Mack Publishing Company[1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaerand L.Hovgaard, Eds., Taylor ﹠amp; Francis[2000]; With Handbook of PharmaceuticalExcipients, 3rd edition, A.Kibbe, Ed., Pharmaceutical Press[2000]).

The present invention relates to conjugate of the present invention on the other hand, variant of the present invention, or pharmaceutical composition of the present invention is used to prepare the purposes of medicine.More specifically, conjugate of the present invention, variant or pharmaceutical composition can be used for preparing the following medicine of treatment: outbreak (stroke); Myocardial infarction; After the phlebothrombosis; Disseminated inravascular coagulation (DIC); Sepsis (sepsis); Septic shock; Embolism (emboli) is as pulmonary infarction; Transplant, as bone marrow transplantation; Burn; Gestation; Great surgical operation/wound or adult respiratory distress syndrome (ARDS) are in particular for the medicine of preparation treatment septic shock.

The invention still further relates to the method for the treatment of or preventing to be selected from down the disease of group: outbreak; Myocardial infarction is after the phlebothrombosis; Disseminated inravascular coagulation (DIC); Sepsis; Septic shock; Embolism is as pulmonary infarction; Transplant, as bone marrow transplantation; Burn; Gestation; Great surgical operation/wound or adult respiratory distress syndrome (ARDS), described method comprises and gives the conjugate of the present invention of significant quantity, variant of the present invention or pharmaceutical composition of the present invention to the patient who needs corresponding treatment, especially treatment or prevention (more particularly treatment) septic shock.

" patient " comprises people and other Mammals among the present invention.Therefore described method can be used for people's treatment or for animals.

Polypeptide variants of the present invention and conjugate can give the patient with effective dose." effective dose " is meant the dosage that controlling morbid state is enough to produce desired result herein.The accurate dosage of administration depends on the disease of controlling, and can be determined by known method by those skilled in the art.As mentioned above, treatment is during severe sepsis, and with the amount administration of 24 μ g/kg/h people APC 96 hours, it was corresponding to the albumen of the about 230mg of patient's administration total amount of the about 100kg of body weight.Therefore conjugate of the present invention and variant are considered to have stronger effectiveness because blood plasma half life increase makes its time lengthening that plays a role in blood plasma.This enhanced is renderd a service passable, for example, assesses by calculating in " human plasma inactivation test II " the following area (AUC) of curve, perhaps assesses by measuring the serum half life.Enhanced is renderd a service and to be meant, obtains the necessary effective dose of required effect than the effective dose of people the APC little proteic amount of administration (need still less) at disease specific.In addition, blood plasma half life increase also makes and can use APC variant or conjugate to treat regularly in the given time period.Therefore, these new features cause can with a small amount of and/or and less administration number of times (as bolus injection (bolus injection)) use compound of the present invention.For example, compound of the present invention can or be inculcated agent or the form administration of their combination with bolus (bolus), the scope of dosage is from the bolus of per second hour administration 1 μ g/kg body weight, continue a few days (as 96 hours) to the bolus of per the 4th day administration 1mg/kg body weight once.Preferably with the least possible number of times of as far as possible little dosed administration, for example, every 4-96 hour, for example, every 8-96 hour, as every 16-96, every 24-96 hour, every 40-96 hour, every 48-96 hour, every 56-96 hour, every 72-96 hour, according to 1-500 μ g/kg body weight, preferred 1-250 μ g/kg body weight, as 1-100 μ g/kg body weight, the more preferably amount administration bolus of 1-50 μ g/kg body weight.

The preferred compound of the present invention is that when detecting with this paper embodiment 13 described " human plasma inactivation test II ", the ratio of the AUC of the AUC of its activity form and people APC is at least those of 1.25.Preferred described ratio is at least 1.5, as is at least 2, for example is at least 3, and more preferably described ratio is at least 4, as is at least 5, for example is at least 6, and also more preferably described ratio is at least 7, as is at least 8, for example is at least 9, and most preferably described ratio is at least 10.

Polypeptide variants of the present invention or conjugate can " with its forms originally " and/or are used with the form of its salt.Suitable salt includes but not limited to, with basic metal or alkaline-earth metal, and sodium for example, potassium, the salt of calcium and magnesium and the salt that forms of zinc for example.These salt or mixture can exist with crystallization and/or unbodied structure.

Pharmaceutical composition of the present invention can be individually dosed or with other therapeutical agent administration.These preparations can be blended in in a kind of pharmaceutical composition, perhaps can with polypeptide of the present invention or conjugate separate administration, can be administration simultaneously, perhaps according to another kind of treatment time table administration.In addition, polypeptide of the present invention, conjugate or pharmaceutical composition can be used as the supplementary form of other treatment.

Pharmaceutical composition of the present invention can be mixed with various forms, liquid for example, glue, dried frozen aquatic products, or compression solid.Preferred form depends on the concrete indication of being treated and is conspicuous to those skilled in the art.

Preparaton of the present invention administration in many ways includes but not limited to, and is oral, subcutaneous, in the intravenously, cerebellum, in the nose, in skin, intraperitoneal, intramuscular, lung, transvaginal, per rectum, intraocular or in any other suitable mode.Although it is acceptable using technology well known in the art (for example pump or implantation) to carry out the bolus injection, described preparaton can be with the mode continuous use of infusion.In some cases, preparaton can directly be used with the form of solution or sprays.

Parenteral route composition (Parental composition)

The example of pharmaceutical composition is the solution that is designed to parenteral administration.Although under many circumstances, the drug solution preparaton can provide to be applicable to the liquid form that uses immediately, and described parenteral route preparaton also can provide with freezing or lyophilized form.Under former instance, described composition must thaw before use.The stability of active ingredient under various storage requirements that back one formulation is generally used in the enhancing composition being comprised, as well known by persons skilled in the art, freeze-dried preparation is more stable than its liquid homologue usually.Described freeze-dried preparation is prepared again by adding one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or stroke-physiological saline solution solution before use.

Under the situation of parenteral administration, they are made into the form of the freeze dried preparaton or the aqueous solution, method is as required, the polypeptide that will have required purity suitably mixes vehicle such as buffer reagent, stablizer, sanitas, isotonic agent, nonionogenic tenside, antioxidant and/or other various additives with pharmaceutically acceptable carrier, vehicle or the stablizer (being referred to as " vehicle ") that one or more this areas are used always.

Buffer reagent helps pH is maintained in the scope near physiological condition.They exist with the concentration range of about 2mM-50mM usually.The suitable buffer reagent that the present invention uses comprises organic and mineral acid and salt thereof such as citrate buffer agent (as, sodium dihydrogen citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-sodium dihydrogen citrate mixture etc.), the succinate buffer reagent (as, succsinic acid-succsinic acid one sodium mixture, succsinic acid-sodium hydroxide mixture, succsinic acid-disodium succinate mixture etc.), the tartrate buffer reagent (as, tartrate-sodium tartrate mixture, tartrate-soluble tartrate mixture, tartrate-sodium hydroxide mixture etc.), the fumarate buffer reagent (as, fumaric acid-fumaric acid one sodium mixture, fumaric acid-Disodium fumarate mixture, fumaric acid one sodium-Disodium fumarate mixture etc.), gluconate (gluconate) buffer reagent (as, gluconic acid-Sunmorl N 60S mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-Potassium Gluconate mixture etc.), the oxalate buffer reagent (as, oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactate buffer agent (as, lactic acid-Sodium.alpha.-hydroxypropionate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture etc.) and acetate buffer (as, acetate-sodium acetate mixture, acetate-sodium hydroxide mixture etc.).Other possible buffer reagent is phosphate buffer, histidine buffer and front three amine salt such as Tris.

Can add sanitas and block microorganism growth, the amount of adding is about 0.2%-1% (w/v) usually.The suitable preservatives that the present invention uses comprises phenol, phenylcarbinol, meta-cresol, oxybenzene formic acid (paraben) methyl esters, propylparaben, octadecyl dimethyl benzene ammonio methacrylate, Ben Yajiaji halogenide (as Ben Yajiaji muriate, bromide or iodide), chlorination hexane diamine, oxybenzene alkyl formate such as methyl esters or propyl ester, catechol, Resorcinol, hexalin and 3-amylalcohol.

Can add isotonic agent and guarantee the isotonicity of liquid composition, isotonic agent comprises polyhydric sugar-alcohol, and preferred three hydroxyls or more senior sugar alcohol are as glycerine, tetrahydroxybutane, arabitol, Xylitol, Sorbitol Powder and mannitol.The amount of polyhydroxy-alcohol can be the 0.1%-25% weight ratio, is generally 1%-5%, with the amount calculating of other component relatively.

Stablizer relates to a big class vehicle, its envelop of function from swelling agent to the dissolution treatment agent or help to prevent sex change or with the adherent additive of wall of container.Common stablizer can be polyhydric sugar-alcohol (above enumerating); Amino acid such as arginine, Methionin, glycine, glutamine, l-asparagine, Histidine, L-Ala, omithine, L-leucine, 2-phenylalanine, L-glutamic acid, Threonine etc., organic sugar or sugar alcohol, as lactose, trehalose, stachyose, mannitol, Sorbitol Powder, Xylitol, ribitol, inositol, melampyrum, glycerine etc., comprise cyclic alcohol such as nucite; Polyoxyethylene glycol; Aminoacid polymers; The sulfur-bearing reductive agent, as urea, gsh, Thioctic Acid, Thioglycolic acid sodium salt, thioglycerin, α-single thioglycerin and Sulfothiorine; Low molecular weight polypeptide (residue promptly＜10); Protein such as human serum albumin, bovine serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer such as polyvinylpyrrolidone; Monose such as wood sugar, seminose, fructose and glucose; Disaccharides such as lactose, maltose and sucrose; Trisaccharide such as raffinose and polysaccharide such as dextran.Calculate based on active protein weight, the scope that exists that stablizer is common is the 0.1-10000 weight part.

Can exist nonionogenic tenside or stain remover (being also referred to as " wetting agent ") to assemble to avoid stirring inductive to help dissolution treatment agent and protection therapeutical peptide, it also allows preparaton to be exposed to shear surface pressure and does not cause the polypeptide sex change.Suitable ionic surfactant pack is drawn together polysorbate (polysorbate) (20,80 etc.), polyoxamer (184,188 etc.), Pluronic  polyvalent alcohol, polyoxyethylene sorbitan monoether (tween  20, tween  80 etc.).

Other various vehicle comprise swelling agent or weighting agent (as starch), sequestrant (as EDTA), antioxidant (as xitix, methionine(Met), vitamin-E) and cosolvent.

