CN100522988C

CN100522988C - Glycopegylation methods and proteins/peptides produced by the methods

Info

Publication number: CN100522988C
Application number: CNB2004800159188A
Authority: CN
Inventors: S·德弗里斯; D·措普夫; R·拜尔; C·鲍; D·哈克斯; 陈希
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS; Neose Technologies Inc
Priority date: 2003-04-09
Filing date: 2004-04-09
Publication date: 2009-08-05
Anticipated expiration: 2024-04-09
Also published as: CN1863458A; ZA200508028B

Abstract

The invention includes methods and compositions for remodeling a peptide molecule, including the addition or deletion of one or more glycosyl groups to a peptide, and/or the addition of a modifying group to a peptide.

Description

Glycopegylated method and the proteins/peptides of producing by this method

Background of invention

Most of naturally occurring peptides contain on the length of one-level peptide chain by the amino acid whose specific bonding with selected number and are attached to carbohydrate part on the peptide.Thereby many naturally occurring peptides are called " glycopeptide ".The mutability of the glycosylation pattern on any given peptide has huge meaning for the function of this peptide.For example, the structure of the glycan of N-connection can influence the various features of peptide on the peptide, comprises the interior transportation of proteolytic enzyme susceptibility, cell, secretion, tissue orientation, biological half time and the antigenicity of peptide in cell or the biology.The change of one or more of these features has influenced the effect of peptide in its natural surroundings widely, and has also influenced peptide as the effect of therapeutical agent in certain situation, and this peptide promptly generates for this purpose in this situation.

The carbohydrate structure that is attached on the peptide chain is known as " glycan " molecule.The specific glycan structures that exists on peptide influences resistance that the folding and function of the solubility of this peptide and aggregation characteristic, one-level peptide chain or enzymic activity, this peptide attack proteolysis and the proteoclastic control that the inactive form that causes peptide is changed to activity form.Importantly, be present in the length that terminal sialic acid residues on the glycan molecule influences the transformation period of peptide in the Mammals recycle system.The peptide that its glycan does not contain terminal sialic acid residues will be removed from circulation fast by liver, and this is the incident that any potential treatment benefit of peptide was lost efficacy.

The glycan structures that natural discovery is present on the glycopeptide generally is divided into two classes, and promptly N-connects is connected with O-.

The peptide of in eukaryotic cell, expressing generally be on the site of peptide primary structure on asparagine residue N-glycosylated, the sequence of l-asparagine-X-serine/threonine is contained in this site, wherein X can be any amino acid except that proline(Pro) and aspartic acid.Carbohydrate in this peptide partly is known as the glycan that N-connects.The glycosylated early stage incident of N-betides in the endoplasmic reticulum (ER) and is identical in Mammals, plant, insect and other higher eucaryotes.At first, the oligonucleotide chain that comprises 14 saccharide residues makes up on lipid carrier.When nascent peptide translation and transposition entered ER, whole oligonucleotide chain was then transferred on the amide group of asparagine residue in the catalytic reaction of membrane-bound glycosyltransferase.The glycan that N-connects is further processed in ER and golgi body.Further processing need be removed some saccharide residues usually and be added other saccharide residue in by Glycosylase and the catalytic reaction of glycosyltransferase, wherein said enzyme is specific for the saccharide residue of removing and add.

Usually, the final structure of the glycan of N-connection depends on the biology that produces this peptide.For example, bacteriogenic peptide complete sugar basedization normally.Contain oligonucleotide chain that high seminose is connected with paunci-seminose N-and other at the peptide of expressed in insect cells.The peptide that produces in the mammalian cell cultures depends on usually as species and cell culture condition and glycosylation differentially.Even in identical species,, also run into some heterogeneity in the glycosyl chain sometimes with under identical condition.Further, the peptide that produces in the vegetable cell comprise significantly with those zooblasts in the different glycan structures that produces.Predicament in the field that produces recombinant peptide (particularly when this peptide is used as therapeutical agent) is to generate correct glycosylated peptide, promptly can generate have with the natural form that is present in peptide in the peptide of similar or identical glycan structures.The peptide that most of recombination methods produce comprises the glycan structures different with naturally occurring glycan.

Proposed the glycosylation pattern that several different methods customizes peptide in the art, comprised that those are described in WO 99/22764, WO 98/58964, WO 99/54342 and the U.S. Patent No. 5,047,335, and other.Basically, the necessary many enzymes of the external glycosylation of peptide are cloned and check order.In some cases, these enzymes add specific sugar on the peptide incomplete glycan molecule in external being used for.In other situations, cell has carried out the combination of genetically engineered processing with expression enzyme and the peptide of wanting, thus the interpolation of the sugar moieties that can in cell, want in the peptide of expressing.

Peptide also can be modified by the glycan that adds the O-connection, and this glycan is because its ubiquity on the Saliva Orthana glycopeptide is also referred to as the glycan of Saliva Orthana type.Be connected to asparagine residue on and to shift the N-glycan that oligosaccharides forms by integral body from lipid bonded intermediate different, the O-glycan mainly is connected on Serine and the threonine residues and by the sugar that progressively adds from the Nucleotide carbohydrate and forms (people such as Tanner, Biochim.Biophys.Acta.906:81-91 (1987); With people such as Hounsell, Glycoconj.J.13:19-26 (1996)).The O-that the peptide function can be subjected to existing thereon connects the influence of glycan structures.For example, P-selects the activity of protein ligands to be subjected to the influence of the O-connection glycan structures of existence on it.For the summary of O-connection glycan structures, referring to Schachter and Brockhausen, The Biosynthesis ofBranched O-Linked Glycans, 1989, Society for ExperimentalBiology, pp.1-26 (Britain).Other glycosylation patterns form (people such as Takeda, TrendsBiochem.Sci.20:367-371 (1995) by connect glycosyl-phosphatidyl inositol to protein C-terminal carboxylic group; With people such as Udenfriend, Ann.Rev.Biochem.64:593-591 (1995)).

Although exist the N-of various modified peptides to connect the technology of glycan at present, this area need generate the universal method of peptide, and this peptide has the i.e. glycosylation pattern of wanting of customization.This area needs the external glycosylation of the customization (Customize) of peptide especially, and wherein the peptide of gained can be produced on technical scale as a result.The present invention can satisfy this and other needs.

Giving glycosylation and nonglycosylated peptide is that field of medicaments is well-known to produce specific physiological responses.One of the most well-known peptide that is used for this purpose is a Regular Insulin, and it is used for the treatment of diabetes.Enzyme also uses because of its treatment benefit.The principal element that has limited the application of therapeutic peptide is the immunogenicity characteristic of most of peptides.In the patient, to the immunogenic response of the peptide that gives can in and peptide and/or cause allergic development among the patient.Other deficiencies of therapeutic peptide comprise inferior optimum effectiveness and clearance rate fast.Have recognized that peptide therapeutics inherent problem in the art, and studied the method for the described problem of various eliminations.For the peptide therapeutics of solubility is provided, the synthetic polymkeric substance is attached on the peptide main chain.

Poly-(ethylene glycol) (" PEG ") is the exemplary polymkeric substance of puting together with peptide.Proved and used PEG to make the peptide therapeutics derivatize can reduce the immunogenicity and the clean-up time of prolongation from circulation of peptide.For example, U.S.Pat.No.4,179,337 (people such as Davis) relate to the peptide of non-immunogenicity, as with polyoxyethylene glycol (PEG) or polypropylene glycol link coupled enzyme and peptide hormone.Every mole of peptide has used 10～100 moles polymkeric substance and has kept at least 15% physiologically active.

WO 93/15189 (people such as Veronese) relates to by keeping the method for polyethyleneglycol modified hydrolase of proteolysis on the inhibitor that proteolytic ferment is connected to macromoleization.This conjugate is intended for use in medical use.

The main pattern that PEG and derivative thereof adhere to peptide is the unspecific connection by the peptide ammino acid residue.For example, U.S. Patent No. 4,088,538 disclose the enzymic activity polymkeric substance-enzyme conjugate with the covalently bound enzyme of PEG.Similarly, U.S. Patent No. 4,496,689 disclose the mixture of α-1 proteinase inhibitor and polymkeric substance such as PEG or poly-(ethylene glycol) (" mPEG ") covalent attachment of methoxyl group.People such as Abuchowski (J.Biol.Chem.252:3578 (1977)) disclose the covalent attachment of the amine groups of mPEG and bovine serum albumin.U.S. Patent No. 4,414,147 disclose by with the acid anhydrides of itself and the dicarboxylic acid method that makes the Interferon, rabbit hydrophobicity lower of puting together as poly-(ethene succinyl oxide).PCT WO 87/00056 discloses puting together of PEG and polyoxy ethylization polyvalent alcohol and proteins such as interferon-beta, interleukin II and immunotoxin.EP 154,316 discloses and asks for protection the lymphokine of chemically modified as containing the IL-2 of the PEG that directly is connected with at least one one-level amino group of lymphokine.U.S. Patent No. 4,055,635 disclose the pharmaceutical composition of the proteolytic ferment water-soluble compound that is connected with polymeric material such as polysaccharide covalent.

The another kind of pattern that PEG and peptide adhere to is the unspecific oxidation by glycosyl residue on the peptide.The sugar of oxidation can be used as peg moiety is attached to seat on the peptide.For example, M ' Timkulu (WO94/05332) discloses hydrazine-PEG or amino-PEG and has added PEG on the sugar-protein application.Glycosyl part is oxidized to corresponding aldehyde randomly, and subsequently with amino-PEG coupling.Also referring to people such as Bona (WO 96/40731), wherein glycan is contacted with amino-PEG molecule and PEG is added on the immunoglobulin molecules by the glycan on the enzymatic oxidn immunoglobulin (Ig).

In above-mentioned each method, poly-(ethylene glycol) is to add on the reactive residue of peptide main chain in nonspecific mode at random.For the peptide of manufacture of therapeutic, what obviously want is a kind of derivatize strategy, and this strategy causes the formation of the product of specific markers, that be easy to characterize and basic homogeneous.

In carbohydrate is synthetic, use the main enzyme of two classes, i.e. glycosyltransferase (as sialyltransferase, oligosaccharyl transferase, N-acetyl-glucosamine transferring enzyme) and Glycosylase.Glycosylase is further divided into exoglycosidase (as beta-Mannosidase, beta-glucosidase enzyme) and endoglycosidase (as Endo-A, Endo-M).Each successfully syntheticly is used to prepare carbohydrate these several zymoid.For general summary, referring to people such as Crout, Curr.Opin.Chem.Biol.2:98-111 (1998).

Oligosaccharide structure on the glycosyltransferase modified peptides.Glycosyltransferase is effective for producing specific product with good stereochemistry and regional chemistry (regiochemical) control.Glycosyltransferase has been used to prepare oligosaccharides and has been used to modify terminal N-or carbohydrate structure that O-is connected, particularly on the peptide of mammalian cell generation.For example, the sialylated fully and/or fucosylation of the terminal oligosaccharides of glycopeptide provides firmer sugared structure, and this has improved the pharmacokinetics and various other the biological characteristics of glycopeptide.For example, β-1,4-galactosyltransferase can be used for synthetic lactose amine, and this is that glycosyltransferase is used for example of carbohydrate synthetic (referring to as people such as Wong, J.Org.Chem.47:5416-5418 (1982)).In addition, many synthesis programs use α-sialyltransferases with sialic acid from Cytidine-5 '-single phosphoric acid-N-n acetylneuraminic acid n transfers to the 3-OH of semi-lactosi or 6-OH (referring to as people such as Kevin, Chem.Eur.J.2:1359-1362 (1996)).Fucosyltransferase can be used for route of synthesis so that fucose unit is transferred on the specific hydroxyl of saccharide acceptor from guanosine-5 '-bisphosphate Fucose.For example, Ichikawa has prepared saliva acid Lewis-X people such as (, J.Am.Chem.Soc.114:9283-9298 (1992)) Ichikawa by the method that relates to sialylated lactose amine and carry out fucosylation by clone's fucosyltransferase.For the glycoconjugate synthetic that the is used for the treatment of purposes discussion of progress recently, referring to people such as Koeller, NatureBio technology 18:835-841 (2000).Also referring to U.S. Patent No. 5,876,980,6,030,815,5,728,554,5,922,577 and WO/9831826.

Glycosylase also can be used for preparing carbohydrate.The hydrolysis of the common catalysis glycosidic link of Glycosylase.Yet under proper condition, they can be used for forming this key.Being used for the most of glucosides enzymes of carbohydrate synthetic is exoglycosidases; Glycosyl shifts the non-reduced end that betides substrate.In conjunction with glycosyl donor, hydrolysate be got and be produced to this intermediate can by the water misfortune to Glycosylase, or get generation new glucosides or oligosaccharides by the acceptor misfortune in glycosyl-enzyme intermediate.An exemplary approach using exoglycosidase is core trisaccharide synthetic of the glycopeptide that connects of all N-, comprises β-seminose glycosidic bond of being formed by beta-Mannosidase effect people such as (, Chem.Commun.993-994 (1996)) Singh.

In using Glycosylase another exemplary application with the formation glycosidic link, prepared the Glycosylase of sudden change, wherein the normal nucleophilic amino acid change of avtive spot is non-nucleophilic amino acid.The enzyme of this sudden change is the hydrolysis sugar glycosidic bond not, but still can form this key.The Glycosylase of this sudden change can be used for preparing oligosaccharides (people such as Withers, U.S. Patent No. 5,716,812) with alpha-glycosyl fluorochemical donor and glucosides acceptor molecule.

Be not so good as the general of exoglycosidase although it is used, endoglycosidase also is used to prepare carbohydrate.Has the advantage of transferable oligosaccharides rather than monose based on the method for using endoglycosidase.Oligose fragment has been added on the substrate to (people such as Wang, Tetrahedron Lett.37:1975-1978) with endo-β-N-acetyl-glucosamine such as endo-F, endo-M; With people such as Haneda, Carbohydr.Res.292:61-70 (1996)).

Except that its application of preparation carbohydrate, above-mentioned enzyme also can be applicable to synthetic glycopeptide.Delivered synthetic people such as (, J.Am.Chem.Soc.119:2114-2118 (1997)) the Witte K. of the homogeneous sugar form (glycoform) of ribonuclease B.The high mannose core of ribonuclease B can be cut by handling glycopeptide with endoglycosidase H.This cutting specifically betides between two core GlcNAc residues.Then by using β-1 successively, 4-galactosyltransferase, α-2,3-sialyltransferase and α-1, the 3-fucosyltransferase, tetrose saliva acid Lewis X can rebuild by enzymatic on remaining GlcNAc anchored site on the protein of homogeneous.Yet although each enzyme catalysis step is all carried out with fabulous output, this program can not be applicable to and generate glycopeptide on technical scale.

Method in conjunction with chemistry and enzymic synthesis element also is as known in the art.For example, Yamamoto and colleague (Carbohydr.Res.305:415-422 (1998)) have reported with endoglycosidase synthetic to the chemical enzymatic (chemoenzymatic) of glycopeptide, glycosylated peptide T.The N-acetyl-glucosamine peptide is fully by the chemical process synthetic.This peptide carries out the enzyme processing with the oligosaccharides of human transferrin peptide subsequently meticulously.Sugar moieties adds on the peptide by handling with endo-beta-N-acetyl glycosamine enzyme (acetylglucosaminidase).The glycosylated peptide of gained and peptide T compare with N-acetyl-glucosamine peptide T and are high stability and proteolysis had resistance as a result.

Also probed into the application of the glycosyltransferase that is used to modify peptide structure with reporter group.For example, people such as Brossmer (U.S. Patent No. 5,405,753) application in the fluorescent mark of measuring sialyltransferase active and cell surface, glycoprotein and peptide of the formation of sialic fluorescently-labeled cytosine riboside monophosphate (" CMP ") derivative and fluorescence glucosides is disclosed.People such as Gross (Analyt.Biochem.186:127 (1990)) have described similar mensuration.People such as Bean (U.S. Patent No. 5,432,059) disclose and have utilized the mensuration of insufficient glycosylated proteinic glycosylation again to the glycosylation defect illness.This deficient protein matter is glycosylated again with fluorescently-labeled CMP glucosides.Each fluorescence sialic acid derivative all partly replaces on common acetylizad 9-position or the amine in sialic acid with fluorescence.The method of using the fluorescence sialic acid derivative is the mensuration whether nonglycosylated or incorrect glycosylated glycoprotein of glycosyltransferase is existed.This mensuration is in the sample of biological origin a spot of enzyme or glycoprotein to be carried out.Sialic acid still unexposed or that proposition utilization is modified carries out the enzymatic derivatize with preparation or technical scale to glycosylated or nonglycosylated peptide in the prior art field.

Also existing considerable effort relates to by changing the glycosyl residue that is presented by cell surface modifies this cell surface.For example, Fukuda and colleague have been developed the method that the glucosides that will have limiting structure is attached to cell surface.This method is utilized the undemanding substrate specificity of fucosyltransferase, the transferable Fucose of this enzyme and have Fucose analogue people such as (, J.Biol.Chem.271:27213 (1996)) Tsuboi of different sugar substrate.

Enzymatic means also has been used to activate glycosyl residue on the glycopeptide to help chemical treatment subsequently.Glycosyl residue generally is to use the galactose oxidase activatory, and this enzyme changes terminal galactose residues into corresponding aldehyde.This aldehyde subsequently with the modification group coupling that contains ammonia.For example, people (Nature Biotech.19:142 (2001)) such as Casares has been attached to Zorubicin on the galactose residue of the chimeric oxidation of MHCII-peptide of reorganization.

Also glycosyl residue is modified so that it contains ketone groups.For example, Mahal and colleague (Science 276:1125 (1997)) have prepared N-acetyl-propionyl mannosamine (" ManLev "), and the position that it is occupied by acetyl group in natural substrate usually has ketone.Handle cell with ManLev, thereby ketone groups is integrated into cell surface.Also referring to people such as Saxon, Science 287:2007 (2000); People such as Hang, J.Am.Chem.Soc.123:1242 (2001); People such as Yarema, J.Biol.Chem.273:31168 (1998); With people such as Charter, Glycobiology 10:1049 (2000).

The method of modifying cell surface is not applied to modify glycosylated or nonglycosylated peptide as yet when not having cell.Further, the cell surface modifying method glycosyl donor part enzymatic that is not used for modifying is integrated into peptide.In addition, not having a kind of cell surface modifying method is feasible in the peptide glycosyl modified with industrial-scale production.

Although the effort that relates to sugared structure enzymically treat is arranged, but still need industrial feasible method to be used for glycosylated and nonglycosylated peptide being modified this modification group such as water-soluble polymers, treatment part, biomolecules etc. with modification group.Interested especially is the method that the peptide wherein modified has the characteristic of improvement, and the characteristic of this improvement has strengthened the application of this peptide as treatment or diagnostic reagent.The present invention has satisfied the needs of these and other.

Summary of the invention

The present invention includes and a plurality of peptide is reconstructed so that it has the method for glycan structures attached to it.Although described specific glycan structures herein, the present invention should not be construed and is limited to any one specific structure.In addition, although described specific peptide herein, the present invention should not be subjected to the restriction of the characteristic of described peptide, and should comprise any or all suitable peptide and variant thereof.

Description subsequently discloses the preferred embodiment of the invention and the written description of additional claim is provided.The present invention comprises any and variants all these embodiments, and this variant after having read specification sheets of the present invention is or will will be conspicuous.

The present invention includes the acellular in vitro method that the peptide that comprises polyoxyethylene glycol is reconstructed, this peptide has following molecular formula:

Wherein

AA is the end or the internal amino acid residue of peptide;

X ¹-X ²Be the sugar covalently bound, wherein with AA

X ¹It is first glycosyl residue; And

X ²Be and X ¹Covalently bound second glycosyl residue, wherein X ¹And X ²Be selected from monose and oligosaccharides residue, this method comprises:

(a) from peptide, remove X ²Or its sugared subunit, thereby the glycan of formation brachymemma.

On the one hand, the present invention further comprises by removing the glycan that the Sia residue forms brachymemma.

In one embodiment of the invention, peptide has molecular formula:

Wherein

X ³, X ⁴, X ⁵, X ⁶, X ⁷And X ¹⁷Be independent monose or the oligosaccharides residue of selecting; With

A, b, c, d, e and x are independently selected from

integer

0,1 and 2.

In one aspect of the invention, the oligosaccharides residue is the member who is selected from GlcNAc-Gal-Sia and GlcNAc-Gal.On the other hand, at least one oligosaccharides member is selected from a, b, c, d, and e and x are 1 or 2.On the other hand, removal in the step (a) produced wherein a, b, c, e and x at least one be the glycan of 0 brachymemma.

The present invention includes the method that peptide is reconstructed, wherein X ³, X ⁵And X ⁷Be independently to be selected from (seminose) _z(seminose) _z-(X ⁸) the member

Wherein

X ⁸Be to be selected from single-and the glycosyl part of widow-sugar; With

Z is the integer between 1 and 20, wherein

When z is 3 or when bigger, each (seminose) _zBe independently selected from linear and ramose structure.

On the one hand, X ⁴Be selected from GlcNAc and wood sugar.On the other hand, X ³, X ⁵And X ⁷Be (seminose) _u, wherein u is selected from the integer between 1 and 20, and when u be 3 or when bigger, each (seminose) _uBe independently selected from linear and ramose structure.

The present invention also comprises the method that peptide is reconstructed, and wherein said peptide has molecular formula:

Wherein

R, s and t independently are selected from 0 and 1 integer.

In one embodiment of the invention, peptide has molecular formula:

Wherein

X ⁹And X ¹⁰Be independent monose or the oligosaccharides residue of selecting; With

M, n and f independently are selected from 0 and 1 integer.

On the one hand, peptide has molecular formula:

Wherein

X ¹⁶Be the member who is selected from following molecular formula:

With

Wherein,

S and i are independently selected from 0 and 1 integer.

On the other hand, peptide has molecular formula:

Wherein

X ¹³, X ¹⁴And X ¹⁵Be independently selected from glycosyl residue; With

G, h, i, j, k and p are independently selected from

integer

0 and 1.

In another aspect of this invention, at least one of g, h, i, j, k and p is 1.On the other hand, X ¹⁴And X ¹⁵Be the member who independently is selected from GlcNAc and Sia, and i and k are independently selected from integer 0 and 1.On the other hand, at least one of i and k is 1, and if k be 1, g, h and j are 0 so.

The present invention also comprises the method that peptide is reconstructed, wherein this method comprises that the glycan that makes brachymemma and at least a glycosyltransferase and at least a glycosyl donor contact under the condition of the glycan that is suitable for described at least a glycosyl donor is transferred to brachymemma, thereby the peptide that comprises polyoxyethylene glycol is reconstructed.

On the one hand, glycosyl donor comprises covalently bound modification group thereon.

The present invention also comprises the method that peptide is reconstructed, and this method comprises removes X ¹Thereby, expose AA.On the one hand, this method comprises makes AA and at least a glycosyltransferase and at least a glycosyl donor contact being suitable for described at least a glycosyl donor is transferred under the condition of AA, thereby the described peptide that comprises polyoxyethylene glycol is reconstructed.

On the one hand, at least one glycosyl donor comprises covalently bound modification group thereon.On the other hand, modification group is a polyoxyethylene glycol.In one embodiment, polyoxyethylene glycol has basic single molecular weight distribution of disperseing (homodisperse).

The present invention includes the method that peptide is reconstructed, wherein, be suitable for described at least a glycosyl donor is transferred at the glycan that makes brachymemma and at least a glycosyltransferase and at least a glycosyl donor under the condition of glycan of brachymemma and contacting, thereby before the peptide that comprises polyoxyethylene glycol is reconstructed, will in the posttranslational modification process, add the group of sugar to and remove.

On the one hand, the group of removal is the member who is selected from phosphoric acid, sulfuric acid, carboxylic acid and ester thereof.

The present invention includes the method that peptide is reconstructed, wherein said peptide has molecular formula:

Wherein

Z is the member who is selected from O, S, NH and linking agent.

Wherein

X ¹¹And X ¹²It is the glycosyl part of selecting independently; And

R and x independently are selected from 0 and 1 integer.

In one aspect of the invention, X ¹¹And X ¹²Be (seminose) _q, wherein q is the integer that is selected from 1-20, and when q be 3 or when bigger, (seminose) _qBe selected from linear and ramose structure.

The present invention includes pharmaceutical composition, it comprises the medicine acceptable diluent and according to the peptide of the acellular in vitro method reconstruct that the peptide that comprises polyoxyethylene glycol is reconstructed, described peptide has molecular formula:

Wherein

AA is the end or the internal amino acid residue of peptide;

X ¹-X ²Be the glycosyl residue covalently bound, wherein with AA

X ¹It is first glycosyl residue; And

X ²Be and X ¹Covalently bound second glycosyl residue, wherein X ¹And X ²Be selected from monose and oligosaccharides residue;

Described method comprises:

(a) remove X from described peptide ²Or its sugared subunit, thereby the glycan of formation brachymemma.

The present invention also comprises the acellular in vitro method that the peptide that comprises polyoxyethylene glycol is reconstructed, and described peptide has molecular formula:

Wherein

AA is the end or the internal amino acid residue of peptide;

X ¹Be the glycosyl residue covalently bound, be selected from monose and oligosaccharides residue with AA; And

U is selected from 0 and 1 integer,

This method comprises:

Peptide and at least a glycosyltransferase and at least a glycosyl donor are contacted under the condition of the glycan that is suitable for described at least a glycosyl donor is transferred to brachymemma, thereby peptide is reconstructed.

On the one hand, at least one glycosyl donor comprises covalently bound modification group thereon.On the other hand, modification group is a polyoxyethylene glycol.On the other hand, polyoxyethylene glycol has basic single molecular weight distribution of disperseing (homodisperse).

The present invention also comprises pharmaceutical composition, and it comprises the medicine acceptable diluent and according to the peptide of the acellular in vitro method reconstruct that the peptide that comprises polyoxyethylene glycol is reconstructed, described peptide has molecular formula:

Wherein

AA is the end or the internal amino acid residue of peptide;

U is selected from 0 and 1 integer,

This method comprises:

The accompanying drawing summary

In order to illustrate purpose of the present invention, certain embodiments of the present invention are described in the accompanying drawings.Yet the present invention is not limited to the accurate arrangement and the instrument of the embodiment that is described in the drawings.

Fig. 1 is the synoptic diagram of the enzyme method (the right) of description three mannose group core glycan (left side) and the glycan that generates (bisecting) GlcNAc with five equilibrium.

Fig. 2 is a synoptic diagram of describing the compound catenary of three basic mannose group core textures and various performance levels.Shown that three basic mannose group core textures never contain the external enzymatic generation method of complicated carbohydrate glycan structures of GlcNAc residue of five equilibrium and the generation of glycan structures that contains the GlcNAc of five equilibrium.Symbol: square: GlcNAc; Light color circle: Man; Black circles: Gal; Trilateral: NeuAc.

Fig. 3 is the synoptic diagram that generates sialylated glycan structures (the right) from GlcNAc (left side) the beginning enzymatic with three mannose group cores and five equilibrium.

Fig. 4 is the typical glycan structures (left side) of high mannose content and is the synoptic diagram of the enzyme method of basic three mannose group core textures with this construction recovery.In this synoptic diagram, X is the seminose as monose, oligosaccharides or polysaccharide.

Fig. 5 is the glycan structures synoptic diagram that the general N-that contains Fucose and wood sugar that produces in vegetable cell is connected.

Fig. 6 is the glycan structures synoptic diagram that the general N-that contains Fucose that produces in insect cell connects.Notice that glycan can not have the core Fucose, it can have the core Fucose of any one key, and perhaps it can have the dominant core Fucose of a kind of key.

Fig. 7 describe to prune the various approach of high mannose structures and from the synoptic diagram of synthetic complicated sugar chain wherein.Symbol: square: GlcNAc; Circle: Man; Rhombus: Fucose; Pentagon: wood sugar.

Fig. 8 is the synoptic diagram of describing from the external strategy of the synthetic complex construction of three basic mannose group core textures.Symbol: square: GlcNAc; Light color circle: Man; Black circles: Gal; Black triangle: NeuAc; The GnT:N-acetylgucosamine transferase; GalT: galactosyltransferase; ST: sialyltransferase.

Fig. 9 describes synthetic single feeler glycan and the optional synoptic diagram that this glycan is carried out two kinds of Glycopegylated external strategies.Black squares: GlcNAc; Black circles: Man; Light color circle: Gal; Black triangle: sialic acid.

Figure 10 describes synthetic single feeler glycan and the optional synoptic diagram that this glycan is carried out two kinds of Glycopegylated external strategies.Black squares: GlcNAc; Black circles: Man; Light color circle: Gal; Black triangle: sialic acid.

Figure 11 is that description can be from the synoptic diagram of the basic various complex constructions of three mannose group core texture synthetic.Symbol: square: GlcNAc; Light color circle: Man; Black circles: Gal; Trilateral: NeuAc; Rhombus: Fucose; FT and FucT: fucosyltransferase; GalT: galactosyltransferase; ST: sialyltransferase; Le:Lewis antigen; SLe: sialylated Lewis antigen.

Figure 12 is the illustrative diagram that preparation is derived from the O-connection glycopeptide of Serine or Threonine.Randomly, water-soluble polymers (WSP) is added in the final glycan structures as polyoxyethylene glycol.

Figure 13 is the diagram of a series of description 4 class O-glycan structures, is called core 1～4.The core texture with dashed lines is described.

Figure 14, comprise Figure 14 A and Figure 14 B, be the synoptic diagram of a series of expressions exemplary of the present invention, the saccharide residue that wherein will comprise complicated sugared structure and/or high mannose structures is trimmed to the structure of the two feelers (the first generation biantennary) of the first-generation.Randomly, only after reacting, add Fucose with GnTI.The sugar that makes modification then with water-soluble polymers (WSP) with put together by the one or more saccharide residues that expose in the pruning process.

Figure 15 is and similar synoptic diagram shown in Figure 4, be the core of seminose β-connection wherein with high mannose structures (highmannose structure) or composite structure " pruning ", and the sugar that makes modification then with water-soluble polymers with put together by the one or more saccharide residues that expose in the pruning process.Sugar adds in order with glycosyltransferase.

Figure 16 is and similar synoptic diagram shown in Figure 4, wherein " pruning " high mannose structures or composite structure be until the accompanying GlcNAc of first seminose, and make modification sugar with water-soluble polymers then and puted together by the one or more saccharide residues that expose in the pruning process.Sugar adds in order with glycosyltransferase.

Figure 17 is and similar synoptic diagram shown in Figure 4, and wherein " pruning " high mannose structures or composite structure are attached to GlcNAc on the Asn of peptide until first, and water-soluble polymers and the one or more saccharide residues that added are subsequently puted together.Sugar adds in order with glycosyltransferase.

Figure 18 comprises Figure 18 A and Figure 18 B, is the synoptic diagram that derivatize was pruned and carried out with the modification sugar moieties (Gal or GlcNAc) with water-soluble polymers subsequently to the optional sugar that N-is connected from high mannose structures or composite structure.

Figure 19 comprises Figure 19 A and Figure 19 B, is the synoptic diagram of pruning and partly carrying out with the sialic acid with water-soluble polymers subsequently derivatize from the sugar that high mannose structures or composite structure connect N-.Sugar adds in order with glycosyltransferase.

Figure 20 is that the optional sugar that N-is connected from high mannose structures or composite structure is pruned and partly carried out derivatize with one or more sialic acids subsequently, and with the sialic acid terminated synoptic diagram of water-soluble polymers derivatize.Sugar adds in order with glycosyltransferase.

Figure 21 carries out the synoptic diagram that " prunings " also puts together with the modification sugar with water-soluble polymers subsequently with the sugar that O-connects.In exemplary synoptic diagram, " pruning " sugar moieties is until the two feeler structures of the first-generation.

The exemplary demonstration of Figure 22, the sugar moieties of the glycopeptide that O-is connected are pruned with generation and be can be used for the seminose puted together with the modification sugar that has adhered to water-soluble polymers thereon.

Figure 23 comprises Figure 23 A～Figure 23 C, is a series of illustrative diagram.Figure 23 A illustrates sugar that adds PEGization and the synoptic diagram that adds literalness sugar subsequently.Figure 23 B illustrates the synoptic diagram that will add to above a kind of sugar of modification on a kind of glycan.Figure 23 C illustrates sugar with different modifications to add synoptic diagram on the glycan that O-connects and the glycan that N-is connected to.

Figure 24 is the diagram of the whole bag of tricks that improves (comprise and puting together) the treatment function of peptide by glycan reconstruct.

Figure 25 is that a cover is to treating height and have a rest the synoptic diagram that (Gaucher) sick treatment peptide carries out glycan reconstruct.

Figure 26 carries out glycan reconstruct has the glycan of terminal Man-6-P part with generation synoptic diagram.

Figure 27 illustrates the glucocerebrosidase (Cerezyme that produces at CHO-after sialylated ^TM) on the glycan structures diagram of arranging.

Figure 28 comprises Figure 28 A～28Z and Figure 28 AA～28CC, is peptide tabulation used in the inventive method.

Figure 29 comprises Figure 29 A～29G, provides to be used for illustrative diagram that the glycan structures on the granulocyte colony-stimulating factor (G-CSF) is reconstructed.Figure 29 A describes G-CSF peptide and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown glycan institute bonded amino-acid residue.Figure 29 B～29G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and expection reconstruction step that the glycan of peptide among Figure 29 A is carried out.

Figure 30 comprises Figure 30 A～30EE, has provided the illustrative diagram that the glycan structures on the interferon alpha is reconstructed.Figure 30 A is the plain α isotype of description disturbance 14c peptide and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown glycan institute bonded amino-acid residue.Figure 30 B～30D is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 30 A is carried out.Figure 30 E is the plain α isotype of description disturbance 14c peptide and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown the amino-acid residue that glycan connected.Figure 30 F～30N is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 30 E is carried out.Figure 30 O is the plain α isotype 2a of description disturbance or 2b peptide and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown the amino-acid residue that glycan connected.Figure 30 P～30W is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 30 O is carried out.Figure 30 X is the diagram of the molecular formula of the plain α of description disturbance-Saliva Orthana fusogenic peptide and the exemplary glycan that connects thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 30 Y～30AA is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 30 X is carried out.Figure 30 BB is the diagram of the molecular formula of the plain α of description disturbance-Saliva Orthana fusogenic peptide and interferon alpha peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 30 CC～30EE is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 30 BB is carried out.

Figure 31 comprises Figure 31 A～31S, has provided the illustrative diagram that the glycan structures on the interferon beta is reconstructed.Figure 31 A is plain β peptide of description disturbance and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown the amino-acid residue that glycan connected.Figure 31 B～31O is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 31 A is carried out.Figure 31 P is plain β peptide of description disturbance and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown the amino-acid residue that glycan connected.Figure 31 Q～31S is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 31 P is carried out.

Figure 32 comprises Figure 32 A～32D, has provided the illustrative diagram that the glycan structures on proconvertin and the proconvertin a is reconstructed.Figure 32 A is the diagram of describing the molecular formula of proconvertin and proconvertin a peptide A (solid line) and B (dotted line) and glycan, has shown to be attached to the residue of expecting on the glycan that is reconstructed.Figure 32 B～32D is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 32 A is carried out.

Figure 33 comprises Figure 33 A～33G, has provided the illustrative diagram that the glycan structures on the plasma thromboplastin component is reconstructed.Figure 33 A is the diagram of describing the molecular formula of plasma thromboplastin component peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 33 B～33G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 33 A is carried out.

Figure 34 comprises Figure 34 A～34J, has provided the illustrative diagram that the glycan structures on the follicle stimulating hormone (FSH) (comprising α and β subunit) is reconstructed.Figure 34 A describes follicle stimulating hormone peptide FSH α and FSH β and the diagram of the molecular formula of the exemplary glycan of bonded thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 34 B～34J is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 34 A is carried out.

Figure 35 comprises Figure 35 A～35AA, has provided the illustrative diagram that the glycan structures on the erythropoietin (EPO) is reconstructed.Figure 35 A is the diagram of describing the molecular formula of EPO peptide and glycan, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 35 B～35S is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 35 A is carried out.Figure 35 T is the diagram of describing the molecular formula of EPO peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 35 U～35W is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 35 T is carried out.Figure 35 X is the diagram of describing the molecular formula of EPO peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 35 Y～35AA is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 35 X is carried out.

Figure 36 comprises Figure 36 A～36K, has provided the illustrative diagram that the glycan structures on the rHuGM-CSF (GM-CSF) is reconstructed.Figure 36 A is the diagram of describing the molecular formula of GM-CSF peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 36 B～36G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 36 A is carried out.Figure 36 H is the diagram of describing the molecular formula of GM-CSF peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 36 I～36K is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 36 H is carried out.

Figure 37 comprises Figure 37 A～37N, has provided the illustrative diagram that the glycan structures on the interferon-gamma peptide is reconstructed.Figure 37 A is the diagram of the molecular formula of plain γ peptide of description disturbance and the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 37 B～37G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 37 A is carried out.Figure 37 H is the diagram of the molecular formula of plain γ peptide of description disturbance and the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 37 I～37N is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 37 H is carried out.

Figure 38 comprises Figure 38 A～38N, has provided α ₁The illustrative diagram that glycan structures on the-antitrypsin (ATT or α-1 proteinase inhibitor) is reconstructed.Figure 38 A describes the ATT peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 38 B～38F is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 38 A is carried out.Figure 38 G describes the ATT peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 38 H～38J is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 38 G is carried out.Figure 38 K describes the ATT peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 38 L～38N is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 38 K is carried out.

Figure 39 comprises Figure 39 A～39J, has provided the illustrative diagram that the glycan structures on the glucocerebrosidase is reconstructed.Figure 39 A describes the glucocerebrosidase peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 39 B～39F is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 39 A is carried out.Figure 39 G describes the glucocerebrosidase peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 39 H～39K is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 39 G is carried out.

Figure 40 comprises Figure 40 A～40W, has provided the illustrative diagram that the glycan structures on the tissue type plasminogen activation factor (TPA) is reconstructed.Figure 40 A is the diagram of describing the molecular formula of TPA peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 40 B～40G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 40 A is carried out.Figure 40 H is the diagram of describing the molecular formula of TPA peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 40 I～40K is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 40 H is carried out.Figure 40 L is the diagram of describing the molecular formula of the TPA peptide of sudden change and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 40 M～40O is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 40 L is carried out.Figure 40 P is the diagram of describing the molecular formula of the TPA peptide of sudden change and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 40 Q～40S is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 40 P is carried out.Figure 40 T is the diagram of describing the molecular formula of the TPA peptide of sudden change and glycan, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 40 U～40W is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 40 T is carried out.

Figure 41 comprises Figure 41 A～41G, has provided the illustrative diagram that the glycan structures on the interleukin II (IL-2) is reconstructed.Figure 41 A describes interleukin II peptide and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown glycan institute bonded amino-acid residue.Figure 41 B～41G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 41 A is carried out.

Figure 42 comprises Figure 42 A～42M, has provided the illustrative diagram that the glycan structures on the blood coagulation factor VIII is reconstructed.Figure 42 A is the molecular formula that the N-that is attached to the blood coagulation factor VIII peptide connects the glycan of glycosylation site (A and A ') and O-connection site (B).Figure 42 B～42F is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 42 A is carried out.Figure 42 G is the molecular formula that the N-that is attached to the blood coagulation factor VIII peptide connects the glycan of glycosylation site (A and A ') and O-connection site (B).Figure 42 H～42M is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 42 G is carried out.

Figure 43 comprises Figure 43 A～43M, has provided the illustrative diagram that the glycan structures on the urokinase is reconstructed.Figure 43 A describes the urokinase peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 43 B～43F is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 43 A is carried out.Figure 43 G describes the urokinase peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 43 H～43L is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 43 G is carried out.

Figure 44 comprises Figure 44 A～44J, has provided the illustrative diagram that the glycan structures on the people DNase (hDNase) is reconstructed.Figure 44 A describes people DNase peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 44 B～44F is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 44 A is carried out.Figure 44 G describes people DNase peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 44 H～44J is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 44 F is carried out.

Figure 45 comprises Figure 45 A～45L, has provided the illustrative diagram that the glycan structures on the Regular Insulin is reconstructed.Figure 45 A contains the insulin peptide of N glycosylation site and the diagram of the molecular formula of the exemplary glycan of connection thereon after describing sudden change.Figure 45 B～45D is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 45 A is carried out.Figure 45 E describes Regular Insulin-Saliva Orthana fusogenic peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 45 F～45H is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 45 E is carried out.Figure 45 I is the diagram of describing the molecular formula of Regular Insulin-Saliva Orthana fusogenic peptide and insulin peptide and glycan, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 45 J～45L is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 45 I is carried out.

Figure 46 comprises Figure 46 A～46K, has provided the illustrative diagram that the glycan structures of the proteinic M-antigen of HBS (preS and S) on (HbsAg) is reconstructed.Figure 46 A is the diagram of describing the molecular formula of M-antigen peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 46 B～46G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 46 A is carried out.Figure 46 H is the diagram of describing the molecular formula of M-antigen peptide and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 46 I～46K is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 46 H is carried out.

Figure 47 comprises Figure 47 A～47K, has provided the illustrative diagram that the glycan structures on the human growth hormone (comprising N, V and variant thereof) is reconstructed.Figure 47 A describes the human growth hormone peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 47 B～47D is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 47 A is carried out.Figure 47 E is the diagram of describing 3 the part or all of fusogenic peptides comprise human growth hormone peptide and Saliva Orthana peptide and the molecular formula of the exemplary glycan that is connected thereon, has shown to be connected to the residue of expecting on the glycan that is reconstructed.Figure 47 F～47K is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 47 E is carried out.

Figure 48 comprises Figure 48 A～48G, has provided TNF acceptor-IgG Fc region fusion protein matter (Enbrel ^TM) on the illustrative diagram that is reconstructed of glycan structures.Figure 48 A describes the diagram can suddenly change with the molecular formula of the TNF acceptor-IgG Fc zone fusogenic peptide that contains extra N glycosylation site and glycan, has shown the residue that is attached on the glycan that expection is reconstructed.TNF acceptor peptide is described with thick line, and IgG Fc zone is described with ordinary lines.Figure 48 B～48G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 48 A is carried out.

Figure 49 comprises Figure 49 A～49D, has provided anti--HER2 monoclonal antibody (Herceptin ^TM) on the illustrative diagram that is reconstructed of glycan structures.Figure 49 A describes through sudden change to contain anti--HER2 monoclonal antibody of N glycosylation site and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue on the heavy chain of antibody that is connected on the glycan that expection is reconstructed.Figure 49 B～49D is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 49 A is carried out.

Figure 50 comprises Figure 50 A～50D, has provided the monoclonal antibody (Synagis to respiratory syncytial virus protein F ^TM) on the illustrative diagram that is reconstructed of glycan structures.Figure 50 A describes the diagram that contains the molecular formula of the monoclonal antibody of protein F peptide of N glycosylation site and the exemplary glycan that connects through sudden change thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 50 B～50D is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 50 A is carried out.

Figure 51 comprises Figure 51 A～51D, has provided the monoclonal antibody (Remicade to TNF-α ^TM) on the illustrative diagram that is reconstructed of glycan structures.Figure 51 A describes the monoclonal antibody of the TNF-α contain the N glycosylation site and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 51 B～51D is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that peptide among Figure 51 A is carried out.

Figure 52 comprises Figure 52 A～52L, has provided the monoclonal antibody (Reopro to glycoprotein iib/iiia ^TM) on the illustrative diagram that is reconstructed of glycan structures.Figure 52 A describes the diagram that contains the molecular formula of the monoclonal antibody of glycoprotein iib/iiia peptide of sudden change of N glycosylation site and the exemplary glycan that connects through sudden change thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 52 B～52D is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of the expection reconstruction step of carrying out.Figure 52 E describes the monoclonal antibody of glycoprotein iib/iiia-Saliva Orthana fusogenic peptide and the diagram of the molecular formula of the exemplary glycan that connects thereon, has shown the residue that is attached on the glycan that expection is reconstructed.Figure 52 F～52H is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of the expection reconstruction step of carrying out.Figure 52 I describes the monoclonal antibody of the monoclonal antibody of glycoprotein iib/iiia-Saliva Orthana fusogenic peptide and glycoprotein iib/iiia peptide and the diagram of the molecular formula of the exemplary glycan that is connected thereon, has shown to be attached to the residue of expecting on the glycan that is reconstructed.Figure 52 J～52L is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of the expection reconstruction step of carrying out.

Figure 53 comprises Figure 53 A～53G, has provided the monoclonal antibody (Rituxan to CD20 ^TM) on the illustrative diagram that is reconstructed of glycan structures.Figure 53 A describes the diagram that contains the molecular formula of the monoclonal antibody of CD20 of N glycosylation site and the exemplary glycan that connects through sudden change thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 53 B～53D is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 53 A is carried out.Figure 53 E describes the diagram that contains the molecular formula of the monoclonal antibody of CD20 of N glycosylation site and the exemplary glycan that connects through sudden change thereon, has shown the residue that is connected on the glycan that expection is reconstructed.Figure 53 F～53G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 53 E is carried out.

Figure 54 comprises Figure 54 A～54O, has provided the illustrative diagram that the glycan structures on the Antithrombin III (AT III) is reconstructed.Figure 54 A describes Antithrombin III peptide and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown the amino-acid residue on the glycan that is connected to the N-connection.Figure 54 B～54G is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 54 A is carried out.Figure 54 H describes Antithrombin III peptide and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown the amino-acid residue on the glycan that is connected to the N-connection.Figure 54 I～54K is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 54 H is carried out.Figure 54 L describes Antithrombin III peptide and the diagram of the molecular formula of the exemplary glycan of connection thereon, has shown the amino-acid residue on the glycan that is connected to the N-connection.Figure 54 M～54O is based on the type of the cell of expressing this peptide and the reconstruct glycan structures wanted and the diagram of expection reconstruction step that the glycan of peptide among Figure 54 L is carried out.

Figure 55 comprises Figure 55 A～55J, has provided the illustrative diagram that the glycan structures on human chorionic gonadotrophin (hCG) α and the β subunit is reconstructed.Figure 55 A is the diagram of describing hCG α and hCG β peptide, has shown the amino-acid residue that is attached on the glycan (B) that glycan (A) that N-that expection is reconstructed connects is connected with O-and the molecular formula of glycan.Figure 55 B～55J is based on the type of the cell of expressing this peptide and the diagram of the expection reconstruction step that the reconstruct glycan structures wanted carries out.

Figure 56 comprises Figure 56 A～56J, has provided alpha-galactosidase (Fabrazyme ^TM) on the illustrative diagram that is reconstructed of glycan structures.Figure 56 A is the diagram of describing the alpha-galactosidase A peptide, has shown the amino-acid residue that is attached on the glycan (A) that N-that expection is reconstructed connects and the molecular formula of glycan.Figure 56 B～56J is based on the type of the cell of expressing this peptide and the diagram of the expection reconstruction step that the reconstruct glycan structures wanted carries out.