Active ingredient also can be wrapped in by, coascervation technology or for example by the prepared micro-capsule of interfacial polymerization, in Walocel MT 20.000PV, gelatin or poly--(methyl methacrylate) micro-capsule, be wrapped in the colloidal medicine and transport in the system (for example liposome, albumin microsphere, micro emulsion, nano particle and Nano capsule), or be wrapped in the big emulsion.These technology are disclosed in Remington ' s Pharmaceutical Sciences (the same).

The employed non-gastrointestinal preparation of vivo medicine-feeding must be aseptic.This process can be passed through, for example, and the filtration of degerming filter membrane and finishing easily.

Extended release preparation

The suitable example of extended release preparation comprise the solid hydrophobic polymkeric substance that contains polypeptide or conjugate semi-permeable matrix, have the matrix of suitable form such as film or micro-capsule.The example that continues release matrix comprises polyester, hydrogel (for example, poly-(2-hydroxyethyl methyl acrylate) or poly-(vinyl alcohol)), polylactide, L-L-glutamic acid and the multipolymer of L-ethyl glutamate, nondegradable ethylene-ethyl acetate, degradable lactic acid-ethanol copolymer such as ProLease  technology or Lupron Depot  (Injectable microspheres that is made of lactic acid-ethanol copolymer and leuprorelin acetate) and poly--D-(-)-3-hydroxybutyric acid.Polymkeric substance such as ethylene-ethyl acetate and lactic acid-ethanol can discharge molecule for a long time as reaching or surpass 100 days, and some hydrogels discharges protein within a short period of time.When the polypeptide in the packing kept in vivo for a long time, they caused loss of bioactivity and may change immunogenicity because of expose sex change or gathering in the environment of 37 ℃ of humidities.Reasonably stable strategy can be according to related mechanism design.For example, if find to assemble mechanism is to exchange mutually by sulfo-disulphide to form intramolecularly S-S key, so can be by modifying sulfydryl, acidic solution freeze-drying, controlling moisture, suitable additive and the exploitation specificity polymer matrix composition of use being reached stable.

Description of drawings

Fig. 1 has shown wild-type people APC and the various conjugate of the present invention and the variant of purifying.These albumen move on gel, have three significantly band be respectively 41,000 and 37,000 heavy chain α-and beta chain corresponding to apparent molecular weight, and apparent molecular weight is 22,000 light chain.Can analyze degree of glycosylation from gel shown in Figure 1: the more close negative electrode of the migration position of the heavy chain of conjugate D214N and M338N is (opposite with variant K251N and S252N, it does not obviously utilize the glycosylation site of introducing), this shows these two kinds of variants all by glycosylation, and all is fully used in the site.Inspection to the migration of heavy chain hypotype (α and β) shows that the molecular weight of the sugared side chain in each site is about 3,000-4,000.

Fig. 2 show the various conjugates of the present invention and variant with the α-1-antitrypsin (16.6 μ M (secret note) and 42.3 μ M (informal voucher)) of different concns 37 ℃ of insulations after 20 hours, remaining acid amides lytic activity.Detailed description is referring to this paper embodiment 11.

Fig. 3 and 4 shows in the human plasma, the function of time of the remaining acid amides lytic activity of various conjugates of the present invention and variant.Details comprises half life in the body in the human plasma that calculates, referring to this paper embodiment 13.

The present invention is further illustrated by following non-limiting example.

Method

Come-at-able surf zone (ASA)

Calculate the come-at-able surf zone (ASA) of each atom in the described structure with computer program Access (B.Lee and F.M.Richards, J.Mol.Biol.55:379-400 (1971)) version 2 ( 1983 Yale University).This method is generally used the probe size of 1.4 and come-at-able surf zone (ASA) is defined as by the zone that is formed centrally in the probe.Before the calculating, from system of coordinates (coordinate set), remove all water moleculess and hydrogen atom, also remove and not directly related other atom of this protein.

The ASA of the branch of side chain (fractional)

On behalf of the ASA value of the side chain atom of this residue type, the ASA of branch of side chain atom by calculate in the ALA-x-ALA tripeptides of ASA summation divided by extension with atom in the side chain.Referring to Hubbard, Campbell ﹠amp; Thornton (1991) J.Mol.Biol.:220,507-530.In this example, the CA atom is considered as glycine residue but not the part of the side chain of other residue.Following train value is used as the standard 100%ASA of side chain:

Ala????69.23?????????????????????Leu??????140.76?????

Arg????200.35????? ²?????????????Lys??????162.50????? ²

Asn????106.25????? ²?????????????Met??????156.08????? ²

Asp????102.06????? ²?????????????Phe??????163.90????? ²

Cys????96.69?????? ²?????????????Pro??????119.65????? ²

Gln????140.58????? ²?????????????Ser??????78.16?????? ²

Glu????134.61????? ²?????????????Thr??????101.67????? ²

Gly????32.28?????? ²?????????????Trp??????210.89????? ²

His????147.00????? ²?????????????Tyr??????176.61????? ²

Ile????137.91????? ²?????????????Val??????114.14????? ²

The residue that does not detect in this structure is defined as 100% and exposes, because can think that these residues are positioned at Hookean region.

Determine the distance between the atom

Distance between the atom use the molecule graphics software, for example InsightIIO v.98.0, MSI Inc. is definite.

Embodiment

Embodiment 1-determines to be exposed to the amino acid on surface

Coordinate (Mather, T., Oganessyan, V., the Hof of the X-line structure of wild-type people APC, P., Huber, R., Foundling, S., Esmon, C., Bode, W., 1996) can be from albumen database (PDB) (Bernstein et.al.J.Mol.Biol. (1977) 112 pp.535) obtaining, can be that the address of 1AUT obtains its electronic version at the login password of http://www.pdb.org/ from The ResearchCollaboratory for Structural Bioinformatics PDB also.Before calculating come-at-able surface-area, from this structure, remove all water moleculess and covalently bound inhibitor.In the present embodiment, β hydroxyl-ASP (AP) of the 71st handles as conventional ASP residue.Residue K156-R169 (Lys-Arg dipeptides and bioactive peptide) is not included in the calculating.

Sequence numbering

The sequence numbering that uses in the present embodiment is equal to the sequence numbering with zymogen protein C shown in the SEQ ID NO:4.

The surface exposes

The APC molecule is carried out the ASA of branch calculate, draw the following residue that accessibility is 0 side chain that has: G67, C89, C98, G103, C105, H107, C109, Y124, G142, G173, V186, L187, A198, V199, I201, V206, L207, T208, A210, C212, V221, E235, I258, A259, L260, L261, L263, A267, V274, I276, L283, V297, C331, M335, A346, G361, M364, T371, F373, L374, G376, L377, V392, I403.

Following residue has the side chain more than 25% to be exposed to the surface: Q49, L51, V52, P54, L55, E56, H57, P58, C59, A60, S61, G65, H66, T68, I70, D71, G72, I73, G74, S75, F76, S77, D79, R81, S82, G83, W84, E85, R87, F88, Q90, R91, E92, F95, L96, N97, S99, L100, D101, L110, E111, E112, V113, G114, W115, R117, S119, P122, G123, K125, G127, D128, D129, L130, L131, Q132, H134, P135, A136, V137, K138, R143, W145, K146, D172, K174, M175, R177, R178, D180, D189, S190, K191, K192, K193, H202, P203, H211, D214, E215, S216, K217, K218, L220, R229, R230, W231, K233, W234, L236, D237, D239, K241, E242, V243, F244, V245, P247, N248, S250, K251, S252, T253, T254, D255, A264, Q265, P266, T268, S270, Q271, D280, S281, G282, E285, R286, E287, Q290, A291, G292, Q293, E294, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, K311, R312, N313, R314, T315, F316, F320, K322, P327, H328, N329, E330, S332, E333, V334, S336, N337, M338, S340, E341, I348, L349, G350, D351, R352, E357, S367, H369, G370, E382, G383, C384, L386, L387, H388, R398, D401, H404, G405, H406, R408, D409.Can find out that avtive spot Histidine (H211) is exposed to the surface.But H211 is not the candidate that the present invention will be modified.And the residue that above listed cysteine residues usually neither the present invention will be modified.

Following residue has the side chain more than 50% to be exposed to the surface: Q49, L51, V52, P54, L55, E56, A60, S61, G65, I70, D71, G72, I73, G74, S75, S77, D79, R81, S82, R87, R91, E92, F95, L96, N97, S99, Bill, V113, G114, W115, R117, P122, K125, D128, D129, L130, Q132, H134, V137, K138, K146, D172, K174, R177, S190, K191, K192, K193, D214, E215, K217, K218, R229, R230, W231, K233, D239, K241, E242, P247, N248, K251, S252, Q265, P266, T268, Q271, S281, E285, Q290, G292, Y302, S305, R306, E307, K308, E309, A310, R312, N313, T315, K322, N329, E330, E333, S336, N337, M338, E341, I348, G350, R352, E357, G370, G383, H388, R398, D401, H404, G405, R408, D409.

Residue A 1, N2, S3, F4, L5, E6, E7, L8, R9, H10, S11, S12, L13, E14, R15, E16, C17, I18, E19, E20, I21, C22, D23, F24, E25, E26, A27, K28, E29, I30, F31, Q32, N33, V34, D35, D36, T37, L38, A39, F40, W41, S42, K43, H44, V45, D46, G47, D48, R147, M148, E149, K150, K151, R152, S153, H154, L155, K410, E411, A412, P413, Q414, K415, S416, W417, A418, P419 are not included in the described structure, and, in this application, be regarded as 100% and be exposed to surperficial type.

Embodiment 2-avtive spot district determines

Use following method to determine avtive spot district: service routine Modeller ' 98, APC reconnected (1AUT) overlapping (superimposing) on the X-line structure of ternary (ternary) mixture, described mixture is by the proconvertin a that is incorporated in the avtive spot, (PDB accession number 1FAK. is referring to Zhang for tissue factor and a kind of BPTI variant composition, E., St Charles, R., Tulinsky, A.:Structure ofExtracellular Tissue Factor Complexed with Factor Viia Inhibited with a BptiMutant J.Mol.Biol.285 pp.2089 (1999)), thereby " avtive spot district " can be defined as during APC reconnects, have any residue that is no more than the atom of 12 with institute eclipsed BPTI molecule apart.In addition, observe and find, be positioned at the integral part that this district ring (residue 306-314) in addition also is considered to the avtive spot district.