Figure 57 comprises Figure 57 A～57J, has provided α-idose glycosides enzyme (iduronidase) (Aldurazyme ^TM) on the illustrative diagram that is reconstructed of glycan structures.Figure 57 A is the diagram of describing α-idose glycosides enzyme peptide, has shown the amino-acid residue that is attached on the glycan (A) that N-that expection is reconstructed connects and the molecular formula of glycan.Figure 57 B～57J is based on the type of the cell of expressing this peptide and the diagram of the expection reconstruction step that the reconstruct glycan structures wanted carries out.

Figure 58 comprises Figure 58 A and 58B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:1 and 2) of granulocyte colony-stimulating factor (G-CSF).

Figure 59 comprises Figure 59 A and 59B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:3 and 4) of interferon alpha (IFN-α).

Figure 60 comprises Figure 60 A and 60B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:5 and 6) of interferon beta (IFN-β).

Figure 61 comprises Figure 61 A and 61B, is Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:7 and 8) of proconvertin a.

Figure 62 comprises Figure 62 A and 62B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:9 and 10) of plasma thromboplastin component.

Figure 63 comprises Figure 63 A～63D, is respectively the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ IDNO:11～14) of follicle stimulating hormone (FSH).

Figure 64 comprises Figure 64 A and 64B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:15 and 16) of erythropoietin (EPO).

Figure 65 is i.e. 165 the amino acid whose aminoacid sequences (SEQ ID NO:73) of ripe EPO.

Figure 66 comprises Figure 66 A and 66B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ IDNO:17 and 18) of granulocyte-macrophage colony stimutaing factor (GM-CSF).

Figure 67 comprises Figure 67 A and 67B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:19 and 20) of interferon-gamma (IFN-γ).

Figure 68 comprises Figure 68 A and 68B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:21 and 22) of α-1 proteinase inhibitor (A-1-PI or alpha antitrypsin).

Figure 69 comprises Figure 69 A-1～69A-2 and 69B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:23 and 24) of glucocerebrosidase.

Figure 70 comprises Figure 70 A and 70B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:25 and 26) of tissue type plasminogen activation factor (TPA).

Figure 71 comprises Figure 71 A and 71B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:27 and 28) of interleukin II (IL-2).

Figure 72 comprises Figure 72 A-1～71A-4 and 72B-1～72B-4, is respectively the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:29 and 30) of blood coagulation factor VIII.

Figure 73 comprises Figure 73 A and 73B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:33 and 34) of urokinase.

Figure 74 comprises Figure 74 A and 74B, is recombinate Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:39 and 40) of DNase (hrDNase) of people.

Figure 75 comprises Figure 75 A and 75B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:43 and 44) of insulin molecule.

Figure 76 comprises Figure 76 A and 76B, is Exemplary core thuja acid and corresponding aminoacid sequence (being respectively SEQ ID NO:45 and 46) from the S-protein (HBsAg) of hepatitis B virus.

Figure 77 comprises Figure 77 A and 77B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:47 and 48) of human growth hormone (hGH).

Figure 78 comprises Figure 78 A and 78D, is the Exemplary core thuja acid and the corresponding aminoacid sequence of Antithrombin III.78A and 78B are the Exemplary core thuja acid and the corresponding aminoacid sequences (being respectively SEQ ID NO:63 and 64) of " wild-type (WT) " Antithrombin III.

Figure 79 comprises Figure 79 A～79D, is the Exemplary core thuja acid and the corresponding aminoacid sequence of human chorionic gonadotrophin (hCG) α and β subunit.79A and 79B are the Exemplary core thuja acid and the corresponding aminoacid sequences (being respectively SEQ ID NO:69 and 70) of human chorionic gonadotrophin α subunit.79C and 79D are the Exemplary core thuja acid and the corresponding aminoacid sequences (being respectively SEQ ID NO:71 and 72) of human chorion gonadotrophic hormone beta subunit.

Figure 80 comprises Figure 80 A and 80B, is the Exemplary core thuja acid and the corresponding aminoacid sequence of α-idose glycosides enzyme.(being respectively SEQ ID NO:65 and 66).

Figure 81 comprises Figure 81 A and 81B, is the Exemplary core thuja acid and the corresponding aminoacid sequence of alpha-galactosidase.(being respectively SEQ ID NO:67 and 68).

Figure 82 comprises Figure 82 A and 82B, is to comprise Enbrel ^TMThe Exemplary core thuja acid and the amino acid sequence corresponding (being respectively SEQ ID NO:31 and 32) of 75kDa tumour necrosis factor (TNF-R) of part (Tumor Necrosis Factor Receptors (TNF-R)/IgG fusions).

Figure 83 comprises Figure 83 A and 83B, is respectively Herceptin ^TMThe Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:35 and 36) of the light and heavy chain of (monoclonal antibody of anti-Human epidermal growth factor receptor Her-2 (MAb)).

Figure 84 comprises Figure 84 A and 84B, is respectively Synagis ^TMThe weight of (MAb of respiratory syncystial virus F peptide) and the Exemplary core thuja acid of light chain and corresponding aminoacid sequence (being respectively SEQIDNO:37 and 38).

Figure 85 comprises Figure 85 A and 85B, is Remicade ^TMThe Exemplary core thuja acid of the inhuman variable region of (MAb of anti-TNF alpha) and corresponding aminoacid sequence (being respectively SEQ ID NO:41 and 42).

Figure 86 comprises Figure 86 A and 86B, is the Exemplary core thuja acid and the corresponding aminoacid sequence (being respectively SEQ ID NO:49 and 50) of the Fc part of human IgG.

Figure 87 is the exemplary amino acid sequence (SEQ ID NO:52) of the ripe variable region of the murine antibody light chain of anti-glycoprotein iib/iiia.

Figure 88 is the exemplary amino acid sequence (SEQ ID NO:54) of the ripe variable region of the murine antibody heavy chain of anti-glycoprotein iib/iiia.

Figure 89 is the exemplary amino acid sequence (SEQ ID NO:51) of human IgG variable region light chain.

Figure 90 is the exemplary amino acid sequence (SEQ ID NO:53) of human IgG variable region heavy chain.

Figure 91 is the exemplary amino acid sequence (SEQ ID NO:55) of human IgG light chain.

Figure 92 is the exemplary amino acid sequence (SEQ ID NO:56) of human IgG heavy chain.

Figure 93 comprises Figure 93 A and 93B, is the Exemplary core thuja acid and the amino acid sequence corresponding (being respectively SEQ ID NO:59 and 60) of ripe variable region of the murine antibody light chain of anti-CD 20.

Figure 94 comprises Figure 94 A and 94B, is the Exemplary core thuja acid and the amino acid sequence corresponding (being respectively SEQ ID NO:61 and 62) of ripe variable region of the murine antibody heavy chain of anti-CD 20.

Figure 95 comprises Figure 95 A～95E, is the nucleotide sequence (SEQ ID NO:57) of series connection chimeric antibody expression vector TCAE8.

Figure 96 comprises Figure 96 A～96E, is the nucleotide sequence (SEQ ID NO:58) that comprises the series connection chimeric antibody expression vector TCAE8 of the murine antibody light chain of anti-CD 20 and weight chain variable structural domain.

Figure 97 comprises Figure 97 A～97C, is the graphic representation of describing the 2-AA HPLC analysis of the glycan that the Cri-IgG1 antibody of expressing from myelomatosis by PNGaseF is discharged.The structure of glycan is determined by retention time: G0 sugar form (glycoform) was at 30 minutes wash-outs, and the G1 sugar form was at～33 minutes wash-outs, and the G2 sugar form was at about 37 minutes wash-outs, and the S1-G1 sugar form was at～70 minutes wash-outs.Figure 97 A has described the analysis to DEAE antibody sample.Figure 97 B has described the analysis to SPA antibody sample.Figure 97 C has described the analysis to Fc antibody sample.In table 14, summed up the area percent under the peak in these graphic representations.

Figure 98 comprises Figure 98 A～98C, is the graphic representation of describing the maldi analysis of the glycan that the Cri-IgG1 antibody of expressing from myelomatosis by PNGaseF is discharged.Glycan carries out derivatize with 2-AA and analyzes by MALDI then.Figure 98 A has described the analysis to DEAE antibody sample.Figure 98 B has described the analysis to SPA antibody sample.Figure 98 C has described the analysis to Fc antibody sample.

Figure 99 comprises Figure 99 A～99D, is the graphic representation of describing the capillary electrophoresis analysis of the glycan that discharges from Cri-IgG1 antibody, this antibody has been carried out sugared reconstruct (glycoremodeled) so that it contains the M3N2 sugar form.In Figure 99 A, shown graphic representation to the capillary electrophoresis analysis of the glycan standard substance that carries out derivatize with APTS.Figure 99 B has described the analysis to DEAE antibody sample.Figure 99 C has described the analysis to SPA antibody sample.Figure 99 D has described the analysis to Fc antibody sample.In table 15, summed up the area percent under the peak in these graphic representations.

Figure 100 comprises Figure 100 A～100D, is the graphic representation of describing the capillary electrophoresis analysis of the glycan that discharges from Cri-IgG1 antibody, this antibody has been carried out sugared reconstruct so that it contains the G0 sugar form.In Figure 100 A, shown the graphic representation of capillary electrophoresis analysis that carries out the glycan standard substance of derivatize with APTS.Figure 100 B has described the analysis to DEAE antibody sample.Figure 100 C has described the analysis to SPA antibody sample.Figure 100 D has described the analysis to Fc antibody sample.In table 16, summed up the area percent under the peak in these graphic representations.

Figure 101 comprises Figure 101 A～101C, is to describe the graphic representation that the 2-AA HPLC to the glycan that discharges from Cri-IgG1 antibody analyzes, and this antibody has been carried out sugared reconstruct so that it contains the G0 sugar form.The glycan that discharges carries out mark with 2AA, and separates on the NH2P-504D nh 2 column by HPLC.Figure 101 A has described the analysis to DEAE antibody sample.Figure 101 B has described the analysis to SPA antibody sample.Figure 101 C has described the analysis to Fc antibody sample.In table 16, summed up the area percent under the peak in these graphic representations.

Figure 102 comprises Figure 102 A～102C, is the graphic representation of describing the maldi analysis of the glycan that discharges from Cri-IgG1 antibody, this antibody has been carried out sugared reconstruct so that it contains the G0 sugar form.The glycan that discharges carries out derivatize with 2-AA and analyzes by MALDI then.Figure 102 A has described the analysis to DEAE antibody sample.Figure 102 B has described the analysis to SPA antibody sample.Figure 102 C has described the analysis to Fc antibody sample.

Figure 103 comprises Figure 103 A～103D, is the graphic representation of describing the capillary electrophoresis analysis of the glycan that discharges from Cri-IgG1 antibody, this antibody has been carried out sugared reconstruct so that it contains the G2 sugar form.In Figure 103 A, shown the graphic representation of capillary electrophoresis analysis that carries out the glycan standard substance of derivatize with APTS.Figure 103 B has described the analysis to DEAE antibody sample.Figure 103 C has described the analysis to SPA antibody sample.Figure 103 D has described the analysis to Fc antibody sample.In table 17, summed up the area percent under the peak in these graphic representations.

Figure 104 comprises Figure 104 A～104C, is to describe the graphic representation that the 2-AA HPLC to the glycan that discharges from Cri-IgG1 antibody analyzes, and this antibody has been carried out sugared reconstruct so that it contains the G2 sugar form.The glycan that discharges carries out mark with 2AA, and separates on the NH2P-504D nh 2 column by HPLC.Figure 104 A has described the analysis to DEAE antibody sample.Figure 104 B has described the analysis to SPA antibody sample.Figure 104 C has described the analysis to Fc antibody sample.In table 17, summed up the area percent under the peak in these graphic representations.

Figure 105 comprises Figure 105 A～105C, is the graphic representation of describing the maldi analysis of the glycan that discharges from Cri-IgG1 antibody, this antibody has been carried out sugared reconstruct so that it contains the G2 sugar form.The glycan that discharges carries out derivatize with 2-AA and analyzes by MALDI then.Figure 105 A has described the analysis to DEAE antibody sample.Figure 105 B has described the analysis to SPA antibody sample.Figure 105 C has described the analysis to Fc antibody sample.

Figure 106 comprises Figure 106 A～106D, is the graphic representation of describing the capillary electrophoresis analysis of the glycan that discharges from Cri-IgG1 antibody, handles this antibody has been carried out sugared reconstruct by the M3N2 sugar form being carried out GnT-I.In Figure 106 A, shown the graphic representation of capillary electrophoresis analysis that carries out the glycan standard substance of derivatize with APTS.Figure 106 B has described the analysis to DEAE antibody sample.Figure 106 C has described the analysis to SPA antibody sample.Figure 106 D has described the analysis to Fc antibody sample.

Figure 107 comprises Figure 107 A～107C, is to describe the graphic representation that the 2-AA HPLC to the glycan that discharges from Cri-IgG1 antibody analyzes, and handles this antibody has been carried out reconstruct by the M3N2 sugar form being carried out GnT-I.The glycan that discharges carries out mark with 2AA, and separates on the NH2P-504D nh 2 column by HPLC.Figure 107 A has described the analysis to DEAE antibody sample.Figure 107 B has described the analysis to SPA antibody sample.Figure 107 C has described the analysis to Fc antibody sample.

Figure 108 comprises Figure 108 A～108C, is the graphic representation of describing the maldi analysis of the glycan that discharges from Cri-IgG1 antibody, handles this antibody has been carried out sugared reconstruct by the M3N2 sugar form being carried out GnT-I.The glycan that discharges carries out derivatize with 2-AA and analyzes by MALDI then.Figure 108 A has described the analysis to DEAE antibody sample.Figure 108 B has described the analysis to SPA antibody sample.Figure 108 C has described the analysis to Fc antibody sample.

Figure 109 comprises Figure 109 A～109D, is the graphic representation of describing the capillary electrophoresis analysis of the glycan that discharges from Cri-IgG1 antibody, handles this antibody has been carried out sugared reconstruct by the M3N2 sugar form being carried out GnT-I, II and III.In Figure 109 A, shown the graphic representation of capillary electrophoresis analysis that carries out the glycan standard substance of derivatize with APTS.Figure 109 B has described the analysis to DEAE antibody sample.Figure 109 C has described the analysis to SPA antibody sample.Figure 109 D has described the analysis to Fc antibody sample.In table 18, summed up the area percent under the peak in these graphic representations.

Figure 110 comprises Figure 110 A～110C, is to describe the graphic representation that the 2-AA HPLC to the glycan that discharges from Cri-IgG1 antibody analyzes, and handles this antibody has been carried out sugared reconstruct by the M3N2 sugar form being carried out GnT-I, II and III.The glycan that discharges carries out mark with 2AA, and separates on the NH2P-504D nh 2 column by HPLC.Figure 110 A has described the analysis to DEAE antibody sample.Figure 110 B has described the analysis to SPA antibody sample.Figure 110 C has described the analysis to Fc antibody sample.In table 18, summed up the area percent under the peak in these graphic representations.

Figure 111 comprises Figure 111 A～111C, is the graphic representation of describing the maldi analysis of the glycan that discharges from Cri-IgG1 antibody, handles this antibody has been carried out sugared reconstruct by the NGA2F sugar form being carried out galactosyltransferase.The glycan that discharges carries out derivatize with 2-AA and analyzes by MALDI then.Figure 111 A has described the analysis to DEAE antibody sample.Figure 111 B has described the analysis to SPA antibody sample.Figure 111 C has described the analysis to Fc antibody sample.

Figure 112 comprises Figure 112 A～112D, is that (Figure 112 A and 112C) and GalT1 handle the graphic representation of back (Figure 112 B and 112D) to the 2-AA HPLC analysis of the glycan that discharges from the Cri-IgG1 antibody that contains the NGA2F isotype before being described in GalT1 and handling.Figure 112 A and 112B have described the analysis to DEAE antibody sample.Figure 112 C and 112D have described the analysis to Fc antibody sample.The glycan that discharges carries out mark with 2AA, and separates on the NH2P-504D nh 2 column by HPLC.

Figure 113 comprises Figure 113 A～113C, is to describe the graphic representation that the 2-AA HPLC to the glycan that discharges from Cri-IgG1 antibody analyzes, and handles this antibody has been carried out sugared reconstruct by the G2 sugar form being carried out ST3Gal3.The glycan that discharges carries out mark with 2AA, and separates on the NH2P-504D nh 2 column by HPLC.Figure 113 A has described the analysis to DEAE antibody sample.Figure 113 B has described the analysis to SPA antibody sample.Figure 113 C has described the analysis to Fc antibody sample.In table 19, summed up the area percent under the peak in these graphic representations.

Figure 114 comprises Figure 114 A～114C, is the graphic representation of describing the maldi analysis of the glycan that discharges from Cri-IgG1 antibody, handles this antibody has been carried out sugared reconstruct by the G2 sugar form being carried out ST3Gal3.The glycan that discharges carries out derivatize with 2-AA, analyzes by MALDI then.Figure 114 A has described the analysis to DEAE antibody sample.Figure 114 B has described the analysis to SPA antibody sample.Figure 114 C has described the analysis to Fc antibody sample.

Figure 115 comprises Figure 115 A～115D, is the graphic representation of describing the capillary electrophoresis analysis of the glycan that discharges from Cri-IgG1 antibody, handles this antibody has been carried out sugared reconstruct by the G2 sugar form being carried out ST6Gal1.In Figure 115 A, shown the graphic representation of capillary electrophoresis analysis that carries out the glycan standard substance of derivatize with APTS.Figure 115 B has described the analysis to DEAE antibody sample.Figure 115 C has described the analysis to SPA antibody sample.Figure 115 D has described the analysis to Fc antibody sample.

Figure 116 comprises Figure 116 A～116C, is to describe the graphic representation that the 2-AA HPLC to the glycan that discharges from Cri-IgG1 antibody analyzes, and handles this antibody has been carried out sugared reconstruct by the G2 sugar form being carried out ST6Gal1.The glycan that discharges carries out mark with 2AA, and separates on NH2P-50 4D nh 2 column by HPLC.Figure 116 A has described the analysis to DEAE antibody sample.Figure 116 B has described the analysis to SPA antibody sample.Figure 116 C has described the analysis to Fc antibody sample.

Figure 117 comprises Figure 117 A～117C, is the graphic representation of describing the maldi analysis of the glycan that discharges from Cri-IgG1 antibody, handles this antibody has been carried out sugared reconstruct by the G2 sugar form being carried out ST6Gal1.The glycan that discharges carries out derivatize with 2-AA and analyzes by MALDI then.Figure 117 A has described the analysis to DEAE antibody sample.Figure 117 B has described the analysis to SPA antibody sample.Figure 117 C has described the analysis to Fc antibody sample.

Figure 118 comprises Figure 118 A～118E, and the SDS-PAGE that has described under non-reduced condition the Cri-IgG1 antibody that carries out sugared reconstruct with the different sugar form analyzes image.Bovine serum albumin (BSA) moves as quantitation standard under non-reduced condition.Shown the protein molecular weight standard thing, and its size shows with kDa.Figure 118 A describes and to analyze with the DEAE, the SPA that contain G0 and G2 sugar form and the SDS-PAGE of Fc Cri-IgG1 antibody having carried out sugared reconstruct.Figure 118 B describes having carried out sugared reconstruct with the DEAE, the SPA that contain NGA2F (binary) and GnT-I-M3N2 (GnT1) sugar form and the SDS-PAGE analysis of Fc Cri-IgG1 antibody.Figure 118 C describes and to analyze with the DEAE, the SPA that contain S2G2 (ST6Gal1) sugar form and the SDS-PAGE of Fc Cri-IgG1 antibody having carried out sugared reconstruct.Figure 118 D describes and to analyze with the DEAE, the SPA that contain the M3N2 sugar form and the SDS-PAGE of Fc Cri-IgG1 antibody and BSA having carried out sugared reconstruct.Figure 118 E describes and to analyze with the DEAE, the SPA that contain Gal-NGA2F (Gal-is binary) sugar form and the SDS-PAGE of Fc Cri-IgG1 antibody and BSA having carried out sugared reconstruct.

Figure 119 is an acrylamide gel image of describing the FACE analytical results of sialylated front and back TP10.BiNA ₀Kind does not have sialic acid residues.BiNA ₁Kind has 1 sialic acid residues.BiNA ₂Kind has 2 sialic acid residueses.The two feelers of Bi=; The NA=neuraminic acid.

Figure 120 is plasma concentration (μ the g/ml)-time plot that is injected into the TP10 of the sialylated front and back in the rat.

Figure 121 is a graphic representation of describing the area (AUC) under plasma concentration-time curve for the TP10 of sialylated front and back with μ g/hr/ml.

Figure 122 is an acrylamide gel image of describing the FACE glycan analysis result of the FACE glycan analysis of fucosylated front and back TP10 and the TP-20 that Chinese hamster ovary celI produces.BiNA ₂F ₂Kind has 2 neuraminic acid (NA) residues and 2 fucosyl residues (F).

Figure 123 is described in external (rhombus) and (square) glycosylated TP20 (sCR1sLe in the body in the Lec11 Chinese hamster ovary celI ^X) external bonded graphic representation.

Figure 124 is the graphic representation of describing from the 2-AA HPLC analysis of the sugar form of the GlcNAcization of EPO.

Figure 125 comprises Figure 125 A and 125B, is the graphic representation of describing the 2-AA HPLC analysis of 2 crowdes of EPO, has added N-acetyl-glucosamine in this EPO.Figure 125 A has described the analysis that A is criticized, and Figure 125 B has described the analysis that B is criticized.

Figure 126 describes introduce the graphic representation of the 2-AA HPLC analysis of the 3rd glycan ramose reaction product to EPO with GnT-V.

Figure 127 describes with GnT-I, GnT-II, GnT-III, GnT-V and GalT1 and suitable donor to handle afterwards the MALDI-TOF of the glycan of the EPO preparation line chart of setting a song to music.

Figure 128 is a graphic representation of describing the glycan MALDI spectrum of natural EPO.

Figure 129 is the SDS-PAGE gel images with the PEGization reaction product of CMP-SA-PEG (1kDa) and CMP-SA-PEG (10kDa).

Figure 130 is a graphic representation of describing the EPO vitro bioassay result of PEGization.The rhombus representative is from the data of the sialylated EPO that does not have the PEG molecule.The data that the square representative obtains with the EPO with PEG (1kDa).The data that the trilateral representative obtains with the EPO with PEG (10kDa).

Figure 131 is the synoptic diagram of the EPO of CHO-expression.EPO peptide length is 165 amino acid, and not having glycosylated molecular weight is 18kDa.The EPO molecular weight of the glycosylation form that produces in Chinese hamster ovary celI is about 33kDa～39kDa.The shape of sugar provides in the frame on figure base in the expression polysaccharide chains.

Figure 132 is the synoptic diagram of the EPO of insect cell expression.The shape of sugar provides in the frame on Figure 131 base in the expression polysaccharide chains.

Figure 133 is the bar chart of molecular weight that is described in the EPO peptide of expressed in insect cells, that this EPO peptide is carried out reconstruct is single completely to form-, two-and three feeler glycan, and optionally carry out Glycopegylated with 1kDa, 10kDa or 20kDa PEG.Epoetin ^TMBe to be expressed in the mammalian cell and not carry out that further glycan is modified or the EPO of PEGization.NESP (Aranesp ^TM, Amgen, Thousand Oaks CA) is the EPO form with glycan site that 5 N-connect, it also is expressed in the mammalian cell and does not carry out further glycan and modify or PEGization.

Figure 134 comprises Figure 134 A and 134B, and the EPO that describes insect cell expression is reconstructed and Glycopegylated a kind of scheme.Figure 134 A describes reconstruct and the Glycopegylated step that the glycan that insect is expressed is reconstructed into the Glycopegylated glycan of single feeler.Figure 134 B is described in the EPO peptide that glycan site that each N-of polypeptide connects all has the reconstruct of Glycopegylated completely single feeler glycan.The shape of sugar provides in the frame on Figure 131 base in the expression polysaccharide chains, and just trilateral is represented sialic acid.

Figure 135 describes EPO-SA and the active graphic representation of EPO-SA-PEG construct external biological.External test is measured the propagation of keeping 48 hours TF-1 erythroleukemia cell after adding 10.0,5.0,2.0,1.0,0.5 and 0 μ g/ml EPO construct in RBMI+FBS 10%+GM-CSF (12ng/ml).Tri-SA refers to that glycan is three feelers and EPO construct that have SA.Tri-SA1K PEG refer to glycan be three feelers and have Gal and use the Glycopegylated EPO construct of SA-PEG1kDa then.Di-SA 10K PEG refer to glycan be two feelers and have Gal and use the Glycopegylated EPO construct of SA-PEG 10kDa then.Di-SA 1K PEG refer to glycan be two feelers and have Gal and use the Glycopegylated EPO construct of SA-PEG 1kDa then.Di-SA refers to that glycan is two feeler and EPO constructs that be building up to SA.Epogen ^TMBe to be expressed in not have the EPO that further glycan is modified in the Chinese hamster ovary celI.

Figure 136 is a graphic representation of describing the pharmacokinetics of EPO construct in the rat.Rat is used [I ¹²⁵Glycopegylated and the non-Glycopegylated EPO of]-mark injects.Graphic representation shows the concentration of radiolabeled EPO in the rat blood flow of injection back 0～about 72 minutes." Biant-10K " refers to have the EPO of two feeler glycan structures, and this pair feeler glycan structures has terminal 10kDa peg moiety." Mono-20K " refers to have the EPO of single feeler glycan structures, and this list feeler glycan structures has terminal 20kDa peg moiety.NESP refers to the commercial Aranesp that buys." Biant-1K " refers to have the EPO of two feeler glycan structures, and this pair feeler glycan structures has terminal 1kDa peg moiety." Biant-SA " refers to have the EPO of two feeler glycan structures, and this pair feeler glycan structures has terminal 1kDa part.In the time of 72 hours in the blood flow concentration of EPO construct as follows: Biant-10K, 5.1cpm/ml; Mono-20K, 3.2cpm/ml; NESP, 1cpm/ml; And Biant-1K, 0.2cpm/ml; Biant-SA, 0.1cpm/ml.Relative area in the EPO construct under the curve is as follows: Biant-10K, 2.9; Mono-20K, 2.1; NESP, 1; Biant-1K, 0.5; And Biant-SA, 0.2.

Figure 137 describes the bar chart that the EPO construct stimulates reticulosis (reticulocytosis) ability in vivo.Each treatment group is made up of 8 mouse.Give the single subcutaneous injection of mouse 10 μ g protein/kg body weight.In the time of 96 hours, measure reticulosis percentage ratio.Three feelers-SA2,3 (6) constructs have with 2,3 or 2,6 key bonded SA molecules (referring to the preparation embodiment 18 of this paper), and wherein the glycan on the EPO is the three feeler glycan that are attached with SA-PEG 10K thereon.Similarly, two feelers-10K PEG is the EPO with two feeler N-glycan of the SA-PEG that is attached with 10K PEG thereon.

Figure 138 describes the graphic representation that the EPO construct increases the hematocrit ability of mouse blood in vivo.The CD-1 female mice is carried out intraperitoneal (i.p.) injection with 2.5 μ g protein/kg body weight.The hematocrit of measuring mouse on the 15th after the EPO injection.Bi-1K refer to glycan be two feelers and be building up to (built out to) Gal and use the Glycopegylated EPO construct of SA-PEG 1kDa then.Mono-20K refer to glycan be single feeler and be building up to Gal and use the Glycopegylated EPO construct of SA-PEG 20kDa then.

Figure 139 comprises Figure 139 A and 139B, describes from the EPO of expressed in insect cells (Protein Sciences, Lot#060302) analysis of the glycan that discharges of enzymatic.The HPLC that Figure 139 A describes the glycan that discharges analyzes.Figure 139 B describes the maldi analysis to the glycan that discharges.Rhombus is represented Fucose, square expression GlcNAc, and circle is represented seminose.

Figure 140 is described in the maldi analysis of GnT-I/GalT-1 reaction back from the glycan of EPO release.The structure of glycan by with the standard glycan carry out peak spectrum relatively come to determine.The structure delineation of glycan is on the next door at peak.Rhombus is represented Fucose, and square expression GlcNAc, and circle is represented seminose, and star is represented semi-lactosi.

Figure 141 be described in GnT-I/GalT-1 reaction, Superdex 75 purifying, with the ST3Gal3 reaction of SA-PEG (10kDa) and SA-PEG (20kDa) after, to the SDS-PAGE analysis of EPO.

Figure 142 describes the biological result who measures of the TF-1 cells in vitro of the single feeler EPO of PEGization.

Figure 143 comprises Figure 143 A and 143B, is described in the analysis of GnT-I/GnT-II reaction back from the glycan of EPO release.The HPLC that Figure 143 A describes the glycan that discharges analyzes, wherein the two feeler GlcNAc glycan of peak 3 expressions.Figure 143 B describes the maldi analysis to the glycan that discharges.The structure of glycan by with the standard glycan carry out peak spectrum relatively come to determine.The structure delineation of glycan is on the next door at peak.Rhombus is represented Fucose, and square expression GlcNAc, and circle is represented seminose.

Figure 144 comprises Figure 144 A and 144B, is described in the HPLC analysis of GalT-1 reaction back from the glycan of EPO release.Figure 144 A is described in the glycan of GalT-1 reaction back release on a small scale.Figure 144 B is described in the glycan that extensive GalT-1 reaction back discharges.In two figure, peak 1 is the two feeler glycan with terminal galactose part, and peak 2 is the two feeler glycan with terminal galactose part.

Figure 145 is described in Superdex 75 chromatographic separation of GalT-1 reaction back to the EPO kind.Peak 2 contains the EPO with two feeler glycan, and this glycan has terminal galactose moiety.

Figure 146 describes the SDS-PAGE analysis of each product of sugared restructuring procedure that preparation is had two feeler glycan of terminal galactose part.

Figure 147 be described in ST3Gal3 sialylated or carry out PEGization with SA-PEG (1kDa) and SA-PEG (10kDa) after, the SDS-PAGE of EPO is analyzed.

Figure 148 is described in the HPLC analysis of GnT-I/GnT-II reaction back from the glycan of EPO release.The structure of glycan relatively comes to determine by what carry out with the standard glycan that the peak is detained.The structure delineation of glycan is on the next door at peak.Rhombus is represented Fucose, and square expression GlcNAc, and circle is represented seminose.

Figure 149 is described in the HPLC analysis of GnT-V reaction back from the glycan of EPO release.The structure of glycan relatively comes to determine by what carry out with the standard glycan that the peak is detained.The structure delineation of glycan is on the next door at peak.Rhombus is represented Fucose, and square expression GlcNAc, and circle is represented seminose.

Figure 150 is described in the HPLC analysis of GalT-1 reaction back from the glycan of EPO release.The structure of glycan relatively comes to determine by what carry out with the standard glycan that the peak is detained.The structure delineation of glycan is on the next door at peak.Rhombus is represented Fucose, and square expression GlcNAc, and circle is represented seminose, and empty circles is represented semi-lactosi, and trilateral is represented sialic acid.

Figure 151 is described in the HPLC analysis of ST3Gal3 reaction back from the glycan of EPO release.The structure of glycan relatively comes to determine by what carry out with the standard glycan that the peak is detained.The structure delineation of glycan is on the next door at peak.Rhombus is represented Fucose, and square expression GlcNAc, and circle is represented seminose, and empty circles is represented semi-lactosi, and trilateral is represented sialic acid.

Figure 152 is described in the HPLC analysis of ST6Gal1 reaction back from the glycan of EPO release.The structure of glycan relatively comes to determine by what carry out with the standard glycan that the peak is detained.The structure delineation of glycan is on the next door at peak.

Figure 153 describes the biological result who measures of the TF-1 cells in vitro of the EPO with two feelers and three feeler glycan." Di-SA " refers to the terminal sialic EPO with two feeler glycan that is." Di-SA10K PEG " refers to the terminal sialic EPO with two feeler glycan with PEG (10kDa) derivatize that is." Di-SA 1K PEG " refers to the terminal sialic EPO with two feeler glycan with PEG (1kDa) derivatize that is." Tri-SA ST6+ST3 " refers to that terminal is that 3-SA adds 2 of cap, the EPO with three feeler glycan of 6-SA with 2." Tri-SA ST3 " refers to that end is 2, the EPO with three feeler glycan of 3-SA.

Figure 154 is the IEF gel images of pI of describing the product of asialylated

program.Swimming lane

1 and 5 is IEF standard substances.Swimming lane 2 is a plasma thromboplastin component protein.Swimming lane 3 is a rfactorIX protein.Swimming lane 4 be rFactor IX protein 20 hours take off the sialic acid reaction.

Figure 155 is the SDS-PAGE gel images that is described in the molecular weight of the plasma thromboplastin component of puting together with CMP-SA-PEG reaction back and SA-PEG (1kDa) or SA-PEG (10kDa

).Swimming lane

1 and 6 is SeeBlue+2 molecular weight standards.Swimming lane 2 is rF-IX.Swimming lane 3 is asialylated rF-IX.Swimming lane 4 is the rFactor IX that put together with SA-PEG (1kDa).Swimming lane 5 is the rFactor IX that put together with SA-PEG (10kDa).

Figure 156 is the SDS-PAGE gel images of sialic acid capping product of describing the direct sialylated and plasma thromboplastin component-SA-PEG of plasma thromboplastin component.Swimming lane 1 is the protein standard substance, and swimming lane 2 is blank; Swimming lane 3 is rFactor-IX; Swimming lane 4 is rFactor-IX-SA-PEG (10kDa) that SA adds cap; Swimming lane 5 is rFactor-IX-SA-PEG (10kDa); Swimming lane 6 is ST3Gal1; Swimming lane 7 is ST3Gal3; Swimming lane the 8,9, the 10th does not carry out the pretreated rFactor-IX-SA-PEG of sialidase (10kDa).

Figure 157 is the image that takes off the isoelectrofocusing gel (pH3-7) of sialic acid (asialo)-proconvertin a.Swimming lane 1 is rFactor VIIa; Swimming lane 2-5 takes off sialic acid-proconvertin a.

Figure 158 is the graphic representation of the MALDI spectrum of proconvertin a.

Figure 159 is the graphic representation of the MALDI spectrum of proconvertin a-PEG (1kDa).

Figure 160 is a graphic representation of describing the MALDI spectrum of proconvertin a-PEG (10kDa).

Figure 161 is the SDS-PAGE gel images of the proconvertin a of PEGization.Swimming lane 1 is to take off sialic acid-proconvertin a.Swimming lane 2 is to take off sialic acid-proconvertin a and CMP-SA-PEG (1kDa) and the product of ST3Gal3 after reacting 48 hours.Swimming lane 3 is to take off sialic acid-proconvertin a and CMP-SA-PEG (1kDa) and the product of ST3Gal3 after reacting 48 hours.Swimming lane 4 is to take off sialic acid-proconvertin a and CMP-SA-PEG (10kDa) and the product of ST3Gal3 after reacting 96 hours.

Figure 162 is an image of describing isoelectrofocusing (IEF) gel of the asialylated reaction product of people hypophysis

FSH.Swimming lane

1 and 4 is standard substances of isoelectrofocusing (IEF).Swimming lane 2 is natural FSH.Swimming lane 3 is asialylated FSH.

Figure 163 is the SDS-PAGE gel images of product of the reaction of the sialylated rFSH of preparation PEG

-.Swimming lane

1 and 8 is SeeBlue+2 molecular weight standard things.Swimming lane 2 is the natural FSH of 15 μ g.Swimming lane 3 is that 15 μ g take off sialic acid-FSH (AS-FSH).Swimming lane 4 is products of 15 μ gAS-FSH and CMP-SA reaction.Swimming lane 5 is products of 15 μ g AS-FSH and CMP-SA-PEG (1kDa) reaction.Swimming lane 6 is products of 15 μ g AS-FSH and CMP-SA-PEG (5kDa) reaction.Swimming lane 7 is products of 15 μ g AS-FSH and CMP-SA-PEG (10kDa) reaction.

Figure 164 is the isoelectrofocusing gel images of product of the reaction of the sialylated FSH of preparation PEG

-.Swimming lane

1 and 8 is IEF standard substances.Swimming lane 2 is the natural FSH of 15 μ g.Swimming lane 3 is that 15 μ g take off sialic acid-FSH (AS-FSH).Swimming lane 4 is products of 15 μ g AS-FSH and CMP-SA reaction.Swimming lane 5 is products of 15 μ g AS-FSH and CMP-SA-PEG (1kDa) reaction.Swimming lane 6 is products of 15 μ g AS-FSH and CMP-SA-PEG (5kDa) reaction.Swimming lane 7 is products of 15 μ g AS-FSH and CMP-SA-PEG (10kDa) reaction.

Figure 165 is the SDS-PAGE gel images of the natural non-Recombinant FSH that produces in people's

pituicyte.Swimming lane

1,2 and 5 is SeeBlue ^TM+ 2 molecular weight standard

things.Swimming lane

3 and 4 is respectively the natural FSH of 5 μ g and 25 μ g.

Figure 166 is isoelectrofocusing gel (pH3-7) image of product of describing the asialylated reaction of

rFSH.Swimming lane

1 and 4 is IEF standard substances.Swimming lane 2 is natural rFSH.Swimming lane 3 is to take off sialic acid-rFSH.

Figure 167 is a SDS-PAGE gel images of describing the sialylated result of the PEG-take off sialic acid-rFSH.Swimming lane 1 is natural rFSH.Swimming lane 2 is to take off sialic acid-FSH.Swimming lane 3 is the products that take off sialic acid-FSH and CMP-SA reaction.Swimming lane 4-7 takes off sialic acid-FSH and 0.5mM CMP-SA-PEG (10kDa) respectively at the product of 2 hours, 5 hours, 24 hours and reaction in 48 hours.Swimming lane 8 is to take off sialic acid-FSH and 1.0mM CMP-SA-PEG (10kDa) product reaction in 48 hours.Swimming lane 9 is to take off sialic acid-FSH and 1.0mM CMP-SA-PEG (1kDa) product reaction in 48 hours.

Figure 168 is that sialic acid-rFSH carries out the sialylated product of PEG-with CMP-SA-PEG (1kDa) isoelectrofocusing gel images is taken off in expression.Swimming lane 1 is natural rFSH.Swimming lane 2 is to take off sialic acid-rFSH.Swimming lane 3 is to take off sialic acid-rFSH and the CMP-SA product reaction in 24 hours.Swimming lane 4-7 takes off sialic acid-rFSH and 0.5mM CMP-SA-PEG (1kDa) respectively at the product of 2 hours, 5 hours, 24 hours and reaction in 48 hours.Swimming lane 8 is

blank.Swimming lane

9 and 10 be take off sialic acid-rFSH respectively with 0.5mM and 1.0mM CMP-SA-PEG (10kDa) product reaction in 48 hours.

Figure 169 is the graphic representation of the pharmacokinetics of rFSH and rFSH-SA-PEG (1KDa and 10KDa).This graphic representation has been illustrated Glycopegylated rFSH and has been compared with the rFSH of non-PEGization, the rFSH compound in the rat blood flow time and the relation between the mean concns of rFSH compound in blood.

Figure 170 is the graphic representation that utilizes the FSH bioassay results of sustenticular cell.This graphic representation illustrated the concentration of FSH in the sustenticular cell incubation substratum and the 17-β estradiol amount that discharges from sustenticular cell between relation.

Figure 171 is the graphic representation of describing the Steelman-Pohley bioassay results of Glycopegylated and non-Glycopegylated FSH.FSH with human chorionic gonadotrophin and various amounts carried out subcutaneous injection to rat in 3 days, in the average ovary weight of definite treatment group on the 4th.RFSH-SA-PEG refers to use PEG (1kDa) to carry out Glycopegylated Recombinant FSH.RFSH refers to non-Glycopegylated FSH.Each treatment group contains 10 rats.

Figure 172 comprises Figure 172 A and 172B, describes from the chromatogram of the INF-β wash-out of Superdex-75 post.Figure 172 A describes whole chromatogram.Figure 172 B describes the blocked areas that contains

peak

4 and 5 among Figure 172 A in more detail.

Figure 173 comprises Figure 173 A and 173B, describes from the maldi analysis of the glycan of INF-β enzymatic release.Figure 173 A describes the maldi analysis to the glycan that discharges from natural INF-β.Figure 173 B describes the maldi analysis to the glycan that discharges from asialylated INF-β.The structure of glycan by with the standard glycan carry out peak spectrum relatively come to determine.The structure delineation of glycan is on the next door at peak.Square expression GlcNAc, trilateral is represented Fucose, and circle is represented seminose, and rhombus represents that semi-lactosi and star represent sialic acid.

Figure 174 describes asialylated INF-β is carried out sialylated lectin engram analysis.The trace on right side is with having carried out (DIG) (RocheApplied Science of digoxigenin (digoxogenin), Indianapolis, IL) the bosom Chinese scholartree of mark (Maackia amurensis) lectin (MAA) is surveyed, and to survey α 2,3-is sialylated.(CA) survey for Vector Laboratories, Burlingame, to survey the galactose residue that exposes by the Erthrinacristagalli lectin (ECL) of mark with having carried out vitamin H for the trace in left side.The SDS-PAGE that Figure 175 describes PEG (10kDa) the PEGization reaction product of INF-β analyzes." PEG " refers to the INF-β before the PEGization reaction."+PEG " refers to the INF-β after the PEGization reaction.

The SDS-PAGE that Figure 176 describes PEG (20kDa) the PEGization reaction product of INF-β analyzes." unmodified " refers to the INF-β before the PEGization reaction." PEGization " refers to the INF-β after the PEGization reaction.

Figure 177 describes from the chromatogram of the INF-β wash-out of PEG (10kDa) PEGization of Superdex-200 post.

Figure 178 describes the result of the INF-β peak fraction biological assay that is shown in PEG (10kDa) PEGization in the figure INF-PEG6 chromatogram.

Figure 179 describes from the chromatogram of the INF-β wash-out of PEG (20kDa) PEGization of Superdex-200 post.

Figure 180 comprises Figure 180 A and 180B, is to describe the MALDI-TOF spectrum (Figure 180 A) of RNaseB and two graphic representations of the HPLC feature (Figure 180 B) of the oligosaccharides that cut by the N-glycanase from RNaseB.Most of N-glycosylation sites of this peptide are to use the high mannose oligosaccharides of being made up of 5～9 mannose residues to modify.

Figure 181 describes the synoptic diagram that high mannose N-glycan changes to heterozygosis N-glycan.Enzyme 1 is the α 1 from Trichodermareesei (Trichodoma reesei) or Aspergillus saitoi, the 2-mannosidase.Enzyme 2 is GnT-I (β-1,2-N-acetylgucosamine transferase I).Enzyme 3 is GalT-I (β-1,4-galactosyltransferases 1).Enzyme 4 is α 2,3-sialyltransferase or α 2,6-sialyltransferase.

Figure 182 comprises Figure 182 A and 182B, be to describe with reorganization trichoderma reesei alpha 1, two graphic representations of the HPLC feature (Figure 182 B) of the MALDI-TOF spectrum (Figure 182 A) of the RNaseB that the 2-mannosidase is handled and the oligosaccharides that from the RNaseB that modifies, cuts by the N-glycanase.

Figure 183 describes the commercial α that buys 1 that uses from the A.saitoi purifying, 2-mannosidase (Glyko ﹠amp; CalBioChem) graphic representation of the MALDI-TOF of the RNaseB of Chu Liing spectrum.

Figure 184 describes the graphic representation that the MALDI-TOF of the RNaseB by the modification that obtains with the product shown in reorganization GnT-I (GlcNAc transferase I) processing Figure 182 composes.

Figure 185 describes the graphic representation that the MALDI-TOF of the RNaseB by the modification that obtains with the product shown in reorganization GalT-1 (galactosyltransferase 1) processing Figure 184 composes.

Figure 186 is a graphic representation of describing the MALDI-TOF spectrum of the RNaseB by handling the modification that the product shown in Figure 185 obtains with reorganization ST3Gal III (α 2,3-sialyltransferase III) as the transferring enzyme donor with CMP-SA.

Figure 187 is a graphic representation of describing the MALDI-TOF spectrum of the RNaseB by handling the modification that the product shown in Figure 185 obtains with reorganization ST3Gal III (α 2,3-sialyltransferase III) as the transferring enzyme donor with CMP-SA-PEG (10kDa).

Figure 188 is a series of schemes that high mannose N-glycan changes to compound N-glycan.Enzyme 1 is the α 1 from Trichodermareesei or Aspergillus saitoi, the 2-mannosidase.Enzyme 2 is GnT-I.Enzyme 3 is GalT1.Enzyme 4 is α 2,3-sialyltransferase or α 2,6-sialyltransferase.Enzyme 5 is α mannosidase II.Enzyme 6 is α mannosidases.Enzyme 7 is GnT-II.Enzyme 8 is α 1, the 6-mannosidase.Enzyme 9 is α 1, the 3-mannosidase.

Figure 189 is by the N-acetyl-glucosamine transferase I～VI (GnT-I～VI) diagram of catalytic connection.R＝GlcNAcβ1，4GlcNAc-Asn-X。

Figure 190 is the image of SDS-PAGE gel: standard substance (swimming lane 1); Natural transferrin (swimming lane 2); Take off sialic acid transferrin (swimming lane 3); Take off sialic acid transferrin and CMP-SA (swimming lane 4);

Swimming lane

5 and 6 takes off sialic acid transferrin and the CMP-SA-PEG (1kDa) that is respectively 0.5mM and 5mM;

Swimming lane

7 and 8 takes off sialic acid transferrin and the CMP-SA-PEG (5kDa) that is respectively 0.5mM and 5mM;

Swimming lane

9 and 10 takes off sialic acid transferrin and the CMP-SA-PEG (10kDa) that is respectively 0.5mM and 5mM.

Figure 191 is the image of IEF gel: natural transferrin (swimming lane 1); Take off sialic acid transferrin (swimming lane 2); Take off sialic acid transferrin and CMP-SA, 24 hours (swimming lane 3); Take off sialic acid transferrin and CMP-SA, 96 hours (swimming lane 4);

Swimming lane

5 and 6, respectively 24 and 96 hours take off sialic acid transferrin and CMP-SA-PEG (1kDa);

Swimming lane

7 and 8, respectively 24 and 96 hours take off sialic acid transferrin and CMP-SA-PEG (5kDa);

Swimming lane

9 and 10, respectively 24 and 96 hours take off sialic acid transferrin and CMP-SA-PEG (10kDa).

Detailed Description Of The Invention

The present invention includes the acellular in vitro method and the composition that in peptide molecule, add and/or from peptide molecule, remove sugar by this way, thereby the molecule of the glycopeptide with glycosylation pattern specific customized or that want is provided, and wherein this glycopeptide is with industrial-scale production.In the preferred embodiment of the invention, Zhi Bei glycopeptide has adhered to the sugar of modifying thereon like this, and the sugar of this modification adds on the peptide by enzymatic reaction.The characteristic of a key of the present invention is to obtain the peptide that is produced by any cell type and generate the core glycan structures on this peptide, subsequently with glycan structures at the external glycopeptide that has the glycosylation pattern that the treatment that is suitable in Mammals uses with generation that is reconstructed.More specifically, possible according to the present invention is that preparation has the glycan molecule of modification or the glycopeptide molecule of other compounds of puting together thereon, thereby the molecule of puting together gives this peptide useful feature.According to the present invention,, conjugate molecule enzymatic ground is added in the peptide because the conjugate molecule has regioselectivity and stereoselective advantage to the interpolation based on enzyme of peptide.Glycoconjugate can add to before or after glycosylation is finished on the glycan on the peptide.In other words, sugar is puted together with glycosylated order and can be changed like that as described elsewhere herein.Therefore possible is utilizes the method and composition that provides herein that peptide is reconstructed giving the glycan that this peptide is wanted, and this glycan is preferably to be attached with thereon modifies sugar.Same possible be utilize method and composition of the present invention on technical scale, to generate to have want and or the peptide molecule of the glycan structures modified, therefore, for the first time to this area provide effective generation improvement therapeutic peptide put into practice solution.

Definition

Unless definition is arranged in addition, all used herein technology and scientific terminology generally have with the present invention under the field in the identical meaning generally understood of technician.Usually, used herein title and the laboratory procedure in cell cultures, molecular genetics, organic chemistry and nucleic acid chemistry and the hybridization are those well known in the art and generally employings.Standard techniques is applied to nucleic acid and peptide is synthetic.This technology and program normally according to the ordinary method of this area and various common reference carry out (as people such as Sambrook, 1989, MolecularCloning:A Laboratory Manual, 2d ed.Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY), this method and reference provide in presents full text.Used herein title is those well known in the art and generally employings with the laboratory procedure that is used for following analytical chemistry and organic synthesis.Standard techniques or its modified method are used for chemosynthesis and chemical analysis.

Article " a " and " an " are used in reference to a kind of herein or surpass the grammar object of a kind of (promptly at least a) this article.As an example, " an element " refers to an element or surpasses an element.

As used herein, term " antibody " refer to can be specifically with antigen on specific epitope bonded immunoglobulin molecules.Antibody can be the complete immunoglobulin (Ig) that is derived from natural origin or recombinant sources, and also can be the immunoreactivity part of complete immunoglobulin (Ig).Antibody generally is the tetramer of immunoglobulin molecules.Antibody among the present invention can exist by various forms, comprises as polyclonal antibody, monoclonal antibody, Fv, Fab and F (ab) ₂And single-chain antibody and humanized antibody (people such as Harlow, 1999, Using Antibodies:ALaboratory Manual, Cold Spring Harbor Laboratory Press NY; People such as Harlow, 1989, Antibodies:A Laboratory Manual, Cold Spring Harbor, New York; People such as Houston, 1988, Proc.Natl.Acad.Sci.USA 85:5879-5883; People such as Bird, 1988, Science 242:423-426).

As used herein, term " synthetic antibody " refers to the antibody that generates with recombinant DNA technology, for example by the antibody of described phage expression herein.This term also may be interpreted as the antibody that refers to that the aminoacid sequence by the dna molecular (this dna molecular expressing antibodies protein) of composite coding antibody or this antibody of encoding generates, and wherein this DNA or aminoacid sequence are with the technology acquisition of this area available and well-known synthetic DNA or aminoacid sequence.

As used herein, term " has function ", and biological molecule is the biological molecule of a definite form, wherein under this form this molecular display it is characterized the feature of institute's foundation.For example, the enzyme of function being arranged is to show the enzyme that this enzyme is characterized the characteristic catalytic activity of institute's foundation.

As used herein, structure It is the point that amino acid or amino acid side chain are connected with glycan structures in the peptide chain.

" N-connects " oligosaccharides is those oligosaccharides that are connected with the peptide main chain with l-asparagine-N-acetyl-glucosamine ways of connecting by l-asparagine.The oligosaccharides that N-connects is also referred to as " N-glycan ".The oligosaccharides that all N-connect all has common pentasaccharides core Man ₃GlcNAc ₂Their difference existence that the periphery sugar props up (being also referred to as feeler) whether with number in, as N-acetyl-glucosamine, semi-lactosi, N-acetylgalactosamine, Fucose and sialic acid.Selectively, this structure also can contain the Fucose molecule and/or the wood sugar molecule of core.

" three basic seminose core textures " refer to only comprise the glycan part of three seminose core textures, do not adhere to and there is extra sugar thereon.When term " basic " was not included in the description of " three seminose core textures ", this glycan comprised the three seminose core textures that are attached with extra sugar thereon so.Selectively, this structure also can contain the Fucose molecule and/or the wood sugar molecule of core.

Term " three basic seminose core glycopeptides " is used in reference to the glycopeptide with the glycan structures that mainly comprises three basic seminose core textures herein.Selectively, this structure also can contain the Fucose molecule and/or the wood sugar molecule of core.

To be those be connected to oligosaccharides on the peptide main chain by Threonine, Serine, oxyproline, tyrosine or other amino acid that contains hydroxyl to " O-connects " oligosaccharides.

All oligosaccharides of describing herein all are to represent with the title of non-reducing sugar or abbreviation (being Gal), it is the configuration (α or β) of glycosidic link thereafter, ring key (1 or 2), the ring position of the reducing sugar that relates in the key (2,3,4,6 or 8) are the title of reducing sugar or abbreviation (being GlcNAc) then.Each sugar all is preferably pyranose.For standard sugar biology nomenclature summary, referring to people eds. such as Essentials of Glycobiology Varki, 1999, CSHL Press.

Term " sialic acid " refers to any member of sugared family of the carboxylation of nine carbon.Prevailing member is N-n acetylneuraminic acid n (2-ketone-5-acetylaminohydroxyphenylarsonic acid 3, the two deoxidations of 5--D-glycerine-D-galactononulopyranos-1-onic acid (often being abbreviated as Neu5Ac, NeuAc or NANA)) in the sialic acid family.Second member of this family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), and wherein the N-ethanoyl of NeuAc is hydroxylated.The 3rd sialic acid family member is 2-ketone-3-deoxidation-nonulosonic acid (KDN) (people such as Nadano, (1986) J.Biol.Chem.261:11550-11557; People such as Kanamori, J.Biol.Chem.265:21811-21819 (1990)).That comprise equally is sialic acid such as the 9-O-C that 9-replaces ₁-C ₆Acyl group-Neu5Ac is as 9-O-lactoyl-Neu5Ac or 9-O-ethanoyl-Neu5Ac, 9-deoxidation-9-fluoro-Neu5Ac and 9-nitrine-9-'-deoxy-n eu5Ac.For the summary of sialic acid family, referring to Varki, Glycobiology 2:25-40 (1992); Sialic Acids:Chemistry, Metabolismand Func tion, R.Schauer, Ed. (Springer-Verlag, NewYork (1992)).Synthetic and the application of sialylated compound in sialylated process is disclosed in the International Application No. WO of delivering on October 1st, 1,992 92/16640.

As used herein, the peptide with " glycosylation of wanting " is the peptide that comprises necessary one or more oligosaccharide moleculars of the effective biologic activity of this peptide.

" disease " is a kind of healthy state of animal, and wherein animal can not be kept homeostasis, and if wherein disease mustn't go to improvement, the health of animal will continue to worsen so.

Used as giving in the peptide medicine to the patient herein, " area under a curve " or " AUC " is defined as the total area under the curve, and this curve description conduct is from the concentration of patient's systemic circulation Chinese traditional medicine of the function of 0 to infinitely-great time.

Used as giving in the peptide medicine to the patient herein, the plasma concentration that term " transformation period " or " t1/2 " are defined as patient's Chinese traditional medicine reduces half required time.Depend on multiple purge mechanism, redistribute and other mechanism well known in the art, the transformation period relevant with the peptide medicine of surpassing can be arranged.Usually, defined α transformation period and β transformation period, thus the α phase with redistribute relevant, and the β phase with remove relevant.Yet the pharmaceutical grade protein that is limited in the blood flow for the overwhelming majority has at least 2 to remove the transformation period.For some glycosylated peptides, fast the β phase remove can be by identification terminal galactose, N-acetylgalactosamine, N-acetyl-glucosamine, seminose or Fucose endotheliocyte or scavenger cell on receptor-mediated.The β phase is at a slow speed removed glomerular filtration and/or specificity or nonspecific picked-up and the metabolism mediation in tissue that can pass through the molecule (about 68kD) of effective radius＜2nm.Glycopegylatedly can add cap, thereby blocking-up was removed by the fast alpha phase of these sugared acceptors of identification to terminal sugar (as semi-lactosi or N-acetylgalactosamine).It also can give bigger effective radius, thereby has reduced dispensed volume and tissue picked-up, thereby has prolonged the late β phase.Thereby Glycopegylated accurate influence to α phase and β transformation period phase will depend on size, glycosylation state and other parameters and change, as be well known in the art.To the further explanation of " transformation period " can be found in Pharmaceutical Biotechnology (1997, DFA Crommelin and RDSindelar, eds., Harwood Publisher, Amsterdam, pp101-120).

Used as giving in the peptide medicine to the patient herein, term " retention time " is defined as the mean time that retains at the back medicine of taking medicine in patient's body.

" isolating nucleic acid " refer to from when the naturally occurring state the sequence that both sides join with it isolating nucleic acid segment or fragment, as the dna fragmentation from just often the sequence that side and this fragment are joined, taking out, the sequence of this sequence as in naturally occurring genome, joining in the side with this fragment.This term also is applied to substantially the nucleic acid of purifying from other compositions of following with nucleic acid natively, as natural RNA or DNA or the protein of following with nucleic acid in cell.Therefore this term also comprise as be integrated in the carrier or be integrated in autonomously replicating plasmid or the virus or be integrated into recombinant DNA among prokaryotic organism or the eukaryotic gene group DNA or that exist as the independent molecule that does not rely on other sequences (as cDNA or genome or the cDNA fragment that produces by PCR or Restriction Enzyme digestion).It also comprises the recombinant DNA as the part of the hybrid nucleic acid of the extra peptide sequence of coding.

" polynucleotide " refer to strand or the parallel and nucleic acid antiparallel chain.Thereby polynucleotide can be strand or double-stranded nucleic acid.

Term " nucleic acid " refers generally to big polynucleotide.Term " oligonucleotide " refers generally to short polynucleotide, is no more than about 50 Nucleotide usually.

Herein ordinary symbol is used to describe polynucleotide sequence: the left hand end of strand polynucleotide sequence is 5 '-end; The left-hand of double-stranded polynucleotide sequence is to being called 5 '-direction.From 5 ' to the 3 ' direction to nascent RNA transcript interpolation Nucleotide is called transcriptional orientation.The DNA chain that has identical sequence with mRNA is called " coding strand "; The sequence that is positioned at the last reference point 5 ' of DNA on the DNA chain is called " upstream sequence "; The sequence that is positioned at the last reference point 3 ' of DNA on the DNA chain is called " downstream sequence ".

" coding " refers to that the middle specific nucleotide sequence of polynucleotide (as gene, cDNA or mRNA) serves as template and is used in synthetic other polymkeric substance of biological procedures and macromolecular inherent feature, and this polymkeric substance and macromole have nucleotide sequence (being rRNA, tRNA and mRNA) or the aminoacid sequence of qualification and the biological property that therefrom produces of qualification.Thereby, if produced protein corresponding to transcribing and translate in cell or other biological system of the mRNA of nucleic acid, this protein of this nucleic acid sequence encoding then.Coding strand and noncoding strand all can be called protein or other products of coding this nucleic acid or cDNA, and wherein the sequence of the nucleotide sequence of coding strand and mRNA is identical and provide in sequence table usually, and noncoding strand is used as template with open gene or cDNA.

Unless definition is arranged in addition, " nucleotide sequence of encoding amino acid sequence " comprises the phase nucleotide sequence of degeneracy form each other of all coding same acid sequences.The nucleotide sequence of coded protein and RNA can comprise intron.

As used herein, " homology " refers to two subunit sequence similarities between the polymer molecule, between two nucleic acid molecule, and between two dna moleculars or two RNA molecules, or between two peptide molecules.When the subunit position in two molecules was occupied by identical monomer subunit, for example, if when a certain position in two dna moleculars is all occupied by VITAMIN B4, then they were homologous in this position.Homology between two sequences is the positive function of coupling number or homology positional number, for example, if the position of half in two compound sequences (being 5 positions in the polymkeric substance of 10 subunits as length) is homologous, two sequences are 50% homologous so, if 90% position is coupling or homologous as 9 in 10, then two sequences have 90% homology.For example, dna sequence dna 3 ' ATTGCC5 ' and 3 ' TATGGC have 50% homology.

As used herein, " homology " used with " consistence " synonym.

The conforming definite available mathematical algorithm of per-cent between two Nucleotide or the aminoacid sequence is realized.For example, the mathematical algorithm that is used for two sequences of comparison is Karlin and Altschul (1990, Proc.Natl.Acad.Sci.USA 87:2264-2268) algorithm, further revised by Karlin and Altschul (1993, Proc.Natl.Acad.Sci.USA90:5873-5877).This algorithm is integrated into people such as Altschul (1990, J.Mol.Biol.215:403-410) in NBLAST and the XBLAST program, and can be from obtaining as NCBI (National Center for Biotechnology Information) Global Internet website (NCBI), this website has universal resource location http://www.ncbi.nlm.nih.gov/BLAST/.The BLAST nucleotide search can carry out with NBLAST program (called after on the NCBI internet sites " blastn "), uses following parameter: breach point penalty=5; Breach extends point penalty=2; Mispairing point penalty=3; The coupling prize divides=1; Desired value 10.0; With word string size=11, to obtain and the nucleic acid homologous nucleotide sequence of describing herein.The BLAST protein search can carry out with " blastp " program of XBLAST program (called after on the NCBI internet sites " blastn ") or NCBI, uses following parameter: desired value 10.0; The BLOSUM62 matrix of keeping the score is to obtain and the protein molecule homologous aminoacid sequence of describing herein.In order to obtain to be used for the comparison jaggy of comparison purpose, can use as Gapped BLAST in the middle description of people such as Altschul (1997, Nucleic Acids Res.25:3389-3402).Selectively, PSI-Blast or PHI-Blast can be used to carry out the multiple search, relation (Id.) far away and have the intermolecular relation of common mode between this repeat search molecular detection.When using BLAST, Gapped BLAST, PSI-Blast and PHI-Blast program, can use the default parameters of each program (as XBLAST and NBLAST).Referring to http://www.ncbi.nlm.nih.gov.

Per-cent consistence between two sequences can be used to above-mentioned those similar permissions or not allow the technology of breach to determine.When calculating the per-cent consistence, calculate typical accurately coupling.

" the heterologous nucleic acids ceneme " of encoded peptide is defined as the nucleic acid that has operationally the target peptide encoding sequence that is connected with one or more expression control sequencs such as promotor and/or inhibition sequence, and wherein at least one sequence is allogenic, promptly is not found in the host cell usually.

Two polynucleotide " are operably connected " and refer to that two polynucleotide arrange by this way in nucleic acid moieties, thereby at least one of two polynucleotide can be brought into play its distinctive physiological role to another.For example, the promotor that is operably connected to the nucleic acid encoding district can start transcribing of coding region.

As used herein, term " promotor/adjusting sequence " refers to operationally the necessary nucleotide sequence of expression with this promotor/gene product that the adjusting sequence is connected.In some instances, this sequence can be the core promoter sequence, and in other examples, this sequence also can comprise the necessary enhancer sequence of gene product expression and other regulatory elements.For example, promotor/adjusting sequence can be the sequence with tissue specificity mode expressing gene product.

" composing type " promotor is the promotor that starts the expression of gene that is operably connected with it in cell in constant mode.For example, the expression promoter that starts the house-keeping gene of cell can be thought constitutive promoter.

" induction type " promotor is a kind of nucleotide sequence, when this nucleotide sequence is operably connected with the polynucleotide of coding or qualification gene product, only when being present in the cell, the inductor corresponding to this promotor can cause gene product in viable cell, to produce basically.

" tissue specificity " promotor is a nucleotide sequence, when this nucleotide sequence and coding or the polynucleotide that limit gene product are operably connected, only when being cell corresponding to the types of organization of this promotor, this cell can cause gene product in viable cell, to produce basically.

" carrier " is to comprise isolating nucleic acid and can be used for isolating nucleic acid sent to pass into the material of cell interior forming.Known in this area have many carriers, including, but not limited to linear polynucleotide, polynucleotide, plasmid and the virus relevant with ionic or amphiphilic compound.Thereby term " carrier " comprises the plasmid or the virus of self-replicating.This term also may be interpreted as and comprises and can promote nucleic acid to transcellular non-plasmid and non-virus compound, as many Methionin compound, liposome etc.The example of virus vector is including, but not limited to adenovirus carrier, adeno-associated virus vector, retrovirus vector etc.

" expression vector " refers to comprise the carrier of recombination of polynucleotide, and this recombination of polynucleotide comprises the expression control sequenc that operationally is connected with the nucleotide sequence that will express.Expression vector comprises enough cis-acting elements that is used to express; Other elements that are used for expressing can provide by host cell or in the vivoexpression system.Expression vector comprises that all that is as known in the art, as has integrated clay, plasmid (as exposed or be contained in the liposome) and the virus of recombination of polynucleotide.

" genetically engineered processing " or " reorganization " cell are the cells that the genetic stocks of pair cell has carried out one or more modifications.Can this modification can be including, but not limited to inserting genetic stocks, deletion genetic stocks and inserting no matter stablize the extrachromosomal genetic stocks of maintenance.

" peptide " is oligopeptides, polypeptide, peptide, protein or glycoprotein.When glycan molecule adhered to thereon, the application of term " peptide " herein comprised the peptide with the glycan molecule that adheres to thereon.

As used herein, " natural form " refers to the form by cell and/or biogenic peptide, and wherein described peptide is found in described cell or the biology under native state.When this peptide was produced by a plurality of cells and/or biology, this peptide can have various natural forms.

" peptide " refers to that monomer wherein is amino acid and the polymkeric substance that links together by amido linkage, and this amido linkage selectively is called peptide bond.Selectively, also comprise non-natural amino acid such as Beta-alanine, phenylglycine and homoarginine.The amino acid of non-nucleic acid encoding also can be used among the present invention.In addition, also can use in the present invention and modified and comprise the amino acid of reactive group, glycosylation site, polymkeric substance, treatment part, biomolecules etc.All used in the present invention amino acid can be its D-or L-isomer.The L-isomer is normally preferred.In addition, also can use other peptide mimicses in the present invention.As used herein, " peptide " refers to glycosylated and nonglycosylated peptide.What comprise equally is by the incomplete glycosylated peptide of system of expressing this peptide.For general summary, referring to Spatola, AF., Chemistry and Biochemistryof Amino Acids, Peptides and Proteins, B.Weinstein, eds., MarcelDekker, New York, p.267 (1983).

Term " peptide conjugate " refers to kind of the present invention (species), and wherein peptide is puted together with the sugar of the modification that proposes herein.

Term " amino acid " refers to naturally occurring and synthetic amino acid, and to bring into play the amino acid analogue and the amino acid analog thing of function with the mode of naturally occurring amino acid similarity.Naturally occurring amino acid is that those are encoded by genetic code, and those amino acid of modifying afterwards, as oxyproline, Gla and O-phosphoserine.Amino acid analogue refers to have with natural amino acid the compound of identical basic chemical structure, and the α carbon that promptly is connected with hydrogen, carboxyl, amino and R group are as homoserine, nor-leucine, methionine sulfoxide, methionine(Met) methyl sulfonium.This analogue has the R group (as nor-leucine) of modification or the peptide main chain of modifying, but has kept the basic chemical structure identical with naturally occurring amino acid.The amino acid analog thing refers to have the chemical compound of the structure different with amino acid whose general chemical structure, but this compound is to bring into play function with the mode of naturally occurring amino acid similarity.

As used herein, amino acid is with its complete name, corresponding trigram coding or corresponding single-letter coded representation, as shown in the following Table 1:

Table 1. amino acid and trigram and single-letter coding

Complete name trigram coding single-letter coding

Aspartic acid Asp D

L-glutamic acid Glu E

Methionin Lys K

Arginine Arg R

Histidine His H

Tyrosine Tyr Y

Halfcystine Cys C

L-asparagine Asn N

Glutamine Gln Q

Serine Ser S

Threonine Thr T

Glycine Gly G

L-Ala Ala A

Xie Ansuan Val V

Leucine Leu L

Isoleucine Ile I

Methionine(Met) Met M

Proline(Pro) Pro P

Phenylalanine Phe F

Tryptophane Trp W

The present invention also provides the analogue that comprises above-mentioned proteinic protein or peptide.Analogue and naturally occurring protein or peptide difference are the difference of the aminoacid sequence guarded or do not influence the modification of sequence, perhaps be both.For example, can the amino acid change of guarding although this change has changed the primary sequence of protein or peptide, but does not change its function usually.Substituting below conservative amino acid replacement generally comprises in the group:

Glycine, L-Ala;

Xie Ansuan, Isoleucine, leucine;

Aspartic acid, L-glutamic acid;

L-asparagine, glutamine;

Serine, Threonine;

Methionin, arginine;

Phenylalanine, tyrosine.

Modify the interior or external chemical derivatization of body that (not changing primary sequence usually) comprises peptide, as acetylize or carboxylation.What comprise equally is glycosylation modified, carries out as by and processing synthetic at it or in the further procedure of processing glycosylation pattern of peptide being modified; As being undertaken by peptide being exposed to glycosylated enzyme of influence such as Mammals glycosylation or de-glycosylation enzyme.What comprise equally is the sequence with amino-acid residue of phosphorylation, as Tyrosine O-phosphate, phosphoserine or phosphothreonine.

Certainly, it should be understood that peptide can be integrated has carried out modifying but has not influenced active amino-acid residue.For example; can carry out derivatize to comprise blocking groups to end; promptly be suitable for the chemical substituting group that protection and/or stable N-and C-end are not subjected to " undesired degraded "; the enzymatic to compound, chemistry or the biological chemistry that should " undesired degraded " be included in any kind that may influence the compound function that end carries out are destroyed, i.e. the degraded of carrying out successively at the end of compound.

Blocking groups comprises the blocking group that is used for the chemistry of peptides field routinely, and this group does not have a negative impact to the activity in vivo of peptide.For example, suitable N-end closure group can be introduced by alkylation or acylations to the N-end.The example of suitable N-end closure group comprises C ₁-C ₅The form of branch or unbranched alkyl group, carboxyl groups such as formyl radical and Acetyl Groups and replacement thereof is as acetylamino methyl (Acm), Fmoc or Boc group.Amino acid whose deamination analogue also is useful N-end closure group, and can or be used for substituting the N-terminal residue with the terminal coupling of the N-of peptide.Suitable C-end closure group comprises ester, ketone and acid amides, has wherein integrated or do not integrated the carboxylic group of C-end.Form the alkyl group, particularly low-grade alkyl group of ester or ketone such as the amino group such as the primary amine (NH of methyl, ethyl and propyl group and formation acid amides ₂), and single-and two-alkylamino group such as methylamino, ethylamino, dimethylamino, diethylamino, methylethyl amino etc. be the example of C-end closure group.Decarboxylized amino acid analogue such as spermine also are useful C-end closure groups and can be used for the terminal coupling of the C-of peptide or be used for substituting this end.Further, it should be understood that terminal free amine group and carboxylic group can be removed from peptide does not together influence the peptide activity to obtain its deaminizating and decarboxylized form.

Also can integrate other modification and not influence activity unfriendly, and these are modified including, but not limited to D-heterogeneous amino acid one or more natural amino acid whose replacements of L-heterogeneous.Thereby peptide can comprise one or more D-amino-acid residues, and maybe can comprise all is the amino acid of D-form.Counter-rotative type (retro-inverso) according to peptide of the present invention also can use, for example the peptide (wherein all amino acid is all replaced by the D-amino acid form) of reverse (inverted).

Expect that also acid salt of the present invention is a function equivalent.Thereby handle peptide of the present invention with mineral acid or organic acid and be applicable to the present invention with the water-soluble salt that peptide is provided, wherein mineral acid such as spirit of salt, Hydrogen bromide, sulfuric acid, nitric acid, phosphoric acid etc., organic acid such as acetate, propionic acid, oxyacetic acid, pyruvic acid, oxalic acid, oxysuccinic acid, succsinic acid, toxilic acid, fumaric acid, tartrate, citric acid, phenylformic acid, TRANSCINNAMIC ACID, amygdalic acid, methylsulfonic acid, ethyl sulfonic acid, tosic acid, sialic acid etc.

What comprise equally is the peptide of modifying with common Protocols in Molecular Biology, thereby improves it to the resistance of proteasome degradation or make the dissolving properties optimization or it is more suitable in as therapeutical agent.The analogue of this peptide comprises that those contain residue except that naturally occurring L-amino acid, the synthesizing amino acid that exists as D-amino acid or non-natural.Peptide of the present invention is not limited to the product of listed herein any specific illustrative methods.

As used herein, term " MALDI " is the abbreviation of the auxiliary laser desorption ionisation of matrix.In ionization, SA-PEG (sialic acid-poly-(ethylene glycol)) can partly remove from the N-glycan structures of glycoprotein.

As used herein, term " glycosyltransferase " refers to have any enzyme/protein of the ability on the acceptor portion that donor sugar is transferred to.

As used herein, term " sugar of modification " refers to that enzymatic in the method for the invention adds natural on the glycosyl residue of amino acid or peptide or sugar that non-natural exists to.The sugar of modifying is selected from many enzyme substratess, including, but not limited to sugar nucleotide (single-, two-and three-phosphoric acid), the sugar of activatory sugar (as glycosyl halide, glycosyl methylsulfonic acid) and both inactive also non-nucleotide.

" sugar of modification " is with " modification group " functionalization covalently.Useful modification group is including, but not limited to water-soluble polymers, treatment part, diagnosis part, biomolecules etc.Thereby be not chosen such that with the position that modification group carries out functionalization and prevent that " sugar of modification " enzymatic ground from adding on the peptide.

Term " water-soluble " refers to have the part of some detectable solvability degree in water.Detection and/or quantitative water miscible method are well known in the art.Exemplary water-soluble polymers comprises peptide, sugar, poly-(ether), many (amine), poly-(carboxylic acid) etc.Peptide can have the blended sequence or only be made up of single seed amino acid, as poly-(Methionin).Similarly, sugar can be the blended sequence or is only planted sugared subunit and is formed by single, as dextran, amylose starch, chitosan with gather (sialic acid).Exemplary poly-(ether) is poly-(ethylene glycol).Poly-(ethyleneimine) is exemplary polyamines, and poly-(asparagus fern ammonia) acid is representational poly-(carboxylic acid).

" polyalkylene oxide " refers to have a compounds of polyether backbone.The polyalkylene oxide kind of Ying Yonging comprises as straight chain and side chain kind in the present invention.In addition, the end of exemplary polyalkylene oxide can be one or more reactive, activable or inert parts.For example, the polyalkylene oxide that polyalkylene oxide is made up of multiple oxyethane subunit, it comprises or does not comprise extra reactive, activable or inert part at each end.Useful polyalkylene oxide kind comprises those kind that end " is added cap " by inertia group, as mono methoxy-polyalkylene oxide.When molecule is a ramose kind time-like, can comprise a plurality of reactive, activable or inert groups at the end of oxirane chain, and reactive group can be identical or different.The derivative of the straight chain polyalkylene oxide kind of isodigeranyl function also is as known in the art.

As used herein, term " glycosyl linking group " refers to the glycosyl residue of reagent (as water-soluble polymers, treatment part, biomolecules) covalent attachment.In the method for the invention, " glycosyl linking group " is covalently attached on the glycosylated or nonglycosylated peptide, thereby reagent is connected on the amino acid and/or glycosyl residue on the peptide." glycosyl linking group " usually adheres to the amino acid on the peptide and/or the enzymatic on the glycosyl residue by " sugar of modification " and is derived from " sugar of modification ".More specifically, as used herein, " glycosyl linking group " refers to the covalent attachment part of the amino-acid residue of " modification group " and peptide as discussed here.Glycosyl linking group-modification group affixture has the structure as enzyme substrates.With glycosyl linking group-modification group affixture be the enzyme of substrate normally those can transfer to glycosyl part the enzyme on the amino-acid residue of peptide, as glycosyltransferase, Ntn hydrolase, Glycosylase, trans sialidase etc.Between the amino-acid residue of " glycosyl linking group " insertion " modification group " and peptide and with its covalent attachment.

" complete glycosyl linking group " refers to be derived from the linking group of glycosyl part, and wherein the single sugar monomer that is connected with conjugate is not degraded, as (as passing through sodium metaperiodate) of oxidation." complete glycosyl linking group " of the present invention can be derived from naturally occurring oligosaccharides by adding glycosyl unit or removing one or more glycosyl units from parent sugar structure.Exemplary " complete glycosyl linking group " comprises (as non-degraded) glycosyl part that at least one is complete, and this part is covalently attached on the amino-acid residue on the peptide.The remainder of " linking group " can have basic any structure.For example, the optional direct and complete glycosyl part of modification group is connected.Selectively, modification group is connected in complete glycosyl part by the linker arm.The linker arm can have the basic any structure in the embodiment that can be used for selecting.In an exemplary, the linker arm is one or more complete glycosyl parts, that is, and and " complete glycosyl linking group " similar oligosaccharides.Another exemplary complete glycosyl linking group be the glycosyl part that wherein directly or indirectly is attached to complete glycosyl part be degraded or derivatize (as, periodate oxidation is reductive amination subsequently) group.Another linker arm comprises the modification group that directly or indirectly is attached to complete glycosyl part by linking agent, as those described herein group and analogue thereof.

As used herein, " degraded " refers to remove one or more carbon atoms from glycosyl part.

As used herein, term " targeting part " and " guiding reagent " refer to optionally be positioned the particular organization of health or the kind in zone.This location is by to mediations such as the molecular size of the specific identification of molecule determinative, guiding reagent or conjugate, ionic interaction, hydrophobic interactions.Other are well known to a person skilled in the art with the mechanism that reagent is directed to particular organization or zone.

As used herein, " treatment part " refers to any reagent that is used for the treatment of, including, but not limited to microbiotic, anti-inflammatory agent, antitumor drug, cytotoxin and radioreagent." treatment part " comprises the prodrug of biologically active agent, and promptly a kind of construct wherein surpasses a kind of treatment part and is connected on carrier such as the multivalence reagent.The treatment part also comprises peptide and comprises the construct of this peptide.Exemplary peptide comprises that those are disclosed in Figure 28 and table 6 and 7 herein.Thereby " treatment part " meaning is for any reagent that is used for the treatment of, including, but not limited to microbiotic, antiphlogistic, antitumor drug, cytotoxin and radioreagent." treatment part " comprises the prodrug of bioactive agents, promptly wherein surpasses a kind of construct such as multivalence reagent that part is connected in carrier for the treatment of.

As used herein, " antitumor drug " refers to any reagent that is used to resist cancer, including, but not limited to cytotoxin with as the reagent of metabolic antagonist, alkylating agent, anthracycline, microbiotic, antimitotic agent, procarbazine, hydroxyl urea, asparaginase, reflunomide, Interferon, rabbit and radioreagent and so on.Be contained in equally in term " antitumor drug " category is the conjugate of peptide and anti-tumor activity thing such as TNF-α.Conjugate includes, but are not limited to that those form between therapeutic protein and sugar-protein of the present invention.Representational conjugate forms between PSGL-1 and TNF-α.

As used herein, " cytotoxin or cytotoxic agent " refers to the deleterious reagent of any pair cell.Example comprises taxol, cytochalasin B, Gramicidin D, ethidium bromide, ipecamine, mitomycin, etioposide, teniposide (tenoposide), vincristin, vincaleucoblastine, colchicine, Zorubicin, daunorubicin, dihydroxy anthracinedione, mitoxantrone, Plicamycin, dactinomycin, 1-dehydrogenation testosterone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lidocaine, Proprasylyte and puromycin and analogue thereof or homologue.Other toxin comprise as ricin, CC-1065 and analogue times ganmycin.Other toxin comprises diphtheria toxin and snake venom (as cobra venom) in addition.

As used herein, " radioreagent " comprises any in diagnosis or destroy efficient emission isotropic substance in the tumour.Example is including, but not limited to indium-111, cobalt-60 and technetium.In addition, the naturally occurring radioelement of general proxy radio isotope mixture such as uranium, radium and thorium are the suitable examples of radioreagent.Metal ion generally with organic chelated part chelating.

Many useful chelation groups, crown ether, cryptand etc. are as known in the art and can be integrated in the compound of the present invention (as EDTA, DTPA, DOTA, NTA, HDTA etc. and phosphoric acid salt analogue thereof such as DTPP, EDTP, HDTP, NTP etc.).Referring to as people such as Pitt, " TheDesign of Chelating Agents for the Treatment of Iron Overload ", Inorgnic Chemistry in Biology and Medicine; Martell, Ed.; American Chemistry Society, Washington, D.C., 1980, pp.279-312; Lindoy, The Chemistry of Macrocyclic Ligand Complexes; CambridgeUniversity Press, Cambridge, 1989; Dugas, Bioorganic Chemistry; Springer-Verlag, New York, 1989, and the reference that contains therein.

In addition, make that sequestrant, crown ether and cyclodextrin are that those skilled in the art are obtainable to the number of ways of other molecule attached.Referring to as people such as Meares, " Properties of InVivo Chelate-Tagged Proteins and Polypeptides ", Modification ofProteins:Food, Nutritional and Pharmacological Aspects; People such as Feeney, Eds., American Chemical Society, Washington, D.C., 1982, pp.370-387; People such as Kasina, Bioconjugate Chem., 9:108-117 (1998); People such as Song, Bioconjugate Chem., 8:249-255 (1997).

As used herein, " medicine acceptable carrier " comprises any material, and this material keeps the activity of conjugate when combining with conjugate, and reactive with subject's immunity system right and wrong.Example is including, but not limited to the pharmaceutical carrier of any standard, as salts solution, water, emulsion such as oil/aqueous emulsion and various types of wetting agent of phosphoric acid buffer.Other carriers also can comprise aseptic solution, tablet (tablet that comprises quilt) and capsule.General this carrier contains vehicle, as clay, gelatin, stearic acid and salt thereof, Magnesium Stearate or calcium stearate, talcum, vegetation fat or oil, natural gum, ethylene glycol or other known vehicle of starch, milk, sugar, some type.This carrier also can comprise spices and color additives or other compositions.The composition that comprises this carrier prepares with well-known ordinary method.

As used herein, " give " to point to the subject orally give, as in (intralesional) of the giving of suppository, local contact, intravenous, endoperitoneal, intramuscular, intralesional, the nose or subcutaneous give, give or the slowly implantation of releasing arrangement in the sheath, as little osmotic pump (mini-osmotic pump).

Term " isolating " refers to be substantially free of the material of the composition that is useful on this material of preparation.For peptide conjugate of the present invention, term " isolating " refers to be substantially devoid of usually the material of the composition that accompanies at the mixture that is used for preparing peptide conjugate and this material." isolating " and " pure " is used interchangeably usually, and the isolating peptide conjugate of the present invention has the purity level that preferably is expressed as a scope.The lower limit of this purity range is about 60%, about 70% or about 80%, and the upper limit of this purity range is about 70%, about 80%, about 90% or surpasses about 90%.

When peptide conjugate for surpassing about 90% when pure, its purity also preferably is expressed as a scope.The lower limit of this purity range is about 90%, about 92%, about 94%, about 96% or about 98%, and the upper limit of this purity range is about 92%, about 94%, about 96%, about 98% or about 100%.

Purity is (the dying band brightness on the gel, polyacrylamide gel electrophoresis, HPLC or similar method as silver) determined with the analytical procedure of any this area approval.

As used from here, " commercial size " refers to produce about 1 gram or a few gram end product in method.

As used herein, " basic each member of colony " refers to the feature of peptide conjugate of the present invention colony, wherein the sugar that adds the modification of the selected per-cent on the peptide to added on the peptide on a plurality of identical acceptor sites.On the peptide that " basic each member of colony " mentions and the sugar modified is puted together " homogeneity " in site, and relate to the present invention at least about 80%, be preferably at least about 90% and more preferably be at least about the conjugate of 95% homogeneity.

Structural integrity in the acceptor portion colony that the sugar that " homogeneity " refers to modify is puted together.Thereby, in peptide conjugate of the present invention, wherein the sugar moieties of each modification and acceptor site are puted together, and this receptor site has the identical structure of acceptor site that the sugar of other modification with each is puted together, and this peptide conjugate then is called and is about 100% homogeneity.Homogeneity generally is expressed as a scope.The lower limit of peptide conjugate homogeneity scope is about 60%, about 70% or about 80%, and the upper limit of purity range is about 70%, about 80%, about 90% or surpasses about 90%.

When peptide conjugate for surpassing or when equaling about 90% homogeneity, its homogeneity also preferably is expressed as a scope.The lower limit of this homogeneity scope is about 90%, about 92%, about 94%, about 96% or about 98%, and the upper limit of this purity range is about 92%, about 94%, about 96%, about 98% or about 100%.The purity of peptide conjugate generally well known to a person skilled in the art that with one or more method is definite, as liquid chromatography (LC)-mass spectrometry (LC-MS), the auxiliary laser desorption flying time mass spectrum analysis of matrix method (MALDI-TOF), capillary electrophoresis etc.

When relating to glycopeptide, " unified substantially sugar form " or " unified substantially glycosylation pattern " refers to be undertaken by target glycosyltransferase (as fucosyltransferase) per-cent of glycosylated acceptor portion.For example, at α 1, under the situation of 2-fucosyltransferase, if basic all (as definition below) Gal β 1 in the peptide conjugate of the present invention, then there are unified substantially fucosylation pattern in 4-GlcNAc-R and sialylated analogue thereof all by fucosylation.It will be appreciated by those skilled in the art that parent material can contain glycosylated acceptor portion (as the Gal β 1 of fucosylation, 4-GlcNAc-R part).Thereby, the glycosylation per-cent of calculating will comprise by the glycosylated acceptor portion of method of the present invention and in parent material glycosylated those acceptor portions.

The above-mentioned acceptor portion that term " substantially " in " basic unified " definition is often referred to specific glycosyltransferase at least about 40%, at least about 70%, at least about 80% or more preferably at least about 90% and be glycosylated more preferably at least about 95%.

Invention is described

I. the method for reconstruct polysaccharide chains

The present invention includes by this way and add sugar and/or the sugared method and composition of deletion from the glycopeptide molecule to the glycopeptide molecule external, thereby the peptide molecule with glycosylation pattern specific customized or that want is provided, is preferably incorporated in and adds the sugar of modifying on it.Therefore a key feature of the present invention is to obtain the peptide that is produced by any cell type and generate the core glycan structures on this peptide, subsequently with glycan structures at the external peptide that has the glycosylation pattern that the treatment that is suitable in Mammals uses with generation that is reconstructed.

The importance of the glycosylation pattern of peptide is well known in the art, also is well-known as the limitation of method in the body of the suitable glycosylated peptide of existing production, particularly when these peptides are produced with recombinant DNA method.In addition, up to the present invention, still can not generation have the glycopeptide of the glycan structures of wanting thereon, wherein this peptide can be produced on technical scale.

In the present invention, the peptide that is produced by cell is carried out the enzyme processing external by systematically adding suitable enzyme and substrate thereof, thereby the sugar moieties that should not be present on the peptide can be removed, the sugar moieties (sugar that selectively comprises modification) that should add on the peptide then adds on the peptide by this way, has as the glycopeptide of defined " glycosylation of wanting " elsewhere thereby provide.

A. reconstruct N-connects the method for glycan

On the one hand, the present invention utilizes the following fact, promptly the peptides of great majority with commercial significance or medicine meaning comprise the pentasaccharides structure that common is called three seminose cores herein, and the l-asparagine on this structure and the peptide chain in the Asn-X-Ser/Thr sequence carries out N-and is connected.Three basic seminose cores are made up of 2 N-acetyl-glucosamines (GlcNAc) and 3 seminoses (Man) residue that are attached on the peptide substantially, and promptly it comprises these 5 saccharide residues and does not have extra sugar, and exception is that it selectively can comprise a fucosyl residues.First GlcNAc is attached on the amide group of l-asparagine, and second GlcNAc passes through β 1, and the 4-key is attached on first.1 mannose residue is by β 1, and the 4-key is attached on second GlcNAc, and 2 seminoses are attached on this seminose by

α

1,3 and

α

1,6 key respectively.Schematic description to three seminose core textures is shown in Fig. 1 left side.Although the glycan structures on most of peptides comprises other sugar except that three seminose cores, three seminose core textures have been represented the essential characteristic of the glycan that N-connects on the mammalian-derived peptides.

The present invention includes generation with the peptide of three seminose core textures thereon as the basic structure element of the glycan molecule that contains.Suppose the various cell systems that are used to produce peptide are arranged, no matter this system itself is the naturally occurring recombinant DNA method that also relates to, the present invention all provides the glycan structures that makes on the peptide that produces in any cell type reducible method for three basic seminose core textures.In case generated three basic seminose core textures, the glycan structures that so possible is utilizes the method described herein to want on external generation peptide, this structure give this peptide one or more strengthen the character of its therapeutic efficiency.

In the discussion from here as can be seen term " three seminose cores " be used to be described in the glycan structures shown in Fig. 1 left side.Glycopeptide with three seminose core textures also has the extra sugar that adds thereon, and largely, has the supernumerary structure that adds thereon, and no matter whether this sugar makes peptide have the glycan structures of wanting.Term " three basic seminose core textures " defines elsewhere.When term " basic " was not included in the description of " three seminose core textures ", this glycan was included in the three seminose core textures that are attached with extra sugar on the seminose so.

Term " three basic seminose core glycopeptides " is used in reference to the glycopeptide with the glycan structures that mainly comprises three basic seminose core textures herein.Yet it also selectively contains the fucosyl residues that adheres to thereon.As discussing herein, three basic seminose core glycopeptides are the suitableeest, and are the parent materials of glycan restructuring procedure preferred for the present invention therefore.

The suitableeest another kind of parent material that is used for glycan restructuring procedure of the present invention is the glycan structures with three seminose cores, wherein one or more extra GlcNAc residues add the α 1 of mannose residue to, 3 and α 1, on each of 6 (referring to as the structure on the second line among Fig. 2, from second structure in the left side of figure).This structure is called " Man3GlcNAc4 " herein.When this structure was single feeler, this structure was called " Man3GlcNAc3 " selectively herein, and this structure also can contain core Fucose molecule.In case generated Man3GlcNAc3 or Man3GlcNAc4 structure, the glycan structures that so possible is utilizes the method described herein to want on external generation peptide, this structure give this peptide one or more strengthen the character of its therapeutic efficiency.

In its natural form, the glycopeptide that N-of the present invention connects is to carry out N-with the three seminose core textures that adhere to one or more sugar thereon to be connected glycosylated especially for Mammals of the present invention and human glycopeptide.

Term " glycopeptide " and " sugared polypeptide " are used in reference to the peptide chain with the sugar moieties that adheres to thereon with the free burial ground for the destitute herein.Herein little sugared polypeptide or glycopeptide and big sugared polypeptide or glycopeptide are distinguished.Thereby hormone molecule and other very large peptides of having considerably less amino acid (as usual few to 3 amino acid) in its peptide chain are included in generic term " sugared polypeptide " and " glycopeptide ", as long as they have the sugar moieties that adheres to thereon.Yet the using of term " peptide " do not got rid of this peptide and is glycopeptide.

Example with glycosylated N-connection glycopeptide of wanting is the peptide with glycan of N-connection, and this glycan has the three seminose cores of adhering at least one GlcNAc residue thereon.This residue adds on the three seminose cores with N-acetyl-glucosamine transferase I (GnT-I).If add second GlcNAc, then use N-acetyl-glucosamine transferase I I (GnT-II).Selectively, available GnT-IV and/or GnT-V add extra GlcNAc residue, and the GlcNAc residue of the 3rd five equilibrium can be attached on β 1,4 seminose of three seminose cores with N-acetyl-glucosamine transferase I II (GnT-III).Selectively, this structure can be handled to extend with the GlcNAc to each non-five equilibrium and go up the interpolation galactose residue with β 1,4 galactosyltransferase, and further selectively, utilize α 2,3 or α 2, the 6-sialyltransferase can add sialic acid residues on each galactose residue to.The GlcNAc of five equilibrium is not to add semi-lactosi subsequently and sialic acid residues is necessary to the interpolation of glycan; Yet for the substrate avidity of rat and human GnT-III enzyme, the interpolation of the GlcNAc of five equilibrium has been got rid of in the existence of one or more galactose residues on the glycan, and this is because the glycan that contains semi-lactosi is not the substrate of the GnT-III of these forms.Thereby, want to exist and use under the situation of GnT-III of these forms at the GlcNAc of five equilibrium, importantly contain the semi-lactosi and/or the sialic acid of interpolation as Polylevulosan, they should be removed before the GlcNAc that adds five equilibrium so.The activity of other forms of GnT-III may not need this specific substrate order.In preferred reaction, the mixture of GnT-I, GnT-II and GnT-III is joined in the reaction mixture, thereby the GlcNAc residue can add with any order.

Representative has in the accompanying drawing that the example of glycan structures of all respects of the peptide of " glycosylation of wanting " provides herein and shows.External generation has the accurate process of the peptide of " glycosylation of wanting " and describes elsewhere.Yet the present invention never should be interpreted as just being confined to disclosed herein any one glycan structures.The present invention more suitably should be interpreted as comprising any one or the whole glycan structures that can be used on method provided herein preparation.

In some cases, basic three seminose cores can constitute the glycosylation of wanting of peptide separately.For example, shown that the peptide that only has three seminose cores is useful component (people such as Mistry, 1966, the Lancet 348:1555-1559 that is used for the treatment of the enzyme of gaucher's disease; People such as Bijsterbosch, 1966, Eur.J.Biochem.237:344-349).

According to the present invention, generation is described below has the program of the glycosylated peptide of wanting.

A) originate in glycopeptide with one or more glycan molecules, the common trait of this glycan molecule is to have three seminose core textures and the mixture that is at least a or multiple heterogeneous or homogeneity of one or more sugar of adding thereon, possible is increase have three basic seminose core textures as unique glycan structures or have Man3GlcNAc3 or Man3GlcNAc4 a ratio as the glycopeptide of unique glycan structures.This is by systematically realizing that to the enzyme of glycopeptide interpolation proper number the mixture heterogeneous or homogeneity that this enzyme can cut the sugar on the glycan structures reduces to basic three seminose cores or Man3GlcNAc3 or Man3GlcNAc4 structure up to it with suitable order external.These specific examples how to realize will depend on various factors, comprise cell type that produces peptide and the complicacy degree of the glycan structures that therefore exists on very most of on the peptide that is produced by cell at first.How complicated glycan structures is reduced to basic three seminose cores or Man3GlcNAc3 or Man3GlcNAc4 example of structure shows in Fig. 2, or has a detailed description elsewhere.