Find that in this way following residue is included in " avtive spot district ":

L170, I171, D172, G173, Q184, V185, V186, L187, L188, D189, S190, K191, K192, K193, L194, A195, C196, G197, A198, T208, A209, A210, H211, C212, M213, D214, E215, S216, K217, K218, L219, L220, L228, I240, V243, V245, N248, Y249, S250, K251, S252, T253, T254, D255, N256, D257, I258, A259, L261, T295, L296, V297, T298, G299, W300, G301, Y302, H303, S304, S305, R306, E307, K308, E309, A310, K311, R312, N313, R314, T315, F316, I321, I323, P324, V326, C331, V334, M335, S336, N337, M338, V339, M343, L344, C345, A346, G347, I348, L349, D351, R352, Q353, D354, A355, C356, E357, G358, D359, S360, G361, G362, P363, M364, G376, L377, V378, S379, W380, G381, E382, G383, C384, G385, L386, L387, H388, N389, Y390, G391, V392, Y393 and T394.Avtive spot residue (H211, D257 and S360) is although also be listed in wherein, and they are not the candidates that the present invention will modify.And the candidate that above-mentioned listed cysteine residues usually neither the present invention will be modified.

Embodiment 3-determines the surface exposure type amino acid in the avtive spot district

To have the amino acid whose tabulation (seeing embodiment 1) that 25% above side chain is exposed to the surface and make up in avtive spot the district in, find that following amino-acid residue not only had been positioned at the avtive spot district, but also has had at least 25% side chain to be exposed to the surface with the amino acid whose tabulation (seeing embodiment 2) that is included in:

D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, H211, L220, V243, V245, N248, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, K311, R312, N313, R314, T315, F316, V334, S336, N337, M338, I348, L349, D351, R352, E357, E382, G383, C384, L386, L387 and H388.Avtive spot Histidine (H211) although also this row in, it is not the candidate that the present invention will modify.The candidate that C384 usually neither the present invention will modify.

Embodiment 4-makes up the PROTEIN C expression vector

The gene of coding human protein C precursor is by making up with PCR assembling synthesis type oligonucleotide, and method therefor is similar to Stemmer etc., (1995) Gene 164, and pp.49-53 is described.The signal sequence of natural (native) PROTEIN C is retained so that the secretion of gene product.Synthetic gene has the NheI site through design at 5 ' end, at 3 ' end the XbaI site is arranged, with these sites with its subclone to the middle CMV promotor of pcDNA3.1/Hygro (Invitrogen).Gained plasmid called after pCR4, PROTEIN C precursor sequence is wherein seen SEQ ID NO:1.

In addition,, synthetic gene is cloned in the KpnI-XbaI site of pcDNA3.1/Hygro, makes in the 5 ' non-translational region of this gene to comprise an intron (from pCI-Neo (Promega)) in order to check the genetic expression of higher level.With gained plasmid called after pRC2.

Embodiment 5-site-directed mutagenesis

Make up all mutant of PROTEIN C with Quick-Change (Stratagene).Primer is available from TAGTechnology (Copenhagen), and they contain suitable sudden change.Carry out the PCR reaction according to producer's handbook, plasmid is converted in the TG1 competent cell.Preparation plasmid preparation on single clone is with dna sequencing instrument 3100 genetic Ahalyser (ABI) checking sequences.

Primer:

D172N

POF003：

CAAGTAGATCCGCGGCTCATTAACGGGAAGATGACCAGGCGGGG

POF004：

CCCCGCCTGGTCATCTTCCCGTTAATGAGCCGCGGATCTACTTG

D214N

EKO001：

CTGACAGCGGCCCACTGCATGAACGAGTCCAAGAAGCTCCTTGTC

EKO002：

GACAAGGAGCTTCTTGGACTCGTTCATGCAGTGGGCCGCTGTCAG

D214A

EK0048：

CTGACAGCGGCCCACTGCATGGCCGAGTCCAAGAAGCTCCTTGTC

EK0049：

GACAAGGAGCTTCTTGGACTCGGCCATGCAGTGGGCCGCTGTCAG

K251N

EK0003：

CTTCGTCCACCCCAACTACAGCAACAGCACCACCGACAATGACATC

EK0004：

GATGTCATTGTCGGTGGTGCTGTTGCTGTAGTTGGGGTGGACGAAG

S252N

EKO005：

CGTCCACCCCAACTACAGCAAGAACACCACCGACAATGACATCGC

EK0006：

GCGATGTCATTGTCGGTGGTGTTCTTGCTGTAGTTGGGGTGGACG

Y302N

EK0007：

CCCTCGTGACGGGCTGGGGCAACCACAGCAGCCGAGAGAAGGAGGCC

EK0008：

GGCCTCCTTCTCTCGGCTGCTGTGGTTGCCCCAGCCCGTCACGAGGG

M338N

EK0011：

CAGCGAGGTCATGAGCAACAACGTGTCTGAGAACATGC

EK0012：

GCATGTTCTCAGACACGTTGTTGCTCATGACCTCGCTG

M338A

EK0046：

GCAGCGAGGTCATGAGCAACGCCGTGTCTGAGAACATGC

EK0047：

GCATGTTCTCAGACACGGCGTTGCTCATGACCTCGCTGC

D189N+K191N

EK0019：

CCCCTGGCAGGTGGTCCTGCTGAACTCAAACAAGAAGCTGGCCTGCG

GGG

EK0020：

CCCCGCAGGCCAGCTTCTTGTTTGAGTTCAGCAGGACCACCTGCCAGG

GG

D189N+K191T

EK0033：

CCCCTGGCAGGTGGTCCTGCTGAACTCAACCAAGAAGCTGGCCTGCG

GGG

EK0034：

CCCCGCAGGCCAGCTTCTTGGTTGAGTTCAGCAGGACCACCTGCC

S190N+K192T

EK0044：

GGCAGGTGGTCCTGCTGGACAACAAGACCAAGCTGGCCTGCGGGGCA

GTGC

EK0045：

GCACTGCCCCGCAGGCCAGCTTGGTCTTGTTGTCCAGCAGGACCACCT

GCC

K191N+K193T

EKO050：

GTCCTGCTGGACTCAAACAAGACCCTGGCCTGCGGGGCAGTG

EK0051：

CACTGCCCCGCAGGCCAGGGTCTTGTTTGAGTCCAGCAGGAC

K217N+L219T

EK0029：

GCATGGATGAGTCCAACAAGACCCTTGTCAGGCTTGGAGAGTATGACC

EK0030：

GGTCATACTCTCCAAGCCTGACAAGGGTCTTGTTGGACTCATCCATGC

T253N+D255T

EK0031：

CCAACTACAGCAAGAGCAACACCACCAATGACATCGCACTGCTGCACC

TGGC

EK0032：

GCCAGGTGCAGCAGTGCGATGTCATTGGTGGTGTTGCTCTTGCTGTAGT

TGG

S305N+E307T

EK0023：

GGCTGGGGCTACCACAGCAACCGAACCAAGGAGGCCAAGAGAAACC

GC

EK0024：

GCGGTTTCTCTTGGCCTCCTTGGTTCGGTTGCTGTGGTAGCCCCAGCC

E307N+E309T

EK0025：

GGCTACCACAGCAGCCGAAACAAGACCGCCAAGAGAAACCGCACCTT

CG

EK0026：

CGAAGGTGCGGTTTCTCTTGGCGGTCTTGTTTCGGCTGCTGTGGTAGCC

S336N+M338T

EK0027：

GCAGCGAGGTCATGAACAACACCGTGTCTGAGAACATGCTGTGTGCGG

G

EK0028：

CCCGCACACAGCATGTTCTCAGACACGGTGTTGTTCATGACCTCGCTG

C

L386N+H388T

EK0017：

GGTGAGCTGGGGTGAGGGCTGTGGGAACCTTACCAACTACGGCGTTTA

CACC

EK0018：

GGTGTAAACGCCGTAGTTGGTAAGGTTCCCACAGCCCTCACCCCAGCT

CACC

Embodiment 6-preparation

The transient expression of wild-type protein C and PROTEIN C variant carries out in the COS7 cell with Fugene transfection reagent (Roche), described cell cultures is being added 10% foetal calf serum, the 2mM L-glutaminate, 100U/ml penicillin is among the DMEM (Gibco21969035) of 100 μ g/ml Streptomycin sulphates and 5 μ g/ml vitamin Ks.Transfection same day, substratum is replaced by fresh culture, carry out transfection after 4-5 hour.The next day of transfection, the serum-free that substratum is replaced by based on DMEM (Gibco 31053-028) prepares substratum, wherein be added with the 2mM L-glutaminate, the 1mM Sodium.alpha.-ketopropionate, 1/500 Ex-cyte (serologicals), 1/100 ITSA (Gibco 51300-044), 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates and 5 μ g/ml vitamin Ks.Be incubated after 2 days, gather in the crops substratum, analyze the generation and the activity (seeing following examples 9) of expressed variant.

Embodiment 7-purifying

According to specification sheets, about 15mg Ca monoclonal antibody specific is coupled on the 5ml CNBr-activatory Sepharose FF (Pharmacia).Will about 1ml link coupled matrix be filled in HR 10 posts, with buffer A (20mM Tris, 0.3M NaCl, 5mM CaCl ₂, pH7.5) wash post with the flow velocity of 1ml/min.Make about 90ml the substratum of filtration sterilization contain 0.3M NaCl and 5mM CaCl ₂, load in the post with identical flow velocity then.Before the wash-out, wash post with the buffer A of 20 times of column volumes.(20mM Tris and 10mM EDTA pH7.5) carry out wash-out, carry out fractional separation according to the fraction of 1ml with buffer B.Pass through OD ₂₈₀, western trace and SDS-PAGE determine to contain the fraction of PROTEIN C, they are collected in together and in-80 ℃ of storages.Above-mentioned purification process be one of numerous methods that may be used for purifying protein C (referring to, Kiesel for example, J.Clin.Invest. (1979) 64 pp.761-769).

Purified albumen activates (seeing following examples 8) with the activation scheme.All proteic purity are checked by polyacrylamide gel electrophoresis (PAGE).In addition, degree of glycosylation is assessed from these gel analysis things, and method is the monitoring change of molecular weight.Have the apparent molecular weight of increase with respect to wild-type people APC, the glycosylation of APC variant just is described.

The visible Fig. 1 of the example of wild-type APC and APC variant.Albumen moves on gel, have three significantly band be respectively 41,000 and 37,000 heavy chain α-and beta chain corresponding to: apparent molecular weight, and apparent molecular weight is 22,000 light chain.Degree of glycosylation also can be studied in this PAGE analyzes.Fig. 1 comprises two kinds of APC variants, the glycosylation site generation glycosylation that they are being introduced.The more close negative electrode of the migration position of the heavy chain of APC modification D 214N and M338N, this shows these two kinds of variants all by glycosylation, and all is fully used in the site.Inspection to the migration of heavy chain hypotype (α and β) shows that the molecular weight of the sugared side chain in each site is about 3,000-4,000.

Embodiment 8-activation

PROTEIN C variant and conjugate activate (Nakagaki etc., Thrombosis Research 58:593-602,1990) with venom PROTEIN C activator ACC-C.With the 50mMTris-HCl (pH7.5) of zymogen forms at 37 ℃, 100mM NaCl is the about 60min of ACC-C insulation of 1ng/ml with final concentration among the 5mM EDTA.Reactivation process is checked with APC acid amides lytic activity test (seeing following examples 9) and polyacrylamide gel electrophoresis.

Embodiment 9-measures the acid amides lytic activity

APC acid amides breaking test

The acid amides lytic activity of people APC and The compounds of this invention is measured with peptide substrates SPECTROZYMEPCa, described peptide substrates has general formula H-D-Lys (γ-Cbo)-Pro-Arg-pNA.2AcOH (American Diagnostica Inc, production code member 336), final concentration is 0.5mM.Test is at 23 ℃ 50mM Tris-HCl (pH8.3), 100mM NaCl, 5mM CaCl ₂In carry out.The PCa substrate is write down 3min in 405nm by the speed of people APC and The compounds of this invention hydrolysis in reading plate instrument (plate reader), the record form is the change of absorbance unit/min.

The result

Analyze activity expressed and all conjugates of activatory and variant.As mentioned above 4 μ l (unpurified) cell cultures are analyzed.The activity that is obtained depends on expression level at least, thereby does not reflect specific activity, but but whether its indicator protein expresses and whether they have activity.

Obtained following activity:

Table 1

Compound milliOD ₄₀₅/ min

Wild-type COS 7 APC 41

D214N??????????????????????????????28

D214A (contrast) 10

K251N ^*????????????????????????????19

S252N ^*????????????????????????????16

Y302N ^*????????????????????????????14

M338N??????????????????????????????53

M338A (contrast) 33

D189N+K191T????????????????????????8

D189N+K191N (contrast) 12

S190N+K192T ^*??????????????????????29

K191N+K193T????????????????????????4

K217+L219T?????????????????????????16

T253N+D255T????????????????????????2

S305N+E307T????????????????????????6

E307N+E309T????????????????????????30

S336N+M338T????????????????????????4

L386N+H388T????????????????????????13

^*: fail to detect the sugar component that is connected with the glycosylation site of introducing by SDS-PAGE

With selected candidate purifying, the albumen that with concentration is 30nM is through their specificity acid amides lytic activity of above-mentioned test determination.Find following active:

Table 1b

Compound milliOD ₄₀₅/ min % wild-type APC

Wild-type COS 7 APC 48.9-

D214N????????????????????34.8??????????????71

K251N ^*??????????????????45.2??????????????92

S252N ^*??????????????????43.1??????????????88

M338N????????????????????44.8??????????????92

S336N+M338T??????????????41.5??????????????85

L386N+H388T??????????????23.0??????????????47

^*: fail to detect the sugar component that is connected with the glycosylation site of introducing by SDS-PAGE

As can be seen, the specificity acid amides lytic activity of conjugate of being tested and variant and wild-type people APC molecule is in par.

Embodiment 10-measures anticoagulating active

The APC thrombotest

Anticoagulating active is assessed by the prolongation in monitoring clotting time, described monitoring is with Nycoplastin (Nycomed in activatory partial thromboplastin time (APTT) test, production code member 1002448) and conventional hemostasis with reference to blood plasma (Normal Hemostasis Reference Plasma, American Diagnostica Inc., catalog number (Cat.No.) 258N) carry out.Blood coagulation is incubated at 37 ℃ with reference to blood plasma with the routine hemostasis by the APTT reagent that will contain APC or The compounds of this invention and starts, and measures the clotting time by hand mix.The clotting time of people APC and the clotting time of The compounds of this invention are compared, so that calculate anticoagulating active, and expression is the per-cent of people APC anticoagulating active.

The result

Use above-mentioned test to find following anticoagulating active:

Table 2

Compound anticoagulating active (% people APC)

D214N???????????????????????22.4

K251N ^*?????????????????????24.5

S252N ^*?????????????????????24.5

M338N???????????????????????34.7

L386N+H388T?????????????????14.3

^*: fail to detect the sugar component that is connected with the glycosylation site of introducing by SDS-PAGE

These results show that the anticoagulating active of conjugate of the present invention and variant is kept to a great extent.This clearly illustrates that, might design have remarkable increase at the inhibiting resistance of blood plasma (seeing following examples) and keep the APC variant and the conjugate of anticoagulating active.

The antitryptic deactivation of embodiment 11-α-l-

α-l-antitrypsin inactivation test

People APC or The compounds of this invention and 16.6 or 42.3 μ M people α-1-antitrypsin (Sigma) are at 10mM Tris-HCl (pH7.5), 150mM NaCl, 5mM CaCl ₂37 ℃ of insulations in (containing 0.1%BSA).Be incubated after 20 hours, from the mixture of insulation, get the 50mM Tris-HCl (pH8.3) that 15 μ l samples are added into 110 μ l in the droplet plate, 100mM NaCl, 5mM CaCl ₂In, described in " APC acid amides breaking test ", analyze APC acid amides lytic activity.Calculate residual activity, carry out stdn with reference to the activity that sample obtained that lacks α-1-antitrypsin but still be incubated under the same conditions.

The result

Obtain following result with above-mentioned test:

Table 3

% remnants' acid amides lytic activity

Compound

16.6 μ M inhibitors 4 2.3 μ M inhibitor

Wild-type plasma A PC 10 2

Wild-type COS 7 APC 7＜1

D214N?????????????????????????80??????????????81

D214A (contrast) 21 1

K251N ^*???????????????????????62??????????????53

S252N ^*???????????????????????62??????????????34

Y302N ^*???????????????????????50??????????????30

M338N?????????????????????????38??????????????12

M338A (contrast) 92

D189N+K191T???????????????????90??????????????77

D189N+K191N (contrast) 12＜1

S190N+K192T ^*?????????????????28??????????????5

K191N+K193T???????????????????59??????????????24

K217+L219T????????????????????20??????????????4

T253N+D255T???????????????????56??????????????38

S305N+E307T???????????????????42??????????????9

E307N+E309T???????????????????10??????????????＜1

S336N+M338T???????????????????72??????????????40

L386N+H388T???????????????????68??????????????44

^*: fail to detect the sugar component that is connected with the glycosylation site of introducing by SDS-PAGE

Above data also are shown in Fig. 2.The result shows that in fact all conjugates strengthen the antitryptic inhibiting resistance of α-1-.Especially, even D214N and D189N+K191T have also kept the acid amides lytic activity more than 70% in the highest α-1-antitrypsin concentration.The glycosylated effect of these compounds is shown when these two kinds of conjugates are compared with glycosylated D214A of shortage and D189N+K191N.These variants are subjected to more significant inhibition than their glycosylation Equivalent, illustrate that glycosylation is very important at α-inhibiting resistance of 1-antitrypsin for strengthening.And it should be noted that, variant K251N, S252N, (they obviously do not utilize the glycosylation site of their introducing for Y302N and S190+K192T, judge as SDS PAGE) compare with wild-type people APC, significantly strengthened them at α-inhibiting resistance of 1-antitrypsin.

The deactivation of embodiment 12-human plasma

Human plasma inactivation test I

People APC or compound of the present invention are containing 50mM Tris-HCl (pH7.5), 100mM NaCl, 5mM CaCl ₂90% human normal plasma (Sigma Diagnostics, Accuclot ^TMWith reference to blood plasma) in 37 ℃ of insulations.Behind the 200min, get sample aliquot, as analysis APC acid amides lytic activity as described in " APC acid amides breaking test ".Remaining APC activity behind the 200min is expressed as the active per-cent of APC when beginning to test.

The result

Use above-mentioned test to obtain following result:

Table 4

In 90% human normal plasma behind the 200min

Compound

% remnants' acid amides lytic activity

Wild-type plasma A PC 5

Wild-type COS 7 APC 7

D214N?????????????????????????????80

K251N ^*???????????????????????????57

S252N ^*???????????????????????????45

M338N?????????????????????????????22

S336N+M338T???????????????????????45

L386N+H388T???????????????????????72

^*: fail to detect the sugar component that is connected with the glycosylation site of introducing by SDS-PAGE

Above result clearlys show that conjugate of the present invention and variant have higher resistance to the deactivation of human plasma.

External half life in the embodiment 13-human plasma

Human plasma inactivation test II

People APC or compound of the present invention are containing 50mM Tris-HCl (pH7.5), 100mM NaCl, 5mM CaCl ₂90% human normal plasma (Sigma Diagnostics, Accuclot ^TMWith reference to blood plasma) in 37 ℃ of insulations.Get sample aliquot in different time points, as analysis APC acid amides lytic activity as described in " APC acid amides breaking test ".The remaining APC activity of different time points is expressed as the active per-cent of APC when beginning to test.Calculate external half life (in minute), be expressed as and still keep the active time of 50%APC.

The result

Obtain following external half life:

Table 5

Compound external half life, is with respect to wild-type people APC

(min) multiple increases

Wild-type plasma A PC 40-

Wild-type COS 7 APC 42-

D214N????????????????????＞400??????????????＞10

K251N ^*??????????????????255????????????????6.4

S252N ^*??????????????????155????????????????3.9

M338N????????????????????85?????????????????2.1

S336N+M338T??????????????185????????????????4.6

L386N+H388T??????????????＞400??????????????＞10

^*: fail to detect the sugar component that is connected with the glycosylation site of introducing by SDS-PAGE

Above experimental data also is found in Fig. 3 and 4.These results show that APC variant and conjugate have the external half life of remarkable increase in human plasma.Especially D214N and L386N+H388T conjugate demonstrate the external half life (increasing more than 10 times) of remarkable increase.