B) possible is to generate by the cell that separating natural exists to have the peptide of three basic seminose core textures as unique glycan structures, and wherein the glycosylation machine of this cell produces this peptide.The DNA transfection of the target peptide of will encoding then in cell, therein DNA transcribe, translation and glycosylation, thereby target peptide has three basic seminose core textures as unique glycan structures.For example, shortage has the cell of the GnT-I enzyme of function will produce the glycopeptide of several types.In some cases, these will be not have the glycopeptide that is attached to the extra sugar on the three seminose cores.Yet under other situation, the peptide of generation can have 2 extra mannose residues that are attached on the three seminose cores, thereby the result obtains the Man5 glycan.This also is the parent material of wanting that is used for restructuring procedure of the present invention.The specific examples that generates this glycan structures is described herein.

C) selectively, possible is that pair cell carries out genetically engineered processing to give this cell specific glycosylation machine, has basic three seminose cores or Man3GlcNAc3 or the Man3GlcNAc4 structure peptide as unique glycan structures on the peptide thereby can produce.The DNA transfection of the target peptide of will encoding then in cell, therein DNA transcribe, translation and glycosylation, thereby target peptide has the glycan that comprises three basic seminose core textures separately that increases number.For example, the cell that carries out genetically engineered processing and lack some type of GnT-I has generation the glycan of three basic seminose core textures, perhaps depend on this cell, can produce the glycan (Man5) of 2 extra mannose residues that have three seminose cores and adhere to thereon.When cell produces the Man5 glycan structures, but pair cell further carries out genetically engineered processing to express

mannosidase

3, and 2 extra mannose residues of these mannosidase 3 cuttings are to generate three seminose cores.Selectively, the Man5 glycan can carry out incubation to reach identical effect with mannosidase 3 external.

D) when peptide during in expressed in insect cells, the glycan on the peptide comprises part compound chain.Insect cell is also expressed hexosaminidase in cell, this enzyme is trimmed to three seminose core textures with part compound chain, and this structure is reconstructed as described in herein then.

E) from b), c) and d) discussion see and it is evident that cell does not need only to produce the peptide that is attached with basic three seminose cores or Man3GlcNAc3 or Man3GlcNAc4 structure thereon.Unless that more suitable is b) and c) in the cell described produce and have 100% basic three seminose core textures (promptly not having the extra sugar that adheres to thereon) or 100% Man3GlcNAc3 or Man3GlcNAc4 structure, cell in fact produces the heterogeneous mixture of peptide, this peptide except that the combination with basic three seminose core textures or Man3GlcNAc3 or Man3GlcNAc4 structure as unique glycan structures, also have these structures that are attached with other sugar thereon.Peptide with the three seminose cores that are attached with extra sugar thereon or Man3GlcNAc3 or Man3GlcNAc4 structure will depend on the cell that produces them to those ratios that only have a kind of peptide of structure and change.The complicacy of glycan (being which kind of and how much sugar are attached on the three seminose cores) also will depend on the cell that produces them.

F) in case pass through a) according to the present invention, b) or c) produced the glycopeptide that has three basic seminose cores or be attached with three seminose cores of one or more GlcNAc residues thereon, have the glycosylated peptide of wanting (promptly having peptide) external extra glycan molecule can being added on the three seminose core textures with generation so at the glycan structures of external customization.

G) yet, be attached with some thereon but when not being the peptide of the basic three seminose cores of the sugar all wanted or Man3GlcNAc4 structure when having produced to have, so only need to add the remaining sugar of wanting, and glycan structures need not be reduced to basic three seminose cores or Man3GlcNAc4 structure.Therefore, in some cases, the peptide with glycan structures of the three seminose core textures that are attached with extra sugar thereon will be the suitable substrate that is reconstructed.

The separation of basic three seminose core glycopeptides

If desired, basic three seminose cores of the present invention or Man3GlcNAc3 or Man3GlcNAc4 glycopeptide can separate and purifying with the well-known technology in peptide purification field.Suitable technology comprises chromatographic technique, isoelectric focusing technique, ultra-filtration technique etc.Utilize any such technology, can prepare composition of the present invention, glycopeptide wherein of the present invention is to be typically found at component separating in the cell culture medium with other peptides with other.With respect to other peptide, the degree of purifying can be as 90% or 95% or higher, as 98%.Referring to as people such as Deutscher (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).

Be present in the heterogeneous separation that only allows small portion target glycopeptide usually that N-in the glycopeptide that is produced by the method in prior art field connects glycan, this target glycopeptide can be modified the glycopeptide of wanting with generation.In the method for the invention, can produce a large amount of basic three seminose core glycopeptides and other glycopeptides of wanting, comprise Man3GlcNAc3 or Man3GlcNAc4 glycopeptide, these glycopeptides can further be modified then to generate has the glycosylated peptide of wanting in a large number.

The specific enrichment that is connected to the glycan of any particular type on the peptide can usually be realized with the aggegation that the glycan of wanting is had avidity.This technology is well-known in the glycobiology field.

In a single day key feature of the present invention in greater detail below is to have generated glycan structures on any peptide, this glycan structures can have the glycosylated peptide of wanting external being reconstructed with generation so, and this peptide has the treatment of improvement and uses in Mammals.Mammals can be the suitable Mammals of any kind, and preferably is the people.

Various situations and accurate method and composition that generation has the glycosylated peptide of wanting will become conspicuous in disclosure subsequently.

The final purpose that production is used for the peptide of Mammals treatment application is that this peptide should comprise the glycan structures that promotes rather than cancel the treatment benefit of this peptide.As it is disclosed to run through this specification sheets, the peptide that produces in cell can be handled at the various enzymes of external use, this enzyme catalysis should not be present in the cutting of the sugar on the glycan and should be present in the interpolation of the sugar on the glycan, has the glycosylation wanted and thereby is suitable for the peptide that the treatment in the Mammals is used thereby then produced.Being created on above of the different sugar form of peptide described in the cell.Described the mechanism that various generations have the glycosylated peptide of wanting now, wherein parent material is promptly variant according to cell type by the peptide that cell produces.As from disclosure of the present invention with conspicuous, aspect its glycan composition, it is unified that parent material needs not to be.Yet preferably the parent material of some sugar form of enrichment is to produce a large amount of end products, promptly correct glycosylated peptide.

In according to the preferred embodiment of the invention, the result obtain having the degraded of the glycosylated peptide of wanting and compound event some the time relate to the generation of basic three seminose core textures on the peptide or Man3GlcNAc3 or Man3GlcNAc4 structure.

The present invention also provides the method for adding the glycosyl residue of one or more selections to peptide, and the sugar that will modify is conjugated on the glycosyl residue of at least one selection on the peptide thereafter.The present embodiment is useful when the glycosyl residue of sugar of for example wanting to modify and selection is puted together, and wherein the glycosyl residue of this selection is not present on the peptide or not and exists with the amount of wanting.Thereby, before the sugar that makes modification and peptide carry out coupling, glycosyl residue and the peptide selected are puted together by enzymatic or chemical coupling.In another embodiment, the glycosylation pattern of peptide is to change by remove saccharide residue from peptide before the puting together of the sugar of modifying.Referring to as WO 98/31826.

Be present in the interpolation of any sugar moieties on the peptide or removal and be chemically or enzymatic is realized.The chemistry de-glycosylation preferably takes place by the compound that the peptide variant is exposed to compound trifluoromethanesulfonic acid or equivalence.This processing causes great majority or all cuttings of sugar except that connecting sugar (N-acetyl-glucosamine or N-acetylgalactosamine), and remaining complete peptide.Chemical degradation is described in people such as Hakimuddin, and 1987, people such as Arch.Biochem.Biophys.259:52 and Edge, 1981, Anal.Biochem.118:131.On the peptide variant enzymatic of sugar moieties cutting can by use various inscribes-and circumscribed-Glycosylase realize, as by people such as Thotakura, 1987, Meth.Enzymol.138:350 is described.

The chemistry interpolation of glycosyl part is to implement by the method for any this area approval.The enzymatic of glycosyl part adds preferably to use to be realized the amending method of the method that proposes herein, promptly with the alternative sugar that is used for the present invention's modification of natural glycosyl unit.Other methods of adding sugar moieties are described in U.S. Patent No. 5,876,980,6,030,815,5,728,554 and 5,922,577.

The exemplary attachment site of the glycosyl residue of selecting is including, but not limited to (a) N-and O-glycosylation site; (b) as the terminal saccharide part of glycosyltransferase acceptor; (c) arginine, l-asparagine and Histidine; (d) free carboxy; (e) free thiohydroxy group is as halfcystine; (f) free hydroxyl group is as Serine, Threonine or oxyproline; (g) aromatic moieties is as those phenylalanines, tyrosine or tryptophane; Or (h) amide group of glutamine.Be used for illustrative methods of the present invention and be described in WO 87/05330 and Aplin and the Wriston that delivered on September 11st, 1987, CRC Crit.Rev.Biochem. is among the pp.259-306 (1981).

Example shown in the several figure that provide herein specifically is provided, provides now on peptide, producing the description of the external enzymatic reaction sequence of the glycan structures of wanting.The accurate reaction conditions that each enzymatic that is described below changes is that the technician in glycobiology field is well-known and therefore do not carry out repetition at this.For the summary of the reaction conditions of these type reaction, referring to people such as Sadler, 1982, Methods in Enzymology 83:458-514 and the reference of quoting therein.

The left side has shown the structure of three basic seminose core glycan in Fig. 1.Possible is by GnT-I subsequently for GnT-II and when further existing for GnT-III and the saccharide donor that comprises UDP-GlcNAc subsequently three basic seminose core textures of incubation be complete glycan structures with this structural transformation with GlcNAc of five equilibrium, wherein GlcNAc is added successively to the three seminose cores that have the GlcNAc of five equilibrium on the basic three seminose core textures with generation.In some cases, for example when when the Fc glycan being reconstructed as described in herein, add GnT-I, GnT-II and GnT-III order can with document in report opposite.Can produce the GlcNAc structure of five equilibrium by in reaction mixture, adding GnT-I, GnT-II and GnT-III and UDP-GlcNAc.

In Fig. 3, shown of the transformation of the three seminose core glycan of the GlcNAc that contains five equilibrium to the complicated glycan structures that comprises semi-lactosi and N-n acetylneuraminic acid n.At first will contain the three seminose core glycan of GlcNAc of five equilibrium and galactosyltransferase and carry out incubation, wherein two galactose residues be added on the periphery GlcNAc residue on the molecule as the UDP-Gal of donor molecule.Then the NeuAc-transferring enzyme is used for respectively adding a NeuAc, totally two NeuAc to each galactose residue.

In Fig. 4, shown of the transformation of high mannose glycans structure to three basic seminose core glycan.Make high mannose glycans (Man9) carry out incubation successively to generate the Man5 structure when mannosidase 1 exists, carry out incubation then when mannosidase 3 exists, wherein all mannose residues except that 3 mannose residues will be removed from glycan.Selectively, the incubation of Man9 structure can only be trimmed to three seminose core textures by carry out incubation when mannosidase 3 exists.According to the scheme that provides in above-mentioned Fig. 1 and 3, these three basic seminose core glycan then are possible to the transformation of the glycan molecule of complexity then.

The glycan structures that in Fig. 5, has shown the typical complicated N-connection that produces in the vegetable cell.Be important to note that wood sugar and Fucose can not add in the glycan when vegetable cell disappearance GnT-I enzymic activity.Thereby, the application of GnT-I gene knockout cell provides special advantage among the present invention, be that these cells can produce the peptide with three basic seminose cores, on these three basic seminose cores, can add extra sugar and need not carry out any " pruning " reaction.Similarly, the structure that produces in vegetable cell is that if do not have GnT-I in these cells, wood sugar and Fucose then can not add in this structure so under the situation of Man5 variant of glycan.In this case, the Man5 structure can be trimmed to three basic seminose cores (Man3) with mannosidase 3.According to the method that provides herein, at present possible is to add sugar moieties to glycan structures that three seminose cores are wanted with generation.

The glycan structures that in Fig. 6, has shown the typical complicated N-connection that produces in the insect cell.Be apparent that extra sugar such as Fucose also can exist.Although further do not show herein, insect cell can produce has nearly 9 mannose residues and high mannose glycans that can be attached with extra sugar thereon.Same situation is in insect cell, and the cell of GnT-I gene knockout has prevented the interpolation of Fucose on glycan.Thereby the generation of peptide preferably can realize in GnT-I gene knockout cell in the insect cell.Thereby if desired, the glycan that produces like this can be used on any method described herein then and scheme is carried out external pruning, and extra sugar also can be used on method described herein and scheme is added on it external.

The glycan structures that in Fig. 2, has shown various performance levels.Specifically, shown that the external enzymatic of complicated sugared glycan structures that three basic seminose core textures never contain the GlcNAc residue of five equilibrium generates.It is same that what show is the glycan structures of the GlcNAc that contains five equilibrium that generates thus.Several producible middle glycan structures have been shown.These structures can be produced by cell, or produce in the external pruning reaction that can describe herein.Sugar moieties can add on the three basic seminose core textures external, or adds the glycan of wanting with generation on any suitable intermediate structure to.

Shown a series of possible vitro reactions in Fig. 7, this reaction can be carried out the pruning of the glycan that originates in high mannose structures and interpolation.For example, the Man9 glycan can be pruned generating the Man5 glycan with mannosidase 1, or its available mannosidase 3 or one or more microorganism mannosidases are trimmed to three seminose cores.Then can with GnT-I and or GnT-II be used for extra GlcNAc residue is transferred to glycan.Further, shown the situation (referring to dash box) that when the glycan molecule produces, does not take place in the cell that does not have GnT-I.For example, Fucose and wood sugar only can add on the glycan when GnT-I is active and promotes GlcNAc to the transfer of molecule.

Fig. 8 describes and to originate in synthetic two feeler, three feelers and even the well-known strategies of the glycan structures of four feelers of three seminose core textures.The method according to this invention, possible is with each in external synthetic these structures of well-known suitable enzyme and reaction conditions in the glycobiology field.

Fig. 9 describes two kinds of methods that begin synthetic single feeler glycan structures from high mannose (6～9 seminose parts) glycan structures.According to Glycopegylated method described herein, can add terminal sialic acid-peg moiety to substitute the sialic acid part.In first method, endo-H is used for the glycan structures on the peptide is cut to first GlcNAc residue.Add semi-lactosi with galactosyltransferase then, and add sialylated PEG as described elsewhere herein.In second method, mannosidase I is used for the glycan structures cutting mannose residue from peptide.Galactose residue is added on the arm of remaining mannose residue, this mannose residue excises from glycan with the sword bean alpha-Mannosidase.The PEG with sialylated as described adds on this structure then.

Figure 10 describes the other two kinds of methods that begin synthetic single feeler glycan structures from high mannose (6～9 seminose parts) glycan structures.The same with among Fig. 9 according to Glycopegylated method described herein, added terminal sialic acid-peg moiety to substitute the sialic acid part.In the described herein situation, never add the arm of sialylated PEG and remove some mannose residues.

In Figure 11, shown the scheme that originates in the synthetic more complicated sugared structure of three seminose core textures.For example, the sialylated or non-sialylated Lewis x of produced in vitro and the scheme of Lewis a antigenic structure have been shown.This structure can give peptide immunologic advantage on being present in peptide the time, regulates immune response to be used for incremental adjustments or decrement.In addition, this structure can be used for the guiding of peptide to specific cells, and this is because this class formation participates in and the combining of cell attachment peptide etc.

Figure 12 is the exemplary arrangement that is used to prepare the peptide array of the O-connection that is derived from Serine or Threonine.

Figure 13 is the diagram that a series of descriptions are called the glycan structures that 4 class O-of core 1～4 connect.Core texture is described with dotted line.The steamed bun stuffed with sugar that also can be included in this structure is drawn together sialic acid residues that adds on the galactose residue and the fucosyl residues that adds on the GlcNAc residue.

Thereby, in preferred embodiments, the invention provides the method for the glycosylation glycopeptide of preparation N-connection, this method is: the glycopeptide of the isolating and purifying that has adhered to three basic seminose cores or Man3GlcNAc4 structure thereon is provided, glycopeptide and glycosyltransferase and the donor molecule with glycosyl part are contacted under the condition that is suitable for glycosyl part transferred in the glycopeptide.Customize three basic seminose cores or Man3GlcNAc4 structure then and have the peptide of the glycosylation pattern of wanting with generation, this can realize by adding the sugar moieties of wanting successively with technology well known in the art.

Determining of glycan primary structure

When the glycopeptide of N-connection is produced by cell, as mentioning elsewhere, it can comprise the heterogeneous mixture of glycan structures, and this glycan structures must be reduced to total and common basic three seminose cores or Man3GlcNAc4 structure before the sugar moieties that adds other on it.Which sugar should be removed from specific glycan structures in order accurately to determine, essential sometimes is to identify the one-level glycan structures.The technology of determining the glycan primary structure is well known in the art, and be described in detail in as Montreuil, " Structure and Biosynthesis ofGlycopeptides ", Polysaccharides in Medicinal Applications, pp.273-327,1996, Eds.Severian Damitriu, Marcel Dekker, NY.Therefore separation is a simple thing by peptide colony and the definite glycan structures that adheres to thereon that cell produces for the technician in the glycobiology field.For example, for lower part the available effective ways are arranged: (i) make the glycosidic link fracture by chemical chop such as hydrolysis, diacetyl oxide decomposition, hydrazinolysis or by the nitrous acid deamination; (ii) exhaustive methylation is hydrolyzed or methanolysis and carry out gas liquid chromatography and mass spectrometry analysis to the monose of part methylization subsequently subsequently; (iii) with exoglycosidase to the determining of different key between the monose, this also provides understanding to the one-level glycan structures by degraded successively.Especially, mass spectrometry and nucleus magnetic resonance (NMR) spectrum assay method particularly the NMR of High-Field can successfully be used for determining the glycan primary structure.

Be used for the test kit of glycan analysis and equipment is also commercial buys.The auxiliary sugared electrophoresis of fluorophore (

) can be available from Glyko, Inc. (Novato, CA).In FACE analyzes, glycoconjugate used at N-connect the inscribe H of glycan or N-glycanase (PNGase F) or from peptide, discharge at the hydrazine that Ser/Thr connects glycan.The mode that this glycan is distinguished with non-structure with fluorophore is carried out mark in reduction end then.Then with the glycan of fluorophore mark based on lotus/matter of sugar than and hydrokinetics volume and on polyacrylamide gel, separating.Under UV light, obtain gel images, and the composition of glycan is to determine by the migration distance of comparing with standard substance.Oligosaccharides can check order by analyzing owing to the migration and variation of removing sugar successively with exoglycosidase digestion by this way.

Exemplary

The glycosylated reconstruct that N-connects has been carried out best illustrating with reference to following molecular formula 1

X wherein ³, X ⁴, X ⁵, X ⁶, X ⁷And X ¹⁷Be (the independent selection) monose or oligosaccharides residue; And

A, b, c, d, e and x are (independent select) 0,1 or 2, and prerequisite is that at least one member who is selected from a, b, c, d, e and x is 1 or 2.

Molecular formula 1 has been described the glycan structures that comprises three seminose cores, and this structure optimization ground is on the covalently bound asparagine residue to the peptide main chain.Preferred expression system will be expressed justacrine and be had the exogenous peptide that the N-that comprises three seminose cores connects glycan.Utilize reconstructing method of the present invention, the glycan structures on these peptides can be reconstructed into any glycan structures of wanting easily.Exemplary reaction conditions can be found in embodiment and the document.

In preferred embodiments, glycan structures has been carried out reconstruct, thereby the structure of describing has specific determinant (determinate) in molecular formula 1.The structure that can select glycan with the biologic activity that strengthens peptide, give the new biologic activity of peptide, the biologic activity of removing peptide or the glycosylation pattern of approximation better (approximate) native peptides and other.

In first embodiment preferred, the glycan that peptide N-is connected has carried out the glycosylation pattern of reconstruct with the proteinoid of approximation natural human better.In this embodiment, the glycan structures that is described in the molecular formula 1 has been carried out reconstruct to have following part:

X ³And X ⁵=|-GlcNAc-Gal-SA;

A and c=1;

D=0 or 1;

B, e and x=0.

This embodiment is particularly advantageous for the human peptide that is expressed in the allos cell expression system.Be reconstructed into this configuration by the glycan structures that N-is connected, this peptide can be in people patient than reduced immunogenicity and/or more stable, and has other characteristic.

In second embodiment preferred, the glycan that peptide N-is connected has carried out reconstruct to have the GlcNAc of five equilibrium on three seminose cores.In this embodiment, the glycan structures that is described in the molecular formula 1 has been carried out reconstruct to have following part:

X ³And X ⁵Be |-GlcNAc-Gal-SA;

A and c=1;

X ⁴Be GlcNAc;

b＝1；

D=0 or 1;

E and x=0.

This embodiment is particularly advantageous for the recombinant antibodies that is expressed in the allos cell system.When antibody molecule comprises the cytotoxicity of Fc-mediation, be well known that the existence of the oligosaccharides of the five equilibrium that is connected with the Fc structural domain has increased antibody dependent cellular cytotoxicity hugely.

In the 3rd embodiment preferred, the glycan that peptide N-is connected has carried out reconstruct to have sialylated Lewis X part.In this embodiment, the glycan structures that is described in the molecular formula 1 has been carried out reconstruct to have following part:

X ³And X ⁵Be

A, c and d=1;

B, e and x=0;

X ⁶It is Fucose.

This embodiment is particularly advantageous when the peptide that is reconstructed is wanted to be directed in the cell of selecting protein molecular and displaying same molecular.

In the 4th embodiment preferred, the glycan that peptide N-is connected has carried out reconstruct to have the part of puting together.This part of puting together can be PEG molecule, another peptide, small molecules such as medicine, and other.In this embodiment, the glycan structures that is described in the molecular formula 1 has been carried out reconstruct to have following part:

X ³And X ⁵Be |-GlcNAc-Gal-SA-R;

A and c=1 or 2;

D=0 or 1;

B, d, e and x=0.

Wherein R=puts together group.

The part of puting together can be PEG molecule, another peptide, small molecules such as medicine, and other.Therefore this embodiment can be used for peptide and the PEG molecule that the peptide that slows down is removed from patient's blood flow are puted together, and two peptides is directed to specific tissue or the peptide in the cell is puted together, or with have another peptide that the complementary treatment uses and put together.

It will be apparent to one skilled in the art that the present invention is not limited to above-mentioned preferred glycan molecule.Embodiment preferred only is the minority in the many useful glycan molecule of available reconstructing method of the present invention preparation.Skilled in the art will recognize that and how to design other useful glycan.

In first exemplary embodiment, peptide is expressed in CHO (Chinese hamster ovary cell) according to method well known in the art.When the peptide in the consistent site of glycan with N-connection is expressed justacrine in Chinese hamster ovary celI, the glycan that this N-connects will have the structure that is described in Fig. 2 top line but also comprises the core Fucose.Although may there be all these structures, the most general so far structure is on the right two.In the term of molecular formula 1,

X ³And X ⁵Be |-GlcNAc-Gal-(SA);

A and c=1;

B, e and x=0 and

D=0 or 1.

Therefore, in an exemplary embodiment, the N-that is expressed in the peptide in the Chinese hamster ovary celI is connected glycan be reconstructed into preferred humanized glycan by making this peptide and contacting of glycosyltransferase, this glycosyltransferase is specific for galactosylated acceptor molecule and sialic acid donor molecule.This process is set forth among Fig. 2 and the embodiment 17.In another exemplary embodiment, the N-that will express the peptide of justacrine in Chinese hamster ovary celI connects the structure that glycan is reconstructed into preferred PEGization.Make this peptide at first with the specific Glycosylase of sialic acid is contacted to remove terminal SA part, contact when existing at PEG-sialic acid-Nucleotide donor molecule with the specific glycosyltransferase of sialic acid acceptor portion then with to the semi-lactosi acceptor portion.Selectively, this peptide is contacted to guarantee that the SA that finishes all glycan molecules adds cap when existing at sialic acid-Nucleotide donor molecule with glycosyltransferase, wherein glycosyltransferase is specific to semi-lactosi acceptor portion and sialic acid acceptor portion.

In another exemplary embodiment, peptide according to method well known in the art in expressed in insect cells, as sf9 clone.When the peptide in the consistent site of glycan with N-connection was expressed justacrine in the sf9 cell, the glycan that this N-connects often had the structure that is described in Fig. 6 top line.In the term of molecular formula 1:

X ³And X ⁵Be |-GlcNAc;

A and c=0 or 1;

b＝0；

X ⁶It is Fucose;

D=0,1 or 2; And

E and x=0.

Three seminose cores are present in the glycan of the most N-connections that prepared by insect cell, and also have feeler GlcNAc and/or fucosyl residues sometimes.Notice that glycan can not have the core Fucose, it can have the core Fucose of any one key, and perhaps it can have the preponderate core Fucose of (perponderance) of a kind of key.In an exemplary embodiment, the glycan that N-at the peptide of expressed in insect cells justacrine is connected is reconstructed into preferably humanized glycan, this realizes by following steps: at first make glycan and the specific Glycosylase of Fucose is contacted, glycan is contacted when existing at Nucleotide-GlcNAc molecule with the glycosylation transferring enzyme, and this glycosyltransferase is specific to the mannose receptor molecule on each feeler of three seminose cores, GlcNAc donor molecule; Make glycan then and GlcNAc acceptor molecule, the specific glycosyltransferase of Gal donor molecule are contacted when existing at Nucleotide-Gal molecule; Glycan is contacted when existing at Nucleotide-SA molecule with glycosyltransferase to semi-lactosi acceptor molecule, sialic acid donor molecular specificity.It should be appreciated by those skilled in the art that if there are some Fucose molecules they can be in the removal whenever of this process so, and if the core Fucose have and identical α 1,6 key of in people's glycan, finding, it is kept perfectly.In another exemplary embodiment, further by glycan is further contacted when existing being reconstructed into sialylated Lewis X glycan at Nucleotide-Fucose molecule with glycosyltransferase, this glycosyltransferase is specific to GlcNAc acceptor molecule, Fucose donor molecule with humanized glycan in the previous examples.This process is illustrated in Figure 11 and embodiment 39.

In the exemplary embodiment of another one, peptide is expressed in yeast according to method well known in the art, as Saccharomyces cerevisiae (Saccharomyces cerevisiae).When the peptide in the consistent site of glycan with N-connection is expressed justacrine in the Saccharomyces cerevisiae cell, the glycan that this N-connects will have the structure that is described in the left side among Fig. 4.The glycan that N-connects will usually have three seminose cores, this will be usually by seminose or nearly the relevant polysaccharide of 1000 residues describe in detail.In the term of molecular formula 1:

X ³And X ⁵Be |-Man-Man-(Man) _0-1000

A and c=1 or 2;

B, d, e and x=0.

In an exemplary embodiment, to in yeast cell, express the glycan that the N-of the peptide of justacrine connects and be reconstructed into three basic seminose cores, this realizes by following steps: at first make glycan and the specific Glycosylase of α 2 mannose molecules is contacted, make glycan then and the specific Glycosylase of α 6 mannose molecules is contacted.This process is illustrated in Fig. 4 and embodiment 38.

In another exemplary embodiment, the further reconstruct of glycan that N-is connected is with the preparation glycan, this glycan is suitable for having the recombinant antibodies of the cytotoxicity function of Fc-mediation, above-mentioned reconstruct realizes by following steps: three basic seminose core glycan are contacted when existing at Nucleotide-GlcNAc molecule with glycosyltransferase, and wherein this glycosyltransferase is specific to the mannose receptor molecule on each feeler of three seminose cores, GlcNAc donor molecule.Make glycan then and acceptor mannose molecules, the specific glycosyltransferase of GlcNAc donor molecule in the middle part of the three seminose cores are contacted when existing at Nucleotide-GlcNAc molecule, and further make glycan and GlcNAc acceptor molecule, the specific glycosyltransferase of Gal donor molecule are contacted when existing at Nucleotide-Gal molecule; Randomly make then glycan with to the semi-lactosi acceptor molecule, and further optional glycosyltransferase to the sialic acid donor molecular specificity contact when existing at Nucleotide-SA molecule.This process is illustrated in Fig. 1,2 and 3.

In the exemplary embodiment of another one, peptide is expressed in bacterium according to method well known in the art, particularly in intestinal bacteria (E.coli) cell.When the peptide in the consistent site of glycan with N-connection is expressed in Bacillus coli cells, the consistent site that this N-connects will not be glycosylated.In an exemplary embodiment, humanized glycan molecule is constructed from the peptide main chain, and this realizes by following steps: peptide is contacted when Nucleotide-GlcNAc exists with the specific glycosyltransferase of GlcNAc donor molecule with the consistent site that N-is connected; And the glycan that further makes growth successively with the special glycosyltransferase of acceptor and donor part is contacted the glycan structures of wanting up to finishing when essential donor partly exists.When the peptide with glycan that N-connects is expressed in eukaryotic cell, but do not have when nascent peptide is directed to suitable leading peptide in the golgi body, sophisticated peptide may be by glycosylation.Equally in this case, this peptide can be by the glycosylation that connects from the consistent site structure N-that connects as above-mentioned peptide N-.When protein carried out chemically modified with sugar moieties, it can be as construct above-mentionedly.

These examples are wanted to illustrate the present invention rather than are limited.It will be appreciated by those skilled in the art that the step of carrying out can carry out with different orders in some cases and obtain identical result in each example.Those skilled in the art also will understand different steps also can produce identical glycan.Preferably the glycan of reconstruct is specific to the expression system of expressing this peptide never.The glycan of reconstruct is the property illustrated only, and those skilled in the art will know that the peptide of how obtaining principle and be applied to produce from these examples is to prepare herein the not glycan of specific description in different expression systems.

B. reconstruct O-connects the method for glycan

The O-glycosylation is characterised in that with the O-glycosides key has adhered to various monose to hydroxy-amino-acid.The O-glycosylation is posttranslational modification general in animal and plant circle.The structural complexity of the glycan that is connected with protein O-is considerably beyond the glycan of N-connection.The Serine or the threonine residues of the peptide of new translation are modified by peptidyl GalNAc transferring enzyme in the compartment of the outside substantially in the inboard of golgi body.O-is not only in glycosylated site by the sequence-specific of glycosyltransferase and determines, and be by the competition between the different substrate sites and and other outer heredity of being responsible for forming the competition mediation between the glycosyltransferase of glycan to regulate (epigenetic regulation) definite.

The glycan that O-is connected is defined as artificially and has 3 zones: core, main chain and outer regions." core " zone that O-connects glycan is near inner most 2 or 3 sugar of the polysaccharide chains of peptide.The main length of forming by the unified polysaccharide chains that extends to form in main chain zone.Outer regions is showed the structural complexity of height.The structural complexity that O-connects glycan starts from core texture.In most of the cases, connecting first saccharide residue that adds in the consistent site of glycan at O-is GalNAc; Yet should also can be GlcNAc, glucose, seminose, semi-lactosi or Fucose etc. for sugar.Figure 12 is the diagram that some known O-connect synthetic enzyme in glycan core texture and responsible its body.

In mammalian cell, found the core texture that 8 different O-connect at least, all be based on core-α-GalNAc residue.4 core textures describing in Figure 13 are the most general.Core 1 and core 2 are abundant structures in the mammalian cell, and core 3 and core 4 are found in the expression system of more restricted and organ characteristic's property.The summary that O-connects glycan has Montreuil, Structure and Synthesis ofGlycopeptides, Polysaccharides in Medicinal Applications, pp.273-327,1996, Eds.Severian Damitriu, Marcel Dekker, NY, with Schachter and Brockhausen, The Biosynthesis of Branced O-LinkedGlycans, 1989, Society for Experimental Biology, pp.1-26 (Britain).

See that from disclosure of the present invention the glycan structures that it is evident that the glycosylated peptide of O-can be reconstructed with the similar technology with the glycan description that N-is connected.O-glycan and the difference of N-glycan are that they are connected on Serine or Threonine rather than the asparagine residue.As described about the reconstruct of N-glycan herein, lytic enzyme can be used for cutting O-and connects undesired sugar moieties on the glycan, and the extra sugar of wanting can add on it then, thus on peptide the O-glycan structures (referring to Figure 12 and 13) of structure customization.

The glycosylated initial step of O-is to adhere to N-acetylgalactosamine (GalNAc) with any one of at least 11 known α-N-acetylgalactosamine transferring enzyme families in the mammalian cell, and each of this enzyme all has strict acceptor peptide specific.Usually, form at least 10 amino acid whose sequences by the acceptor peptide of each enzyme identification.If in the cell of expressing this enzyme, express and they are when roughly being positioned on the golgi body that UDP-GalNAc also exists then to become O-glycosylated if contain by the peptide of the aminoacid sequence of a specific GalNAc-transferring enzyme identification.

Yet under the situation of recombinant protein, initial the adhering to of GalNAc may not can take place.Natural α-N-acetylgalactosamine the transferring enzyme of express cell can have and the different concensus sequence specificity of expressing of recombinant peptide.

The recombinant peptide of wanting can cannot not be expressed in syntheticly in the bacterial cell of polysaccharide chains, in intestinal bacteria.In these cases, partly be favourable at the initial GalNAc of external interpolation.In case recombinant peptide reclaims with the form of solubility, then the GalNAc part can contact when UDP-GalNAc exists and be incorporated on the peptide external with suitable GalNAc transferring enzyme by making peptide.

In one embodiment, can have extra aminoacid sequence, this aminoacid sequence is formed the effective acceptor that is used to shift the sugar that O-connects.This aminoacid sequence is by the dna sequence encoding that merges with the encoding sequence of peptide in frame, perhaps selectively can be introduced by chemical process.This peptide may lack polysaccharide chains in addition.Selectively, this peptide may have the polysaccharide chains of N-and/or O-connection, but needs extra glycosylation site, as when needing extra glycan substituting group.

In an exemplary embodiment, add aminoacid sequence PTTTK-COOH as merging mark, this aminoacid sequence is the natural GalNAc receptor sequence among people's Saliva Orthana MUC-1.Make fused protein at expression in escherichia coli and purifying then.This peptide when existing, UDP-GalNAc is contacted so that the GalNAc residue is transferred in the peptide external with the people GalNAc-transferring enzyme T3 or the T6 of reorganization then.

The method that polysaccharide chains on the peptide can further be described with the glycan that connects for N-herein or O-connects is then extended.Selectively, the reaction of GalNAc transferring enzyme can be carried out when UDP-GalNAc exists, and the PEG covalency has replaced O-3,4 or 6 positions or N-2 position on UDP-GalNAc.Sugar is conjugated in other places and has a detailed description.Any antigenicity of introducing by new peptide sequence can be easily by the PEGization of relevant glycan is covered.The acceptor site integration technology can be used for not only introducing peg moiety, and introduce other glycan and non-glycan part, including, but not limited to the sequestrant of toxin, anti-infective, cytotoxic agent, radioactive nuleus thuja acid with have the glycan of other functions such as tissue guide.

Exemplary

It is to carry out best illustrating with following molecular formula 2 that O-connects glycosylated reconstruct:

Molecular formula 2 described comprise preferably with the peptide main chain on Serine or the glycan structures of the covalently bound GalNAc of threonine residues.Although this structure is used to illustrate the O-of common format and connects glycan, should not be construed as it the present invention only is confined to the glycan that these O-connect.Other forms of O-connects glycan and is illustrated among Figure 12.Be used for preferred expression system of the present invention and express the exogenous peptide that justacrine has the O-connection glycan that comprises the GalNAc residue.Utilize reconstructing method of the present invention, can be reconstructed easily to generate any glycan structures of wanting the glycan structures on these peptides.It should be appreciated by those skilled in the art that O-connect glycan can with this area in case to be provided by disclosure of the present invention be that the identical principle of available, enzyme and reaction conditions are reconstructed.Exemplary reaction conditions can be found in embodiment all the time.

In preferred embodiments, thus the structure that glycan structures has been carried out in the reconstruct molecular formula 2 describing has specific part.The structure that can select glycan with the biologic activity that strengthens peptide, give the new biologic activity of peptide, removal or change the biologic activity of peptide or the glycosylation pattern of approximation native peptides better, and other.

In first embodiment preferred, the glycan that peptide O-is connected has carried out the glycosylation pattern of reconstruct with the proteinoid of approximation natural human better.In this embodiment, the glycan structures that is described in the molecular formula 2 has been carried out reconstruct to have following part:

X ²Be |-SA; Or |-SA-SA;

F and n=0 or 1;

X ¹⁰Be SA;

m＝0。

This embodiment is particularly advantageous for the human peptide that is expressed in the allos cell expression system.Be reconstructed into by the glycan structures that O-is connected and have this configuration, can make this peptide in people patient for than reduced immunogenicity and/or more stable.

In another preferred embodiment, the glycan that peptide O-is connected has carried out reconstruct to show sialylated Lewis X antigen.In this embodiment, the glycan structures that is described in the molecular formula 2 has been carried out reconstruct to have following part:

X ²Be |-SA;

X ¹⁰Be Fuc or |-GlcNAc (Fuc)-Gal-SA;

F and n=1

m＝0。

To select protein molecular and show in the cell of same molecular be particularly advantageous when the most effective to this embodiment when the peptide that is reconstructed is being directed to.

In the another one embodiment preferred, the glycan that peptide O-is connected has carried out reconstruct to have the part of puting together.This part of puting together can be PEG molecule, another peptide, small molecules such as medicine, and other.In this embodiment, the glycan structures that is described in the molecular formula 2 has been carried out reconstruct to have following part:

X ²Be |-SA-R;

f＝1；

N and m=0.

Wherein R=puts together group.

This embodiment can be used for the peptide and the PEG molecule that peptide removes from patient's blood flow that slows down are puted together, and two peptides is directed to specific tissue or the peptide in the cell is puted together, or with have another peptide that the complementary treatment uses and put together.

It will be apparent to one skilled in the art that the present invention is not limited to above-mentioned preferred glycan molecule.Embodiment preferred only is the minority in the many useful glycan molecule of available reconstructing method of the present invention preparation.In case having read behind the present invention, those skilled in the art knows how to design other useful glycan.

In first exemplary embodiment, peptide is expressed in CHO (Chinese hamster ovary cell) according to method well known in the art.When the peptide with consistent site of glycan that O-connects is expressed justacrine in Chinese hamster ovary celI, the great majority of this O-connection glycan will have following structure usually, promptly in the term of molecular formula 2,

X ²Be |-SA;

f＝1；

M and n=0.

Therefore, the glycan in most of Chinese hamster ovary celIs does not need to be reconstructed to be applicable to people patient.In an exemplary embodiment, the O-that will express the peptide of justacrine in Chinese hamster ovary celI connects glycan and contain sialylated Lewis X structure by this glycan and glycosyltransferase being contacted being reconstructed into when Nucleotide-Fucose exists, and this glycosyltransferase partly is specific to GalNAc acceptor portion and Fucose donor.The N-that this process is set forth in Figure 11 and embodiment 39 is connected in the glycan.

In another exemplary embodiment, peptide is to be according to what method well known in the art was expressed in insect cell such as sf9.When the peptide in the consistent site of glycan with O-connection was expressed justacrine in most of sf9 cells, the great majority of this O-connection glycan had the structure of molecular formula 2, wherein:

X ²＝H；

F=0 or 1;

N and m=0.

Referring to as people such as Marchal, (2001, Biol.Chem.382:151-159).In an exemplary embodiment, the O-at the peptide of expressed in insect cells is connected glycan be reconstructed into humanized glycan: at first make glycan and the glycosyltransferase of GlcNAc acceptor molecule with the galactose donor molecular specificity contacted when Nucleotide-Gal exists by following steps; Make glycan then and the Gal acceptor molecule is contacted when Nucleotide-SA exists with the specific glycosyltransferase of SA donor molecule.In another exemplary embodiment, O-is connected glycan further by glycan and glycosyltransferase are contacted to be reconstructed into sialylated Lewis X-shaped formula from humanized form when Nucleotide-Fucose exists, wherein this glycosyltransferase is specific to GalNAc acceptor molecule and Fucose donor molecule.

In the exemplary embodiment of another one, peptide is expressed in fungi according to method well known in the art, particularly in the Saccharomyces cerevisiae cell.When the peptide in the consistent site of glycan with O-connection was expressed justacrine in the Saccharomyces cerevisiae cell, the great majority that this O-connects glycan had following structure:

|-AA-Man-Man _1-2.

Referring to Gemmill and Trimble (1999, Biochim.Biophys.Acta 1426:227-237).For these O-of reconstruct connect glycan to be used for the mankind, preferably glycan is cut and is reconstructed therefrom at amino acid levels.

In an exemplary embodiment, glycan is that the O-of the peptide of expressing in the fungal cell connects glycan, and be reconstructed into humanized glycan, this realizes by following steps: make glycan and the specific inscribe glycosylase of amino acid-GalNAc key is contacted; Glycan when existing, Nucleotide-GalNAc is contacted with the specific glycosyltransferase of GalNAc donor molecule with the consistent site that O-is connected; Make glycan then and the glycosyltransferase of GalNAc acceptor molecule with the galactose donor molecular specificity contacted when Nucleotide-Gal exists; Make glycan then and the Gal acceptor molecule is contacted when Nucleotide-SA exists with the specific glycosyltransferase of SA donor molecule.

Selectively, in another exemplary embodiment, glycan is that the O-of the peptide of expressing in the fungal cell connects glycan, and be reconstructed into humanized glycan, this realizes by following steps: make glycan and protein O-seminose β-1,2-N-acetylgucosamine transferase (POMGnTI) contacts when GlcNac-Nucleotide exists; Glycan is contacted when Nucleotide-Gal exists with galactosyltransferase; Glycan is contacted when Nucleotide-SA exists with sialyltransferase.

In another exemplary embodiment, peptide is expressed in bacterium according to method well known in the art, particularly in Bacillus coli cells.When the peptide in the consistent site of glycan with O-connection is expressed in Bacillus coli cells, the consistent site that this O-connects will not be glycosylated.In this case, the glycan molecule of wanting must with to above Saccharomyces cerevisiae is expressed described similar mode and from the peptide main chain, constructs.Further, express in eukaryotic cell when having the peptide that O-connects glycan, but do not have when nascent peptide is directed to suitable leading peptide in the golgi body, sophisticated peptide may be by glycosylation.Equally in this case, can the glycosyl structure of O-connection be added to this peptide by directly connecting consistent site structure glycan from peptide O-.Further, when protein carried out chemically modified with sugar moieties, it also can be as be reconstructed above-mentionedly.

These examples are wanted to illustrate the present invention and are limited anything but.It will be appreciated by those skilled in the art that the step of carrying out can carry out with different orders in some cases and obtain identical result in each example.Those skilled in the art also will understand different steps also can produce identical glycan.Further, preferably the glycan of reconstruct is specific to the expression system of expressing this peptide never.The glycan of reconstruct is the property illustrated only, and those skilled in the art will know in the peptide of how obtaining principle from these examples and being applied to produce in different expression systems to prepare herein the not glycan of specific description.

C. sugar is puted together, the general description

The invention provides the method for the conjugate for preparing glycosylated or nonglycosylated peptide.Conjugate of the present invention forms between grading at peptide and different sorts such as water-soluble polymers, treatment part, diagnosis part, guide part.Provide the conjugate that comprises the two or more peptides that link together by the linker arm equally, promptly multi-functional conjugate.Multi-functional conjugate of the present invention can comprise two or more copies of identical peptide or have different structure and/or the set of the different peptides of character.

Conjugate of the present invention also can adhere to the enzymatic of glycosylated or nonglycosylated peptide by the sugar modified and forms.When inserting between the modification group on peptide and sugar, the sugar of this modification then becomes alleged herein " intact glycosyl linking group ".Utilize the accurate selectivity of enzyme such as glycosyltransferase, method of the present invention provides the peptide that has the group of wanting at one or more specific positions.Thereby according to the present invention, the sugar of modification is directly to be attached to the position selected on the peptide chain, or selectively, and the sugar of modification is to append on the sugar moieties of peptide.Wherein the sugar of Xiu Shiing be connected on the sugar of peptide and be directly connected on the amino-acid residue of peptide main chain peptide also within the scope of the invention.

Opposite with enzymatic peptide processing policy with known chemistry, method of the present invention makes may assemble the peptide and the glycopeptide of the derivatize pattern with basic homogeneity; Being used for enzyme of the present invention is optionally to the combination or the specific glycan structures of specified amino acid residues on the peptide or amino-acid residue usually.This method also is feasible for the peptide of modifying and the scale operation of glycopeptide.Thereby method of the present invention provides the practical approach of the peptide of the unified substantially derivatize pattern that mass preparation has preliminary election.This method is particularly suitable for the modification to therapeutic peptide, and this therapeutic peptide is including, but not limited to incomplete glycosylated peptide in the cell (as mammalian cell, insect cell, fungal cell, vegetable cell, yeast cell or prokaryotic cell prokaryocyte) of cell cultures or the production process in transgenic plant or the zooblast.

Method of the present invention also provides the glycosylated and nonglycosylated peptide conjugate of the transformation period with increase, the transformation period of this increase owing to as the clearance rate that reduces by immunity or reticuloendothelial system (RES) or the uptake rate of reduction.In addition, method of the present invention provides the method for covering antigenic determinant on the peptide, thereby reduces or eliminated the immune response of host to peptide.The selectivity of directed agents is adhered to and also be can be used for the peptide guiding this specific particular organization of specific directed agents or cell surface receptor.In addition, the peptide that provides a class specifically to modify with the treatment part.

1. conjugate

In first aspect, the invention provides the conjugate between the part of peptide and selection.Connection between the part of peptide and selection is included in the intact glycosyl linking group that inserts between the part of peptide and selection.As discussing herein, the part of selection is any kind of that can be attached in the sugared unit substantially, and the result causes can be by " sugar of modification " of suitable transferring enzyme identification, and the sugar that this enzyme will be modified appends on the peptide.When inserting between the part of peptide and selection, the sugared composition of the sugar of this modification then becomes " intact glycosyl linking group ".This glycosyl linking group forms from any list or oligosaccharides, and this list or oligosaccharides are the substrate of suitable transferring enzyme after modifying with the part of selecting.

Conjugate of the present invention generally will be corresponding to following common structure:

Non-0 positive integer of symbol a, b, c, d and s representative wherein; And t is 0 or positive integer." reagent " is therapeutical agent, bioactive agents, detectable mark, water-soluble portion etc.Should " reagent " can be peptide, as enzyme, antibody, antigen etc.Linker can be hereinafter any one of linking group widely.Selectively, this linker can be singly-bound or " zero level linker (zero order linker) ".The feature of this peptide is unrestricted.Exemplary peptide provides in Figure 28.