Sequence table

＜110〉Maxygen Inc. (Maxygen Aps); Maxigen Holdings Co., Ltd (Maxygen Holding)

＜120〉PROTEIN C or activatory PROTEIN C-sample molecule

<130>0219wo310-protein?C

<140>

<141>

<160>40

<170>Patent?In?Ver.2.1

<210>1

<211>1383

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(1383)

<220>

<221>mat_peptide

<222>(127)..(1383)

<400>1

atg?tgg?cag?ctc?aca?agc?ctc?ctg?ctg?ttc?gtg?gcc?acc?tgg?gga?att???48

Met?Trp?Gln?Leu?Thr?Ser?Leu?Leu?Leu?Phe?Val?Ala?Thr?Trp?Gly?Ile

-40?????????????????-35?????????????????-30

tcc?ggc?aca?cca?gct?cct?ctt?gac?tca?gtg?ttc?tcc?agc?agc?gag?cgt???96

Ser?Gly?Thr?Pro?Ala?Pro?Leu?Asp?Ser?Val?Phe?Ser?Ser?Ser?Glu?Arg

-25?????????????????-20?????????????????-15

gcc?cac?cag?gtg?ctg?cgg?atc?cgc?aaa?cgt?gcc?aac?tcc?ttc?ctg?gag??144

Ala?His?Gln?Val?Leu?Arg?Ile?Arg?Lys?Arg?Ala?Asn?Ser?Phe?Leu?Glu

-10?????????????????-5??????????????-1????1???????????????5

gag?ctc?cgt?cac?agc?agc?ctg?gag?cgg?gag?tgc?ata?gag?gag?atc?tgt??192

Glu?Leu?Arg?His?Ser?Ser?Leu?Glu?Arg?Glu?Cys?Ile?Glu?Glu?Ile?Cys

10??????????????????15??????????????????20

gac?ttc?gag?gag?gcc?aag?gaa?att?ttc?caa?aat?gtg?gat?gac?aca?ctg??240

Asp?Phe?Glu?Glu?Ala?Lys?Glu?Ile?Phe?Gln?Asn?Val?Asp?Asp?Thr?Leu

25??????????????????30??????????????????35

gcc?ttc?tgg?tcc?aag?cac?gtc?gac?ggt?gac?cag?tgc?ttg?gtc?ttg?ccc??288

Ala?Phe?Trp?Ser?Lys?His?Val?Asp?Gly?Asp?Gln?Cys?Leu?Val?Leu?Pro

40??????????????????45??????????????????50

ttg?gag?cac?ccg?tgc?gcc?agc?ctg?tgc?tgc?ggg?cac?ggc?acg?tgc?atc??336

Leu?Glu?His?Pro?Cys?Ala?Ser?Leu?Cys?Cys?Gly?His?Gly?Thr?Cys?Ile

55??????????????????60??????????????????65??????????????????70

gac?ggc?atc?ggc?agc?ttc?agc?tgc?gac?tgc?cgc?agc?ggc?tgg?gag?ggc??384

Asp?Gly?Ile?Gly?Ser?Phe?Ser?Cys?Asp?Cys?Arg?Ser?Gly?Trp?Glu?Gly

75??????????????????80??????????????????85

cgc?ttc?tgc?cag?cgc?gag?gtg?agc?ttc?ctc?aat?tgc?tcg?ctg?gac?aac??432

Arg?Phe?Cys?Gln?Arg?Glu?Val?Ser?Phe?Leu?Asn?Cys?Ser?Leu?Asp?Asn

90??????????????????95?????????????????100

ggc?ggc?tgc?acg?cat?tac?tgc?cta?gag?gag?gtg?ggc?tgg?cgg?cgc?tgt????480

Gly?Gly?Cys?Thr?His?Tyr?Cys?Leu?Glu?Glu?Val?Gly?Trp?Arg?Arg?Cys

105?????????????????110?????????????????115

agc?tgt?gcg?cct?ggc?tac?aag?ctg?ggg?gac?gac?ctc?ctg?cag?tgt?cac????528

Ser?Cys?Ala?Pro?Gly?Tyr?Lys?Leu?Gly?Asp?Asp?Leu?Leu?Gln?Cys?His

120?????????????????125?????????????????130

ccc?gca?gtg?aag?ttc?cct?tgt?ggg?agg?ccc?tgg?aag?cgg?atg?gag?aag????576

Pro?Ala?Val?Lys?Phe?Pro?Cys?Gly?Arg?Pro?Trp?Lys?Arg?Met?Glu?Lys

135?????????????????140?????????????????145?????????????????150

aag?cgc?agt?cac?ctg?aaa?cga?gac?aca?gaa?gac?caa?gaa?gac?caa?gta????624

Lys?Arg?Ser?His?Leu?Lys?Arg?Asp?Thr?Glu?Asp?Gln?Glu?Asp?Gln?Val

155?????????????????160?????????????????165

gat?ccg?cgg?ctc?att?gat?ggg?aag?atg?acc?agg?cgg?gga?gac?agc?ccc????672

Asp?Pro?Arg?Leu?Ile?Asp?Gly?Lys?Met?Thr?Arg?Arg?Gly?Asp?Ser?Pro

170?????????????????175?????????????????180

tgg?cag?gtg?gtc?ctg?ctg?gac?tca?aag?aag?aag?ctg?gcc?tgc?ggg?gca????720

Trp?Gln?Val?Val?Leu?Leu?Asp?Ser?Lys?Lys?Lys?Leu?Ala?Cys?Gly?Ala

185?????????????????190?????????????????195

gtg?ctc?atc?cac?ccc?tcc?tgg?gtg?ctg?aca?gcg?gcc?cac?tgc?atg?gat????768

Val?Leu?Ile?His?Pro?Ser?Trp?Val?Leu?Thr?Ala?Ala?His?Cys?Met?Asp

200?????????????????205??????????????????210

gag?tcc?aag?aag?ctc?ctt?gtc?agg?ctt?gga?gag?tat?gac?ctg?cgg?cgc????816

Glu?Ser?Lys?Lys?Leu?Leu?Val?Arg?Leu?Gly?Glu?Tyr?Asp?Leu?Arg?Arg

215?????????????????220?????????????????225?????????????????230

tgg?gag?aag?tgg?gag?ctg?gac?ctg?gac?atc?aag?gag?gtc?ttc?gtc?cac????864

Trp?Glu?Lys?Trp?Glu?Leu?Asp?Leu?Asp?Ile?Lys?Glu?Val?Phe?Val?His

235?????????????????240?????????????????245

ccc?aac?tac?agc?aag?agc?acc?acc?gac?aat?gac?atc?gca?ctg?ctg?cac????912

Pro?Asn?Tyr?Ser?Lys?Ser?Thr?Thr?Asp?Asn?Asp?Ile?Ala?Leu?Leu?His

250?????????????????255?????????????????260

ctg?gcc?cag?ccc?gcc?acc?ctc?tcg?cag?acc?ata?gtg?ccc?atc?tgc?ctc????960

Leu?Ala?Gln?Pro?Ala?Thr?Leu?Ser?Gln?Thr?Ile?Val?pro?Ile?Cys?Leu

265?????????????????270?????????????????275

ccg?gac?agc?ggc?ctt?gca?gag?cgc?gag?ctc?aat?cag?gcc?ggc?cag?gag????1008

Pro?Asp?Ser?Gly?Leu?Ala?Glu?Arg?Glu?Leu?Asn?Gln?Ala?Gly?Gln?Glu

280?????????????????285??????????????????290

acc?ctc?gtg?acg?ggc?tgg?ggc?tac?cac?agc?agc?cga?gag?aag?gag?gcc????1056

Thr?Leu?Val?Thr?Gly?Trp?Gly?Tyr?His?Ser?Ser?Arg?Glu?Lys?Glu?Ala

295????????????????300?????????????????305?????????????????310

aag?aga?aac?cgc?acc?ttc?gtc?ctc?aac?ttc?atc?aag?att?ccc?gtg?gtc????1104

Lys?Arg?Asn?Arg?Thr?Phe?Val?Leu?Asn?Phe?Ile?Lys?Ile?Pro?Val?Val

315?????????????????320?????????????????325

ccg?cac?aat?gag?tgc?agc?gag?gtc?atg?agc?aac?atg?gtg?tct?gag?aac????1152

Pro?His?Asn?Glu?Cys?Ser?Glu?Val?Met?Ser?Asn?Met?Val?Ser?Glu?Asn

330?????????????????335?????????????????340

atg?ctg?tgt?gcg?ggc?atc?ctc?ggg?gac?cgg?cag?gat?gcc?tgc?gag?ggc????1200

Met?Leu?Cys?Ala?G1y?Ile?Leu?Gly?Asp?Arg?Gln?Asp?Ala?Cys?Glu?Gly

345?????????????????350?????????????????355

gac?agt?ggg?ggg?ccc?atg?gtc?gcc?tcc?ttc?cac?ggc?acc?tgg?ttc?ctg????1248

Asp?Ser?Gly?Gly?Pro?Met?Val?Ala?Ser?Phe?His?Gly?Thr?Trp?Phe?Leu

360?????????????????365?????????????????370

gtg?ggc?ctg?gtg?agc?tgg?ggt?gag?ggc?tgt?ggg?ctc?ctt?cac?aac?tac????1296

Val?Gly?Leu?Val?Ser?Trp?Gly?Glu?Gly?Cys?Gly?Leu?Leu?His?Asn?Tyr

375?????????????????380?????????????????385?????????????????390

ggc?gtt?tac?acc?aaa?gtc?agc?cgc?tac?ctc?gac?tgg?atc?cat?ggg?cac????1344

Gly?Val?Tyr?Thr?Lys?Val?Ser?Arg?Tyr?Leu?Asp?Trp?Ile?His?Gly?His

395?????????????????400?????????????????405

atc?aga?gac?aag?gaa?gcc?ccc?cag?aag?agc?tgg?gca?cct????????????????1383

Ile?Arg?Asp?Lys?Glu?Ala?Pro?Gln?Lys?Ser?Trp?Ala?Pro

410?????????????????415

<210>2

<211>461

<212>PRT

＜213〉people (Homo sapiens)