In an exemplary embodiment, the part of selection is water miscible polymkeric substance.This water-soluble polymers is covalently attached on the peptide by the intact glycosyl linking group.This glycosyl linking group is to be covalently attached on the amino-acid residue of peptide or the glycosyl residue.Selectively, the glycosyl linking group is attached in one or more glycosyl units of glycopeptide.The present invention also provides glycosyl linking group wherein to be attached to conjugate on amino-acid residue and the glycosyl residue.

Except that the conjugate that provides the intact glycosyl linking group that adds by enzymatic to form, the invention provides the conjugate of its substitute mode height homogeneity.Utilize method of the present invention, possible is forms following peptide conjugate, and the sugar moieties of wherein basic all modifications in conjugate of the present invention colony is all attached to structure on a plurality of copies of identical amino acid or glycosyl residue.Thereby, in second aspect, the invention provides peptide conjugate with water-soluble polymers partial mass, this part is covalently bound to peptide by the intact glycosyl linking group.In the preferred conjugate of the present invention, each group member all is to be connected on the glycosyl residue of peptide by the glycosyl linking group substantially, and each glycosyl residue of the accompanying peptide of glycosyl linking group all has identical structure.

Provide the peptide conjugate that has by the covalently bound water-soluble polymers partial mass of intact glycosyl linking group equally thereon.In preferred embodiments, basic each member of water-soluble polymers partial mass is connected on the amino-acid residue of peptide by the intact glycosyl linking group, and each amino-acid residue that is attached with the intact glycosyl linking group thereon all has identical structure.

The present invention also provides and above-mentioned similar conjugate, and wherein peptide is puted together by intact glycosyl linking group and treatment part, diagnosis part, targeting part, toxin moiety etc.Each above-mentioned part can be small molecules, natural polymer (as peptide) or synthetic polymkeric substance.

In an exemplary embodiment, interleukin II (IL-2) is puted together by bifunctional linker and transferrin, and this bifunctional linker includes intact glycosyl linking group (scheme 1) at each end of peg moiety.For example, an end of PEG linker uses the complete sialic acid linker that adheres to transferrin functionalized, and another end uses the complete GalNAc linker that adheres to IL-2 functionalized.

In another exemplary embodiment, EPO and transferrin are puted together.In another exemplary embodiment, EPO and colloid deutero-neurotrophic growth factor (GDNF) are puted together.In these embodiments, each is puted together all by aforementioned bifunctional linker and realizes that this bifunctional linker includes the intact glycosyl linking group at each end of peg moiety.Transferrin translocator matter is passed through blood brain barrier.

As proposition in the accompanying drawings, conjugate of the present invention can comprise the intact glycosyl linking group of list or multivalence (as the feeler structure), referring to Figure 14-22.Conjugate of the present invention comprises that also the O-that is derived from Serine or Threonine connects the glycosyl linking group (Figure 11) of glycan.Thereby conjugate of the present invention comprises two kinds, and wherein the part of Xuan Zeing is attached on the peptide by unit price glycosyl linking group.Comprise equally among the present invention to be the part that wherein surpasses a selection be attached to conjugate on the peptide by the multivalence linking group.One or more protein can be conjugated in together to utilize its biophysics and biological property.

In other further embodiment, the invention provides owing to optionally be positioned conjugate in the particular organization as the existence of the directed agents of conjugate composition.In an exemplary embodiment, this directed agents is a protein.Exemplary protein comprises transferrin (brain; blood storehouse (blood pool)); human serum (HS)-glycoprotein (bone; brain; the blood storehouse); antibody (brain; has the antigenic tissue of antibodies specific, the blood storehouse); coagulation factors V-XII (damaged tissue, grumeleuse; cancer; the blood storehouse); serum protein such as α-Suan Xingtangdanbai; Pp63 glycophosphoproteins; α-fetus protein (brain, blood storehouse); beta 2-glycoprotein (liver, atherosclerotic plaque; brain; the blood storehouse); G-CSF; GM-CSF; M-CSF and EPO (immunostimulation, cancer, blood storehouse; the red blood cell excess is produced, neuroprotective) and albumin (increase of transformation period).

Except that conjugate discussed above, the invention provides the method for the conjugate for preparing these and other.Thereby, further, the invention provides the method that between part of selecting and peptide, forms covalent conjugates.In addition, the invention provides particular organization or the regional method that conjugate of the present invention is directed to health.

In exemplary embodiment, conjugate forms between water-soluble polymers, treatment part, targeting part or biomolecules and glycosylation or nonglycosylated peptide.Polymkeric substance, treatment part or biomolecules are puted together by intact glycosyl linking group and peptide, and this intact glycosyl linking group inserts between peptide and modification group (as water-soluble polymers) and covalently is connected with both.This method comprises that the mixture that the sugar that makes peptide and contain modification and sugar with this modification are the glycosyltransferase of substrate contacts.This reaction is to carry out under the condition that forms covalent linkage between sugar that is enough to modifying and the peptide.The sugar moieties of the sugar of modifying preferably is selected from nucleotide sugar, activatory sugar and neither the also sugar of inactive sugar of nucleotide sugar.

In one embodiment, the invention provides the method that connects two or more peptides by linking group.Linking group has any useful structure and can be selected from the structure of straight or branched.Preferably, each end that is attached to the linker on the peptide all comprises the sugar (i.e. Xin Sheng intact glycosyl linking group) of modification.

In the exemplary method of the present invention, two peptides are partly to link together by the linker that comprises the PEG linker.This construct is consistent with the general structure that proposes in last texts and pictures.As described herein, construct of the present invention comprises two intact glycosyl linking groups (being s+t=1).The emphasis description comprises that the PEG linker of two glycosyl groups is purposes for the sake of simplicity, and should not be construed as the feature that restriction is used for the linker arm of the embodiment of the present invention.

Thereby peg moiety is terminal functionalized with first glycosyl unit at first, and terminal functionalized with second glycosyl unit at second.First is preferably the substrate of different transferring enzymes with second glycosyl unit, thereby makes first peptide and second peptide can be attached to squarely respectively on first and second peptide.In practice, make (glycosyl) ¹-PEG-(glycosyl) ²Linker contacts with first transferring enzyme that with first glycosyl unit is substrate with first peptide, thereby forms (peptide) ¹-(glycosyl) ¹-PEG-(glycosyl) ²Selectively first transferring enzyme and/or unreacted peptide are removed from reaction mixture then.With second peptide with second glycosyl unit is that second transferring enzyme of substrate joins (peptide) ¹-(glycosyl) ¹-PEG-(glycosyl) ²In the conjugate, thereby form (peptide) ¹-(glycosyl) ¹-PEG-(glycosyl) ²-(peptide) ²It should be appreciated by those skilled in the art that above-mentioned method also can be applicable to by with forming conjugate as ramose PEG, dendrimer, poly-(amino acid), polysaccharide etc. surpassing between two peptides.

As previously described, in an exemplary embodiment, interleukin II (IL-2) is puted together by bifunctional linker and transferrin, and this bifunctional linker includes intact glycosyl linking group (scheme 1) at each end of peg moiety.This IL-2 conjugate utilizes the bigger molecular size of conjugate and has the transformation period in the body that increases than IL-2 self.In addition, puting together optionally of IL-2 and transferrin is directed to conjugate in the brain.For example, of PEG linker is terminal functionalized with cmp sialic acid, and another is functionalized with UDP-GalNAc.Make this linker when the GalNAc transferring enzyme exists and IL-2 combination, thereby cause GalNAc on the linker arm to be attached on the Serine and/or threonine residues on the IL-2.

In another exemplary embodiment, transferrin and nucleic acid are puted together to be used for gene therapy

Scheme 1

Said process can repeatedly circulate, and is not limited to single linker forms conjugate between two peptides.In addition, it should be appreciated by those skilled in the art that the reaction of the intact glycosyl linking group functionalization that makes the PEG that is arranged in peptide (or other) linker end can take place simultaneously at identical reaction vessel, perhaps can mode progressively carry out.When this reaction was carried out in mode progressively, the conjugate that produces in each step was a purifying from one or more reacted constituents (as enzyme, peptide) selectively.

Further exemplary proposes in scheme 2.Scheme 2 expression preparations are with protein such as EPO guiding bone of selecting and the method that increases the conjugate of the proteinic circulating half-life of selecting.

Scheme 2

It is within the scope of the present invention that the reactive derivatives of application PEG (or other linkers) is attached to one or more peptide moieties on the linker.The present invention is not subjected to the restriction of the feature of reactive PEG analogue.Many activatory derivatives of poly-(ethylene glycol) all be commercial and document in obtainable.Same is to select and the PEG derivative of synthetic suitableization if desired the time in technician's limit of power, utilizes this PEG derivative to prepare and is used for substrate of the present invention.Referring to people such as Abuchowski, Cancer Biochem.Biophys., 7:175-186 (1984); People such as Abuchowski, J.Biol.Chem., 252:3582-3586 (1977); People such as Jackson, Anal.Biochem., 165:114-127 (1987); People such as Koide, Biochem.Biophys.Res.Commun., 111:659-667 (1983)), difluoro ethyl sulfonic acid (people such as Nilsson, Methods Enzymol., 104:56-69 (1984); People such as Delgado, Biotechnol.Appl.Biochem., 12:119-128 (1990)); N-hydroxy-succinamide deutero-active ester (people such as Buckmann, Makromol.Chem., 182:1379-1384 (1981); People such as Joppich, Makromol.Chem., 180:1381-1384 (1979); People such as Abuchowski, Cancer Biochem.Biophys., 7:175-186 (1984); People such as Katre, Proc.Natl.Acad.Sci.U.S.A., 84:1487-1491 (1987); People such as Kitamura, Cancer Res., 51:4310-4315 (1991); People such as Boccu, Z.Naturforsch., 38C:94-99 (1983)), carbonate (people such as Zalipsky, Poly (ethylene glycol) Chemistry:Biotechnical andBiomedical Applications, Harris, Ed., Plenum Press, New York, 1992, pp.347-370; People such as Zalipsky, Biotechnol.Appl.Biochem., 15:100-114 (1992); People such as Veronese, Appl.Biochem.Biotech., 11:141-152 (1985)), imidazolyl manthanoate (people such as Beauchamp, Anal.Biochem., 131:25-33 (1983); People such as Berger, Blood, 71:1641-1647 (1988)), 4-two thiopyridines (people such as Woghiren, Bioconjugat Chem., 4:314-318 (1993)), isocyanate (people such as Byun, ASAIO Journal, M649-M-653 (1992)) and epoxide (are authorized people such as Noishiki the U.S.Pat.No.4 of (1989), 806,595).Other linking groups comprise that the urethanum between amino group and the activatory PEG connects.Referring to people such as Veronese, Appl.Biochem.Biotechnol., 11:141-152 (1985).

Utilize in the exemplary of reactive PEG derivative at another, the invention provides the blood circulation transformation period of the peptide of extend selecting and basically peptide is directed to method in the blood storehouse, this is by peptide and size being enough to hinder renal glomerulus that protein (as albumin) filtering synthesized or natural polymkeric substance is puted together and realized.Embodiment of the present invention are illustrated in scheme 3, and wherein erythropoietin (EPO) utilizes chemistry and enzymatically modifying to put together by PEG linker and albumin.

Scheme 3

Thereby as shown in the scheme 3, albuminous amino-acid residue is modified with reactive PEG derivative, as X-PEG-(cmp sialic acid)), wherein X is activating group (as active ester, a lsothiocyanates etc.).Make PEG derivative and EPO combination and with contact with the transferring enzyme of cmp sialic acid as substrate.In the further property illustrated embodiment, the N-hydroxy-succinamide ester reaction that makes the epsilon-amino of Methionin and PEG-linker is to form the albumin conjugate.Cmp sialic acid and the EPO last suitable residue such as the Gal enzymatic ground of linker are puted together, thereby formed conjugate.The technician will understand aforesaid method and be not limited to the reactant that is proposed.In addition, in fact this method can be utilized as having the branch's linker formation that surpasses two ends and comprise above two proteinic conjugates.

2. the sugar of Xiu Shiing

The glycosyl donor kind (" sugar of modification ") of modifying preferably is selected from the sugar nucleotide of modification, sugar that activatory is modified and the both sugar of the also non-activated modification monose of non-nucleotide.The all available method of the present invention of any sugared structure of wanting is added on the peptide.Usually, this structure is a monose, but the present invention is not limited to the monose of using modification; Oligosaccharides and polysaccharide also can be used.

The group of modifying is attached on the sugar moieties by enzyme method, chemical process or its combination, thereby produces the sugar of modifying.Sugar is to replace in any position that makes it possible to adhere to the part of modification, and this sugar that still makes this sugar to serve as to be used for modifying is connected to the substrate of the enzyme on the peptide.In a preferred embodiment, when sialic acid is this sugar, make sialic acid on the Pyruvyl side chain the 9-position or the 5-position on amine moiety replace with modification group, this amine moiety is normally acetylizad in sialic acid.

In certain embodiments of the invention, the sugar that the sugar nucleotide of modifying is used for modifying adds peptide to.The exemplary sugar nucleotide that is used for the present invention with the form of its modification comprises list, two or triphosphopyridine nucleotide or its analogue.In a preferred embodiment, the sugar nucleotide of modification is selected from UDP-glucosides, CMP-glucosides or GDP-glucosides.Even more preferably, the sugar nucleotide of modification is selected from UDP-semi-lactosi, UDP-GalN, UDP-glucose, UDP-glycosamine, GDP-seminose, GDP-Fucose, cmp sialic acid or CMP-NeuAc.The N-acetamide derivative of sugar nucleotide also is used for method of the present invention.

The present invention also provides the method with the peptide of the sugared synthetic modification of modifying, as semi-lactosi, the Fucose of modification and the sialic acid of modifying of modification.When using the sialic acid of modifying, sialyltransferase or trans sialidase (only at α 2, the sialic acid that 3-connects) can be used in these methods.

In another embodiment, the sugar of modification is activatory sugar.The sugar that is used for activatory modification of the present invention is generally to synthesize and changes to comprise the glucosides of activatory leavings group.As used herein, term " activatory leavings group " refers to those easy-to-displaceable parts in the nucleophilic substitution reaction that enzyme is regulated.Many activatory sugar are as known in the art.Referring to as people such as Vocadlo, Carbohydrate Chemistry and Biology, volume 2, people such as Ernst, Wiley-VCH Verlag:Weinheim, Germany, 2000; People such as Kodama, TetrahedronLett.34:6419 (1993); People such as Lougheed, J.Biol.Chem.274:37717 (1999)).

The example of activating group (leavings group) comprises fluorine, chlorine, bromine, tosylate (tosylate), methanesulfonates (mesylate), trifluoromethane sulfonic acid ester etc.Be used for preferred activatory leavings group of the present invention and be those indistinctively the steric restriction glucosides shift to the enzymatic of acceptor.Therefore, the preferred embodiment of activatory glycosides derivatives comprises glycosyl fluorochemical and glycosyl methanesulfonates, and wherein the glycosyl fluorochemical is particularly preferred.In the glycosyl fluorochemical, most preferably α-galactosyl fluorochemical, α-mannose group fluorochemical, alpha-glucosyl fluorochemical, α-fucosido fluorochemical, α-xylosyl fluorochemical, α-sialic acid fluorochemical, α-N-acetyl-glucosamine fluorochemical, α-N-acetylgalactosamine fluorochemical, beta galactose base fluorochemical, β-mannose group fluorochemical, β-glucosyl fluorochemical, β-fucosido fluorochemical, β-xylosyl fluorochemical, β-sialic acid fluorochemical, β-N-acetyl-glucosamine fluorochemical, β-N-acetylgalactosamine fluorochemical.

For instance, the glycosyl fluorochemical can be by at first making sugared acetylize and with the HF/ pyridine it being handled then and prepare from free sugar.This has generated the anomer (being the alpha-glycosyl fluorochemical) of (acetylizad) glycosyl fluorochemical of the most stable protection on the thermodynamics.If want more unsettled anomer (being β-glycosyl fluorochemical), it can be by with HBr/HOAc or will cross bromide or the muriate that acetylizad sugar changes different head into HCl and prepare so.Make the reaction of this intermediate and fluoride salt such as silver fluoride to generate the glycosyl fluorochemical.Acetylizad glycosyl fluorochemical can by with methyl alcohol in gentle (catalytic) alkali (as NaOMe/MeOH) reaction go protection.In addition, many glycosyl fluorochemicals are commercial buying.

Other activatory glycosyl derivatives can be with well known to a person skilled in the art the ordinary method preparation.For example, the glycosyl methanesulfonates can prepare by with the methylsulfonyl muriate hemiacetal form of the complete henzylate of sugar being handled and being removed benzyl group by catalytic hydrogenation subsequently.

In further exemplary, the sugar of modification is the oligosaccharides with feeler structure.In a preferred embodiment, one or more ends of feeler have the modification part.When one or more modification parts were attached on the oligosaccharides with feeler structure, this oligosaccharides can be used for " amplification " and modifies part; Each be conjugated on the peptide oligosaccharide unit all with a plurality of copies of modification group attached on the peptide.Structure comprises the multivalence kind as the inner complex of the present invention that proposes among the figure in the above, and the result obtains this multivalence kind the conjugate of the present invention from preparing with the feeler structure.Many feeler sugar structures are as known in the art, and method of the present invention can be implemented them without restriction.

Exemplary modification group is discussed hereinafter.This modification group can be selected according to one or more character of wanting.Exemplary character distributes, provides water-soluble, enhanced or the lipotropy that reduces and the tissue guide of multivalence kind, improvement including, but not limited to the biology of enhanced pharmacokinetics, enhanced pharmacokinetics, improvement.

D. peptide conjugate

A) water-soluble polymers

The wetting ability of the peptide of selecting is by puting together and enhanced as containing amine, ester, hydroxyl and polyhydric molecule with polar molecule.Representational example is including, but not limited to polylysine, polymine, poly-(ethylene glycol) and poly-(propylene glycol).Preferred water-soluble polymers is non-fluorescence substantially, or only radiates a spot of fluorescence, thereby they are not suitable for as the fluorescent mark in measuring.Can use is the polymkeric substance of natural non-existent sugar.In addition, also can use other by other entities of covalent attachment (as, poly-(ethylene glycol), poly-(propylene glycol), poly-(aspartic acid), biomolecules, treatment part, diagnosis part etc.) and the naturally occurring sugar modified.In another exemplary, curative sugar moieties is conjugated on the linker arm, and should be conjugated on the peptide by method of the present invention subsequently by sugar-linker arm.

The method that makes water-soluble polymers and sugared activatory method and chemicals and be used for making sugar and polymkeric substance and each kind to put together is described in document.The method of activated polymer commonly used comprises with cyanogen bromide, periodates, glutaraldehyde, diepoxide (biepoxides), Epicholorohydrin, divinyl sulfone, carbodiimide, sulfonic acid halide, three chlorotriazines etc. the activation of functional group (referring to R.F.Taylor, (1991), Protein Immobilisation:Fundamentals andApplications, Marcel Dekker, N.Y.; S.S.Wong, (1992) Chemistryof Protein Conjugation and Crosslinking, CRC Press, Boca Raton; People such as G.T.Hermanson, (1993), Immobilized Affinity LigandTechniques, Academic Press, N.Y.; Dunn, people such as R.L., Eds.PolymericDrugsand Drug Delivery Systems, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C.1991).

Preparation feedback PEG molecule and be as known in the art with the approach that reactive molecule forms conjugate.For example, U.S. Patent No. 5,672,662 disclose the water-soluble and separable conjugate of the active ester of polymeric acid, this polymeric acid is selected from linearity or ramose polyalkylene oxide, poly-(oxygen ethylization polyvalent alcohol), poly-(enol), gathers (propylene morpholine), and wherein polymkeric substance has about 44 or more repeating unit.

U.S. Patent No. 6,376,604 have proposed to prepare the method for water-soluble 1-benzotriazole base carbonic ether of the polymkeric substance of water-soluble and non-peptide, and this method is reacted in organic solvent by terminal hydroxyl and two (the 1-benzotriazole base) carbonic ether that makes polymkeric substance and finished.This active ester can be used for and biologic activity agent such as protein or peptide formation conjugate.

WO 99/45964 has described the conjugate that comprises biologic activity agent and activatory water soluble (CO) polymers, described polymkeric substance comprises the main polymer chain with at least one end that is connected with main polymer chain by stable keys, wherein at least one end comprises the component with the near-end reactive group that is connected with component, and biologic activity agent therein is connected at least one near-end reactive group.Other ramose poly-(ethylene glycol) are described among the WO 96/21469, U.S. Patent No. 5,932, and 462 have described the conjugate that forms with the PEG of the branch molecule that comprises branches end, and this branches end comprises reactive functional group.The free reactive group can react with biologic activity kind such as protein or peptide, thereby forms conjugate between poly-(ethylene glycol) and biologic activity kind.U.S. Patent No. 5,446,090 has described bifunctional PEG linker and the application in forming conjugate thereof, and this conjugate all has peptide at each PEG linker end.

Comprise conjugate that degradable PEG connects be described among the WO 99/34833 and WO99/14259 and U.S. Patent No. 6,348,558 in.This degradable connection can be applicable among the present invention.

Although reactive PEG derivative and the conjugate that forms with this derivative all are known in the art, but up to the present invention, people do not recognize as yet between PEG (or other polymkeric substance) and other kinds such as peptide or glycopeptide can form conjugate by the intact glycosyl linking group.

Many water-soluble polymerss are to well known to a person skilled in the art and can be used in the practice of the present invention.The term water-soluble polymers comprises following kind, as sugar (as dextran, amylose starch, hyaluronic acid, poly-(sialic acid), heparitin, heparin etc.); Poly-(amino acid) is as poly-(L-glutamic acid); Nucleic acid; Synthetic polymkeric substance (as poly-(vinylformic acid)); Poly-(ether) is as poly-(ethylene glycol); Peptide; Protein etc.The present invention can carry out with any water miscible polymkeric substance, and unique restriction is that this polymkeric substance must comprise the site that the remainder of conjugate can adhere to.

Make the method for polymer activation also be found in WO 94/17039, U.S.Pat.No.5,324,844, WO 94/18247, WO 94/04193, U.S.Pat.No.5,219,564, U.S.Pat.No.5,122,614, WO 90/13540, U.S.Pat.No.5,281, in 698, and more be WO 93/15189, and it is at the conjugate between activatory polymkeric substance and the peptide, as coagulation factors VIII (WO 94/15625) that method is also arranged, oxyphorase (WO94/09027), carry molecule (U.S.Pat.No.4,412 of oxygen, 989), rnase and superoxide-dismutase (people such as Veronese, Appl.Biochem.Biotech.11:141-145 (1985)).

Preferred water-soluble polymers be those wherein in the polymer samples a large amount of ratio of polymer molecules have approximately identical molecular weight; This polymkeric substance is " monodispersed ".

The present invention further illustrates with poly-(ethylene glycol) conjugate.Is obtainable to the functionalized of PEG with several summaries and the monograph puted together.Referring to as Harris, Macronol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology135:30-65 (1987); People such as Wong, Enzyme Microb.Technol.14:866-874 (1992); People such as Delgado, Critical Reviews in Therapeutic DrugCarrier Systems 9:249-304 (1992); Zalipsky, Bioconjugate Chem.6:150-165 (1995); With people such as Bhadra, Pharmazie, 57:5-29 (2002).

Be applicable to that poly-(ethylene glycol) of the present invention is described by following molecular formula 3 including, but not limited to those:

Molecular formula 3

R=H, alkyl, benzyl, aryl, acetal, OHC-, H ₂N-CH ₂CH ₂-, HS-CH ₂CH ₂-,

-sugar-Nucleotide, protein, methyl, ethyl;

X, Y, W, U (independent selection)=O, S, NH, H-R ';

R ', R " ' (the independent selection)=alkyl, benzyl, aryl, alkylaryl, pyridyl, the aryl of replacement, arylalkyl, acyl group alkyl;

n＝1～2000；

M, q, p (independent selection)=0～20;

o＝0～20；

Z=HO, NH ₂, halogen, S-R " ', Acibenzolar,

-sugar-Nucleotide, protein, imidazoles, HOBT, tetrazolium, halogenide; With

V=HO, NH ₂, halogen, S-R " ', Acibenzolar, activating terephthalamide amine ,-sugar-Nucleotide, protein.

In preferred embodiments, poly-(ethylene glycol) molecule is selected from following:

Poly-(ethylene glycol) that is used to form conjugate of the present invention is linear or ramose.Be applicable to what ramose of the present invention poly-(ethylene glycol) was described by following molecular formula including, but not limited to those:

Molecular formula 4

R ', R ", R " ' (the independent selection)=H, alkyl, benzyl, aryl, acetal, OHC-, H ₂N-CH ₂CH ₂-, HS-CH ₂CH ₂-,-(CH ₂) _qCY-Z ,-sugar-Nucleotide, protein, methyl, ethyl, heteroaryl, acyl group alkyl, acyl group aryl, acyl group alkylaryl;

X, Y, W, A, B (independent selection)=O, S, NH, H-R ', (CH ₂) ₁

N, p (independent selection)=1～2000;

M, q, o (independent selection)=0～20;

Z=HO, NH ₂, halogen, S-R " ', Acibenzolar,

-sugar-Nucleotide, protein;

Also used water soluble polymer such as polyoxyethylene glycol (PEG) and polypropylene glycol (PPG) strengthen for transformation period, area under curve and/or retention time in the body of therapeutical agent.For example, with PEG proteinic chemically modified (PEGization) has been increased its molecular size and reduced its surperficial accessibility and functional group accessibility, this all depends on the size of the PEG that adheres on protein.This has caused reduction (people such as Chaffee, the J.Clin.Invest.89:1643-1651 (1992) of the improvement of plasma half-life and proteolysis stability and immunogenicity and liver picked-up; People such as Pyatak, Res.Commun.Chem.Pathol Pharmacol.29:113-127 (1980)).The PEGization of having reported interleukin II can be increased in intravital anti-tumor capacity (people such as Katre, Proc.Natl.Acad.Sci.USA.84:1487-1491 (1987)), and is derived from the F (ab ') of monoclonal antibody A7 ₂PEGization can improve its tumor-localizing (people such as Kitamura, Biochem.Biophys.Res.Commun.28:1387-1394 (1990)).

In a preferred embodiment, increased with respect to the transformation period in the body of the peptide of non-derivative with the transformation period in the body of the peptide of water-soluble polymers derivatize by method of the present invention.In another preferred embodiment, increased with the area under curve of the peptide of water-soluble polymers derivatize area under curve by method of the present invention with respect to the peptide of non-derivative.In another preferred embodiment, increased with the retention time of the peptide of water-soluble polymers derivatize retention time by method of the present invention with respect to the peptide of non-derivative.The technology of determining transformation period, area under curve and retention time in the body is well known in the art.The description of this technology can be referring to J.G.Wagner, and 1993, Pharmacokinetics for thePharmaceutical Scientist, Technomic Publishing Company, Inc.Lancaster PA.

The increase of transformation period preferably is expressed as the per-cent increase scope of this amount in the peptide body.The lower limit that this per-cent increases scope is about 40%, about 60%, about 80%, about 100%, about 150% or about 200%.The upper limit of this scope is about 60%, about 80%, about 100%, about 150% or surpasses about 250%.

In an exemplary embodiment, the invention provides the follicle stimulating hormone (embodiment 23 and 24) of PEGization.In a further preferred embodiment, the invention provides the transferrin (embodiment 42) of PEGization.

Other examples that are used for water-soluble polymers of the present invention are including, but not limited to linearity or ramose polyalkylene oxide, (polyoxy ethylization polyvalent alcohol), poly-(enol), poly-(propylene morpholine), dextran, starch, poly-(amino acid) etc.

B) insoluble polymer

Conjugate of the present invention also can comprise one or more water-insoluble polymkeric substance.Embodiment of the present invention are by using conjugate as sending the carrier of passing therapeutic peptide to illustrate in the mode that control is arranged.It is as known in the art that polymeric medicine send delivery system.Referring to as people such as Dunn, Eds.Polymeric Drugs and Drug Delivery Systems, ACS SymposiumSeries Vol.469, American Chemistry Society, Washington, D.C.1991.It should be appreciated by those skilled in the art that any basically known drug send delivery system all to can be applicable to conjugate of the present invention.

Representational insoluble polymer is including, but not limited to poly-phosphine piperazine, polyvinyl alcohol, polymeric amide, polycarbonate, poly-alkylene class, polyacrylamide, polyalkylene glycol, polyalkylene oxide, the polyalkylene terephthalate, polyvinyl ether, polyvinyl ester, polyvinylhalide, polyvinylpyrrolidone, polyglycolide, polysiloxane, polyurethane(s), polymethylmethacrylate, polyethyl methacrylate, poly-n-butyl methacrylate, polyisobutyl methacrylate, the own ester of polymethyl acrylic acid, polymethyl acrylic acid isodecyl ester, polylauryl methacrylate, the polymethyl acid phenenyl ester, polymethyl acrylate, the polyacrylic acid isopropyl ester, polyisobutyl acrylate, polyoctodecyl acrylate, polyethylene, polypropylene, polyoxyethylene glycol, polyethylene oxide, polyethylene terephthalate, polyvinyl acetate, polyvinyl chloride, polystyrene, polyvinylpyrrolidone, pluronics and polyvinyl phenol and multipolymer thereof.

The natural polymer of synthetic sex modification that is used for conjugate of the present invention is including, but not limited to alkylcellulose, hydroxy alkyl cellulose, ether of cellulose, cellulose ester and nitrocellulose.Particularly preferred member is including, but not limited to the polymkeric substance of methylcellulose gum, ethyl cellulose, hydroxypropylcellulose, Vltra tears, hydroxy butyl methyl cellulose, rhodia, cellulose propionate, cellulose acetate butyrate, Cellacefate, carboxymethyl cellulose, cellulose triacetate, sulfate cellulose sodium salt and vinylformic acid and methacrylic ester and alginic acid in the big class of natural polymer of synthetic sex modification.

These and other polymkeric substance of Tao Luning can be easy to buy from commercial source herein, as SigmaChemical Co. (St.Louis, MO.), Polysci ences (Warrenton, PA.), Aldrich (Milwaukee, WI.), Fluka (Ronkonkoma, NY) and BioRad (Richmond, CA), perhaps the available standards technology is synthesized from the monomer that obtains from these suppliers.

The representative Biodegradable polymeric that is used for conjugate of the present invention is including, but not limited to polylactide, polyglycolide and multipolymer thereof, polyethylene terephthalate, poly-butyric acid, poly-valeric acid, poly-(rac-Lactide-altogether-caprolactone), poly-(rac-Lactide-altogether-second lactone), polyanhydride, poe and composition thereof and multipolymer.What have special application is the composition that forms gel, comprises collagen, pluronics etc. as those.

Be used for polymkeric substance of the present invention and comprise " heterozygosis " polymkeric substance with water-insoluble material, this water-insoluble material has at least a portion of its structure can biological resorbent molecule.An example of this polymkeric substance is as follows: it comprises that have can biological water-insoluble copolymer, the hydrophilic region of absorption region again, and every polymer chain has a plurality of crosslinkable functional groups.

For purpose of the present invention, " water-insoluble material " is included in the water or contains undissolved substantially material in the environment of water.Thereby, although some zone of multipolymer or section can be hydrophilic or even water miscible, polymer molecule integral body can not be in any measurable substantially degree dissolving in water.

For purpose of the present invention, term " can biological resorbent material " comprises the zone that can carry out metabolism or decomposition and be absorbed and/or get rid of by normal excretion pathway by health.This metabolite or degradation production are nontoxic substantially to health preferably.

Can biological resorbent zone can be hydrophobicly or hydrophilic, be water dissolvable as long as make polyalcohol integral.Thereby, can biological resorbent zone be based on that polyalcohol integral keeps water-insoluble and preferred the selection.Therefore, select relevant character (kind and this regional relative proportion and hydrophilic region of the functional group that can biological resorbent zone contains) to guarantee useful can to keep water-insoluble by biological resorbent composition.

Exemplary can resorbent polymkeric substance comprise as synthetic produce can resorbent many Alpha-hydroxy-carboxylic acids/polyoxyalkylene segmented copolymer (referring to people such as Cohn, U.S. Patent No. 4,826,945).These multipolymers are not crosslinked and are water miscible, thereby health can be drained the block copolymer composition of degraded.Referring to people such as Younes, J.Biomed.Mater.Res.21:1301-1316 (1987); With people such as Cohn, J.Biomed.Mater.Res.22:993-1009 (1988).

At present preferred biology can comprise that one or more are selected from the composition of polyester, polyhydroxy-acid, polylactone, polymeric amide, polyesteramide, polyamino acid, polyanhydride, poe, polycarbonate, poly-phosphine piperazine, poly phosphate, polythioester, polysaccharide and composition thereof by resorbent polymkeric substance.This can more preferably comprise the polyhydroxy-acid composition by biological resorbent polymkeric substance.Polyhydroxy-acid, poly(lactic acid), polyglycolic acid, poly-caproic acid, poly-butyric acid, poly-valeric acid and multipolymer thereof and mixture are preferred.

Except that being formed on body systemic (" biological resorbent ") fragment, but the preferred polymer coating thing that is used for method of the present invention also can form excretory and/or metabolizable fragment.

More the high-grade multipolymer also can be used among the present invention.For example, the U.S. Patent No. 4,438,253 delivered on March 20th, 1984 of people such as Casey discloses from the multipolymer of three-block that the transesterification of the polyalkylene glycol of polyglycolic acid and hydroxyl ending is produced.Openly can resorbent monofilament suture with this composition to be used as.The flexibility of this composition is by being integrated into the aromatics orthocarbonic ester in the copolymer structure and controlling as four-right-tolyl orthocarbonic ester.

Also can use other encrusting substances based on lactic acid and/or oxyacetic acid.For example; the U.S. Patent No. 5 that Spinu delivered on April 13rd, 1993; 202; 413 disclose biodegradable the have polylactide of consecutive order and/or the segmented copolymer of polyglycolide block; this segmented copolymer is the ring-opening polymerization on oligomerization glycol or diamines and produce this bifunctional compound such as vulcabond, diacyl chloride or dichlorosilane subsequently by the chain extension that carries out with bifunctional compound by lactide and/or glycollide.

What be used for encrusting substance of the present invention can biological absorption region again can be designed to hydrolyzable and/or enzyme cutting.For the purposes of the present invention, " but water cutting " refer to multipolymer especially can biological absorption region again in water or the susceptibility of hydrolysis in the aqueous environment.Similarly, " but enzyme cutting " be used in reference to herein multipolymer especially can biological absorption region again to susceptibility endogenous or the exogenous enzyme cutting.

In the time of in being in health, but hydrophilic region can be processed as excretory and/or metabolizable fragment.Thereby hydrophilic region can comprise as polyethers, polyalkylene oxide, polyvalent alcohol, poly-(ethene tetramethyleneimine), polyvinyl alcohol, poly-(Wan oxazolin), polysaccharide, sugar, peptide, protein and multipolymer and mixture.In addition, hydrophilic region also can be as polyalkylene oxide.This polyalkylene oxide can comprise as polyethylene oxide, poly(propylene oxide) and composition thereof and multipolymer.

Polymkeric substance as the composition of hydrogel also can be used among the present invention.Hydrogel is the polymer materials that can absorb a large amount of relatively water.Form hydrogel compound example including, but not limited to polyacrylic acid, Xylo-Mucine, polyvinyl alcohol, polyvinylpyrrolidine, gelatin, carrageenan and other polysaccharide, hydroxyl ethylidene methacrylic acid (HEMA) with and derivative etc.Can produce stable, biodegradable and can biological resorbent hydrogel.In addition, hydrogel composition can comprise the subunit of showing one or more these character.

Its integrity can be known by the biocompatible hydrogel composition of crosslinked control and be preferably used at present in the method for the present invention.For example, the U.S. Patent No. 5 that people such as Hubbell delivered April 25 nineteen ninety-five, 410,016 and on June 25th, 1996 deliver 5,529,914 disclose water-soluble system, and this system is the cross-linked block copolymer with water-soluble central block section, and this water-soluble central block section sandwiches in the extension of two hydrolytically unstables.This multipolymer further carries out end with the acrylate-functional groups of photopolymerization and adds cap.After crosslinked, these systems become hydrogel.The water-soluble central block thing of this multipolymer can comprise polyoxyethylene glycol; Yet the extension of hydrolytically unstable can be many alpha hydroxy acid, as polyglycolic acid or poly(lactic acid).Referring to people such as Sawhney, Macromolecules 26:581-587 (1993).

In another preferred embodiment, gel is hot reversible (thermoreversible) gel.Following hot reversible gel is preferred at present, and promptly this hot reversible gel comprises as pluronics, collagen, gelatin, hyaluronic acid, polysaccharide, aqueous polyurethane gel, polyurethane(s)-urea water gel and combination thereof.

In another exemplary, conjugate of the present invention comprises liposome component.Liposome can be according to those skilled in the art's known method preparation, as is described in people such as Eppstein in the U.S. Patent No. of delivering on June 11st, 1,985 4,522,811.For example; liposome can prepare by suitable lipid (as hard acyl fat base phosphatidylethanolamine, stearyl-phosphatidylcholine, peanut acyl group (arachadoyl) phosphatidylcholine and cholesterol) is dissolved in to evaporate then in the inorganic solvent, thereby at vessel surface residue skim exsiccant lipid.The aqueous solution with active compound or the acceptable salt of its medicine is incorporated in the container then.Make container rotation with hand then so that matrix material from the side release of container and disperse the lipid coacervate, thereby form liposome suspension.

The method of above-mentioned microparticle and this microparticle of preparation provides by illustrative method, and they are not to want to limit the scope that is used for microparticle of the present invention.What it will be apparent to those skilled in the art that is that a series of microparticles by the different methods manufacturing all can be used among the present invention.

C) biomolecules

In another preferred embodiment, the sugar of modification has biomolecules.In other further preferred embodiment, this biomolecules is functional protein, enzyme, antigen, antibody, peptide, nucleic acid (as mononucleotide or nucleosides, oligonucleotide, polynucleotide and strand with more than the nucleic acid of a chain), lectin, acceptor or its combination.

Some preferred biomolecules are non-blooming substantially, or only radiate the fluorescence of minimum, thereby they are not suitable for as the fluorescent mark in measuring.The other biological molecule can be fluorescence.It is suitable using other naturally occurring sugar of modifying by another entity of covalent attachment (as PEG, biomolecules, treatment part, diagnosis part etc.).In an exemplary embodiment, make as the sugar moieties and the linker arm of biomolecules and put together, and subsequently sugar-linker arm box and peptide are puted together by method of the present invention.

The biomolecules that is used for practice of the present invention can be derived from any source.This biomolecules can be separated or can be by synthetic method production from natural origin.Peptide can be the natural peptide or the peptide of sudden change.Sudden change can be realized by chemomorphosis, site-directed mutagenesis or other methods that well known to a person skilled in the art induced mutation.Being used to put into practice peptide of the present invention comprises as enzyme, antigen, antibody and acceptor.Antibody can be polyclone or monoclonal; Complete or segmental.Peptide selectively is the product of orthogenesis program.

Be derived from and natural all can be used among the present invention together with synthetic peptide and nucleic acid; These molecules can be attached on glycosyl residue composition or the linking agent by any available reactive group.For example, peptide can adhere to by reactive amine, carboxyl, sulfydryl or oh group.This reactive group can be positioned at the terminal of peptide or be positioned at the site, inside of peptide chain.Nucleic acid can adhere to by available hydroxyl on reactive group on the base (as encircling outer amine) or the sugar moieties (as 3 '-or 5 '-hydroxyl).Peptide and nucleic acid chains can further be carried out derivatize in one or more sites so that suitable reactive group can be attached on the chain.Referring to people such as Chrisey, Nucleic AcidsRes.24:3031-3039 (1996).

In a further preferred embodiment, select biomolecules, pass to sending of this tissue thereby strengthen peptide with respect to the amount of sending the peptide that is delivered to the underivatized in this tissue being directed in the specific tissue with the peptide that method of the present invention is modified.In other further preferred embodiment, the amount of sending the derivatize peptide that is delivered in the particular organization in the time period of selecting has strengthened at least about 20%, more preferably has been at least about 40% by derivatize, and more preferably is at least about 100%.At present, be used to the to lead preferred biomolecules used comprises the part of antibody, hormone and cell surface receptor.Exemplary guiding biomolecules includes, but are not limited to send the specific antibody of the transferrin receptor that is delivered to brain (people such as Penichet, 1999, J.Immunol.163:4421-4426 to being used for molecule; Pardridge, 2002, Adv.Exp.Med.Biol.513:397-430), the peptide (people such as Arap of the prostatic vasculature of identification, 2002, PNAS99:1527-1531) with to the specific antibody of pneumonocyte film caveolae (people such as McIntosh, 2002, PNAS 99:1996-2001).

In present embodiment preferred, modification group is a protein.In an exemplary embodiment, this protein is Interferon, rabbit.This Interferon, rabbit is antiviral glycoprotein, and this glycoprotein is to induce the back excretory with virus or double-stranded RNA by people's elementary inoblast in the people.The Interferon, rabbit useful as therapeutics is as antiviral and to the treatment of multiple sclerosis.For the reference that interferon-beta is discussed, referring to as people such as Yu, J.Neuroimmunol., 64 (1): 91-100 (1996); Schmidt, J.Neurosci.Res., 65 (1): 59-67 (2001); People such as Wender, Folia Neuropathol., 39 (2): 91-93 (2001); People such as Martin, Springer Semin.Immunopathol., 18 (1): 1-24 (1996); People such as Takane, J.Pharmacol.Exp.Ther., 294 (2): 746-752 (2000); People such as Sburlati, Biotechnol.Prog., 14:189-192 (1998); People such as Dodd, Biochimicaet Biophysica Acta, 787:183-187 (1984); People such as Edelbaum, J.Interferon Res., 12:449-453 (1992); People such as Conradt, J.Biol.Chem., 262 (30): 14600-14605 (1987); People such as Civas, Eur.J.Biochem., 173:311-316 (1988); People such as Demolder, J.Biotechnol., 32:179-189 (1994); People such as Sedmak, J.Interferon Res., 9 (Suppl1): S61-S65 (1989); People such as Kagawa, J.Biol.Chem., 263 (33): 17508-17515 (1988); People such as Hershenson, U.S. Patent No. 4,894,330; People such as Jaya ram, J.Interferon Res., 3 (2): 177-180 (1983); People such as Menge, Develop.Biol.Standard., 66:391-401 (1987); People such as Vonk, J.Interferon Res., 3 (2): 169-175 (1983); With people such as Adolf, J.Interferon Res., 10:255-267 (1990).About the reference of interferon-' alpha ', referring to people such as Asano, Eur.J.Cancer, 27 (Suppl 4): S21-S25 (1991); People such as Nagy, AnticancerResearch, 8 (3): 467-470 (1988); People such as Dron, J.Biol.Regul.Homeost.Agents, 3 (1): 13-19 (1989); People such as Habib, Am.Surg., 67 (3): 257-260 (3/2001); With people such as Sugyiama, Eur.J.Biochem., 217:921-927 (1993).

In exemplary conjugates of interferon, interferon beta is puted together by linker arm and second peptide.This linker arm comprises the intact glycosyl linking group, and this linker arm uses method of the present invention to be attached on second peptide by this intact glycosyl linking group.This linker arm also selectively comprises second intact glycosyl linking group, can be attached on the Interferon, rabbit by this intact glycosyl linking group.

In another exemplary embodiment, the invention provides the conjugate of follicle stimulating hormone (FSH).FSH is a glycoprotein hormones.Referring to as people such as Saneyoshi, Biol.Reprod., 65:1686-1690 (2001); People such as Hakola, J.Endocrinol., 158:441-448 (1998); People such as Stanton, Mol.Cell.Endocrinol., 125:133-141 (1996); People such as Walton, J.Clin.Endocrinol.Metab., 86 (8): 3675-3685 (08/2001); People such as Ulloa-Aguirre, Endocrine, 11 (3): 205-215 (12/1999); People such as Castro-Fern á ndez, I.J.Clin.Endocrinol.Metab., 85 (12): 4603-4610 (2000); Prevost, Rebecca R., Pharmacotherapy, 18 (5): 1001-1010 (1998); People such as Linskens, The FASEBJournal, 13:639-645 (04/1999); People such as Butnev, Biol.Reprod., 58:458-469 (1998); People such as Muyan, Mol.Endo., 12 (5): 766-772 (1998); People such as Min, Endo.J., 43 (5): 585-593 (1996); People such as Bo ime, RecentProgress in Hormone Research, 34:271-289 (1999); With people such as Rafferty, J.Endo., 145:527-533 (1995).The FSH conjugate can be to form to the similar mode that Interferon, rabbit is described.

In the another one exemplary, conjugate comprises erythropoietin (EPO).The generation of the reaction of the known mediation hypoxemia of EPO and stimulation red blood cell.For relevant reference, referring to people such as Cerami, Seminars in Oncology, 28 (2) (Suppl8): 66-70 (04/2001).The formation method of exemplary EPO conjugate and the conjugate of Interferon, rabbit are similar.

In further exemplary embodiment, the invention provides the conjugate of Filgrastim (G-CSF).G-CSF stimulates short neural generative nature (neutropoietic) ancester cell breeding, differentiation and activation to form the glycoprotein of sophisticated neutrophil(e) cell on the function.The G-CSF of known injection will remove from health very soon.Referring to as people such as Nohynek, CancerChemother.Pharmacol., 39:259-266 (1997); People such as Lord, ClinicalCancer Research, 7 (7): 2085-2090 (07/2001); People such as Rotondaro, Molecular Biotechnology, 11 (2): 117-128 (1999) and

Deng the people, Bone Marrow Transplatation, 28:259-264 (2001).Exemplary G-CSF conjugate is as mentioned the description of the conjugate of Interferon, rabbit to be prepared like that.It should be appreciated by those skilled in the art that many other protein can put together with method and composition of the present invention and Interferon, rabbit, this wherein provides independent modification protocols including, but not limited to the peptide among table 7 and 8 (providing elsewhere) and Figure 28 and Figure 29-57 is provided.

In in addition further exemplary, provide conjugate with vitamin H.Thereby for example the peptide of selectivity organism elementization can be made by adhering to avidin or streptavidin with one or more modification groups.

In a further preferred embodiment, select biomolecules being directed in the specific intracellular region chamber, pass thereby strengthen peptide sending in this intracellular region chamber with respect to the amount of sending the peptide that is delivered to the underivatized in this tissue with the peptide that method of the present invention is modified.In other further preferred embodiment, in the time period of selecting, send the amount that is delivered to the peptide of the derivatize in the compartment in the specific cells to strengthen at least about 20%, more preferably be at least about 40%, and more preferably be at least about 100% by derivatize.In another particularly preferred embodiment, biomolecules is connected, with peptide by the linker that can cut in case be hydrolyzable after this linker internalization.At present, be used for guiding is used in the cell preferred biomolecules comprise transferrin, lactotransferrin (lactoferrin), melanoma transferrin (melanotranferrin) (p97), ceruloplasmin and divalent cation transporter albumen, and the antibody of anti-specific blood vessel target.The connection of expection is including, but not limited to protein-sugar-linker-sugar-protein, protein-sugar-linker-protein and multivalence form thereof, and protein-sugar-linker-medicine, and wherein medicine comprises small molecules, peptide, lipid etc.

The locus specificity of therapeutical agent and goal-orientedly send that to pass be that purpose in order to treat various human diseasess is wanted, this disease such as various types of malignant tumour and some nervous disorders.This program is to be followed by the low side effect and the higher effect of medicine.Depend on various principles when these send delivery system in design.About summary, referring to Garnett, Advanced DrugDeliveryRe views 53:171-216 (2001).

An important consideration is the destination organization specificity in the design drug delivery system.The discovery of TSA makes and may develop methods of treatment, and the tumour cell of showing the surface antigen that limits in this methods of treatment is to lead specifically and killed.Mainly contain 3 classes and in the experiment of human clinical treatment malignant tumour, prove effective therapeutic monoclonal antibodies (antibody): (1) unconjugated MAb, it is induced growth inhibition and/or apoptosis directly, and the defense mechanism that perhaps activates the host indirectly is with mediation antitumor cell toxicity; (2) MAb that puts together of medicine, it preferably send strong cytotoxin and is delivered in the tumour cell thereby makes usually that the general cytotoxicity relevant with conventional treatment minimizes; (3) MAb that puts together of radio isotope, it send the radiation of aseptic dosage and is delivered in the tumour.Referring to people such as Reff, the summary of CancerControl 9:152-166 (2002).

In order MAb to be had kill the ability of malignant cell, MAb is connected with toxin, this toxin can obtain from plant, bacterium or originated from fungus, thereby forms the chimeric protein that is called immunotoxin.Plant poison commonly used is divided into two classes: (1) holotoxin (or II class ribosome inactivating protein), as ricin, toxalbumin, mistletoe lectin element and modeccin, (2) hemitoxin (I class ribosome inactivating protein) is as Pokeweed antiviral protein (PAP), Escin, Bryodinl, bouganin and gelonin.Bacteriotoxin commonly used comprises diphtheria toxin (DT) and Pseudomonas exotoxin (PE).Kreitman，Current?PharmaceuticalBiotechnology?2：313-325(2001)。Other expections are used for toxin of the present invention and include, but are not limited to the toxin shown in the table 2.

Table 2. toxin.

Conventional immunotoxin contains the MAb chemically conjugated with toxin, and this toxin has been carried out sudden change or chemically modified so that minimize with Normocellular the combination.Example comprises the anti--ricin of B4-blocking-up and the deglycosylated ricin A of the RFB4-chain of guiding CD22 of guiding CD5.The recombinant immunotoxin of latest developments is chimeric proteins, and this chimeric protein is made up of the variable region that is fused to the antibody of the tumor-resistant antigen on the archon with recombinant DNA technology.This toxin also often carries out genetically engineered and modifies with the binding site of removing healthy tissues but keep its cytotoxicity.A large amount of differentiation antigens, the acceptor of overexpression or cancer specific antigen have been accredited as the target of immunotoxin, as CD19, CD22, CD20, IL-2 acceptor (CD25), CD33, IL-4 acceptor, EGF acceptor and mutant, Er β 2, Lewis sugar, mesothelin, transferrin receptor, GM-CSF acceptor, Ras, Bcr-Ab1 and c-Kit, to be used for the treatment of various malignant tumours, comprise hematopoietic cancer, neurospongioma and breast, colon, uterus, bladder and gastrointestinal cancer.Referring to as people such as Brinkmann, ExpertOpin.Biol.Ther.1:693-702 (2001); Perentesis and Sievers, Hematology/Oncology Clinics ofNorth America 15:677-701 (2001).

To be used as the method, particularly hematopoietic cell malignant tumour of another kind of treatment human malignancies with the MAb that radio isotope is puted together, this method has high-caliber specificity and effectiveness.The most frequently used isotropic substance that is used for the treatment of is high-octane missile, as ¹³¹I and ⁹⁰Y.Recently, ²¹³Anti--CD33 humanization the MAb of Bi mark also checks in I phase people clinical experiment.People such as Reff see above.

Many MAb are used for the treatment of purpose.For example, the reorganization inosculating antibody-CD20MAb that is used for the treatment of some hematopoietic cell malignant tumour is rituximab (Rituxan ^TM) application obtained the approval of FDA in 1997.Other MAb that got permission to be used for the treatment of human cancer from that time comprise: a kind of humanized rat anti-CD 52 antibody alemtuzumab (Campath-1H ^TM); MAb gemtuzumabozogamicin (the Mylotarg of the humanization mouse anti CD33 that puts together with a kind of calicheamicin ^TM).At present FDA also checked several other be used for security and the effect that locus specificity send the MAb of delivery cell toxic agents or radiation purpose, as radiolabeled Zevalin ^TMAnd Bexxar ^TMPeople such as Reff see above.

Second important consideration is the accessibility of destination organization to therapeutical agent in the design drug delivery system.This is a situation about will consider especially in the disease of treatment central nervous system (CNS), because blood brain barrier has prevented macromolecular diffusion.Develop several method to get around blood brain barrier and therapeutical agent sent effectively and be delivered to CNS.

Understanding to the iron transfer mechanism from blood plasma to the brain provides the useful tool that gets around blood brain barrier (BBB).The iron of being transported in blood plasma by transferrin is the basal component of nearly all cell type.Brain needs iron to carry out metabolic process and to accept iron by the transferrin receptor that is positioned on the brain capillary endothelial cells through receptor-mediated transcytosis and endocytosis.Moos and Morgan, Cellular and Molecular Neurobiology 20:77-95 (2000).Established effectively peptide, protein and liposome are sent be delivered in the brain based on the interactional delivery system that send of transferrin-transferrin receptor.For example, the MAb coupling that can make peptide and anti-transferrin receptor is to obtain bigger brain capture, and Moos and Morgan see above.Similarly, when with the MAb coupling of anti-transferrin receptor, the transhipment that Basic Fibroblast Growth Factor (bFGF) strides across blood brain barrier has obtained enhancing.People such as Song, The Journal ofPharmacology and Experimental Therapeutics 301:605-610 (2002); People such as Wu, Journal of Drug Targeting 10:239-245 (2002).In addition, reported that the liposome that effectively the chemotherapeutics Zorubicin is transported in the C6 neurospongioma send delivery system, wherein is attached to transferrin the far-end on the liposome PEG chain.People such as Eavarone, J.Biomed.Mater.Res.51:10-14 (2000).Many United States Patent (USP)s also relate to based on the interactional method of passing of sending that gets around blood brain barrier of transferrin-transferrin receptor.Referring to as U.S. Patent No. 5,154,924,5,182,107,5,527,527,5,833,988,6,015,555.

For the pharmaceutical agent that gets around blood brain barrier other suitable conjugate mating partners are arranged.For example, U.S. Patent No. 5,672,683,5,977,307 and WO95/02421 relate to and neurologic agent reagent is striden across blood brain barrier send the method for passing, wherein this reagent is to give with the form with the fusion rotein of part, this part can with the capillary endothelial cells receptor response of brain; WO99/00150 has described a kind of drug delivery system, and the transhipment that its Chinese traditional medicine strides across blood brain barrier is to put together and promoted by the MAb with anti-insulin human's acceptor; WO 89/10134 has described chimeric protein, and this chimeric protein comprises peptide that can stride across blood brain barrier with high relatively speed and the hydrophilic neuropeptide that can not carry out transcytosis, thereby as a kind of hydrophilic neuropeptide is incorporated into method in the brain; WO 01/60411 provides a kind of pharmaceutical composition that can easily active constituents of medicine be transported in the brain.This activeconstituents is attached on the hibernation specific protein as conjugate, and gives with Triiodothyronine or the material that promotes Triiodothyronine to produce.In addition, probe into the selectable medicine that is used to get around blood brain barrier and sent the approach of passing.For example, giving in the nose of the therapeutical agent that need not to put together to show is the promising selectable method of passing (Frey, 2002, Drug Delivery Technology, 2 (5): 46-49) sent.

Except that promoting that medicine strides across the transhipment of blood brain barrier, transferrin-transferrin receptor interaction also can be used for the specificity guiding of some tumour cell, and this is because many tumour cells show the overexpression transferrin at it.This strategy has been used for by the transferrin conjugate bioactive macromolecule being sent to be passed into K562 cell (people such as Wellhoner, The Journal ofBiological Chemistry 266:4309-4314 (1991)), and be used for Regular Insulin being sent and pass into class intestinal cells (enterocyte-like) Caco-2 cell (Shah and Shen, Journal of Pharmaceutical Sciences 85:1306-1311 (1996)) by the transferrin conjugate.

In addition, because proteinic function of iron transfer and expression pattern thereof have been had more understanding, as lactotransferrin acceptor, melanoma transferrin, ceruloplasmin and divalent cation transporter albumen, found that protein (as the melanoma transferrin) that some participate in the iron transfer mechanism or its fragment stride across in the blood brain barrier transhipment or the specific tissue that leads at auxiliary therapeutical agent and have similar efficient (WO 02/13843 A2, WO 02/13873 A2).Send the transferrin that participates in the iron picked-up in passing and related protein summary for medicine as the application of conjugate, referring to Li and Qian, Medical Research Reviews 22:225-250 (2002).

The tissue specificity of therapeutical agent send the notion of passing not to be confined to interaction between transferrin and transferrin receptor or its related protein.For example, described the specific delivery system that send of bone, wherein protein is to put together to improve protein with the amino bisphosphate of close bone to pass to sending of mineralized tissue.Uludag and Yang, Biotechnol.Prog.18:604-611 (2002).About the summary of this theme, referring to people such as Vyas, Critical Reviews inTherapeutic Drug Carrier System 18:1-76 (2001).

Various linkers can be used for generating the specificity that is used for the treatment of agent and send in the method for the bioconjugates of passing purpose.Suitable linker comprises with the difunctional and cross-linking reagent isodigeranyl function, this reagent for can by as the acid catalyzed cutting of dissociating maybe can not cut (referring to as Srinivasachar and Neville, Biochemistry 28:2501-2509 (1989); People such as Wellhoner, The Journal of Biological Chemistry 266:4309-4314 (1991)).Interaction between any known binding partners such as vitamin H and the avidin/streptavidin also can be used for therapeutical agent and conjugate mating partner bonded method, thereby guarantees the specificity of therapeutical agent and effectively send and pass.Utilize method of the present invention, protein can be used for molecule and conjugate sent and is delivered in the intracellular region chamber.Be attached to protein on the specific cell surface receptor, peptide, hormone, cytokine, small molecules etc. and can be used for leading in the cell to the therapeutic compound puted together, but this specific cell surface receptor part in conjunction with after internalization.Usually, the receptor-ligand mixture will depend on that internalization is to sending in the endocytic vesicle that is delivered in the specific cells compartment by position in the cell of receptor-directed, and this cellular compartment is in nuclear, plastosome, golgi body, ER, lysosome and endosome.By receptors ligand and the molecule wanted are puted together, medicine can be carried in the receptor-ligand mixture and send in the intracellular region chamber that is delivered to the normal guiding of this receptor.Therefore, this medicine can send position in the specific cells that is delivered to cell, and it need be in this position treatment disease.

Numerous protein can be used for therapeutical agent is directed in the specific tissue and organ.Guiding protein matter is including, but not limited to somatomedin (EPO, HGH, EGF, nerve growth factor, FGF etc.), cytokine (GM-CSF, G-CSF, Interferon, rabbit family, interleukin-etc.), hormone (FSH, LH, steroid family, oestrogenic hormon, reflunomide, Regular Insulin etc.), serum protein (albumin, lipoprotein, fetoprotein, human serum protein, antibody and antibody fragment etc.) and VITAMIN (folic acid, vitamins C, vitamin A etc.).Directed agents to receptor-specific on the most cell types can be buied.

The connection configuration of expection is including, but not limited to protein-sugar-linker-sugar-protein and multivalence form, protein-sugar-linker-protein and multivalence form thereof, protein-sugar-linker-therapeutical agent, and wherein this therapeutical agent is including, but not limited to small molecules, peptide and lipid.In some embodiments, in case be the hydrolysable linkers of hydrolysis after the application internalization.It is right to dissolve endosome or lysosome with acid pH in protein conjugate, can use sour unsettled linker to gain the upper hand.In case in dissolve in endosome or the lysosome, linker then is hydrolyzed and therapeutical agent discharges from directed agents.

In an exemplary embodiment, transferrin is conjugated to by linker wants to be directed on the nucleic acid carrier of the enzyme in patient's cell or this enzyme of encoding this cell display transferrin receptor.This patient can be to the enzyme replacement treatment of this specific enzyme.In particularly preferred embodiments, this enzyme is to have (referring to table 5) that lacks among the patient of lysosomal storage disease.In case after entering circulation, transferrin-enzyme conjugate can be connected with transferrin receptor and (people such as Xing, 1998, the Biochem.J.336:667 of internalization in the endosome in early days; People such as Li, 2002, Trends in Pharmacol.Sci.23:206; People such as Suhaila, 1998, J.Biol.Chem.273:14355).The directed agents of other expections relevant with transferrin is including, but not limited to lactotransferrin (lactoferrin), melanoma transferrin (p97), ceruloplasmin and divalent cation transporter albumen.

In another exemplary, transferrin-dystrophin conjugate will enter in the endosome by the transferrin approach.In case after in endosome, dystrophin then discharges owing to hydrolyzable linker, they can be taken to then needs in their the intracellular region chamber.This embodiment can be used for treating the patient who suffers from muscular dystrophy by dystrophin gene and/or the protein of using the dystrophin that function is arranged that is connected with transferrin to replenish hereditary defect.

E. treat part

In another preferred embodiment, the steamed bun stuffed with sugar of modification is drawn together the treatment part.It should be appreciated by those skilled in the art that and treating partly and having overlapping between the category of biomolecules; Many biomolecules have therapeutic property or potentiality.

Treatment part can be the reagent of accepting as clinical application, and perhaps they can be the medicine in experiment, or its activity or mechanism of action are among research.The treatment part has certified effect in given morbid state, or can only suppose to show in given morbid state the effect of wanting.In preferred embodiments, the treatment part is a compound, and this compound is to select according to its ability with the tissue interaction of selecting.Be used to put into practice treatment of the present invention and partly comprise medicine from extensive medicament categories, this medicament categories has various pharmacologic activities.In some embodiments, preferably use the treatment part of non-sugar.An exception of this preferred property is to use by being covalently attached to the sugar on another entity, this entity such as PEG, biomolecules, treatment part, diagnosis part etc.In an exemplary embodiment, antisense nucleic acid partly is conjugated on the linker arm that is attached to targeting part.In another exemplary embodiment, the therapeutic sugar moieties is conjugated on the linker arm, and sugar-linker arm box is puted together by method of the present invention and peptide subsequently.

The method that treatment and diagnostic reagent and various other kinds are puted together is that those skilled in the art are well-known.Referring to as Hermanson, Bioconjugate Techniques, AcademicPress, San Diego, 1996; With people such as Dunn, Eds.Polymeric Drugs AndDrug Delivery Systems, ACS Symposium Series Vol.469, AmericanChemical Society, Washington, D.C.1991.

In an exemplary embodiment, treatment part is by on the sugar that is attached to modification at the key that can cut under the condition of selecting.Exemplary condition exists (as esterase, proteolytic enzyme, reductase enzyme, oxydase), light, heat etc. including, but not limited to the pH (as stomach, intestines, endocytic vacuole) that selects, organized enzyme.Many groups that cut are as known in the art.Referring to as people such as Jung, Biochem.Biophys.Acta, 761:152-162 (1983); People such as Joshi, J.Biol.Chem.265:14518-14525 (1990); People such as Zarling, J.Immunol., 124:913-920 (1980); People such as Bouizar, Eur.J.Biochem., 155:141-147 (1986); People such as Park, J.Biol.Chem.261:205-210 (1986); People such as Browning, J.Immunol., 143:1859-1867 (1989).

The kind of useful treatment part comprises as nonsteroidal anti-inflammatory medicaments (NSAIDS).NSAIDS can be selected from following kind: (as propanoic derivatives, acetogenin, fenamicacid derivative, phenylbenzene carboxylic acid derivative and oxicams); The steroid anti-inflammatory medicaments comprises hydrocortisone etc.; Adjuvant; Antihistamine drug (as chlorpheniramine, triprolidine); Antitussive (as dextrorotation methorphan, morphine monomethyl ether, caramiphen and pentoxyverine); Antipruritic medicine (as methdilazine and Trimeprazine); Anticholinergic agents (as scopolamine, coromegine, tropine melate, levodopa); Anti-emetic and antinanseant (as cyclizine, Meclozine, chlorpromazine, buclizine); Apocleisis medicine (as Benzphetamine, phentermine, chlorphentermine, Phenfluoramine); Central authorities' stimulating drug (as amphetamine, desoxyephedrine, Dextrofenfluramine and Methylphenidylacetate); Anti-rhythm disturbance medicine (as Proprasylyte, pronestyl, disopyramide, quinidine, encainide); Beta-adrenergic blockade medicine (as metoprolol, acebutolol, betaxolol, Trate and timolol); Cardiac tonic (as milrinone, amrinone and dobutamine); Antihypertensive drug (as enalapril, cloidine, the bent piperazine of hydrazine, minoxidil, Quanadrel, guanethidine); Diuretic (as guanamprazine and hydrochlorothiazide); Vasodilator drug (as diltiazem, amiodarone, isoxsuprine, Nylidrine, tolazoline and verapamil); Vasoconstriction medicine (as dihydroergotamine, Ergotamine and methysergide (methylsergide)); Anti-ulcer medicament (as Ranitidine HCL and Cimitidine Type A/AB); Anaesthetic (as lidocaine, bupivacaine, chloroprocaine, Percamine); Antidepressant drug (as imipramine, Desipramine, amitriptyline (amitryptiline), nortriptyline (nortryptiline)); Tranquilizer and downern are (as chlorine nitrogen

, benactyzine (benacytyzine), benzquinamide, flurazepam, hydroxyzine, loxapine and promazine; Antipsychotics (as chlorprothixene, Fluphenazine, haloperidol, molindone, thioridazine and trifluoperazine); Antimicrobial agents (as antibacterial, antimycotic, antiprotozoal and antiviral drug).

Useful treatment part kind comprises adjuvant.The optional keyhole limpet hemocyanin freely of this adjuvant conjugate, monophosphoryl lipid A, the lipopeptid MALP-2 that is derived from mycoplasma, b subunit of cholera toxin, intestinal bacteria thermolability toxin, general t helper cell epitope, interleukin 12, CpG oligodeoxynucleotide, dimethyl two (octadecyl) brometo de amonio, cyclodextrin, shark alkene, aluminium salt, meningococcus adventitia vesicle (OMV), montanideISA, TiterMax from Toxoid,tetanus ^TM(available from Sigma, St.Louis MO), nitrocellulose absorbate, immunostimulating complex such as Quil A, Gerbu ^TMAdjuvant (Gerbu Biotechnik, Kirchwald, Germany), threonyl Muramyl dipeptide, extrasin alpha, bupivacaine, GM-CSF, incomplete Freund's adjuvant, MTP-PE/MF59 (Ciba/Geigy, Basel, Switzerland), polyphosphonitrile, the saponin(e that is derived from Quillaia saponaria Quillaja saponaria and Syntex adjuvant formulation (Biocine, Emeryville, CA), and other is well-known in the art.

The antimicrobial agents that preferably is integrated into composition of the present invention comprises the drug acceptable salt as the beta-lactam medicine, the quinlone medicine, Ciprofloxacin, norfloxicin, tsiklomitsin, erythromycin, amikacin, trifluoro is given birth to, doxycycline, capromycin, chlorhexidine, duomycin, hydroxytetracycline, clindamycin, Tibutol, the different thiosulphate of hexamidine (hexamidineisothionate), metronidazole, pentamidine, gentamycin, kantlex, lineomycin, methacycline, hexamine, MINOCYCLINE HCL, Xin Meisu, netilmicin (netilmycin), paromycin, Streptomycin sulphate, tobramycin, mould anti-azoles and Symmetrel.Other drug moieties that are used for practice of the present invention comprise antitumor drug (as, antiandrogen (as leuproside or flutamide), cytocidal reagent (as andriamycin, Zorubicin, taxol, endoxan, busulfan, cisplatin, β-2-Interferon, rabbit), estrogen antagonist material (as tamoxifen), metabolic antagonist (as Fluracil, methotrexate, purinethol, thioguanine).In such, comprise equally be used to diagnose and treat both based on radioisotopic reagent and the toxin puted together, as ricin, geldanamycin, mytansin, CC-1065, C-1027, times ganmycin, thorn saitomycin and dependency structure and analogue, and list in toxin in the table 2.

The treatment part also can be hormone (as 6 Alpha-Methyls-17 α-Progesterone, estradiol, leuproside, megestrol, Sandostatin or somatostatin); Medicine of flaccid muscles (as N-cinnamylephedrine, cyclobenzaprine, flavoxate, orphenadrine, paraverine, mebeverine, idaverine, ritodrine, diphenoxylate, dantrolene and azumolene (azumolen)); The antispastic medicine; Bone pharmacological activation (as diphosphate and phosphine acyl-alkyl phosphinates (phosphonoalkylphosphinate) medical compounds); Endocrine regulation medicine (as contraceptive bian (as ethinodiol, lynoral, Norethisterone, mestranol, desogestrel, 6 Alpha-Methyls-17 α-Progesterone), diabetes conditioning agent (as Glyburide or P-607), anastate such as testolactone or stanozolol, male sex hormone (as methyltestosterone, testosterone or Fluoxymesterone), antidiuretic (as Desmopressin) and thyrocalcitonin).

Being used for of the present invention equally is oestrogenic hormon (as diethylstilbestrol,DES), glucocorticosteroid (as Aristocort, Betamethasone Valerate etc.) and progesterone, as Norethisterone, Norethindrone (ethynodiol), Norethisterone, Levonorgestrel; Tiroidina reagent (as Tetroxin or Levothyroxine) or antithyroid agent (as Thiamazole); Anti-hyperprolactinemia medicine (as Cabergoline); Hormone inhibitors (as danazol or goserelin), pitocin (as Methylergonovine or pitocin) and prostaglandin(PG) are as mioprostol, Prostaglandin E1 or rostaglin E2.

Other useful modification groups comprise immunoregulation druge (as antihistamine, mast cell stabilizers such as lodoxamide and/or Cromoglycic Acid (cromolyn), steroid (as Aristocort, beclometasone (beclomethazone), cortisone, dexamethasone, Ultracortene-H, methyl meticortelone, beclometasone (beclomethasone) or clobetasone), histamine H 2 antagonists (as famotidine, Cimetidine Type AB, Ranitidine HCL), immunosuppressor (as azathioprine, S-Neoral) etc.Also can use kind, as sulindac, R-ETODOLAC, Ketoprofen and ketorolac with anti-inflammatory activity.Being used for the other drug of puting together of the present invention is conspicuous to those skilled in the art.

The kind of useful treatment part comprises as antisense drug, and also comprises naked DNA.The optional Affinitak freely of antisense drug (ISIS, Carlsbad, CA) and Genasense ^TM(available from Genta, Berkeley Heights, NJ).Naked DNA for example can send as gene therapeutic agents with the DNA of coding as haemophiliachemophiliac blood coagulation factor VIII of treatment and IX and pass.

F. the preparation of the sugar of Xiu Shiing

The sugar that is used to form the modification of conjugate of the present invention is discussed herein.Clear for what illustrate, this discussion concentrates on preparation with on the water-soluble polymers decorated sugar.Especially, this discussion concentrates in the preparation of the sugar that comprises poly-(ethylene glycol) modification partly.The technician will understand herein in the preparation of the sugar that the method that proposes can be widely used in modifying, and therefore, this discussion should not thought limitation of the scope of the invention.

Usually, sugar moieties and modification group are that the application by reactive group links together, and this reactive group generally is connected method and is transformed into new organo-functional group or non-reacted kind.This sugar reactive functional group cumularsharolith any position on sugar moieties.Normally those bioconjugates chemical fields are well-known with being used for putting into practice reaction type of the present invention for reactive group.At present dominant is that those carry out under gentle relatively condition to reactive sugars part available reaction type.These are including, but not limited to nucleophilic substitution (as the reaction with acyl halide, active ester of amine and alcohol), electrophilic substitution (as enamine reaction) with to the addition (as MichaelShi reaction, Diels-Alder addition) of carbon-to-carbon and carbon-heteroatoms Multiple Bonds.The discussion of the reaction that these and other are useful is found in as Smith and March, Advanced OrganicChemistry, 5 ^ThEd., John Wiley ﹠amp; Sons, New York, 2001; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; With people such as Feeney, Modification of Proteins; Advances in ChemistrySeries, Vol.198, American Chemical Society, Washington, D.C., 1982.

The useful reactive functional group that stretches out (pendent) in sugared core or modification group includes, but are not limited to:

(a) carboxylic group and various derivative thereof are including, but not limited to N-hydroxy-succinamide ester, N-hydroxybenzotriazole ester, acid halide, acylimidazole, thioesters, p-nitrophenyl ester, alkyl, alkenyl, alkynyl and aromatic base ester;

(b) oh group, this group can change into as ester, ether, aldehyde etc.

(c) alkylhalide group group, wherein the available afterwards nucleophilic group of halogenide substitutes, and as amine, carboxylate anion, mercaptan negatively charged ion, carbanion or alkoxide ion, thereby causes the covalent attachment of new group on the halogen atom functional group;

(d) dienophile group, this group can participate in the Diels-Alder reaction, as dimaleoyl imino (maleimido) group;

(e) aldehydes or ketones group, thus be possible by the formation carbonyl derivative or by deriving subsequently as the mechanism of Grignard addition or lithium alkylide addition, this carbonyl derivative such as imines, hydrazone, semicarbazone or oxime;

(f) be used for reacting to form for example alkylsulfonyl halide group of sulphonamide with amine subsequently;

(g) thiol group, this group can change into as disulphide or with the reaction of alkyl and acyl halide;

(h) amine or sulfhedryl group, this group can carry out as acidylate, alkylation or oxidation;

(i) alkene, this alkene can carry out as cycloaddition, acidylate, Michael addition etc.; With

(j) epoxide, this epoxide can with as the reaction of amine and oxy-compound.

Can select functional group like this, thereby make them not participate in or do not disturb assembling reactive sugars core or the necessary reaction of modification group.Selectively, can be to the reactive functional group by when blocking group exists, protecting to prevent that it from participating in reaction.Those skilled in the art understands how to protect specific functional group, thereby makes it not disturb a selected cover reaction conditions.For the example of useful blocking group, referring to as people such as Greene, Protective Groupsin Organic Synthesis, John Wiley ﹠amp; Sons, New York, 1991.

In the following discussion, proposed the specific example of the sugar of many modifications, the sugar of this modification can be used in the practice of the present invention.In an exemplary embodiment, sialic acid derivative is used as the sugared core of adhering to modification group thereon.Lay particular emphasis on discussion to sialic acid derivative and only be in order to illustrate purpose clearly, and should not be interpreted as limitation of the scope of the invention.It should be appreciated by those skilled in the art that various other sugar moieties can be to activate and derivatize to the similar mode that proposes as an example with sialic acid.For example, the method for many modification semi-lactosis, glucose, N-acetylgalactosamine and Fucose is an available, and these sugar only are the substrates that minority is mentioned, and they can be easy to modify with the method for this area approval.Referring to as people such as Elhalabi, Curr.Med.Chem.6:93 (1999); With people such as Schafer, J.Org.Chem.65:24 (2000).

In an exemplary embodiment, the peptide of modifying by method of the present invention is in mammalian cell (as Chinese hamster ovary celI) or the peptide that produces in transgenic animal, thereby contains the oligonucleotide chain of not exclusively sialylated N-and/or O-connection.The oligonucleotide chain that lacks sialic acid and contain terminal galactose residues of this glycopeptide can carry out PEGization, PPGization or modify with the sialic acid of modification.

In scheme 4, handle mannosamine glucosides 1 with the active ester of amino acid (as the glycine) derivative of protecting, thereby the osamine residue is changed into the amino acid amide adducts of corresponding protection.This adducts is handled to form sialic acid 2 with zymohexase.Effect by the CMP-SA synthetic enzyme makes compound 2 change corresponding C MP derivative into, subsequently by to the shortening of CMP derivative to generate compound 3.Thereby will by form position that amine that glycine adduct introduces adheres to as PEG or PPG make compound 3 respectively with activatory PEG or PPG derivative (as PEG-C (O) NHS, PPG-C (O) NHS) reaction,

form

4 or 5.

Scheme 4

Table 3 has proposed the representative example with the sugared phosplate of PEG or PPG part derivatize.Some compound in the table 3 is the method preparation by scheme 1.Other derivatives are to prepare by the method that this area is approved.Referring to as people such as Keppler, Glycobiology 11:11R (2001); With people such as Charter, Glycobiology 10:1049 (2000)).Reactive PEG of other amine and PPG analogue are commercial buying, or they can prepare by the method that those skilled in the art is easy to obtain.

Table 3. is with the example of the sugared phosplate of PEG or PPG part derivatize

The sugar phosphoric ester that is used to put into practice modification of the present invention can substitute in other positions and in position proposed above." i " can be Na or another kind of salt, and " i " can exchange with Na.Sialic acid preferably substitutes in molecular formula 5 and proposes at present.

Molecular formula 5:

Wherein, X be preferably be selected from-O-,-N (H)-,-S, CH ₂-and N (R) ₂Linking group, wherein each R is and independently is selected from R ¹-R ⁵The member." i " can be Na or another kind of salt, and Na can exchange with " i ".Symbol Y, Z, A and B represent separately and are selected from hereinbefore to the described group of X.X, Y, Z, A and B all select each independently, so they can be identical or different.Symbol R ¹, R ², R ³, R ⁴And R ⁵Represent H, polymkeric substance, water-soluble polymers, treatment part, biomolecules or other parts.Symbol R6 represents H, OH or polymkeric substance.Selectively, these symbologies are connected to the linker on polymkeric substance, water-soluble polymers, treatment part, biomolecules or other parts.

In another exemplary embodiment, mannosamine is acidylate and the activatory that carries out nucleophilic substitution in scheme 5 by the sym-dichloroacetic anhydride of using as propose simultaneously.In each scheme that in this part, provides, i ⁺Or Na ⁺Can exchange, wherein salt can be sodium, perhaps can be any other suitable salt.

Scheme

Make as a result that the glycan of the chlorine derivatize of gained contacts when zymohexase exists with pyruvate salt, thereby form the sialic acid of chlorine derivatize.Corresponding nucleotide sugar is to prepare by sialic acid derivative and suitable Nucleotide triphosphoric acid are contacted with synthetic enzyme.Cl radical on the sialic acid part can replace with nucleophilic PEG derivative such as sulfo--PEG then.

In further exemplary, as showing in scheme 6, mannosamine carries out acidylate with two-HOBT dicarboxylate, thereby produces corresponding amide-alkyl-carboxylic acid, and this acid amides-alkyl-carboxylic acid changes sialic acid derivative subsequently into.Change this sialic acid derivative into nucleotide sugar, and react with the carboxylic acid activation and with nucleophilic PEG derivative such as amino-PEG.

Scheme 6

In another exemplary embodiment, as in scheme 7, proposing, by the primary hydroxyl group is changed into corresponding p-toluenesulfonic esters make amine-and the neuraminic acid of carboxyl-protection activate, and methyl ester is excised.Make the activatory neuraminic acid change corresponding nucleotide sugar into, and activating group is replaced with nucleophilic PEG kind such as sulfo--PEG.

Scheme 7

In other further exemplary, as in scheme 8, proposing, make with nucleophilic PEG such as chloro-PEG that amine-and the primary hydroxyl of the neuraminic acid derivatives of carboxyl-protection is partially alkylated.Excise methyl ester subsequently and change PEG-sugar into nucleotide sugar.

Scheme 8

Glycan outside the desalivation acid can be used on the method that proposes herein and carries out derivatize with PEG.The glycan of derivatize itself also within the scope of the invention.Thereby scheme 9 provides the exemplary route of synthesis of the semi-lactosi nucleotide sugar of PEGization.The primary hydroxyl group activation that makes semi-lactosi is corresponding p-toluenesulfonic esters, changes nucleotide sugar subsequently again into.

Scheme 9

Scheme 10 has proposed the exemplary approach based on the preparation semi-lactosi-PEG derivative of semi-lactosi-6-amine moiety.Thereby, make GalN change nucleotide sugar into, and make the amine moiety of GalN carry out functionalized with active PEG derivative.

Scheme 10

Scheme 11 provides another exemplary approach of preparation galactose derivate.The starting point of scheme 11 is semi-lactosi-2-amine, and this semi-lactosi-2-amine changes nucleotide sugar into.The amine moiety of nucleotide sugar is the position of adhering to the PEG derivative, as methoxyl group-PEG (mPEG) carboxylic acid.

Scheme 11

Be attached to exemplary part on the disclosed herein conjugate including, but not limited to the PEG derivative (as acyl group-PEG, acyl group-alkyl-PEG, alkyl-acyl group-PEG carbamyl-PEG, aryl-PEG, alkyl-PEG), PPG derivative (as acyl group-PPG, acyl group-alkyl-PPG, alkyl-acyl group-PPG carbamyl-PPG, aryl-PPG), poly aspartic acid, polyglutamic acid, polylysine, treatment part, diagnosis part, Man-6-P, heparin, heparitin, SLe ^x, seminose, Man-6-P, Sialyl Lewis X, FGF, VFGF, protein (as transferrin), chrondroitin, keratin, dermatan, dextran, modification dextran, amylose starch, diphosphate, poly-SA, hyaluronic acid, keritan, albumin, integral protein, feeler oligosaccharides, peptide etc.The method that various modification groups and sugar moieties are puted together is that those skilled in the art is easy to obtain (Poly (Ethylene Glycol) Chemistry:Biotechnicaland Biomedical Applications, J.Milton Harris, Ed., Plenum Pub.Corp., 1992; Poly (Ethylene Glycol) Chemical and BiologicalApplications, J.Milton Harris, Ed., ACS Symposium Series No.680, American Chemical Society, 1997; Hermanson, BioconjugateTechniques, Academic Press, San Diego, 1996; With people such as Dunn, Eds.Polymeric Drugs and Drug Delivery Systems, ACS Symposium SeriesVol.469, American Chemical Society, Washington, D.C.1991).

The purifying of sugar, nucleotide sugar and derivative

Nucleotide sugar that is produced by aforesaid method and derivative can be not purified and use.Yet, preferably reclaim product usually.But the well-known technology that is used to reclaim glycosylated sugar of application standard is as thin layer or thick-layer chromatography, column chromatography, ion exchange chromatography or membrane filtration.Applying membrane filtering preferably is more preferably for being used for reverse osmotic membrane or one or more column chromatography technologies to reclaim, as hereinafter and discuss in the reference of quoting herein.For example, available membrane filtration removes molecular weight less than 10, and the protein of the reagent of 000Da, this film have about 3000～about 10,000 weight shutoff point.Membrane filtration or inverse osmosis can be used for removing salt and/or purified product sugar (referring to as WO 98/15581) then.The Nanofilter film be a class can make monovalent salt through and keep polyvalent salt and greater than the reverse osmotic membrane of uncharged solute of about 100～about 2,000 dalton (depending on used film).Thereby in general application, the sugar that is prepared by method of the present invention will be retained on the film, and impurity salt will pass through film.

G. crosslinked group

The preparation of sugar that is used for the modification of method of the present invention comprises modification group adhering to and form stable adducts on saccharide residue, and this adducts is the substrate of glycosyltransferase.Thereby, often preferably use linking agent modification group and sugar are puted together.Can be used for the exemplary dual-function compound that modification group is attached on the sugar moieties is included, but are not limited to difunctional poly-(ethylene glycol), polymeric amide, polyethers, polyester etc.Making carbohydrate is known with the general method that is connected on other molecules in the literature.Referring to as people such as Lee, Biochemistry 28:1856 (1989); People such as Bhatia, Anal.Biochem.178:408 (1989); People such as Janda, people such as J.Am.Chem.Soc.112:8886 (1990) and Bednarski, WO92/18135.In discussion subsequently, the sugar moieties reactive group of the sugar of new life's modification is carried out gentleness handle.The special emphasis of this discussion is in order to illustrate purpose clearly.It should be appreciated by those skilled in the art that this discussion also is applicable to the reactive group on the modification group.

Exemplary strategy comprises and will be integrated in the sugar with the sulfhedryl of isodigeranyl functional cross-link agent SPDP (positive succinimide-3-(2-pyridyl dithio) propionic acid) protection, then to sulfhedryl go to protect with modification group on other sulfhedryls form disulfide linkage.

If SPDP influences the ability of the sugar of modification as the glycosyltransferase substrate nocuously, certain other linking agent can be used to form disulfide linkage so, as 2-imido grpup tetramethylene sulfide or N-succinimide S-ethanoyl thioacetic acid (SATA).2-imido grpup tetramethylene sulfide can with primary amine reaction, existing side by side is about to unprotected sulfhedryl and is integrated on the molecule that contains amine.SATA also with primary amine reaction, but integrate the sulfhedryl of protection, the sulfhedryl of this protection is deacetylated to produce the free sulfhedryl with azanol subsequently.Under each situation, the sulfhedryl of integration can freely react to form required disulfide linkage with other the sulfhedryl or the sulfhedryl such as the SPDP of protection.

Above-mentioned strategy is exemplary for being used for linker of the present invention, rather than restrictive.Other linking agents that can be used for modification group and peptide are carried out in the crosslinked Different Strategies are available.For example; TPCH (S-(2-sulfo-pyridyl)-L-halfcystine hydrazides and TPMPH ((S-(2-sulfo-pyridyl) sulfydryl-propionyl hydrazides) with in advance with the gentle sugar moieties reaction of handling oxidation of periodates, thereby between the aldehyde that the hydrazides part and the periodates of linking agent generates, form the hydrazone key.TPCH and TPMPH have introduced the sulfhedryl group of 2-pyridyl thioketones protection on sugar, this group can go protection also to be used to subsequently put together with DTT, as form disulfide linkage between composition.

If finding disulfide linkage is unsuitable for the sugar that produces stable modification, can be applicable to other linking agents of the more stable key of integration between the composition so.Isodigeranyl functional cross-link agent GMBS (N-gama-malimidobutyryloxy) succinimide) and SMCC (succinimido 4-(N-maleimide-methyl) hexanaphthene) and primary amine reaction, thus maleimide base group is incorporated on the composition.This maleimide base group subsequently can with the sulfhedryl reaction that can introduce by previously described linking agent on other compositions, thereby between composition, form stable thioether bond.If the sterically hindered sugar that has disturbed the active of composition or modified between the composition is as the ability of glycosyltransferase substrate, can use the linking agent that between composition, to introduce than long spacer arm so, and this linking agent comprises the derivative of some previously described linking agents (being SPDP).Thereby, there are a large amount of useful suitable linking agents; They each all is to depend on its effect that sugar of the suitableeest peptide conjugate and modification is produced to select.

Many reagent can be used for modifying the composition of the sugar of the modification with intramolecularly chemically crosslinked (for the summary of linking agent and crosslinked program, referring to Wold, F., Meth.Enzymol.25:623-651,1972; Weetall, H.H. and Cooney, D.A., Enzymes as Drugs (Holcenberg and Roberts, eds.) pp.395-442, Wiley, New York, 1981; Ji, T.H., Meth.Enzymol.91:580-609,1983; People such as Mattson, Mol.Biol.Rep.17:167-183,1993, all be incorporated herein by reference herein).Preferred cross-linking agents can be derived from various distance of zero mark degree, with linking agent bifunctional and the isodigeranyl function.The linking agent of distance of zero mark degree comprises directly puting together of two inner chemical groups, and does not introduce outside material.The reagent that the catalysis disulfide linkage forms belongs to this type.Another example is to induce the condensation of carboxyl and primary amino group and the reagent that forms amido linkage, as carbodiimide, ethyl carbonochloridic acid, Woodward reagent K (2-ethyl-5-phenylisoxazolium-3 '-sulfonate) and carbonyl dimidazoles.Except that these chemical reagent, trans-glutaminases (glutamy-peptide gamma glutamyltransferase; EC 2.3.2.13) can be used as the linking agent of distance of zero mark degree.The acyl group shift reaction of the carboxamide of the glutamine residue that this enzyme catalysis protein connects is rolled into a ball as substrate with primary amino usually.Together preferred-as to contain two identical or different sites respectively with different-bifunctional reagent, this site is reactive for amino, sulfhedryl, guanidine radicals, indoles or unspecific group.

2. preferred specificity site on the linking agent

A. amino-reactive group

In a preferred embodiment, the site on the linking agent is the amino-reactive group.The useful non-limitative example of amino-reactive group comprises N-hydroxy-succinamide (NHS) ester, polyurethane, isocyanic ester, acyl halide, aryl nitride, p-nitrophenyl ester, aldehyde and SULPHURYL CHLORIDE.

The NHS ester preferably with primary (comprising aromatics) amine groups reaction of the sugared composition of modifying.The imidazole group of Histidine known can with the primary amine competing reaction, but its reaction product is unstable and is easy to hydrolysis.This reaction comprises the nucleophillic attack of amine on the acid carboxyl of NHS ester forming acid amides, thereby discharges N-hydroxy-succinamide.Thereby the positive charge of initial amino group has then been lost.

Polyurethane is the most specific acylating reagent that reacts with the amine groups of the sugared composition of modifying.When 7～10 pH, polyurethane only with primary amine reaction.The imines ester is attacked to produce intermediate in primary amine nucleophilic ground, and this intermediate is decomposed into amidine and is decomposed into new imidoether when hanging down pH when high pH.This new imidoether can with another primary amine reaction, thereby make two amino groups crosslinked, this is the situation that single function imidoether of inferring carries out difunctional reaction.With the primary product of primary amine reaction be amidine, this amidine is the alkali stronger than initial amine alkalescence.Positive charge on the therefore initial amino group has then kept.

The primary amine reaction of the sugared composition of isocyanic ester (and lsothiocyanates) and modification is to form stable key.They produce relative unsettled product with the reaction of sulfhedryl, imidazoles and tyrosyl group.

Also as amino specific reagent, wherein the nucleophilic amine of affine composition is attacked acid carboxylic group to the acyl group nitride under the condition of slight alkalinity such as pH8.5.

Aryl halide is as 1, and 5-two fluoro-2,4-dinitrobenzene preferably react with the amino group of the sugared composition of modifying and the phenolic group group of tyrosine, but also react with sulfhedryl and imidazole group.

Single-and the p-nitrophenyl ester of dicarboxylic acid also be useful amino-reactive group.Although the specificity of this reagent is not high especially, α-and the reaction of epsilon-amino group be the fastest.

The primary amine reaction of the sugar of aldehyde such as glutaraldehyde and modification.Although in the reaction of amino group and aldehyde, formed unsettled schiff bases, the sugar that the enough stable crosslinked modifications of glutaraldehyde energy are modified.When typical crosslinked pH condition pH6-8, annular polymeric is dewatered to form the undersaturated aldehyde polymer of alpha-beta.Yet schiff bases is stable when puting together with another pair key.The Resonant Interaction of two two keys has stoped the hydrolysis of Schiff key.In addition, the amine of high local concentrations can be attacked ethylene double bond to form stable Michael adduct.

The a plurality of sites reaction of the sugared composition of aromatics SULPHURYL CHLORIDE and modification, but with the reaction of amino group be most important, the result forms stable sulphonamide key.

B. sulfhedryl reactive group

In another preferred embodiment, this site is the sulfhedryl reactive group.Useful nonrestrictive sulfhedryl reactive group comprises maleimide, alkyl halide, pyridyl disulfide and sulfo-phthalic imidine.

The sulfhedryl radical reaction of the sugared composition of maleimide and modification is to form stable thioether bond.They also react with the imidazole group of low-down speed and primary amino group and Histidine.Yet, can think that when pH7 maleimide is the specific group of sulfhedryl, this is because the speed of reaction of simple mercaptan is higher 1000 times than the speed of reaction of corresponding amine when pH7.

Alkyl halide and sulfhedryl, sulfide, imidazoles and amino group reaction.Yet when the subalkaline pH, alkyl halide is main to react to form stable thioether bond with sulfhedryl in neutrality.When higher pH, with the reaction of amino group be dominant.

Pyridyl disulfide and free sulfhydryl base react to form blended disulphide by disulfide exchange.Pyridyl disulfide is the most specific sulfhedryl reactive group as a result.

Sulfo-phthalic imidine and free sulfhydryl base radical reaction are to form disulphide.

C. carboxyl-reactive residue

In another embodiment, will in water and organic solvent, be used as carboxyl-reactive reagent by soluble carbodiimide.These compounds and free carboxy radical reaction to be to form pseudo-urea, then this pseudo-urea can with the coupling of available amine to form amido linkage.The program of modifying carboxylic group with carbodiimide is (referring to people such as Yamada, Biochemistry 20:4836-4842,1981) well known in the art.

3. preferred non-specific site in the linking agent

Except that practical site specific reaction part, the present invention also relates to use nonspecific reactive group so that sugar is connected with modification group.

Exemplary non-specific linking agent comprises in the dark inert photoactivation type group fully, and this group can change reactive kind into after the photon that absorbs suitable energy.In a preferred embodiment, the photoactivation type group is selected from the nitrene precursor that generates by heating or photodissociation nitride.Electronic defects type nitrene is extremely reactive, and can with various chemical bonding reactions, comprise N-H, O-H, C-H and C=C.Although can use 3 class nitride (aryl, alkyl and acyl derivative), the aryl nitride is preferred at present.The aryl nitride after photodissociation with good than with C-H of the reactivity of N-H and O-H.Electronic defects type aryl nitrene carries out ring extension apace to form the dehydrogenation azepine, and this dehydrogenation azepine tends to and the reaction of nucleophilic thing, rather than forms C-H insertion product.The reactivity of aryl nitride can strengthen by have electron-withdrawing substituent such as nitro or oh group in ring.This substituting group is shifted the maximum absorption of aryl nitride onto bigger wavelength.Unsubstituted aryl nitride has maximum absorption in the scope of 260-280nm, and hydroxyl and nitro aryl nitride significantly absorb the light that surpasses 305nm.Therefore, hydroxyl and nitro aryl nitride are most preferred, and this is because they make it possible to use the photodissociation condition lower to affine composition harm than unsubstituted aryl nitride.

In another preferred embodiment, the photoactivation type group is selected from fluorizated aryl nitride.The photolytic product of fluorizated aryl nitride is the aryl nitrene, and they all carry out efficiently the characteristic reaction with this group, comprises that c h bond inserts people such as (, J.Org.Chem.55:3640-3647,1990) Keana.