<400>2

Met?Trp?Gln?Leu?Thr?Ser?Leu?Leu?Leu?Phe?Val?Ala?Thr?Trp?Gly?Ile

-40?????????????????-35?????????????????-30

Ser?Gly?Thr?Pro?Ala?Pro?Leu?Asp?Ser?Val?Phe?Ser?Ser?Ser?Glu?Arg

-25?????????????????-20?????????????????-15

Ala?His?Gln?Val?Leu?Arg?Ile?Arg?Lys?Arg?Ala?Asn?Ser?Phe?Leu?Glu

-10??????????????????-5??????????????-1???1???????????????5

Glu?Leu?Arg?His?Ser?Ser?Leu?Glu?Arg?Glu?Cys?Ile?Glu?Glu?Ile?Cys

10??????????????????15??????????????????20

Asp?Phe?Glu?Glu?A1a?Lys?Glu?Ile?Phe?Gln?Asn?Val?Asp?Asp?Thr?Leu

25??????????????????30??????????????????35

Ala?Phe?Trp?Ser?Lys?His?Val?Asp?Gly?Asp?Gln?Cys?Leu?Val?Leu?Pro

40??????????????????45??????????????????50

Leu?Glu?His?Pro?Cys?Ala?Ser?Leu?Cys?Cys?Gly?His?Gly?Thr?Cys?Ile

55??????????????????60??????????????????65??????????????????70

Asp?Gly?Ile?Gly?Ser?Phe?Ser?Cys?Asp?Cys?Arg?Ser?Gly?Trp?Glu?Gly

75??????????????????80??????????????????85

Arg?Phe?Cys?Gln?Arg?Glu?Val?Ser?Phe?Leu?Asn?Cys?Ser?Leu?Asp?Asn

90??????????????????95?????????????????100

Gly?Gly?Cys?Thr?His?Tyr?Cys?Leu?Glu?Glu?Val?Gly?Trp?Arg?Arg?Cys

105?????????????????110?????????????????115

Ser?Cys?Ala?Pro?Gly?Tyr?Lys?Leu?Gly?Asp?Asp?Leu?Leu?Gln?Cys?His

120?????????????????125?????????????????130

Pro?Ala?Val?Lys?Phe?Pro?Cys?Gly?Arg?Pro?Trp?Lys?Arg?Met?Glu?Lys

135?????????????????140?????????????????145?????????????????150

Lys?Arg?Ser?His?Leu?Lys?Arg?Asp?Thr?Glu?Asp?Gln?Glu?Asp?Gln?Val

155?????????????????160?????????????????165

Asp?Pro?Arg?Leu?Ile?Asp?Gly?Lys?Met?Thr?Arg?Arg?Gly?Asp?Ser?Pro

170?????????????????175?????????????????180

Trp?Gln?Val?Val?Leu?Leu?Asp?Ser?Lys?Lys?Lys?Leu?Ala?Cys?Gly?Ala

185?????????????????190?????????????????195

Val?Leu?Ile?His?Pro?Ser?Trp?Val?Leu?Thr?Ala?Ala?His?Cys?Met?Asp

200?????????????????205?????????????????210

Glu?Ser?Lys?Lys?Leu?Leu?Val?Arg?Leu?Gly?Glu?Tyr?Asp?Leu?Arg?Arg

215????????????????220?????????????????225?????????????????230

Trp?Glu?Lys?Trp?Glu?Leu?Asp?Leu?Asp?Ile?Lys?Glu?Val?Phe?Val?His

235?????????????????240?????????????????245

Pro?Asn?Tyr?Ser?Lys?Ser?Thr?Thr?Asp?Asn?Asp?Ile?Ala?Leu?Leu?His

250?????????????????255?????????????????260

Leu?Ala?Gln?Pro?Ala?Thr?Leu?Ser?Gln?Thr?Ile?Val?Pro?Ile?Cys?Leu

265?????????????????270?????????????????275

Pro?Asp?Ser?Gly?Leu?Ala?Glu?Arg?Glu?Leu?Asn?Gln?Ala?Gly?Gln?Glu

280?????????????????285?????????????????290

Thr?Leu?Val?Thr?Gly?Trp?Gly?Tyr?His?Ser?Ser?Arg?Glu?Lys?Glu?Ala

295?????????????????300?????????????????305?????????????????310

Lys?Arg?Asn?Arg?Thr?Phe?Val?Leu?Asn?Phe?Ile?Lys?Ile?Pro?Val?Val

315?????????????????320?????????????????325

Pro?His?Asn?Glu?Cys?Ser?Glu?Val?Met?Ser?Asn?Met?Val?Ser?Glu?Asn

330?????????????????335?????????????????340

Met?Leu?Cys?Ala?Gly?Ile?Leu?Gly?Asp?Arg?Gln?Asp?Ala?Cys?Glu?Gly

345?????????????????350?????????????????355

Asp?Ser?Gly?Gly?Pro?Met?Val?Ala?Ser?Phe?His?Gly?Thr?Trp?Phe?Leu

360?????????????????365?????????????????370

Val?Gly?Leu?Val?Ser?Trp?Gly?Glu?Gly?Cys?Gly?Leu?Leu?His?Asn?Tyr

375?????????????????380?????????????????385?????????????????390

Gly?Val?Tyr?Thr?Lys?Val?Ser?Arg?Tyr?Leu?Asp?Trp?Ile?His?Gly?His

395?????????????????400?????????????????405

Ile?Arg?Asp?Lys?Glu?Ala?Pro?Gln?Lys?Ser?Trp?Ala?Pro

410?????????????????415

<210>3

<211>1257

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(1257)

<400>3

gcc?aac?tcc?ttc?ctg?gag?gag?ctc?cgt?cac?agc?agc?ctg?gag?cgg?gag?????48

Ala?Asn?Ser?Phe?Leu?Glu?Glu?Leu?Arg?His?Ser?Ser?Leu?Glu?Arg?Glu

1???????????????5??????????????????10??????????????????15

tgc?ata?gag?gag?atc?tgt?gac?ttc?gag?gag?gcc?aag?gaa?att?ttc?caa?????96

Cys?Ile?Glu?Glu?Ile?Cys?Asp?Phe?Glu?Glu?Ala?Lys?Glu?Ile?Phe?Gln

20??????????????????25??????????????????30

aat?gtg?gat?gac?aca?ctg?gcc?ttc?tgg?tcc?aag?cac?gtc?gac?ggt?gac????144

Asn?Val?Asp?Asp?Thr?Leu?Ala?Phe?Trp?Ser?Lys?His?Val?Asp?Gly?Asp

35??????????????????40??????????????????45

cag?tgc?ttg?gtc?ttg?ccc?ttg?gag?cac?ccg?tgc?gcc?agc?ctg?tgc?tgc????192

Gln?Cys?Leu?Val?Leu?Pro?Leu?Glu?His?Pro?Cys?Ala?Ser?Leu?Cys?Cys

50??????????????????55??????????????????60

ggg?cac?ggc?acg?tgc?atc?gac?ggc?atc?ggc?agc?ttc?agc?tgc?gac?tgc????240

Gly?His?Gly?Thr?Cys?Ile?Asp?Gly?Ile?Gly?Ser?Phe?Ser?Cys?Asp?Cys

65??????????????????70??????????????????75??????????????????80

cgc?agc?ggc?tgg?gag?ggc?cgc?ttc?tgc?cag?cgc?gag?gtg?agc?ttc?ct?c???288

Arg?Ser?Gly?Trp?Glu?Gly?Arg?Phe?Cys?Gln?Arg?Glu?Val?Ser?Phe?Leu

85??????????????????90??????????????????95

aat?tgc?tcg?ctg?gac?aac?ggc?ggc?tgc?acg?cat?tac?tgc?cta?gag?gag????336

Asn?Cys?Ser?Leu?Asp?Asn?Gly?Gly?Cys?Thr?His?Tyr?Cys?Leu?Glu?Glu

100?????????????????105?????????????????110

gtg?ggc?tgg?cgg?cgc?tgt?agc?tgt?gcg?cct?ggc?tac?aag?ctg?ggg?gac????384

Val?Gly?Trp?Arg?Arg?Cys?Ser?Cys?Ala?Pro?Gly?Tyr?Lys?Leu?Gly?Asp

115??????????????????120????????????????125

gac?ctc?ctg?cag?tgt?cac?ccc?gca?gtg?aag?ttc?cct?tgt?ggg?agg?ccc????432

Asp?Leu?Leu?Gln?Cys?His?Pro?Ala?Val?Lys?Phe?Pro?Cys?Gly?Arg?Pro

130?????????????????135?????????????????140

tgg?aag?cgg?atg?gag?aag?aag?cgc?agt?cac?ctg?aaa?cga?gac?aca?gaa????480

Trp?Lys?Arg?Met?Glu?Lys?Lys?Arg?Ser?His?Leu?Lys?Arg?Asp?Thr?Glu

145?????????????????150?????????????????155?????????????????160

gac?caa?gaa?gac?caa?gta?gat?ccg?cgg?ctc?att?gat?ggg?aag?atg?acc????528

Asp?Gln?Glu?Asp?Gln?Val?Asp?Pro?Arg?Leu?Ile?Asp?Gly?Lys?Met?Thr

165?????????????????170?????????????????175

agg?cgg?gga?gac?agc?ccc?tgg?cag?gtg?gtc?ctg?ctg?gac?tca?aag?aag????576

Arg?Arg?Gly?Asp?Ser?Pro?Trp?Gln?Val?Val?Leu?Leu?Asp?Ser?Lys?Lys

180?????????????????185?????????????????190

aag?ctg?gcc?tgc?ggg?gca?gtg?ctc?atc?cac?ccc?tcc?tgg?gtg?ctg?aca????624

Lys?Leu?Ala?Cys?Gly?Ala?Val?Leu?Ile?His?Pro?Ser?Trp?Val?Leu?Thr

195?????????????????200?????????????????205

gcg?gcc?cac?tgc?atg?gat?gag?tcc?aag?aag?ctc?ctt?gtc?agg?ctt?gga????672

Ala?Ala?His?Cys?Met?Asp?Glu?Ser?Lys?Lys?Leu?Leu?Val?Arg?Leu?Gly

210?????????????????215?????????????????220

gag?tat?gac?ctg?cgg?cgc?tgg?gag?aag?tgg?gag?ctg?gac?ctg?gac?atc????720

Glu?Tyr?Asp?Leu?Arg?Arg?Trp?Glu?Lys?Trp?Glu?Leu?Asp?Leu?Asp?Ile

225?????????????????230?????????????????235?????????????????240

aag?gag?gtc?ttc?gtc?cac?ccc?aac?tac?agc?aag?agc?acc?acc?gac?aat????768

Lys?Glu?Val?Phe?Val?His?Pro?Asn?Tyr?Ser?Lys?Ser?Thr?Thr?Asp?Asn

245?????????????????250?????????????????255

gac?atc?gca?ctg?ctg?cac?ctg?gcc?cag?ccc?gcc?acc?ctc?tcg?cag?acc????816

Asp?Ile?Ala?Leu?Leu?His?Leu?Ala?Gln?Pro?Ala?Thr?Leu?Ser?Gln?Thr

260?????????????????265?????????????????270

ata?gtg?ccc?atc?tgc?ctc?ccg?gac?agc?ggc?ctt?gca?gag?cgc?gag?ctc????864

Ile?Val?Pro?Ile?Cys?Leu?Pro?Asp?Ser?Gly?Leu?Ala?Glu?Arg?Glu?Leu

275?????????????????280?????????????????285

aat?cag?gcc?ggc?cag?gag?acc?ctc?gtg?acg?ggc?tgg?ggc?tac?cac?agc???912

Asn?Gln?Ala?Gly?Gln?Glu?Thr?Leu?Val?Thr?Gly?Trp?Gly?Tyr?His?Ser

290?????????????????295?????????????????300

agc?cga?gag?aag?gag?gcc?aag?aga?aac?cgc?acc?ttc?gtc?ctc?aac?ttc????960

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

305?????????????????310?????????????????315?????????????????320

atc?aag?att?ccc?gtg?gtc?ccg?cac?aat?gag?tgc?agc?gag?gtc?atg?agc????1008

Ile?Lys?Ile?Pro?Val?Val?Pro?His?Asn?Glu?Cys?Ser?Glu?Val?Met?Ser

325?????????????????330?????????????????335

aac?atg?gtg?tct?gag?aac?atg?ctg?tgt?gcg?ggc?atc?ctc?ggg?gac?cgg????1056

Asn?Met?Val?Ser?Glu?Asn?Met?Leu?Cys?Ala?Gly?Ile?Leu?Gly?Asp?Arg

340?????????????????345?????????????????350

cag?gat?gcc?tgc?gag?ggc?gac?agt?ggg?ggg?ccc?atg?gtc?gcc?tcc?ttc????1104

Gln?Asp?Ala?Cys?Glu?Gly?Asp?Ser?Gly?Gly?Pro?Met?Val?Ala?Ser?Phe

355?????????????????360?????????????????365

cac?ggc?acc?tgg?ttc?ctg?gtg?ggc?ctg?gtg?agc?tgg?ggt?gag?ggc?tgt????1152

His?Gly?Thr?Trp?Phe?Leu?Val?Gly?Leu?Val?Ser?Trp?Gly?Glu?Gly?Cys

370?????????????????375?????????????????380

ggg?ctc?ctt?cac?aac?tac?ggc?gtt?tac?acc?aaa?gtc?agc?cgc?tac?ctc????1200

Gly?Leu?Leu?His?Asn?Tyr?Gly?Val?Tyr?Thr?Lys?Val?Ser?Arg?Tyr?Leu

385?????????????????390?????????????????395?????????????????400

gac?tgg?atc?cat?ggg?cac?atc?aga?gac?aag?gaa?gcc?ccc?cag?aag?agc????1248

Asp?Trp?Ile?His?Gly?His?Ile?Arg?Asp?Lys?Glu?Ala?Pro?Gln?Lys?Ser

405?????????????????410?????????????????415

tgg?gca?cct????????????????????????????????????????????????????????1257

Trp?Ala?Pro

<210>4

<211>419

<212>PRT

＜213〉people (Homo sapiens)

<400>4

Ala?Asn?Ser?Phe?Leu?Glu?Glu?Leu?Arg?His?Ser?Ser?Leu?Glu?Arg?Glu

1???????????????5??????????????????10??????????????????15

Cys?Ile?Glu?Glu?Ile?Cys?Asp?Phe?Glu?Glu?Ala?Lys?Glu?Ile?Phe?Gln

20??????????????????25??????????????????30

Asn?Val?Asp?Asp?Thr?Leu?Ala?Phe?Trp?Ser?Lys?His?Val?Asp?Gly?Asp

35??????????????????40??????????????????45

Gln?Cys?Leu?Val?Leu?Pro?Leu?Glu?His?Pro?Cys?Ala?Ser?Leu?Cys?Cys

50??????????????????55??????????????????60

Gly?His?Gly?Thr?Cys?Ile?Asp?Gly?Ile?Gly?Ser?Phe?Ser?Cys?Asp?Cys

65??????????????????70??????????????????75??????????????????80

Arg?Ser?Gly?Trp?Glu?Gly?Arg?Phe?Cys?Gln?Arg?Glu?Val?Ser?Phe?Leu

85??????????????????90??????????????????95

Asn?Cys?Ser?Leu?Asp?Asn?Gly?Gly?Cys?Thr?His?Tyr?Cys?Leu?Glu?Glu

100?????????????????105?????????????????110

Val?Gly?Trp?Arg?Arg?Cys?Ser?Cys?Ala?Pro?Gly?Tyr?Lys?Leu?Gly?Asp

115?????????????????120?????????????????125

Asp?Leu?Leu?Gln?Cys?His?Pro?Ala?Val?Lys?Phe?Pro?Cys?Gly?Arg?Pro

130?????????????????135?????????????????140

Trp?Lys?Arg?Met?Glu?Lys?Lys?Arg?Ser?His?Leu?Lys?Arg?Asp?Thr?Glu

145?????????????????150?????????????????155?????????????????160

Asp?Gln?Glu?Asp?Gln?Val?Asp?Pro?Arg?Leu?Ile?Asp?Gly?Lys?Met?Thr

165?????????????????170?????????????????175

Arg?Arg?Gly?Asp?Ser?Pro?Trp?Gln?Val?Val?Leu?Leu?Asp?Ser?Lys?Lys

180?????????????????185?????????????????190

Lys?Leu?Ala?Cys?Gly?Ala?Val?Leu?Ile?His?Pro?Ser?Trp?Val?Leu?Thr

195?????????????????200?????????????????205

Ala?Ala?His?Cys?Met?Asp?Glu?Ser?Lys?Lys?Leu?Leu?Val?Arg?Leu?Gly

210?????????????????215?????????????????220

Glu?Tyr?Asp?Leu?Arg?Arg?Trp?Glu?Lys?Trp?Glu?Leu?Asp?Leu?Asp?Ile

225????????????????230?????????????????235?????????????????240

Lys?Glu?Val?Phe?Val?His?Pro?Asn?Tyr?Ser?Lys?Ser?Thr?Thr?Asp?Asn

245?????????????????250?????????????????255

Asp?Ile?Ala?Leu?Leu?His?Leu?Ala?Gln?Pro?Ala?Thr?Leu?Ser?Gln?Thr

260?????????????????265?????????????????270

Ile?Val?Pro?Ile?Cys?Leu?Pro?Asp?Ser?Gly?Leu?Ala?Glu?Arg?Glu?Leu

275?????????????????280?????????????????285

Asn?Gln?Ala?Gly?Gln?Glu?Thr?Leu?Val?Thr?Gly?Trp?Gly?Tyr?His?Ser

290?????????????????295?????????????????300

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

305?????????????????310?????????????????315?????????????????320

Ile?Lys?Ile?Pro?Val?Val?Pro?His?Asn?Glu?Cys?Ser?Glu?Val?Met?Ser

325?????????????????330?????????????????335

Asn?Met?Val?Ser?Glu?Asn?Met?Leu?Cys?Ala?Gly?Ile?Leu?Gly?Asp?Arg

340?????????????????345?????????????????350

Gln?Asp?Ala?Cys?Glu?Gly?Asp?Ser?Gly?Gly?Pro?Met?Val?Ala?Ser?Phe

355?????????????????360?????????????????365

His?Gly?Thr?Trp?Phe?Leu?Val?Gly?Leu?Val?Ser?Trp?Gly?Glu?Gly?Cys

370?????????????????375?????????????????380

Gly?Leu?Leu?His?Asn?Tyr?Gly?Val?Tyr?Thr?Lys?Val?Ser?Arg?Tyr?Leu

385?????????????????390?????????????????395?????????????????400

Asp?Trp?Ile?His?Gly?His?Ile?Arg?Asp?Lys?Glu?Ala?Pro?Gln?Lys?Ser

405????????????????410?????????????415

Trp?Ala?Pro

<210>5

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>5

caagtagatc?cgcggctcat?taacgggaag?atgaccaggc?gggg??????????44

<210>6

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>6

<210>7

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>7

ctgacagcgg?cccactgcat?gaacgagtcc?aagaagctcc?ttgtc??????????45

<210>8

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>8

gacaaggagc?ttcttggact?cgttcatgca?gtgggccgct?gtcag??????????45

<210>9

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>9

ctgacagcgg?cccactgcat?ggccgagtcc?aagaagctcc?ttgtc??????????45

<210>10

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>10

gacaaggagc?ttcttggact?cggccatgca?gtgggccgct?gtcag??????45

<210>11

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>11

cttcgtccac?cccaactaca?gcaacagcac?caccgacaat?gacatc?????46

<210>12

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>12

gatgtcattg?tcggtggtgc?tgttgctgta?gttggggtgg?acgaag??????46

<210>13

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>13

cgtccacccc?aactacagca?agaacaccac?cgacaatgac?atcgc???????45

<210>14

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>14

gcgatgtcat?tgtcggtggt?gttcttgctg?tagttggggt?ggacg???????45

<210>15

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>15

ccctcgtgac?gggctggggc?aaccacagca?gccgagagaa?ggaggcc??????47

<210>16

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>16

ggcctccttc?tctcggctgc?tgtggttgcc?ccagcccgtc?acgaggg??????47

<210>17

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>17

cagcgaggtc?atgagcaaca?acgtgtctga?gaacatgc????????????????38

<210>18

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>18

gcatgttctc?agacacgttg?ttgctcatga?cctcgctg????????????????38

<210>19

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>19

gcagcgaggt?catgagcaac?gccgtgtctg?agaacatgc???????????????39

<210>20

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>20

gcatgttctc?agacacggcg?ttgctcatga?cctcgctgc????????????????????39

<210>21

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>21

cccctggcag?gtggtcctgc?tgaactcaaa?caagaagctg?gcctgcgggg????????50

<210>22

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>22

ccccgcaggc?cagcttcttg?tttgagttca?gcaggaccac?ctgccagggg?????????50

<210>23

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>23

cccctggcag?gtggtcctgc?tgaactcaac?caagaagctg?gcctgcgggg?????????50

<210>24

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>24

ccccgcaggc?cagcttcttg?gttgagttca?gcaggaccac?ctgcc??????????????45

<210>25

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>25

ggcaggtggt?cctgctggac?aacaagacca?agctggcctg?cggggcagt???????????49

<210>26

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>26

gcactgcccc?gcaggccagc?ttggtcttgt?tgtccagcag?gaccacctgc?c????????51

<210>27

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>27

gtcctgctgg?aetcaaacaa?gaccctggcc?tgcggggcag?tg???????????????????42

<210>28

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>28

cactgccccg?caggccaggg?tcttgtttga?gtccagcagg?ac???????????????????42

<210>29

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>29

gcatggatga?gtccaacaag?acccttgtca?ggcttggaga?gtatgacc?????????????48

<210>30

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>30

ggtcatactc?tccaagcctg?acaagggtct?tgttggactc?atccatgc?????????????48

<210>31

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>31

ccaactacag?caagagcaac?accaccaatg?acatcgcact?gctgcacctg???????????50

<210>32

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>32

gccaggtgca?gcagtgcgat?gtcattggtg?gtgttgctct?tgctgtagtt?gg????????52

<210>33

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>33

ggctggggct?accacagcaa?ccgaaccaag?gaggccaaga?gaaaccgc?????????????48

<210>34

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>34

gcggtttctc?ttggcctcct?tggttcggtt?gctgtggtag?ccccagcc?????????????48

<210>35

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>35

ggctaccaca?gcagccgaaa?caagaccgcc?aagagaaacc?gcaccttcg????????????49

<210>36

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>36

cgaaggtgcg?gtttctcttg?gcggtcttgt?ttcggctgct?gtggtagcc??????????49

<210>37

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>37

gcagcgaggt?catgaacaac?accgtgtctg?agaacatgct?gtgtgcggg??????????49

<210>38

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>38

cccgcacaca?gcatgttctc?agacacggtg?ttgttcatga?cctcgctgc??????????49

<210>39

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>39

ggtgagctgg?ggtgagggct?gtgggaacct?taccaactac?ggcgtttaca?cc??????52

<210>40

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>40

ggtgtaaacg?ccgtagttgg?taaggttccc?acagccctca?ccccagctca?cc??????52

Claims (50)

1. conjugate, it comprises at least one and the covalently bound non-polypeptide fraction of protein c polypeptide, described protein c polypeptide contains following aminoacid sequence, the aminoacid sequence part that this sequence is different from parent's protein c polypeptide is, has introduced and/or has removed at least one and contained the amino-acid residue of the group that can be connected with described non-polypeptide fraction.