In another embodiment, the photoactivation type group is selected from the benzophenone residue.Benzophenone reagent provides higher crosslinked result than aryl nitride reagent usually.

In another embodiment, the photoactivation type group is selected from diazonium compound, and this diazonium compound forms electronic defects type Cabbeen after photodissociation.These Cabbeens can carry out various reactions, addition, the hydrogen that comprises insertion to c h bond, Xiang Shuanjian (comprising aromatic systems) attract and to the coordination of nucleophilic center to provide carbon ion.

In the another one embodiment, the photoactivation type group is selected from the diazonium pyruvate salt.For example, the p-nitrophenyl ester of p-nitrophenyl diazonium pyruvate salt and aliphatic amine reaction form diazonium pyruvic acid acid amides, and this diazonium pyruvic acid acid amides carries out ultraviolet photodissociation and forms aldehyde.The affine composition that photolytic diazonium pyruvate salt is modified will be similar to formaldehyde or glutaraldehyde reacts crosslinked to form.

4. homobifunctional agent

A. with the homobifunctional agent of primary amine reaction

Synthetic, the character and the application of amine reactant cross-linker are described in (summary for crosslinked program and reagent sees above) in the document commercially.Many reagent be can buy (as PierceChemical Company, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR).

Preferred non-limitative example with difunctional NHS ester comprises two succinimido glutarates (DSG), two succinimido suberates (DSS), two (sulfosuccinimide base) suberates (BS), two succinimido tartrates (DST), disulfo succinimido tartrate (sulfo group-DST), two-2-(succinimido oxygen base ketonic oxygen base) ethyl sulfone (BSOCOES), two-2-(sulfosuccinimide base oxygen base ketonic oxygen base) ethyl sulfone (sulfo group-BSOCOES), ethylene glycol bisthioglycolate (succinimido succinate) (EGS), ethylene glycol bisthioglycolate (sulfosuccinimide base succinate) (sulfo group-EGS), dithio two (succinyl phosphorons amino propyl acid ester) (DSP), with dithio two (sulfosuccinimide base propionic esters) (sulfo group-DSP).Preferred non-limitative example with difunctional imido-ester comprises dimethyl propylene imide acid esters (DMM), dimethyl succinimide acid esters (DMSC), dimethyl adipimide acid esters (DMA), dimethyl-g imide acid esters (DMP), dimethyl-octa imide acid esters (DMS), dimethyl-3,3 '-oxygen base dipropyl imide acid esters (DODP), dimethyl-3,3 '-(methylene radical dioxy) dipropyl imide acid esters (DMDP), dimethyl-3 '-(dimethylene dioxy) dipropyl imide acid esters (DDDP), dimethyl-3,3 '-(tetramethylene dioxy) dipropyl imide acid esters (DTDP) and dimethyl-3, the two propionyl imidoethers (DTBP) of 3 '-dithio.

Preferred non-limitative example with difunctional lsothiocyanates comprises: bitoscanate (DITC) and 4,4 '-diisothiocyanic acid-2,2 '-disulfonic acid stilbene (DIDS).

Preferred non-limitative example with difunctional isocyanic ester comprises dimethylbenzene-vulcabond, Toluene-2,4-diisocyanate, 4-vulcabond, Toluene-2,4-diisocyanate-isocyanic ester-4-lsothiocyanates, 3-methoxyl group ditan-4,4 '-vulcabond, 2,2 '-dicarboxyl-4,4 '-azobenzene vulcabond and hexamethylene diisocyanate.

Preferred non-limitative example with difunctional aryl halide comprises 1,5-two fluoro-2,4-dinitrobenzene (DFDNB) and 4,4 '-two fluoro-3,3 '-dinitrophenyl sulfone.

With the preferred non-limitative example of difunctional aliphatic aldehyde reagent comprise oxalic dialdehyde, mda (malondialdehyde) and glutaraldehyde.

The nitro phenyl ester that comprises dicarboxylic acid with the preferred non-limitative example of difunctional acylating reagent.

Preferred non-limitative example with difunctional aromatics SULPHURYL CHLORIDE comprises phenol-2,4-disulfonyl base muriate and naphthyl alcohol-2,4-disulfonyl base muriate.

The preferred non-limitative example of extra amino-reactive homobifunctional agent comprises the tetrahydroxybutane dicarboxylic acid (erythriolbiscarbonate) that generates diurethanes (biscarbamate) with the amine reaction.

B. with the same bi-functional cross-linking agent of free sulfhydryl base radical reaction

Synthetic, the character and the application of this reagent are described in (summary for crosslinked program and reagent sees above) in the document.Many reagent are that commercial buying is (as Pierce ChemicalCompany, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR).

Preferred non-limitative example with difunctional maleimide comprises bismaleimides hexane (BMH), N, N '-(1, the 3-phenylene) bismaleimides, N, N '-(1, the 2-phenylene) bismaleimides, azobenzene bismaleimides and two (N-maleimide methyl) ether.

Preferred non-limitative example with difunctional pyridyl disulfide comprises 1,4-two-3 '-(2 '-pyridyl dithio) propionamido-butane (DPDPB).

Preferred non-limitative example with difunctional alkyl halide comprises 2; 2 '-dicarboxyl-4; 4 '-diiodo-acetamido nitrogen benzide, α; α '-two iodo-p-Xylol sulfonic acid, α; α '-two bromo-p-Xylol sulfonic acid, N; N '-two (b-bromotrifluoromethane) benzylamine, N, N '-two (acetyl bromide) phenyl hydrazine and 1,2-two (acetyl bromide) amino-3-phenyl-propane.

C. with difunctional photoactivation type linking agent

Synthetic, the character and the application of this reagent are described in (summary for crosslinked program and reagent sees above) in the document.Many reagent are that commercial buying is (as Pierce ChemicalCompany, Rockford, I11.; Sigma Chemical Company, St.Louis，Mo.；Molecular?Probes，Inc.，Eugene，OR)。

Preferred non-limitative example with difunctional photoactivation type linking agent comprises two-β-(4-azido-salicyl amide group) ethyl disulphide (BASED), two-N-(2-nitro-4-azido-phenyl)-cystamine-S, S '-dioxide (DNCO) and 4, the two phenyl nitride of 4 '-dithio.

5. heterobifunctional agent

A. the amino-reactive heterobifunctional agent that has pyridyl disulfide moieties

Synthetic, the character and the application of this reagent are described in (summary for crosslinked program and reagent sees above) in the document.Many reagent be can buy (as Pierce Chemica lCompany, Rockford, I11.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR).

Preferred non-limitative example with heterobifunctional agent of pyridyl disulfide moieties and amino-reactive NHS ester comprises N-succinimido-3-(2-pyridyl dithio) propionic acid (SPDP), succinimido 6-3-(2-pyridyl dithio) propionic acid amide caproic acid (LC-SPDP), sulfosuccinimide base 6-3-(2-pyridyl dithio) propionic acid amide caproic acid (sulfo group-LCSPDP), 4-succinimido oxygen base carbonyl-Alpha-Methyl-α-(2-pyridyl dithio) toluene (SMPT) and sulfosuccinimide base 6-Alpha-Methyl-α-(2-pyridyl dithio) toluamide caproic acid (sulfo group-LC-SMPT).

B. the amino-reactive heterobifunctional agent that has the maleimide amine moiety

Synthetic, the character and the application of this reagent are described in the document.Preferred non-limitative example with heterobifunctional agent of maleimide amine moiety and amino-reactive NHS ester comprises succinimide maleimide acetonyl ester (AMAS), succinimide 3-maleimide propionic ester (BMPS), N-γ-maleimide butyryl oxygen succinimide ester (GMBS), N-γ-maleimide butyryl oxygen sulfosuccinimide ester (sulfo group-GMBS), succinimide 6-maleimide capronate (EMCS), succinimide 3-maleimide benzoic ether (SMB), between maleimide benzoyl-N-hydroxy-succinamide ester (MBS), between maleimide benzoyl-N-hydroxysulphosuccinimide ester (sulfo group-MBS), succinimide 4-(N-maleimide methyl)-hexanaphthene-1-carboxylicesters (SMCC), sulfosuccinimide 4-(N-maleimide methyl)-hexanaphthene-1-carboxylicesters (sulfo group-SMCC), succinimide 4-(to maleimide phenyl) butyric ester (SMPB) and sulfosuccinimide 4-(to maleimide phenyl) butyric ester (sulfo group-SMPB).

C. the amino-reactive heterobifunctional agent that has the alkyl halide part

Synthetic, the character and the application of this reagent are described in the document.Preferred non-limitative example with heterobifunctional agent of alkyl halide part and amino-reactive NHS ester comprises N-succinimide-(4-iodo ethanoyl) Aminobenzoate (SIAB); sulfosuccinimide-(4-iodo ethanoyl) Aminobenzoate (sulfo group-SIAB); succinimide-6-(iodo ethanoyl) hexosamine ester (SIAX); succinimide-6-(6-((iodo ethanoyl)-amino) hexanamido) capronate (SIAXX); succinimide-6-(((4-iodo ethanoyl)-amino)-methyl)-hexanaphthene-1-carbonyl) hexosamine ester (SIACX) and succinimide-4 ((iodo acetyl)-amino) methylcyclohexane-1-carboxylicesters (SIAC).

Preferred example with heterobifunctional agent of amino-reactive NHS ester and alkyl dihalide part is a N-hydroxy-succinamide 2,3-dibromo-propionic acid ester (SDBP).SDBP introduces intramolecular crosslinking by puting together its amino group to affine composition.Dibromo propionyl part can be controlled people such as (, Protein Chem.7:581-592 (1988)) McKenzie by temperature of reaction with the reactivity of primary amine.

Preferred non-limitative example with heterobifunctional agent of alkyl halide part and amino-reactive p-nitrophenyl ester comprises p-nitrophenyl iodoacetic acid ester.

Other linking agents are well known to a person skilled in the art.Referring to as people such as Pomato, U.S. Patent No. 5,965,106.To the suitable linking agent of specific application choice is in those skilled in the art's limit of power.

The linker group that d. can cut

In in addition further embodiment, the linker group has can cut the group that discharges from saccharide residue with modification group.Many groups that cut are as known in the art.Referring to as people such as Jung, Biochem.Biophys.Acta 761:152-162 (1983); People such as Joshi, J.Biol.Chem.265:14518-14525 (1990); People such as Zarling, J.Immunol.124:913-920 (1980); People such as Bouizar, Eur.J.Biochem.155:141-147 (1986); People such as Park, J.Biol.Chem.261:205-210 (1986); People such as Browning, J.Immunol.143:1859-1867 (1989).The difunctional linker group that can cut widely in addition, (same or the isodigeranyl function) is commercial can buying from the supplier as Pierce.

The exemplary part available light of cutting, heat or reagent cutting, this reagent such as mercaptan, azanol, alkali, periodates etc.In addition, some preferred group cuts (as cis rhizome of Chinese monkshood base (cis-aconityl) in response to endocytosis in vivo; Referring to people such as Shen, Biochem.Biophys.Res.Commun.102:1048 (1991)).Preferably but the group that can cut comprises the member's who is selected from disulphide, ester, imide, carbonate, nitrobenzyl, phenacyl and st-yrax group cutting part.

E. sugar of Xiu Shiing and peptide puts together

The sugar of modifying is to put together with glycosylation or nonglycosylated peptide by the suitable enzyme that mediation is puted together.Preferably, the concentration of donor sugar, enzyme and the acceptor peptide of select modifying, thus glycosylation can proceed to acceptor when running out.Although propose about sialyltransferase, the factor that is discussed below can be applicable in other the glycosyltransferase reaction usually.

Many methods with the synthetic oligosaccharide structure of wanting of glycosyltransferase are known, and are applied among the present invention usually.Some document descriptions exemplary method, as people such as WO 96/32491, Ito, Pure Appl.Chem.65:753 (1993) and U.S.Pat.No.5,352,670,5,374,541 and 5,545,553.

The present invention puts into practice with the combination of single glycosyltransferase or glycosyltransferase.For example, people can use the combination of sialyltransferase and galactosyltransferase.In use surpassing a kind of embodiment of enzyme, enzyme and substrate preferably make up in the initial reaction compound, and perhaps the enzyme of second enzymatic reaction and reagent are finished or added in the reaction medium when finishing in first enzymatic reaction.By carry out two enzymatic reactions successively in single container, ultimate production has been improved with respect to the program that will separate the intermediate kind.In addition, removing and processing have been reduced to extra solvent and byproduct.

In a preferred embodiment, first kind and second kind of enzyme all are glycosyltransferases.In another preferred embodiment, a kind of enzyme is an endoglycosidase.In another preferred embodiment, a kind of enzyme is an exoglycosidase.In extra embodiment preferred, will be used to assemble modified glucoprotein of the present invention above two kinds of enzymes.This enzyme is used for the sugared structure on any site change peptide before or after the sugar that will modify adds on the peptide.

In another embodiment, at least two kinds of enzymes are glycosyltransferases, and add that the last sugar in the sugared structure is the sugar of non-modification on the peptide to.Perhaps, the sugar of modification is positioned at the inside of glycan structures, therefore needs not to be the last sugar on the glycan.In an exemplary embodiment, but galactosyltransferase catalysis Gal-PEG is from the transfer of UDP-Gal-PEG on glycan, carry out incubation subsequently when ST3Gal3 and CMP-SA exist, this will add " adding cap " sialic acid (Figure 23 A) of unmodified to glycan.

In another embodiment, at least two kinds of used enzymes are glycosyltransferases, and the sugar of at least two modifications is added on the glycan structures of peptide.By this way, two or more different peptide conjugates can add on the peptide on one or more glycan.This process has produced the glycan structures of the sugar with the different modification of two or more functions.In an exemplary embodiment, the incubation of peptide and GnT-I, II and UDP-GlcNAc-PEG can add the GlcNAc-PEG molecule on the glycan to; Incubation with galactosyltransferase and UDP-Gal can add the Gal residue thereon then; And SA-Man-6-P molecule can be added on the glycan with the incubation of ST3Gal3 and CMP-SA-Man-6-phosphoric acid.This serial reaction result produces functional character with PEGization glycan and the Man-6-P active polysaccharide chains (Figure 23 B) that leads.

In another embodiment, at least two kinds of enzymes that are used for reacting are glycosyltransferases, and with the sugar of different modifications add on the peptide N-that connect with glycan that O-is connected on.Add to when sugar on the glycan of peptide, and when importantly spatially the sugar of the above modification of peptide being separated mutually, this embodiment is useful two different modifications.For example, if the steamed bun stuffed with sugar of modifying contains bulky molecule, then this method is preferred, and this bulky molecule is including, but not limited to PEG and other molecules such as linker molecule.The sugar of modifying can add on the glycan of peptide simultaneously, and perhaps they can add successively.In an exemplary embodiment, sialic acid-PEG can be added on the glycan that N-is connected with the incubation of ST3Gal3 and CMP-SA-PEG, and the sialic acid bisphosphate can be added on the glycan that O-is connected (Figure 23 C) with the incubation of ST3Gal1 and CMP-SA-bisphosphate.

In another embodiment, this method is used one or more circumscribed or endoglycosidases.This Glycosylase generally is a mutant, this mutant be through processing to form glycosyl bond rather than to destroy this key.The sudden change glycanase that is sometimes referred to as sugared synthetic enzyme generally comprises the replacement of amino-acid residue to the avtive spot acidic amino acid residue.For example, when the inscribe glycanase was endo-H, the avtive spot of replacement generally was the Asp of position 130, Glu or its combination of position 132.This amino acid normally replaces with Serine, L-Ala, l-asparagine or glutamine.Exoglycosidase also is useful as changeing sialidase (transialylidase).

The enzyme of sudden change is usually with the synthesis step catalyzed reaction, and this is similar with the back reaction of the hydrolysing step of inscribe glycanase.In these embodiments, the glycosyl donor molecule (as the widow who wants-or list-sugared structure) contain leavings group, and reaction is carried out along with add donor molecule on the GlcNAc residue on the protein.For example, leavings group can be halogen, as fluorochemical.In other embodiments, leavings group is Asn or Asn-peptide moiety.In in addition further embodiment, the GlcNAc residue on the glycosyl donor molecule has obtained modification.For example, the GlcNAc residue can comprise 1,2 oxazoline part.

In a preferred embodiment, each enzyme that is used to produce conjugate of the present invention all exists with catalytic amount.The catalytic amount of certain enzyme changes according to the concentration and the reaction conditions of this enzyme substrates, this reaction conditions such as temperature, time and pH value.Determine that given enzyme is that those skilled in the art are well-known at the concentration of substrate of selecting in advance and the method for the catalytic amount under the reaction conditions.

The temperature range of implementing aforesaid method is the temperature that chill point arrives the most responsive enzyme denaturation.Preferred temperature range is about 0 ℃～about 55 ℃, and more preferably is about 20 ℃～about 37 ℃.In another exemplary embodiment, one or more compositions in the inventive method are to carry out at elevated temperatures with thermophilic enzyme.

Make reaction mixture keep time enough so that acceptor can carry out glycosylation, thereby form the conjugate of wanting.Some conjugates a few hour of being everlasting promptly may detect, and callable amount obtains in 24 hours or in the shorter time usually.Those skilled in the art understands speed of reaction and depends on many variable factors (as enzyme concn, donor concentration, acceptor density, temperature, solvent volume), makes these factor optimizations according to the system of selecting.

The present invention also provides the plant-scale production to modified peptides.As used herein, technical scale is produced the conjugate of the final purifying of at least one gram usually.

In discussion subsequently, the present invention carries out example by the sialic acid part of modifying with puting together of glycosylated peptide.The modification sialic acid PEG mark that this is exemplary.The discussion of the application of sialic acid that PEG-is modified and glycosylated peptide is in order to illustrate purpose clearly below, rather than means that the present invention is confined to puting together of these two mating partners.The technician is appreciated that this discussion can be used for adding the modification glycosyl part outside the desalivation acid usually.In addition, this discussion is used for reagent except that PEG equally to the modification of glycosyl unit, and this reagent comprises other water-soluble polymers, treatment part and biomolecules.

A kind of enzymatic means can be used for optionally sugar PEGization or PPGization being incorporated in peptide or the glycopeptide.This method utilization contains PEG, PPG or the sugar of the modification of the reactive functional group covered, and with suitable glycosyltransferase or the combination of sugared synthetic enzyme.Connect and utilize and modify the glycosyltransferase of sugar by selecting to form the sugar of wanting, can be introduced directly into PEG or PPG on the peptide main chain, be incorporated on the glycopeptide on the already present saccharide residue or be incorporated on the saccharide residue that adds on the peptide as the donor substrate.

The acceptor of sialyltransferase is present on the peptide of being modified by method of the present invention, or it is as naturally occurring structure, or recombinate ground, enzymatic ground or chemically placed on it.Suitable acceptor comprises as galactosyl acceptor such as Gal β 1,4GlcNAc, Gal β 1,4GalNAc, Gal β 1,3GalNAc, lacto-N-tetraose, Gal β 1,3GlcNAc, Gal β 1,3Ara, Gal β 1,6GlcNAc, Gal β 1,4Glc (lactose) and the known acceptor of other those skilled in the art (referring to as people such as Paulson, J.Biol.Chem.253:5617-5624 (1978)).

In one embodiment, the acceptor of sialyltransferase is promptly to exist thereon after synthesizing in peptide body to be finished.This peptide can not modified the glycosylation pattern of peptide in advance and carried out sialylated with method of the present invention.Selectively, method of the present invention can be used for carrying out sialylated to the peptide that does not comprise suitable acceptor; People at first modify so that it comprises acceptor peptide by those skilled in the art's known method.In an exemplary embodiment, the GalNAc residue is to add by the effect of GalNAc transferring enzyme.

In an exemplary embodiment, the galactosyl acceptor is upward to assemble as GlcNAc by galactose residue being added to the suitable acceptor that is connected with peptide.This method comprises makes peptide and the reaction mixture that will modify carry out incubation, and this reaction mixture contains the galactosyltransferase (as

gal β

1,3 or gal β 1,4) of appropriate amount and suitable galactosyl donor (as the UDP-semi-lactosi).This reaction carried out fully substantially, or stop when selectively making this be reflected at the galactose residue that has added the amount of selecting in advance.The method of the saccharide acceptor that other assemblings are selected is conspicuous for those skilled in the art.

In the another one embodiment, at first completely or partially the oligosaccharides that peptide is connected carries out " pruning " to expose acceptor or a kind of part of sialyltransferase, and wherein one or more suitable residues can add on this part to obtain suitable acceptor.For example glycosyltransferase and endoglycosidase (referring to as U.S. Patent No. 5,716,812) are useful for adhering to and pruning reaction.The going through elsewhere of the glycan that is connected with O-that " pruning " is connected with reconstruct N-provides.

In discussion subsequently, method of the present invention is example by the sugar of using the modification be attached with water-soluble polymers thereon.The special emphasis of discussing is in order to illustrate purpose clearly.The technician will understand the embodiment that sugar that this discussion is equally applicable to wherein modify carries treatment part, biomolecules etc.

An exemplary embodiment of the present invention proposes in Figure 14, and wherein saccharide residue had carried out " pruning " before adding the sugar of modifying, and this embodiment has proposed high mannose is trimmed to the scheme of the two feeler structures of the first-generation.The sugar that carries the modification of water-soluble polymers is then puted together with the one or more saccharide residues that exposed by " pruning ".In an example, water-soluble polymers partly adds by GlcNAc, and this GlcNAc part is puted together with water-soluble polymers.The GlcNAc that modifies is attached on one or two terminal mannose residues of two feeler structures.Selectively, the GlcNAc of unmodified can add on one or two end of branch's kind.

In another exemplary embodiment, water-soluble polymers is that the sugar by the modification with galactose residue adds on one or two terminal mannose residues of two feeler structures, and the sugar of this modification is to put together with the GlcNAc residue that adds on the terminal mannose residue.Selectively, the Gal of unmodified can add on one or two terminal GlcNAc residue.

In in addition further example, water-soluble polymers is to add on the Gal residue with the sialic acid of modifying.

Another exemplary embodiment proposes in Figure 15, and this figure showed and similar scheme shown in Figure 14, wherein with high mannose structures " pruning " to seminose (two feeler structures are from this seminose branch).In an example, water-soluble polymers is by adding with polymer-modified GlcNAc.Selectively, the GlcNAc of unmodified adds on the seminose, adds the Gal with the water-soluble polymers that adheres to subsequently again.In the another one embodiment, the GlcNAc of unmodified and Gal residue are to add to successively on the seminose, add with water-soluble polymers decorated sialic acid part more subsequently.

Figure 16 has proposed the further exemplary of the similar scheme shown in utilization and Figure 14, wherein with high mannose " pruning " to the accompanying GlcNAc of first seminose.This GlcNAc puts together with the Gal residue that carries water-soluble polymers.Selectively, the Gal of unmodified is added on the GlcNAc, add with water-soluble sugar-modified sialic acid subsequently.In in addition further example, terminal GlcNAc and Gal are puted together, and subsequently GlcNAc is carried out fucosylation with the modification Fucose that carries water-soluble polymers.

Figure 17 is and the similar scheme shown in Figure 14 wherein high mannose to be trimmed to first GlcNAc on the Asn that is attached to peptide.In an example, GlcNAc-(Fuc) _aThe GlcNAc of residue puts together with the GlcNAc that carries water-soluble polymers.In another example, GlcNAc-(Fuc) _aThe GlcNAc of residue modifies with the Gal that carries water-soluble polymers.In in addition further embodiment, GlcNAc modifies with Gal, puts together subsequently with water-soluble polymers decorated sialic Gal.

Other exemplary embodiments propose in Figure 18-22.The explanation of the series reaction type that the present invention put into practice is provided in each aforesaid figure.

The example that provides in the above provides illustrating the strength of the method that proposes herein.Utilize method of the present invention, possible is the saccharide residue that " pruning " and structure have almost any structure of wanting.The sugar of modifying can add to as on the sugar moieties end that proposes in the above, and perhaps it can be between peptide core and sugared end.

In an exemplary embodiment, the sialic acid that exists is removed from glycopeptide with sialidase, thus all or most galactosyl residue below exposing.Selectively, peptide or glycopeptide are to carry out mark with galactose residue or with the oligosaccharides residue that galactose unit finishes.After exposing or having added galactose residue, suitable sialyltransferase is used to add the sialic acid of modification.This method summary is in scheme 12.

Scheme 12

In the method that further is summarized in scheme 13, on sialic acid, there is the reactive functional groups of covering.The sialic acid that this reactive group of covering preferably is not used for modifying is attached to the condition influence on the peptide.After the covalent attachment of the sialic acid of modifying and peptide, this is covered and is removed, and this peptide with as PEG, PPG, treatment partly, biomolecules or other reagent puts together.This reagent is to put together by itself and the reaction and the peptide of reactive group of exposure on the saccharide residue of modifying in a particular manner.

Scheme 13

Depend on the terminal sugar of the oligosaccharides side chain of glycopeptide, can use the sugar glycosyltransferase (table 4) suitable of any modification with it.As mentioned above, the terminal sugar of introducing PEGization or the necessary glycopeptide of PPGization structure can natural introducing in its expression process, perhaps it can be after expression with the mixture generation of suitable Glycosylase, glycosyltransferase or Glycosylase and glycosyltransferase.

The sugar that table 4. is modified

In further exemplary, UDP-semi-lactosi-PEG is and milk β 1 that the 4-galactosyltransferase reacts, thereby the semi-lactosi of modifying is transferred on the suitable terminal N-acetyl-glucosamine structure.Terminal GlcNAc residue on the glycopeptide can produce in the expression process, as what in the expression system of Mammals, insect, plant or fungi, occur, but also can be by with required sialidase and/or Glycosylase and/or glycosyltransferase glycopeptide being handled to produce.

In another exemplary embodiment, GlcNAc transferring enzyme such as GnT-I-IV are used for the GlcNAc of PEGization is transferred to the mannose residue of glycopeptide.In exemplary further, amino acid or terminal saccharide residue that the glycan structures enzymatic ground that N-and/or O-are connected gets on and can put together with the sugar of modification divided by exposing subsequently from glycopeptide.For example, the inscribe glycanase is used to remove structure that the N-of glycopeptide connects with exposed distal ends GlcNAc, makes it be shown as the Asn that GlcNAc connects on the glycopeptide.UDP-Gal-PEG and suitable galactosyltransferase can be used for PEG-or PPG-semi-lactosi functional group are incorporated on the GlcNAc of exposure.

In a selectable embodiment, the sugar of modification directly adds on the peptide main chain with glycosyltransferase, and wherein this enzyme is known can transfer to saccharide residue on the peptide main chain.This exemplary embodiment proposes in scheme 14.Be used to put into practice exemplary glycosyltransferase of the present invention including, but not limited to GalNAc transferring enzyme (GalNAc T1-14), GlcNAc transferring enzyme, fucosyltransferase, Transglucosylase, xylosyltransferase, mannose transferase etc.Using present method makes it possible to directly add to the sugar of modifying on the peptide that lacks any sugar or add on the existing glycopeptide.In both cases, the interpolation of the sugar of modification betides by the specific position on the peptide main chain that substrate specificity limited of glycosyltransferase, rather than the random fashion as taking place in the peptide main chain process with the chemical process modifying protein.By suitable aminoacid sequence is manufactured in the peptide chain, a series of reagent can be incorporated in the protein or glycopeptide of the peptide substrate sequence that lacks glycosyltransferase originally.

Scheme 14

In each exemplary embodiment proposed above, after puting together, the sugar of modifying and peptide can use one or more extra chemistry or enzymatically modifying step.In an exemplary embodiment, enzyme (as fucosyltransferase) can be used for additional glycosyl unit's (as Fucose) on the end modified sugar that is attached on the peptide.In another example, enzymatic reaction can be used for " adding cap " carried out in the site that the sugar of modifying can not be puted together.Selectively, chemical reaction is used to change the structure of the sugar of the modification of puting together.For example, the sugar of the modification of puting together and reagent are reacted, this reagent can make its with the sugared accompanying peptide component of modifying be connected stabilization or stabilization removal.In another example, after puting together with peptide, the composition of the sugar of modification is de-protected.The technician has a series of enzymatic and chemical program to be used for method of the present invention in the stage after the sugar of modifying and peptide are puted together understanding.Further modification to sugar-peptide conjugate of modifying is within the scope of the present invention.

Peptide with the Man-6-P guiding

In an exemplary embodiment, peptide is with at least a Man-6-P part derivatize.This Man-6-P part can be directed to peptide in the lysosome of cell, and can be used for as therapeutic protein being directed in the lysosome with the treatment lysosomal storage disease.

Lysosomal storage disease is the colony that surpasses 40 kinds of illnesss, and they are results of the genetic flaw of the coding enzyme that decomposes glycolipid in the Cytolysosome or polysaccharide refuse.The product of this enzyme can recirculate to new product as sugar and lipid.Each of these illnesss all is derived from the euchromosome or the chain recessive character of X-of heredity, the level of enzyme in this affect trait lysosome.Affected enzyme does not have biology or functionally active in the cell of the individuality of usually, catching an illness and the tissue.Table 5 provide representative thesaurismosis and with the tabulation of the enzyme defect of disease-related.In this disease, the defective of enzyme function causes the deposition of general step by step of lipid in the soma lysosome or carbohydrate substrate, finally causes the forfeiture and the death of organ dysfunction.The inherited pathogenic factor of lysosomal storage disease, clinical manifestation, molecular biology and sickness rate are described in detail in people such as Scriver, eds., The Metabolicand Molecular Basis of Inherited Disease, 7.sup.thEd., Vol.II, McGraw Hill, (1995).

Table 5. lysosomal storage disease and involved enzyme defective

* MPS=mucopolysaccharide

De Duve at first proposes to substitute the feasible method (De Duve, Fed.Proc.23:1045 (1964)) that the lysosomal enzyme that lacks may be the treatment lysosomal storage disease with the enzyme of external source biologic activity.From then on, various researchs proposed enzyme replacement therapy for the treatment various lysosomal storage diseases all be useful.Shown maximum success in the individuality with I type gaucher's disease, this individuality has been used exogenous enzyme (β-glucocerebrosidase) (Ceredase for preparing from placenta ^TM) or the nearest enzyme (Cerezyme of recombinant production ^TM) treat.Also having proposed enzyme alternative all is useful for treatment fabry disease and other lysosomal storage disease.Referring to as people such as Dawson, people such as Ped.Res.7 (8): 684-690 (1973) (external) and Mapes, Science169:987 (1970) (in vivo).The clinical experiment of enzyme replacement therapy is with normal plasma (people such as Mapes, Science169:987-989 (1970)), the alpha-galactosidase A of purifying (people such as Brady from placenta, N.Eng.J.Med.279:1163 (1973)) alpha-galactosidase A (people such as Desnick of purifying or from spleen or blood plasma, Proc.Natl.Acad.Sci., USA 76:5326-5330 (1979)) reports among the fabry disease patient who inculcates, and proved that fabry disease is directly carried out enzyme alternate biomedicine to be renderd a service.These studies show that by the multiple enzyme and substitute elimination or significantly reduce the potentiality that pathological glycolipid is stored up.For example, in a research (people such as Desnick sees above), the intravenous injection of the enzyme of purifying causes the temporary transient reduction of blood plasma level of the lipid substrates globotriasylceramide that stores up.

Therefore, need in this area to provide the lysosomal enzyme of biologic activity of q.s such as the method for human alpha-galactosidase A to deficient cells.Recently, attempted satisfying these needs with recombination method, referring to as u.S.Pat.No.5,658,567,5,580,757; People such as Bishop, Proc.Natl.Acad.Sci., USA, 83:4859-4863 (1986); People such as Medin, Proc.Natl.Acad.Sci., USA93:7917-7922 (1996); Novo, F.J., Gene Therapy 4:488-492 (1997); People such as Ohshima, Proc.Natl.Acad.Sci., USA 94:2540-2544 (1997); With people such as Sugimoto, Human Gene Therapy6:905-915 (1995).The guiding of therapeutic peptide in lysosome by the Man-6-P mediation the invention provides the biologic activity lysosome peptide that is used for q.s and send composition and the method that is delivered to deficient cells.

Thereby, in an exemplary embodiment, the invention provides with the peptide (Figure 24 and Figure 25) in the table 7 of Man-6-P derivatize.This peptide can be reorganization or chemical preparation.In addition, this peptide can be complete native sequences, perhaps can by as block, extend and modify, perhaps it can comprise and substituting or disappearance.Exemplary protein with method reconstruct of the present invention comprises glucocerebrosidase, beta-glucosidase enzyme, alpha-galactosidase A, acidity-alpha-glucosidase (acid maltase).Be used for peptide that the representativeness of clinical application modifies including, but not limited to Ceredase ^TM, Cerezyme ^TMAnd Fabryzyme ^TMThe also available method of the present invention of glycosyl group on that modify and suitable clinically peptide changes.Man-6-P is attached on the peptide by the glycosyl linking group.In an exemplary embodiment, the glycosyl linking group is derived from sialic acid.The exemplary sialic glycosyl linking group that is derived from proposes in table 3, and wherein one or more " R " parts are Man-6-P or the spacer groups that is attached with one or more Man-6-P parts thereon.The sialic acid part of this modification preferably is connected to the terminal residue (Figure 26) of the lip-deep oligosaccharides of peptide.

Except that Man-6-P, peptide of the present invention can further be used such as the part of water-soluble polymers, treatment part or extra targeting part and carry out derivatize.The method that is used to adhere to the group of these and other proposes herein.In an exemplary embodiment, the group except that Man-6-P is to be attached on the peptide by the sialic acid derivative according to the derivatize of table 3, and wherein one or more " R " parts are the groups except that Man-6-P.

In an exemplary embodiment, prepared the sialic acid part of modifying with the linker arm based on glycine of Cbz-protection.Prepared corresponding nucleotide sugar, and the Cbz group has been removed by shortening.The nucleotide sugar of gained has the available reactive amine that contacts with activatory Man-6-P derivative as a result, thereby the nucleotide sugar that can be used for putting into practice Man-6-P derivatize of the present invention is provided.

As shown in scheme (scheme 15) below; the generation type of the Man-6-P derivative of exemplary activated is as follows: change the phosphotriester of 2-bromo-benzyl-protection into corresponding trifluoromethane sulfonic acid ester in position; and make this trifluoromethane sulfonic acid ester and have the linker reaction that reactivity contains the oxygen part, thereby between sugar and linker, form ehter bond.The benzyl protection group is removed by shortening, and with the methyl esters hydrolysis of linker, thereby corresponding carboxylic acid is provided.This carboxylic acid can be by any method activation well known in the art.An exemplary activation procedure depends on the transformation of carboxylic acid to the N-hydroxy-succinamide ester.

Scheme 15

In another exemplary embodiment, as shown in scheme (scheme 16) below, the acetylizad sialic acid of N-is to change amine into by the processing to the pyruvyl part.Thereby, primary hydroxyl is changed into sulphonate and makes it and reaction of sodium azide.With the trinitride catalytic reduction is corresponding amine.Subsequently sugar is changed into its nucleotide analog, and make it by amine groups and the linker arm deutero-Man-6-P coupling of preparation as mentioned above.

Scheme 16

The peptide that is used for the treatment of lysosomal storage disease can carry out derivatize with other targeting parts, this targeting part including, but not limited to transferrin (with peptide stride across blood brain barrier send pass and deliver to endosome), carnitine (send pass peptide) and phosphoric acid salt into muscle cell, as diphosphate (peptide being directed in the tissue of bone and other calcifications).Targeting part and therapeutic peptide are to put together by any method or other method of discussing herein as known in the art.

In an exemplary embodiment, directed agents and therapeutic peptide are by linker part link coupled.In this embodiment, at least one therapeutic peptide or directed agents are that the method according to this invention is passed through intact glycosyl linking group and linker part link coupled.In an exemplary embodiment, linker comprises that partly poly-(ether) is as poly-(ethylene glycol).In another exemplary embodiment, linker partly comprises the key of at least one degradation in vivo, thereby the back discharges therapeutic peptide from directed agents in conjugate being sent the destination organization that is delivered to health or zone.

In the exemplary embodiment of another one, distribution is to change by the sugar form that changes on the treatment part in the body of treatment part, and need not therapeutic peptide and targeting part are puted together.For example, can make therapeutic peptide avoid the picked-up of reticuloendothelial system (Figure 24 and 27) by the terminal galactose of glycosyl group partly being added cap with sialic acid (or derivatives thereof).Cover the sialylated of terminal Gal and avoided of the picked-up of liver asialoglycoprotein (ASGP) acceptor, and extensible this peptide only has the transformation period of the peptide of complicated polysaccharide chains with respect to asialoization peptide.

II. peptide/glycopeptide of the present invention

In one embodiment, the invention provides the composition of the single peptide that comprises multiple copied, this peptide with three basic seminose cores as the main glycan structures that adheres to thereon.In preferred embodiments, this peptide can be therapeutic molecules.The natural form of this peptide can comprise the glycan of complicated N-connection or can be high mannose glycans.This peptide can be mammiferous peptide, and is preferably human peptide.In some embodiments, this peptide is selected from (seeing Figure 28) such as immunoglobulin (Ig), erythropoietin, tissue-type activation factor peptides.

Its glycan can propose in Figure 28 with the exemplary peptides that method of the present invention is reconstructed.

Table 6. is used to carry out the preferred peptide of glycan reconstruct

Table 7. is used to carry out the most preferred peptide of glycan reconstruct

The more detailed tabulation that is used for peptide of the present invention and source thereof proposes at Figure 28.

The member's (as antibody, MHC molecule, TXi Baoshouti etc.), iuntercellular acceptor (as integral protein, hormone or growth factor receptors etc.), lectin and the cytokine (as interleukin-) that comprise immunoglobulin (Ig) family with other exemplary peptides of method modification of the present invention.Extra example comprises tissue type plasminogen activation factor (TPA), feritin, thrombin such as blood coagulation factor VIII and plasma thromboplastin component, bombesin, zymoplasm, hemopoieticgrowth factor, G CFS, virus antigen, the complement peptide, alpha1-antitrypsin, erythropoietin, P-selects albumen glycopeptide part-1 (PSGL-1), rHuGM-CSF, Antithrombin III, interleukin-, Interferon, rabbit, peptide A and C, fibrinogen, herceptin ^TM, leptin, Glycosylase etc.This peptide tabulation is exemplary, and and should not be regarded as exclusive.On the contrary, as conspicuous from the disclosure that provides herein, the inventive method can be applicable in any peptide, can form any glycan structures of wanting in this peptide.

Method of the present invention also is used for the peptide of modified chimeric, and this chimeric peptide is including, but not limited to comprising the chimeric peptide of the part that is derived from immunoglobulin (Ig) such as IgG.

The peptide of modifying with method of the present invention can be the peptide of synthetic or wild-type, and perhaps they can be the mutant peptide with method generation well known in the art, as site-directed mutagenesis.The glycosylation of peptide generally is that N-connects or O-connects.It is sugar the adhering to the asparagine residue side chain of modifying that an exemplary N-connects.Tripeptides structure l-asparagine-X-Serine and l-asparagine-X-Threonine is the sugar moieties recognition sequence that enzymatic adheres on the l-asparagine side chain, and wherein X is any amino acid except that proline(Pro).Thereby the existence of any one these tripeptide sequence has all produced the potential glycosylation site in the peptide.As described elsewhere, the glycosylation that O-connects refers to a sugar (as N-acetylgalactosamine, semi-lactosi, seminose, GlcNAc, glucose, Fucose or wood sugar) adhering to the hydroxyl side chain of hydroxy-amino-acid, be preferably Serine or Threonine, although also can use 5-oxyproline or 5-oxylysine.

The embodiment of several exemplary of the present invention is discussed below.Although the several application of these embodiments has the peptide of trade(brand)name and other specific peptides as exemplary peptide, these examples are not limited to any specific peptide.The following illustrative embodiment comprises the variant of all peptide Equivalents and any peptide.This variant is including, but not limited to adding glycosylation site that is connected with O-that is connected with deletion N-and the fused protein with glycosylation site of interpolation.It should be appreciated by those skilled in the art that following embodiment and disclosed herein basic skills can equally successfully be applied to many peptides.

In an exemplary embodiment, the invention provides the method that is used for modified granulocyte colony stimulating factor (G-CSF).Figure 29 A～29G has proposed some and how to be used in the example that method disclosed herein realizes this modification.In Figure 29 B, the G-CSF peptide that will express in mammalian cell is pruned with sialidase.Like this residue of Bao Luing can by add with its suitable donor and ST3Gal1 sialic acid-poly-(ethylene glycol) partly (peg moiety) modify.Figure 29 C has proposed to be used to be modified at the exemplary arrangement of the G-CSF peptide of expressing on the insect cell.This peptide is to modify by adding galactose moiety with its suitable donor and galactosyltransferase.Galactose residue is to carry out functionalized by the effect of ST3Gal1 and sialic acid-PEG derivative with PEG.In Figure 29 D, the G-CSF of bacterial expression is contacted with the N-acetylgalactosamine transferring enzyme with the N-acetylgalactosamine donor.This peptide is functionalized with PEG by the sialic acid donor of PEGization and sialyltransferase.In Figure 29 E, the G-CSF of mammalian cell expression is contacted with sialic acid donor, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.On adding peptide on the glycosyl residue of glycan after, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 29 F, the G-CSF of bacterial expression is reconstruct by peptide and inscribe-GalNAc enzyme contacts under the condition of its performance complex functionality rather than hydrolysis function, thus from its activatory derivative interpolation PEG-Gal-GalNAc molecule.Figure 29 G provides another approach of the G-CSF of reconstruct bacterial expression.Polypeptide is with the N-acetylgalactosamine residue derivatize of PEGization by this polypeptide is contacted with the N-acetylgalactosamine donor of N-acetylgalactosamine transferring enzyme and suitable substance P EGization.

In another exemplary embodiment, the invention provides the method that is used for modified interferon α-14C (IFN α 14C), shown in Figure 30 A～30N.The various forms of IFN α is open elsewhere.In Figure 30 B, the IFN α 14C that will express in mammalian cell at first handles to prune the sialic acid unit on it with sialidase, with the sialic acid donor of ST3Gal3 and PEGization this molecule carry out PEGization then.In Figure 30 C, on the IFN α 14C that N-acetyl-glucosamine is at first added to express in insect or fungal cell, this reaction is to be undertaken by the effect of GnT-I that utilizes the N-acetyl-glucosamine donor and/or II.With galactosyltransferase and PEG-galactose donor this polypeptide carry out PEGization then.In Figure 30 D, the IFN α 14C that expresses in yeast is at first handled to prune the glycosyl unit on it with Endo-H.With galactosyltransferase and galactose donor this molecule is carried out galactosylation, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 30 F, the IFN α 14C that is produced by mammalian cell is modified so that it has (inched) peg moiety with ST3Gal3 and PEG-sialic acid donor.In Figure 30 G, at first N-acetyl-glucosamine is added among the IFN α 14C that expresses in insect or fungal cell with one or more GnT-I, II, IV and V and N-acetyl-glucosamine donor.With suitable donor and galactosyltransferase this protein is carried out galactosylation subsequently.With ST3Gal3 and PEG-sialic acid donor IFN α 14C carry out PEGization then.In Figure 30 H, at first handle the IFN α 14C of yeast generation to prune the mannose group group with mannosidase.Add N-acetyl-glucosamine with N-acetyl-glucosamine donor and one or more GnT-I, II, IV and V then.Further IFN α 14C is carried out galactosylation with suitable donor and galactosyltransferase.Then, with ST3Gal3 and PEG-sialic acid donor this polypeptide carry out PEGization.In Figure 30 I, by with sialic acid donor suitable terminal residue being added the IFN α 14C that cap is modified the NSO cell expressing, wherein this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 30 J, with PEG-sialic acid donor and α 2, the 8-sialyltransferase is to undertaken PEGization by the IFN α 14C of mammalian cell expression.In Figure 30 K, at first handle the IFN α 14C that produces by mammalian cell to prune terminal sialic acid residues with sialidase, with the sialic acid-lactose mixture of trans sialidase and PEGization this molecule carry out PEGization then.In Figure 30 L, with sialic acid donor and α 2, the 8-sialyltransferase carries out sialylated to the IFN α 14C that is expressed by mammlian system.In Figure 30 M, at first N-acetyl-glucosamine is added among the IFN α 14C that expresses in insect or fungal cell with suitable donor and GnT-I and/or II.This molecule is contacted with galactose donor with galactosyltransferase, this galactose donor by linker with reactive sialic acid derivatize, thereby this polypeptide can be attached on the reactive sialic acid by linker and galactose residue.This polypeptide is contacted with transferrin with ST3Gal1, thereby it is connected with transferrin by sialic acid residues.In Figure 30 N, at first handle the IFN α 14C that expresses among insect or the fungal cell to prune glycosyl group with the inscribe glycanase, it is contacted with galactose donor with galactosyltransferase, this galactose donor by linker with reactive sialic acid derivatize, thereby this polypeptide can be attached on the reactive sialic acid by linker and galactose residue.This molecule is contacted with transferrin with ST3Gal3, thereby it is connected with transferrin by sialic acid residues.

In another exemplary embodiment, the invention provides the method that is used for modified interferon α-2a or 2b (IFN α), shown in Figure 30 O～30EE.In Figure 30 P, the IFN α that will express in mammalian cell at first handles to prune glycosyl unit with sialidase, with the sialic acid donor of ST3Gal3 and PEGization it carry out PEGization then.In Figure 30 Q, at first with suitable donor and galactosyltransferase to carrying out galactosylation at the IFN of expressed in insect cells α, with the sialic acid donor of ST3Gal1 and PEGization it carry out PEGization then.Figure 30 R provides the another kind of method of reconstruct IFN α in bacterium: add in the protein with suitable donor and the N-acetylgalactosamine transferring enzyme N-acetylgalactosamine with PEGization.In Figure 30 S, be modified at the IFN α that expresses in the mammalian cell by suitable terminal residue being added cap with sialic acid donor, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 30 T, the IFN α of bacterial expression carry out PEGization in order to the enzyme inscribe-N-acetylgalactosaminase of the modification of synthetic rather than hydrolysis method performance function with the N-acetylgalactosamine donor of peg moiety derivatize.In Figure 30 U, at first N-acetylgalactosamine is added among the IFN with suitable donor and N-acetylgalactosamine transferring enzyme, with the sialic acid donor of sialyltransferase and PEGization it carry out PEGization then.In Figure 30 V, at first handle the IFN α in mammlian system, express to prune sialic acid residues with sialidase, with suitable donor and ST3Gal1 and/or ST3Gal3 it carry out PEGization then.In Figure 30 W, at first handle the IFN α in mammlian system, express to prune sialic acid residues with sialidase.Make this polypeptide and ST3Gal1 and two reactive sialic acid residues contacts that are connected by linker then, thereby this polypeptide is attached on the reactive sialic acid by linker and second sialic acid residues.This polypeptide is contacted with transferrin with ST3Gal3, thereby it is connected with transferrin by sialic acid residues.In Figure 30 Y, at first handle the IFN α in mammlian system, express to prune sialic acid residues with sialidase, with ST3Gal1 and PEG-sialic acid donor it carry out PEGization then.In Figure 30 Z, the IFN α that is produced by insect cell carry out PEGization with the galactose donor of galactosyltransferase and PEGization.In Figure 30 AA, at first N-acetylgalactosamine is added among the IFN α of bacterial expression with suitable donor and N-acetylgalactosamine transferring enzyme.With sialyltransferase and PEG-sialic acid donor this protein carry out PEGization then.In Figure 30 CC, the IFN α that expresses in the bacterium modifies with another program: promptly the N-acetylgalactosamine donor with PEGization adds in the protein by the N-acetylgalactosamine of N-acetylgalactosamine transferring enzyme with PEGization.In Figure 30 DD, the IFN α that expresses in bacterium is with another kind of scheme reconstruct.At first make this polypeptide and N-acetylgalactosamine transferring enzyme and contact, thereby IFN α is attached on the reactive sialic acid by linker and N-acetylgalactosamine with the N-acetylgalactosamine donor of reactive sialic acid by the linker derivatize.Make IFN α and ST3Gal3 then and take off the sialic acid transferrin and contact, thereby it is connected with transferrin by sialic acid residues.With ST3Gal3 and sialic acid donor IFN α is carried out sialic acid residues then and add cap.The additional method of the IFN α of modification bacterial expression is open in Figure 30 EE, and IFN α is exposed among NHS-CO-linker-SA-CMP, and it is connected with reactive sialic acid by linker.With ST3Gal3 and transferrin itself and transferrin are puted together subsequently.