2. the conjugate of claim 1, wherein said parent's protein c polypeptide has aminoacid sequence or its variant shown in the SEQ ID NO:4.

3. the conjugate of claim 2, wherein said parent's protein c polypeptide has the aminoacid sequence shown in the SEQ ID NO:4.

4. each conjugate of claim 1-3 is its activated form.

5. each conjugate of claim 1-4 has wherein been introduced the group that at least one is connected with non-polypeptide fraction.

6. the conjugate of claim 5 has wherein been introduced at least one glycosylation site.

7. the conjugate of claim 6, wherein said glycosylation site are glycosylation sites in the body.

8. the conjugate of claim 7, wherein said glycosylation site has been introduced in by following amino-acid residue position occupied, and described amino-acid residue has at least 25% side chain to be exposed to surface (as the qualification of this paper embodiment 1).

9. the conjugate of claim 8, wherein the glycosylation site of being introduced is selected from: D172N+K174S, D172N+K174T, D189N+K191S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, K192N+L194S, K192N+L194T, K193N+A195S, K193N+A195T, D214N, D214N+S216T, E215N+K217S, E215N+K217T, S216N+K218S, S216N+K218T, K217N+L219S, K217N+L219T, K218N+L220S, K218N+L220T, L220N+R222S, L220N+R222T, V243N+V245S, V243N+V245T, V245N+P247S, V245N+P247T, S250N, S250N+S252T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, T254N+N256S, T254N+N256T, D255N+D257S, D255N+D257T, L296N, L296N+T298S, Y302N, Y302N+S304T, H303N, H303N+S305T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T, E309N+K311S, E309N+K311T, A310N+R312S, A310N+R312T, R312N+R314S, R312N+R314T, T315N+V317S, T315N+V317T, F316N+L318S, F316N+L318T, V334N, V334N+S336T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, I348N+G350S, I348N+G350T, L349N+D351S, L349N+D351T, D351N+Q353S, D351N+Q353T, R352N+D354S, R352N+D354T, E357N+D359S, E357N+D359T, G383N+G385S, G383N+G385T, L386N+H388S, L386N+H388T, L387N+N389S, L387N+N389T, H388N+Y390S or H388N+Y390T.

10. the conjugate of claim 9, wherein the glycosylation site of being introduced is selected from: D189N+K191S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, D214N, D214N+S216T, K217N+L219S, K217N+L219T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, T253N+D255S, T253N+D255T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S or L386N+H388T.

11. the conjugate of claim 10, wherein the glycosylation site of being introduced is selected from: D189N+K191S, D189N+K191T, K191N+K193T, D214N, D214N+S216T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S305N+E307T, S305N+E307S, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S or L386N+H388T.

12. the conjugate of claim 11, wherein the glycosylation site of being introduced is selected from: D189N+K191T, K191N+K193T, D214N, K251N, S252N, T253N+D255T, Y302N, S305N+E307T, S336N+M338T, V339T, M338N, G383N+G385T or L386N+H388T.

13. the conjugate of claim 12, wherein the glycosylation site of being introduced is selected from: D189N+K191T, K191N+K193T, D214N, T253N+D255T, S305N+E307T, S336N+M338T, M338N, G383N+G385T or L386N+H388T.

14. the conjugate of claim 13, wherein the glycosylation site of being introduced is selected from: D189N+K191T, D214N or L386N+H388T.

15. each conjugate of claim 1-4, the linking group of wherein said introducing and/or removal is selected from lysine residue, glutaminic acid residue, asparagicacid residue, tyrosine residues, serine residue or cysteine residues.

16. the conjugate of claim 15, wherein said linking group has been introduced in by following amino-acid residue position occupied or has removed this linking group from described position, and described amino-acid residue has at least 25% side chain to be exposed to surface (as the qualification of this paper embodiment 1).

17. the conjugate of claim 16, wherein said linking group is introduced into or removed position is selected from: D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, R312, T315, F316, V334, S336, N337, M338, I348, L349, D351, R352, E357, G383, L386, L387 or H388.

18. the conjugate of claim 17, wherein said linking group is introduced into or removed position is selected from: D189, S190, K191, D214, K217, K251, S252, T253, Y302, S305, E307, S336, N337, M338, G383 or L386.

19. the conjugate of claim 18, wherein said linking group is introduced into or removed position is selected from: D189, D214, K251, S252, T253, Y302, S305, S336, N337, M338, G383 or L386.

20. each conjugate of claim 15-19, wherein the linking group of being introduced is a cysteine residues.

21. each conjugate of claim 15-20, wherein said non-polypeptide fraction is a polymer molecule.

22. the conjugate of claim 21, wherein said non-polypeptide fraction are linearity or ramose polyoxyethylene glycol or polyalkylene oxide.

23. each conjugate of claim 5-14, it also comprises the non-polypeptide fraction of each qualification of claim 15-22.

24. each conjugate of claim 1-23, wherein, when it is an activity form, and when detecting with this paper embodiment 9 described " APC acid amides breaking tests ", its activity is wild-type people APC active at least 10%.

25. each conjugate of claim 1-24, wherein, when it is an activity form, and when detecting with this paper embodiment 10 described " APC thrombotests ", its anticoagulating active is at least 10% of a wild-type people APC anticoagulating active.

26. each conjugate of claim 1-25 wherein, when it is activity form, more can be resisted the antitryptic deactivation effect of α-1-than people APC.

27. the conjugate of claim 26, wherein, when it is an activity form, and when with the inhibitor concentration of 16.6 μ M when this paper embodiment 11 described " α-1-antitrypsin inactivation test " detects, residual activity is at least 20%.

28. each conjugate of claim 1-27 wherein, when it is activity form, increases the resistance of the deactivation of human plasma.

29. the conjugate of claim 28 wherein, when it is an activity form, and when detecting with this paper embodiment 12 described " human plasma inactivation test I ", has at least 20% residual activity.

30. the conjugate of claim 28 or 29, when detecting with this paper embodiment 13 described " human plasma inactivation test II ", the ratio of the external half life of the activity form of described conjugate and the external half life of people APC is at least 1.25.

31. each described conjugate of claim 1-30 when it is activity form, is compared with people APC, half life, increase or serum half life increase in the function gonosome.

32. the conjugate of claim 31, the ratio of interior half life of the function gonosome of half life or serum half life and people APC or serum half life is at least 1.25 in the function gonosome of wherein said conjugate.

33. the variant of parent's protein c polypeptide, the position of group comprises replacement a: D172, D189, S190, K191, K192 to described variant being selected from down, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, R312, T315, F316, V334, S336, N337, M338, I348, L349, D351, R352, E357, E382, G383, L386, L387 and H388, condition is, described replacement is not selected from: T254S, T254A, T254H T254K, T254R, T254N, T254D, T254E, T254G, T254Q, Y302S, Y302A, Y302T, Y302H, Y302K, Y302R, Y302N, Y302D, Y302E, Y302G, Y302Q, F316S, F316A, F316T, F316H, F316K, F316R, F316N, F316D, F316E, F316G or F316Q.

34. the variant of claim 33, wherein said parent's protein c polypeptide has aminoacid sequence shown in the SEQ ID NO:4.

35. the variant of claim 33 or 34 is its activity form.

36. each variant of claim 33-35, described variant is the polypeptide portion of each conjugate that is limited of claim 9-20.

37. comprising, the variant of claim 36, wherein said variant be selected from K251N, the replacement of S252N or Y302N.

38. each variant of claim 33-37, it has the characteristic of each qualification of claim 24-32.

39. a nucleotide sequence, the variant of each qualification of its coding claim 33-38.

40. an expression vector, it comprises the nucleotide sequence that claim 39 limits.

41. a host cell, it comprises the nucleotide sequence of claim 39 qualification or the expression vector that claim 40 limits.

42. the host cell of claim 41, it is selected from COS, CHO, BHK or HEK293 cell.

43. a pharmaceutical composition, it comprises the conjugate of each qualification of claim 1-32 or variant and a kind of pharmaceutically acceptable carrier or the vehicle of each qualification of claim 33-38.

44. as medicine, the conjugate of each qualification of claim 1-32, the variant of each qualification of claim 33-38, or the pharmaceutical composition that limits of claim 43.

45. the conjugate of each qualification of claim 1-32, the variant of each qualification of claim 33-38, or the purposes of the pharmaceutical composition of claim 43 qualification are used to prepare the medicine for the treatment of following situation: outbreak, and myocardial infarction is after the phlebothrombosis; Disseminated inravascular coagulation (DIC); Sepsis; Septic shock; Embolism is as pulmonary infarction; Transplant, as bone marrow transplantation; Burn; Gestation; Great surgical operation/wound or adult respiratory distress syndrome (ARDS).

46. the purposes of claim 45 is used to prepare the medicine for the treatment of septic shock.

47. treatment or prevention are selected from down the method for the situation of group: outbreak, myocardial infarction is after the phlebothrombosis; Disseminated inravascular coagulation (DIC); Sepsis; Septic shock; Embolism is as pulmonary infarction; Transplant, as bone marrow transplantation; Burn; Gestation; Great surgical operation/wound or adult respiratory distress syndrome (ARDS), described method comprises and gives claim 1-32 the conjugate of each qualification to the patient who needs corresponding treatment, the variant of each qualification of claim 33-38, or the pharmaceutical composition of claim 43 qualification.

48. the method for claim 47 is used for the treatment of or prevents septic shock.

49. the method for each conjugate that is limited of preparation claim 1-32, this method comprises, cultivates proper host cell under the condition that the polypeptide portion of inducing described conjugate is expressed, and reclaims this polypeptide, wherein

A) described polypeptide comprises at least one N-or O-glycosylation site, and described host cell is to carry out glycosylated eukaryotic host cell in the body, and/or

B) described polypeptide is in external and non-polypeptide fraction coupling.

50. the method for half life or serum half life in the function gonosome that increases parent's protein c polypeptide, this method comprises, the amino-acid residue of forming the linking group of non-polypeptide fraction is introduced in parent's protein c polypeptide, and/or the amino-acid residue that will form described linking group is removed, and make the modified polypeptide and the described non-polypeptide fraction coupling of gained, the position of described introducing comprise with its side chain of at least 25% be exposed to the surface amino-acid residue (such as this paper embodiment 1 qualification) but linking group as described in not containing, described non-polypeptide fraction has the amino-acid residue as linking group that is introduced into and/or removes.

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