The method that IFN ω is reconstructed is basic identical with the method that IFN is provided herein, is the amino-acid residue 101 that betides SEQ ID NO:75 that adheres to of glycan and IFN peptide.The Nucleotide of IFN ω and aminoacid sequence are provided as SEQ ID NO:74 and 75 herein.The method of preparation and application IFN ω is seen U.S. Patent No. 4,917,887 and 5,317,089, and European patent No.0170204-A.

In another exemplary embodiment, the invention provides the method that is used for modified interferon β (IFN β), shown in Figure 31 A～31S.In Figure 31 B, at first handle the IFN β in mammlian system, express to prune terminal sialic acid residues with sialidase.With the sialic acid donor of ST3Gal3 and PEGization this protein carry out PEGization then.Figure 31 C is the scheme of modifying the IFN β that is produced by insect cell.At first with suitable donor and GnT-I and/or-II adds N-acetyl-glucosamine among the IFN β to.With galactose donor and galactosyltransferase this protein is carried out galactosylation then.At last, with ST3Gal3 and PEG-sialic acid donor IFN β carry out PEGization.In Figure 31 D, at first handle the IFN β in yeast, express to prune its glycosyl chain with Endo-H, with galactose donor and galactosyltransferase it is carried out galactosylation then, with the sialic acid donor of ST3Gal3 and PEGization it carry out PEGization then.In Figure 31 E, the IFN β that is produced by mammalian cell is with ST3Gal3 and has carried out the PEGization modification with the sialic acid donor of peg moiety derivatize.In Figure 31 F, at first among the IFN β that N-acetyl-glucosamine is added in expressed in insect cells by one or more GnT-I, II, IV and V with the N-acetyl-glucosamine donor, with galactose donor and galactosyltransferase it is carried out galactosylation then, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 31 G, at first handle the IFN β in yeast, express to prune mannose units with mannosidase, add N-acetyl-glucosamine with the N-acetyl-glucosamine donor by one or more GnT-I, II, IV and V then.This protein is further carried out galactosylation with galactose donor and galactosyltransferase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 31 H, by with sialic acid donor suitable terminal residue being added the IFN β that cap is modified mammalian cell expression, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 31 I, with PEG-sialic acid donor and α 2, the 8-sialyltransferase carry out PEGization to the IFN β that expresses in the mammlian system.In Figure 31 J, at first handle IFN β by mammalian cell expression to prune terminal sialic acid residues with sialidase, with the sialic acid donor of trans sialidase and PEGization it carry out PEGization then.In Figure 31 K, at first handle the IFN β that expresses in the mammalian cell to prune terminal sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and carry out sialylated with ST3Gal3 and sialic acid donor to it then.In Figure 31 L, at first handle the IFN β that expresses in the mammalian cell to prune the glycosyl chain with SG, with galactose donor and alpha-galactosyltransferasactivity it is carried out galactosylation then, and with ST3Gal3 or sialyltransferase and PEG-sialic acid donor it carry out PEGization then.In Figure 31 M, at first handle the IFN β that expresses in the mammalian cell to prune glycosyl unit with sialidase.With ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and carry out sialylated with ST3Gal3 and sialic acid donor to it then.In Figure 31 N, by with sialic acid donor suitable terminal residue being added the IFN β that cap is modified mammalian cell expression, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 31 O, with sialic acid donor and α 2, the 8-sialyltransferase carries out sialylated to the IFN β that expresses in the mammalian cell.In Figure 31 Q, at first among the IFN β that N-acetyl-glucosamine is added in expressed in insect cells by one or more GnT-I, II, IV and V with the N-acetyl-glucosamine donor, and further it carry out PEGization with PEG-galactose donor and galactosyltransferase.In Figure 31 R, at first handle the IFN β that expresses in the yeast to prune glycosyl group with the inscribe glycanase, with galactose donor and galactosyltransferase it is carried out galactosylation then, and with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 31 S, at first make the IFN β and ST3Gal3 and two reactive sialic acid contacts that are connected by linker that in mammlian system, express, thereby this polypeptide is attached on the reactive sialic acid by linker and second sialic acid residues.This polypeptide and ST3Gal3 are contacted with asialylated transferrin, thereby it is connected with transferrin by sialic acid residues.Then, further carry out sialylated to IFN β with sialic acid donor and ST3Gal3.

In another exemplary embodiment, the invention provides the method that is used to modify proconvertin or VIIa, shown in Figure 32 A～32D.In Figure 32 B, at first handle the proconvertin in mammlian system, produce or VIIa to prune terminal sialic acid residues with sialidase, with the sialic acid donor of ST3Gal3 and PEGization it carry out PEGization then.In Figure 32 C, at first handle the proconvertin in mammalian cell, express or VIIa to prune terminal sialic acid residues with sialidase, with the sialic acid donor of ST3Gal3 and PEGization it carry out PEGization then.Further, carry out sialylated with ST3Gal3 and sialic acid donor to this polypeptide.Figure 32 D provides proconvertin that is produced by mammalian cell or the another kind of modification protocols of VIIa: promptly at first handle this polypeptide to prune its sialic acid and galactose residue with SG, with galactosyltransferase and galactose donor it is carried out galactosylation then, and with the sialic acid donor of ST3Gal3 and PEGization it carry out PEGization then.

In another exemplary embodiment, the invention provides the method that is used to modify plasma thromboplastin component, its some examples are included among Figure 33 A～33G.In Figure 33 B, at first handle the plasma thromboplastin component in mammalian cell, produce to prune terminal sialic acid residues with sialidase, be that donor carry out PEGization with ST3Gal3 to it with the PEG-sialic acid then.In Figure 33 C, at first handle the plasma thromboplastin component in mammalian cell, express to prune terminal sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and further carry out sialylated to this polypeptide with ST3Gal1 and sialic acid donor.In Figure 33 D, can find another kind of scheme that the plasma thromboplastin component that mammalian cell produces is reconstructed.At first handle this polypeptide to prune terminal sialic acid residues with sialidase, with galactose donor and galactosyltransferase it is carried out galactosylation then, further it is carried out sialylatedly, and with the sialic acid donor and the ST3Gal1 of PEGization it carry out PEGization then with sialic acid donor and ST3Gal3.In Figure 33 E, the plasma thromboplastin component of in mammlian system, expressing by with the PEG-sialic acid donor with the catalytic sialylated process of ST3Gal3 PEGization.In Figure 33 F, by with sialic acid donor suitable terminal residue being added the plasma thromboplastin component that cap is modified mammalian cell expression, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.Figure 33 G provides the extra method of modifying plasma thromboplastin component.This polypeptide that is produced by mammalian cell is with PEG-sialic acid donor and α 2, and the 8-sialyltransferase carries out PEGization.

In another exemplary embodiment, the invention provides the method that is used to modify follicle stimulating hormone (FSH).Figure 34 A～34J provides some examples.In Figure 34 B, FSH be in mammlian system, express and handle by sialidase and to modify to prune terminal sialic acid residues, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization subsequently.In Figure 34 C, at first handle the FSH in mammalian cell, express to prune terminal sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and carry out sialylated with ST3Gal3 and sialic acid donor to it then.Figure 34 D provides the scheme of modifying the FSH that expresses in the mammlian system.Handle this polypeptide to prune its sialic acid and galactose residue with SG, with galactose donor and galactosyltransferase it is carried out galactosylation then, and with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 34 E, the FSH that expresses in mammalian cell modifies with following procedure: promptly at first handle FSH to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and carry out sialylated with ST3Gal3 and sialic acid donor to it then.Figure 34 F provides another example of modifying the FSH that is produced by mammalian cell: promptly modify this polypeptide by with sialic acid donor suitable terminal residue being added cap, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 34 G, the FSH that is expressed in the mammlian system modifies with another kind of program: promptly by using sialic acid donor and α 2, the 8-sialyltransferase adds sialic acid and this polypeptide is reconstructed.In Figure 34 H, FSH be expressed in the insect cell and modify with following procedure: promptly by one or more GnT-I, II, IV and V N-acetyl-glucosamine is added among the FSH with suitable N-acetyl-glucosamine donor; With PEG-galactose donor and galactosyltransferase FSH carry out PEGization then.Figure 34 I has described the scheme of modifying the FSH that is produced by yeast.According to this scheme, at first handle FSH to prune glycosyl group with the inscribe glycanase, with galactose donor and galactosyltransferase it is carried out galactosylation then, and with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 34 J, at first make the FSH and ST3Gal3 and two reactive sialic acid residues contacts that are connected by linker that in mammalian cell, express, thereby this polypeptide is attached on the reactive sialic acid by linker and second sialic acid residues.This polypeptide is contacted with the asialylated chorionic-gonadotropin hormone (CG) that produces in CHO with ST3Gal1, thereby it is connected with CG by sialic acid residues.Then, carry out sialylated with sialic acid donor and ST3Gal3 and/or ST3Gal1 to FSH.

In another exemplary embodiment, the invention provides the method that is used for modified erythropoietin (EPO).Figure 35 A～35AA has proposed some examples relevant with the EPO peptide of reconstruct wild-type or sudden change.In Figure 35 B, EPO be in various mammlian systems, express and be reconstructed by expressed protein is contacted with sialidase to remove terminal sialic acid residues.The peptide that makes gained as a result and sialyltransferase contact with cmp sialic acid with the peg moiety derivatize.In Figure 35 C, the EPO of expressed in insect cells with GnT-I and/or-II is reconstructed with N-acetyl-glucosamine.With galactosyltransferase semi-lactosi is added on this peptide then.The PEG group is by peptide and sialyltransferase are contacted on the peptide that adds reconstruct to cmp sialic acid with the peg moiety derivatize.In Figure 35 D, the EPO that expresses in the mammal cell line system removes terminal sialic acid residues by the effect of sialyltransferase to be reconstructed.The terminal galactose residues of the glycosyl group that N-connects " adds cap " with ST3Gal3 and sialic acid donor with sialic acid.Carry out functionalized with the sialic acid that carries peg moiety to the terminal galactose residues that O-is connected glycan with ST3Gal1 with suitable sialic acid donor.In Figure 35 E, be expressed in EPO in the mammal cell line system and be reconstruct by the functionalized of the glycosyl residue that N-connected with the sialic acid part of PEG-derivatize.This peptide is contacted with the sialic acid donor of suitably modifying with ST3Gal3.In Figure 35 F, be expressed in the EPO reconstruct by adding at least one N-acetyl-glucosamine residue in insect cell system, yeast or the fungi, these N-acetyl-glucosamine residues add by peptide is contacted with GnT-V with one or more GnT-I, GnT-II with the N-acetyl-glucosamine donor.Then by this peptide being contacted with galactosyltransferase with the galactose donor of PEGization and it carry out PEGization.In Figure 35 G, be expressed in EPO in insect cell system, yeast or the fungi and be by adding at least a N-acetyl-glucosamine residue and be reconstructed with suitable N-acetyl-glucosamine donor and one or more GnT-I, GnT-II and GnT-V.The tilactase of changing into synthetic rather than hydrolysis method performance function is used for adding activatory PEGization galactose donor to the N-acetyl-glucosamine residue.In Figure 35 H, the EPO that is expressed in insect cell system, yeast or the fungi is reconstructed by adding at least one terminal N-acetyl-glucosamine-PEG residue.Peptide and GnT-I are contacted with suitable N-acetyl-glucosamine donor with the peg moiety derivatize.In Figure 35 I, the EPO that is expressed in insect cell system, yeast or the fungi is reconstructed by adding one or more terminal galactose-PEG residue.Peptide and GnT-I are contacted with suitable N-acetyl-glucosamine donor with the peg moiety derivatize.Peptide is contacted with the suitable galactose donor of modifying with peg moiety with galactosyltransferase.In Figure 35 J, the EPO that is expressed in insect cell system, yeast or the fungi is reconstructed by adding a terminal sialic acid-PEG residue again.Peptide is contacted with GnT-I with suitable N-acetyl-glucosamine donor.Peptide is contacted with suitable galactose donor with galactosyltransferase.Peptide and ST3Gal3 are contacted with suitable sialic acid donor with the peg moiety derivatize.In Figure 35 K, the EPO that is expressed in insect cell system, yeast or the fungi is reconstructed by adding terminal sialic acid-PEG residue.Peptide is contacted with GnT-V with one or more GnT-I, GnT-II with suitable N-acetyl-glucosamine donor.Peptide is contacted with suitable galactose donor with galactosyltransferase.Peptide and ST3Gal3 are contacted with suitable sialic acid donor with the peg moiety derivatize.In Figure 35 L, the EPO that is expressed in insect cell system, yeast or the fungi is passed through to add one or more terminal α 2,6-sialic acid-PEG residue is reconstructed.Peptide is contacted with GnT-V with one or more GnT-I, GnT-II with suitable N-acetyl-glucosamine donor.Peptide is contacted with suitable galactose donor with galactosyltransferase.Make peptide and α 2 then, the 6-sialyltransferase contacts with the sialic acid donor of suitably modifying.In Figure 35 M, the EPO that is expressed in the mammal cell line system is reconstructed by adding one or more terminal sialic acids-PEG residue.Peptide is contacted with sialidase to remove terminal sialic acid residues.Peptide and sialyltransferase are contacted with suitable sialic acid donor.Peptide and sialyltransferase are contacted with suitable sialic acid donor with the peg moiety derivatize.In Figure 35 N, the EPO that is expressed in the mammal cell line system is reconstructed by adding one or more terminal sialic acids-PEG residue.Peptide and sialyltransferase are contacted with suitable sialic acid donor with the peg moiety derivatize.In Figure 35 O, the EPO that is expressed in the mammal cell line system is added one or more terminal α 2 by the glycan that connects to main O-, 8-sialic acid-PEG residue is reconstructed.Make peptide and α 2, the 8-sialyltransferase contacts with suitable sialic acid donor with the peg moiety derivatize.In Figure 35 P, the EPO that is expressed in the mammal cell line system is added one or more terminal α 2 by the glycan that is connected with N-that connects to O-, 8-sialic acid-PEG residue is reconstructed.Make peptide and α 2, the 8-sialyltransferase contacts with suitable sialic acid donor with the peg moiety derivatize.In Figure 35 Q, the EPO that is expressed in yeast or the fungi is reconstructed by adding one or more terminal sialic acids-PEG residue.Peptide is contacted with mannosidase to remove terminal mannose residue.Next step makes peptide and GnT-I contact with suitable N-acetyl-glucosamine donor.Peptide and galactosyltransferase are contacted with suitable galactose donor.Peptide and sialyltransferase are contacted with suitable sialic acid donor with the peg moiety derivatize.In Figure 35 R, the EPO that is expressed in yeast or the fungi is reconstructed by adding at least one terminal N-acetyl-glucosamine-PEG residue.Peptide is contacted with mannosidase to remove terminal mannose residue.Peptide and GnT-I are contacted with suitable N-acetyl-glucosamine donor with the peg moiety derivatize.In Figure 35 S, the EPO that is expressed in yeast or the fungi is reconstructed by adding one or more terminal sialic acids-PEG residue.Peptide is contacted with mannosidase-I to remove α 2 mannose residues.Peptide and GnT-I are contacted with suitable N-acetyl-glucosamine donor.Peptide and galactosyltransferase are contacted with suitable galactose donor.Peptide and sialyltransferase are contacted with suitable sialic acid donor with the peg moiety derivatize.In Figure 35 U, the EPO that is expressed in yeast or the fungi is reconstructed by adding one or more semi-lactosis-PEG residue.Peptide is contacted to be trimmed to glycosyl group with endo-H.Peptide and galactosyltransferase are contacted with suitable galactose donor with the peg moiety derivatize.In Figure 35 V, the EPO that is expressed in yeast or the fungi is reconstructed by adding one or more terminal sialic acids-PEG residue.Peptide is contacted to be trimmed to glycosyl group with endo-H.Peptide and galactosyltransferase are contacted with suitable galactose donor.Peptide and sialyltransferase are contacted with suitable sialic acid donor with the peg moiety derivatize.In Figure 35 W, the EPO that is expressed in the insect cell system is reconstructed by adding terminal galactose-PEG residue.Peptide is contacted with mannosidase to remove terminal mannose residue.Peptide and galactosyltransferase are contacted with suitable galactose donor with the peg moiety derivatize.In Figure 35 Y, to be expressed in being called in the NSO rat bone marrow tumour cell BPO of sudden change of " novel red corpuscle generation stimulating protein " or NESP by suitable terminal residue being added cap and reconstruct with sialic acid donor, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 35 Z, be that NESP is reconstructed by adding one or more terminal sialic acids-PEG residue with the sudden change EPO that is expressed in the mammal cell line system.Use sialic acid and α 2 that PEG modifies, the 8-sialyltransferase adds PEG on the glycosyl residue on the glycan to.In Figure 35 AA, the NESP that is expressed in the mammal cell line system is reconstructed by adding terminal sialic acid residues.Sialic acid is that the 8-sialyltransferase adds on the glycosyl residue with sialic acid donor and α 2.

In another exemplary embodiment, the invention provides the method for reconstruct rHuGM-CSF (GM-CSF), shown in Figure 36 A～36K.In Figure 36 B, at first handle the GM-CSF in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization subsequently.In Figure 36 C, at first handle the GM-CSF in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and further carry out sialylated to it then with sialic acid donor and ST3Gal3 and/or ST3Gal1.In Figure 36 D, at first handle the GM-CSF that in the NSO cell, expresses to prune glycosyl group with sialidase and alpha-galactosidase, with sialic acid donor and ST3Gal3 it is carried out sialylatedly then, and with ST3Gal1 and PEG-sialic acid donor it carry out PEGization then.In Figure 36 E, at first handle the GM-CSF in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and carry out sialylated with ST3Gal3 and sialic acid donor to it then.In Figure 36 F, be modified at the GM-CSF that expresses in the mammalian cell by suitable terminal residue being added cap with sialic acid donor, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 36 G, the GM-CSF that is expressed in the mammalian cell is that the 8-sialyltransferase carries out sialylated with sialic acid donor and α 2.In Figure 36 I, the modification that is expressed in the GM-CSF in the insect cell is by adding N-acetyl-glucosamine with suitable donor and one or more GnT-I, II, IV and V, and carries out with the semi-lactosi of suitable donor and galactosyltransferase interpolation PEGization subsequently.In Figure 36 J, the GM-CSF that at first uses inscribe glycanase and/or mannosidase processing yeast expression carry out PEGization with galactosyltransferase and PEG-galactose donor to it subsequently to prune glycosyl unit.In Figure 36 K, at first handle the GM-CSF in mammalian cell, express pruning sialic acid residues, and carry out sialylated with ST3Gal1 and sialic acid donor to it subsequently with sialidase.Make this polypeptide and ST3Gal1 and two reactive sialic acid residues contacts that are connected by linker then, thereby this polypeptide is attached on the reactive sialic acid by linker and second sialic acid residues.This polypeptide is contacted with transferrin with ST3Gal3, thereby it is connected with transferrin.

In another exemplary embodiment, the invention provides the method that is used for modified interferon γ (IFN γ).Figure 37 A～37N contains some examples.In Figure 37 B, at first handle the IFN γ in various mammalian cells, express to prune terminal sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization subsequently.In Figure 37 C, at first handle the IFN γ in mammlian system, express to prune terminal sialic acid residues with sialidase.With ST3Gal3 and PEG-sialic acid donor this polypeptide carry out PEGization then, and further carry out sialylated to it with ST3Gal3 and sialic acid donor.In Figure 37 D, at first handle the IFN γ of mammalian cell expression to prune sialic acid and galactose residue with sialidase and alpha-galactosidase.With galactose donor and galactosyltransferase this polypeptide is carried out galactosylation then.With PEG-sialic acid donor and ST3Gal3 IFN γ carry out PEGization then.In Figure 37 E, at first handle the IFN γ in mammlian system, express to prune terminal sialic acid residues with sialidase.With ST3Gal3 and PEG-sialic acid donor this polypeptide carry out PEGization then, and further carry out sialylated to it with ST3Gal3 and sialic acid donor.Figure 37 F has described the another kind of method that is modified at the IFN γ that expresses in the mammlian system.Modify this protein by with sialic acid donor suitable terminal residue being added cap, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 37 G, the IFN γ that is expressed in the mammalian cell is by usefulness sialic acid donor and α 2, and the 8-sialyltransferase adds sialic acid and is reconstructed.In Figure 37 I, the IFN γ that is expressed among insect or the fungal cell modifies by adding N-acetyl-glucosamine with suitable donor and one or more GnT-I, II, IV and V.This protein is further modified by galactose donor and galactosyltransferase interpolation peg moiety with PEGization.Figure 37 J provides the method that is modified at the IFN γ that expresses in the yeast.At first handle this polypeptide to prune sugar chain, with galactose donor and galactosyltransferase it is carried out galactosylation subsequently with the inscribe glycanase.With the sialic acid donor and the ST3Gal3 of PEGization IFN γ carry out PEGization then.In Figure 37 K, the IFN γ that mammalian cell produces is following the modification: this polypeptide is contacted with sialic acid donor with ST3Gal3, this sialic acid donor by linker with reactive galactose-derivedization, thereby this polypeptide can be attached on the reactive semi-lactosi by linker and sialic acid residues.Make this polypeptide and galactosyltransferase then and contact, thereby it is connected with transferrin by galactose residue with the pretreated transferrin of inscribe glycanase.In the scheme of being described by Figure 37 L, the IFN γ that is expressed in the mammlian system modifies by the effect of ST3Gal3: even the sialic acid of PEGization is transferred on the IFN γ from suitable donor.Figure 37 M modifies the example that is expressed in the IFN γ among insect or the fungal cell, and wherein the PEGization of polypeptide is to realize by with GnT-I and/or II the N-acetyl-glucosamine of PEGization being transferred on the IFN γ from donor.In Figure 37 N, be expressed in IFN γ in the mammlian system and be by with suitable donor and α 2, the 8-sialyltransferase adds the sialic acid of PEGization and reconstruct.

In another exemplary embodiment, the invention provides and be used for modified alpha ₁The method of antitrypsin (Prolastin).Some this examples can be found among Figure 38 A～38O.In Figure 38 B, at first handle the α that in various mammalian cells, expresses with sialidase ₁Antitrypsin is to prune sialic acid residues.Add the sialic acid residues of PEGization with suitable donor such as CMP-SA-PEG and sialyltransferase such as ST3Gal3 then.α has demonstrated among Figure 38 C ₁The another kind of scheme that antitrypsin is modified.At first handle the α that in mammlian system, expresses with sialidase ₁Antitrypsin is to prune sialic acid residues.Add sialic acid residues with suitable donor and sialyltransferase such as ST3Gal3 then with the PEG derivatize.Subsequently, further modify this molecule by adding sialic acid residues with sialic acid donor and ST3Gal3.Randomly, at first handle the α of mammalian cell expression with sialidase and alpha-galactosidase ₁Antitrypsin is to prune the galactose residue of sialic acid and α-be connected.With galactosyltransferase and suitable galactose donor this polypeptide is carried out galactosylation then.Further, the sialic acid with the PEG derivatize is added in the ST3Gal3 effect of the sialic acid donor by utilizing PEGization.In Figure 38 D, at first handle the α that in mammlian system, expresses with sialidase ₁Antitrypsin, thus terminal sialic acid residues pruned.Effect by ST3Gal3 is added PEG on the glycosyl residue that N-connects to then, the sialic acid of this ST3Gal3 mediation PEGization from donor such as CMP-SA-PEG to α ₁Antitryptic transfer.Adhered to more sialic acid residues with sialic acid donor and ST3Gal3 subsequently.Figure 38 E has described reconstruct α ₁Antitryptic another kind of method.Modify the α that is expressed in the mammalian cell by suitable terminal residue being added cap with sialic acid donor ₁Antitrypsin, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 38 F, α is disclosed ₁The another one method that antitrypsin is modified.The α that from mammalian expression system, obtains ₁Antitrypsin is by usefulness sialic acid donor and α 2, and the 8-sialyltransferase adds sialic acid and is reconstructed.In Figure 38 H, α ₁Antitrypsin is expressed in insect or the yeast cell, and is reconstructed by this polypeptide and UDP-N-acetylglucosamine are contacted with one or more GnT-I, II, IV or V to add terminal N-acetyl-glucosamine residue.Then, utilize the galactose donor of PEGization and galactosyltransferase this polypeptide to be modified with peg moiety.In Figure 38 I, at first handle the α that in yeast cell, expresses with the inscribe glycanase ₁Antitrypsin is to prune sugar chain.With galactosyltransferase and galactose donor it is carried out galactosylation then.Then, with ST3Gal3 and PEG-sialic acid donor this polypeptide carry out PEGization.In Figure 38 J, α ₁Antitrypsin is expressed in mammlian system.This polypeptide is contacted with sialic acid donor with ST3Gal3, this sialic acid donor by linker with reactive galactose-derivedization, thereby this polypeptide can be attached on the reactive semi-lactosi by linker and sialic acid residues.Make this polypeptide and galactosyltransferase then and contact, thereby it is connected with transferrin by galactose residue with the pretreated transferrin of inscribe glycanase.In Figure 38 L, at first handle the α that in yeast, expresses with the inscribe glycanase ₁Antitrypsin is to prune its glycosyl group.With galactosyltransferase and galactosyl donor this protein carry out PEGization then with peg moiety.In Figure 38 M, handle the α that in vegetable cell, expresses with hexosaminidase, mannosidase and xylosidase ₁Antitrypsin to be pruning its glycosyl chain, and subsequently with N-acetyl-glucosamine transferring enzyme and suitable donor to modify with the N-acetyl-glucosamine of peg moiety derivatize.In Figure 38 N, be expressed in the α in the mammalian cell ₁Antitrypsin is by modifying with ST3Gal3 with the sialic acid residues that the sialic acid donor of PEG derivatize adds PEGization.

In another exemplary embodiment, the invention provides and be used to modify glucocerebrosidase (beta-glucosidase enzyme, Cerezyme ^TMOr Ceredase ^TM) method, shown in Figure 39 A～39K.In Figure 39 B, at first handle the Cerezyme that in mammlian system, expresses with sialidase ^TMTo prune terminal sialic acid residues, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 39 C, at first handle the Cerezyme that in mammalian cell, expresses with sialidase ^TMTo prune sialic acid residues, adhere to Man-6-P with ST3Gal3 with the reactive sialic acid of Man-6-P derivatize then, and carry out sialylated with ST3Gal3 and sialic acid donor to it then.Randomly, at first handle the Cerezyme of NSO cell expressing with SG ^TMTo prune glycosyl group, with galactose donor and alpha-galactosyltransferasactivity it is carried out galactosylation then.Then, the available ST3Gal3 of Man-6-P part and add on this molecule with the reactive sialic acid of Man-6-P derivatize.In Figure 39 D, at first handle the Cerezyme that in mammalian cell, expresses with sialidase ^TMTo prune sialic acid residues, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and carry out sialylated with ST3Gal3 and sialic acid to it then.In Figure 39 E, modify the Cerezyme that is expressed in the mammalian cell by suitable terminal residue being added cap with sialic acid donor ^TM, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone is with carrying out derivatize as for example part of one or more Man-6-P groups.In Figure 39 F, be expressed in the Cerezyme in the mammalian cell ^TMBe that the 8-sialyltransferase carries out sialylated with sialic acid donor and α 2.In Figure 39 H, with suitable donor and one or more GnT-I, II, IV and the Cerezyme of V in being expressed in insect cell ^TMThe middle N-acetyl-glucosamine that adds carry out PEGization with galactosyltransferase and PEG-galactose donor to it then.In Figure 39 I, at first handle the Cerezyme that in yeast cell, expresses with the inscribe glycanase ^TMTo prune glycosyl group, with galactose donor and galactosyltransferase it is carried out galactosylation then, and with ST3Gal3 and PEG-sialic acid donor this polypeptide carry out PEGization then.In Figure 39 JK, at first make the Cerezyme that is expressed in the mammalian cell ^TMWith ST3Gal3 and two reactive sialic acid residues contacts that are connected by linker, thereby this polypeptide is attached on the reactive sialic acid by linker and second sialic acid residues.This polypeptide and ST3Gal3 are contacted with asialylated transferrin, thereby it is connected with transferrin.Carry out sialylated with sialic acid donor and ST3Gal3 to this polypeptide then.

In another exemplary embodiment, the invention provides the modifying method that is used for tissue type plasminogen activation factor (TPA) and mutant thereof.Several specific modification protocols in Figure 40 A～40W, have been provided.Figure 40 B has illustrated a kind of modification program: after TPA expresses in mammalian cell, with one or more mannosidases and sialidase it is handled to prune mannose group and/or sialic acid residues.By being contacted with one or more GnT-I, II, IV and V, polypeptide and suitable N-acetyl-glucosamine donor add terminal N-acetyl-glucosamine then.Further TPA is carried out galactosylation with galactose donor and galactosyltransferase.Then by PEG being attached on this molecule by ST3Gal3 catalysis with the sialylated of sialic acid donor of peg moiety derivatize.In Figure 40 C, TPA expresses in insect or fungal cell.This modification may further comprise the steps, promptly with suitable N-acetyl-glucosamine donor and GnT-I and/or II interpolation N-acetyl-glucosamine; Carry out galactosylation with galactose donor and galactosyltransferase; With by the sialylated PEG that adheres to the sialic acid donor of ST3Gal3 and peg moiety derivatize.In Figure 40 D, TPA expresses in yeast, and with the inscribe glycanase it is handled to prune sugar chain subsequently.This polypeptide is the PEGization by the effect of galactosyltransferase further, and this galactosyltransferase catalysis PEG-semi-lactosi is from the transfer of donor to TPA.In Figure 40 E, TPA expresses in insect or yeast cell.Then with α-and beta-Mannosidase handle this polypeptide to prune terminal mannose residue.Further, peg moiety is to be attached on this molecule from the transfer of suitable donor to TPA by the PEG-semi-lactosi, and this transfer is mediated by galactosyltransferase.Figure 40 F provides the different methods that the TPA that obtains is modified from insect or Yeast system: to being reconstructed into N-acetyl-glucosamine donor and GnT-I and/or II interpolation N-acetyl-glucosamine of this polypeptide, with the galactose donor of galactosyltransferase and PEGization it carry out PEGization subsequently.Figure 40 G provides the another kind of scheme that the TPA that is expressed in insect or the yeast cell is reconstructed.Terminal N-acetyl-glucosamine is with N-acetyl-glucosamine donor and GnT-I and/or II interpolation.The tilactase that is modified to synthetic rather than hydrolysis method performance function is used for adding the semi-lactosi of PEGization to the N-acetyl-glucosamine residue from suitable donor.In Figure 40 I, at first handle the TPA be expressed in the mammlian system to repair sialic acid and galactose residue with SG.Further modify this polypeptide by with sialic acid donor suitable terminal residue being added cap, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 40 J, be expressed in TPA in the mammlian system and be according to following scheme reconstruct: at first, with α-and beta-Mannosidase handle this polypeptide to prune terminal mannose residue; With sialic acid donor and ST3Gal3 sialic acid residues is attached on the terminal galactose residues then; Further, TPA is by being shifted and PEGization to the N-acetyl-glucosamine residue from donor by the catalytic PEGization semi-lactosi of galactosyltransferase.In Figure 40 K, TPA expresses in botanical system.Modification program in this example is as follows: at first handle TPA to prune its glycosyl group with hexosaminidase, mannosidase and xylosidase; Add among the TPA with suitable donor and N-acetyl-glucosamine transferring enzyme N-acetyl-glucosamine then PEGization.In Figure 40 M, the TPA mutant (TNK TPA) that is expressed in the mammalian cell has been carried out reconstruct.At first prune terminal sialic acid residues with sialidase; Then ST3Gal3 is used for the sialic acid of PEGization is shifted to TNK TPA from donor, thereby makes this polypeptide PEGization.In Figure 40 N, at first handle the TNK TPA be expressed in the mammlian system to prune terminal sialic acid residues with sialidase.Then with CMP-SA-PEG as donor and ST3Gal3 with this protein PEGization, and further carry out sialylated to it with sialic acid donor and ST3Gal3.In Figure 40 O, at first handle the TNK TPA of NSO cell expressing to prune terminal sialic acid and galactose residue with sialidase and alpha-galactosidase.With galactose donor and galactosyltransferase TNK TPA is carried out galactosylation then.Final step in this reconfiguration scheme is with sialyltransferase such as ST3Gal3 the sialic acid of peg moiety derivatize to be transferred to the TNK TPA from donor.In Figure 40 Q, TNK TPA expresses in mammlian system, and at first with sialidase it is handled to prune terminal sialic acid residues.With the sialic acid donor of ST3Gal3 and PEGization this protein carry out PEGization then.Carry out sialylated with sialic acid donor and ST3Gal3 to this protein then.In Figure 40 R, modify the TNK TPA that is expressed in the mammlian system by suitable terminal residue being added cap with sialic acid donor, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 40 S, the TNK TPA that is expressed in the mammalian cell modifies by diverse ways: this polypeptide is by usefulness sialic acid donor and α 2, and the 8-sialyltransferase adds sialic acid and is reconstructed.In Figure 40 U, the TNK TPA that is expressed in the insect cell is reconstructed by adding N-acetyl-glucosamine with suitable donor and one or more GnT-I, II, IV and V.This protein is further modified by galactose donor and galactosyltransferase interpolation peg moiety with PEGization.In Figure 40 V, TNK TPA expresses in yeast.At first handle this polypeptide to prune its glycosyl chain, with the galactose donor and the galactosyltransferase of PEG derivatize it carry out PEGization then with the inscribe glycanase.In Figure 40 W, TNK TPA produces in mammlian system.This polypeptide is contacted with sialic acid donor with ST3Gal3, this sialic acid donor by linker with reactive galactose-derivedization, thereby this polypeptide can be attached on the reactive semi-lactosi by linker and sialic acid residues.The anti-TNF IG mosaic that produces among this polypeptide and galactosyltransferase and the CHO is contacted, thereby it is connected with mosaic by galactose residue.

In another exemplary embodiment, the invention provides the method that is used to modify interleukin II (IL-2).Figure 41 A～41G provides some examples.Figure 41 B provides the modification protocols of two steps: at first handle the IL-2 that produced by mammalian cell to prune terminal sialic acid residues with sialidase, with the sialic acid donor of ST3Gal3 and PEGization it carry out PEGization then.In Figure 41 C, the IL-2 of insect cell expression at first modifies with the galactosylation of galactose donor and galactosyltransferase.With the sialic acid donor of ST3Gal3 and PEGization IL-2 carry out PEGization subsequently.In Figure 41 D, the IL-2 that expresses in bacterium modifies with N-acetyl-glucosamine with suitable donor and N-acetyl-glucosamine transferring enzyme, is the PEGization step with PEG-sialic acid donor and sialyltransferase subsequently.Figure 41 E provides the another kind of scheme of modifying the IL-2 that is produced by mammlian system.Modify this polypeptide by with sialic acid donor suitable terminal residue being added cap, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.Figure 41 F has illustrated the example that the IL-2 by escherichia coli expression is reconstructed.This polypeptide is that reactive N-acetyl-glucosamine mixture and a kind of enzyme with PEG group derivatize carries out PEGization, thereby the function of wherein this enzyme having been carried out modifying it is synthetic enzyme rather than lytic enzyme.In Figure 41 G, be to modify by the N-acetylgalactosamine that adds PEGization with suitable donor and N-acetylgalactosamine transferring enzyme by the IL-2 of bacterial expression.

In another exemplary embodiment, the invention provides the method that is used to modify blood coagulation factor VIII, shown in Figure 42 A～42N.In Figure 42 B, at first handle the blood coagulation factor VIII in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 42 C, at first handle the blood coagulation factor VIII in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal3 and suitable donor it carry out PEGization then, and further carry out sialylated to it with ST3Gal1 and sialic acid donor.

In Figure 42 E, the blood coagulation factor VIII that mammalian cell produces is one to go on foot the PEGization modification by what the sialic acid donor with ST3Gal3 and PEGization carried out.Figure 42 F provides and has modified another example that is expressed in the blood coagulation factor VIII in the mammalian cell.This protein is with the sialic acid donor PEGization of ST3Gal1 and PEGization.In Figure 42 G, the blood coagulation factor VIII of mammalian cell expression is according to following scheme reconstruct: promptly use α 2,8-sialyltransferase and PEG-sialic acid donor carry out PEGization to it.In Figure 42 I, modify blood coagulation factor VIII by suitable terminal residue being added cap by mammalian cell expression with sialic acid donor, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 42 J, at first the blood coagulation factor VIII of mammalian cell expression is handled with Endo-H, to be trimmed to glycosyl group.Carry out PEGization with galactosyltransferase and PEG-galactose donor then.In Figure 42 K, at first carry out sialylated to the blood coagulation factor VIII that is expressed in the mammlian system with ST3Gal3 and sialic acid donor, handle to prune glycosyl group with Endo-H then, carry out PEGization with galactosyltransferase and PEG-galactose donor then.In Figure 42 L, at first handle the blood coagulation factor VIII be expressed in the mammlian system to prune terminal mannose residue with mannosidase, with suitable donor and GnT-I and/or II interpolation N-acetyl-glucosamine group, carry out PEGization with galactosyltransferase and PEG-galactose donor then then.In Figure 42 M, at first handle the blood coagulation factor VIII be expressed in the mammalian cell to prune mannose units with mannosidase, add the N-acetyl-glucosamine group with N-acetyl-glucosamine transferring enzyme and suitable donor then.Further carry out galactosylation, carry out sialylated with ST3Gal3 and sialic acid donor then with galactosyltransferase and galactose donor.In Figure 42 N, produce blood coagulation factor VIII and carry out following modification by mammalian cell: at first handle to prune terminal mannose residue with mannosidase.Add the N-acetyl-glucosamine group of PEGization with GnT-I and suitable substance P EGization N-acetyl-glucosamine donor then.

In another exemplary embodiment, the invention provides the method that is used to modify urokinase, shown in Figure 43 A～43M.In Figure 43 B, at first handle the urokinase in mammalian cell, express to prune sialic acid residues with sialidase, with the sialic acid donor of ST3Gal3 and PEGization it carry out PEGization then.In Figure 43 C, at first handle the urokinase in mammalian cell, express to prune sialic acid residues with sialidase, with the sialic acid donor of ST3Gal3 and PEGization it carry out PEGization then, and carry out sialylated with ST3Gal3 and sialic acid donor to it then.Randomly, at first handle the urokinase of expressing in the mammlian system to prune the glycosyl chain with SG, with galactose donor and alpha-galactosyltransferasactivity it is carried out galactosylation then, and with ST3Gal3 or sialyltransferase and PEG-sialic acid donor it carry out PEGization then.In Figure 43 D, at first handle the urokinase in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and further carry out sialylated to it with ST3Gal3 and sialic acid donor.In Figure 43 E, modify the urokinase that is expressed in the mammalian cell by suitable terminal residue being added cap with sialic acid donor, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 43 F, the urokinase that is expressed in the mammalian cell is that the 8-sialyltransferase carries out sialylated with sialic acid donor and α 2.In Figure 43 H, the urokinase that is expressed in the insect cell is to modify with following step: at first, add N-acetyl-glucosamine with suitable N-acetyl-glucosamine donor and one or more GnT-I, II, IV and V in this polypeptide; Add the semi-lactosi of PEGization with galactosyltransferase and PEG-galactose donor then.In Figure 43 I, at first handle the urokinase be expressed in the yeast to prune glycosyl group with the inscribe glycanase, carry out galactosylation with galactose donor and galactosyltransferase then, and carry out PEGization with ST3Gal3 and PEG-sialic acid donor then.In Figure 41 J, at first make the urokinase and ST3Gal3 and two reactive sialic acid residues contacts that are connected by linker that are expressed in the mammalian cell, thereby this polypeptide is attached on the reactive sialic acid by linker and second sialic acid residues.The asialylated urokinase that produces in this polypeptide and ST3Gal1 and the mammalian cell is contacted, thereby it is connected with second urokinase molecule.Further carry out sialylated to whole molecule then with sialic acid donor and ST3Gal1 and/or ST3Gal3.In Figure 43 K, at first use sulfo group lytic enzyme (sulfohydrolase) to handle isolating urokinase to remove sulfate group, with sialyltransferase and PEG-sialic acid donor it carry out PEGization then.In Figure 43 LM, at first handle isolating urokinase to remove sulfate group and hexosamine group with sulfo group lytic enzyme and hexosaminidase, with galactosyltransferase and PEG-galactose donor it carry out PEGization then.

In another exemplary embodiment, the invention provides the method that is used to modify DNase I, shown in Figure 44 A～44J.In Figure 44 B, DNase I is also modifying with following step of expressing in mammlian system: at first, handle this protein to prune sialic acid residues with sialidase; With ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 44 C, at first handle the DNaseI in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and carry out sialylated with ST3Gal3 and sialic acid donor to it then.Randomly, the DNase I that expresses in the mammlian system is exposed in the SG to prune glycosyl group, with galactose donor and alpha-galactosyltransferasactivity it is carried out galactosylation then, and with ST3Gal3 or sialyltransferase and PEG-sialic acid donor it carry out PEGization then.In Figure 44 D, at first handle the DNaseI in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then, and carry out sialylated with ST3Gal3 and sialic acid donor to it then.In Figure 44 E, modify the DNase I that is expressed in the mammalian cell by suitable terminal residue being added cap with sialic acid donor, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 44 F, the DNase I that is expressed in the mammalian cell is that the 8-sialyltransferase carries out sialylated with sialic acid donor and α 2.In Figure 44 H, at first the DNase I in being expressed in insect cell adds N-acetyl-glucosamine with suitable donor and one or more GnT-I, II, IV and V.With galactosyltransferase and PEG-galactose donor this protein carry out PEGization then.In Figure 44 I, at first handle the DNase I in yeast, express to prune glycosyl unit with the inscribe glycanase, with galactose donor and galactosyltransferase it is carried out galactosylation then, and with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 44 JK, at first make the DNase I and ST3Gal3 and two reactive sialic acid residues contacts that are connected by linker that are expressed in the mammalian cell, thereby this polypeptide is attached on the reactive sialic acid by linker and second sialic acid residues.This polypeptide and ST3Gal1 are contacted with asialylated α-1-proteinase inhibitor, thereby it is connected with this inhibitor by sialic acid residues.Then, further carry out sialylated to this polypeptide with suitable donor and ST3Gal1 and/or ST3Gal3.

In another exemplary embodiment, the invention provides and be used to modify the method that contains the Regular Insulin of N-glycosylation site after the sudden change, shown in Figure 45 A～45L.In Figure 45 B, at first handle the Regular Insulin in mammlian system, express to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 45 C, the Regular Insulin that is expressed in the insect cell is by modifying with the N-acetyl-glucosamine of suitable donor and GnT-I and/or II interpolation PEGization.In Figure 45 D, at first handle the Regular Insulin in yeast, express to prune glycosyl group with Endo-H, with galactosyltransferase and PEG-galactose donor it carry out PEGization then.In Figure 45 F, at first handle the Regular Insulin in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal1 and PEG-sialic acid donor it carry out PEGization then.In Figure 45 G, the Regular Insulin that is expressed in the insect cell is to modify by the semi-lactosi that adds PEGization with suitable donor and galactosyltransferase.In Figure 45 H, at first the Regular Insulin in being expressed in bacterium adds N-acetylgalactosamine with suitable donor and N-acetylgalactosamine transferring enzyme.With sialyltransferase and PEG-sialic acid donor this polypeptide carry out PEGization then.In Figure 45 J, the Regular Insulin that is expressed in the bacterium is modified by diverse ways: promptly add the N-acetylgalactosamine of PEGization to protein with suitable donor and N-acetylgalactosamine transferring enzyme.In Figure 45 K, the Regular Insulin that is expressed in the bacterium is modified according to another scheme: this polypeptide is contacted with reactive N-acetylgalactosamine with the N-acetylgalactosamine transferring enzyme, this N-acetylgalactosamine by linker with reactive sialic acid derivatize, thereby this polypeptide can be by linker and N-acetylgalactosamine attached on the reactive sialic acid.This polypeptide is contacted with asialylated transferrin with ST3Gal3, therefore can be connected, make this polypeptide sialylated with ST3Gal3 and sialic acid donor then with transferrin.In Figure 45 L, the Regular Insulin that is expressed in the bacterium is modified with the another one method: this polypeptide is exposed among NHS-CO-linker-SA-CMP and by linker is connected with reactive sialic acid residues.With ST3Gal3 and asialylated transferrin this polypeptide and transferrin are puted together then.Further make this polypeptide sialylated then with ST3Gal3 and sialic acid donor.

In another exemplary embodiment, the invention provides the method that is used to modify hepatitis B B antigen (M antigen-preS and S), shown in Figure 46 A～46K.In Figure 46 B, M-antigen be expressed in the mammlian system and by handling to prune terminal sialic acid residues and with the reactive sialic acid that is connected with lipid A by linker itself and lipid A to be puted together with ST3Gal3 subsequently to modify at first with sialidase.In Figure 46 C, at first handle the M-antigen in mammalian cell, express to prune terminal sialic acid residues with sialidase, with the reactive sialic acid residues that is connected with toxin by linker itself and tetanus toxin are puted together with ST3Gal1 then, and carried out sialylated with ST3Gal3 and sialic acid donor to it then.In Figure 46 D, at first with the M-antigen of expressing in the galactosidase treatments mammlian system to prune the galactosyl residue, carry out sialylated with ST3Gal3 and sialic acid donor to it then.Add the sialic acid of using the KLH derivatize to this polypeptide with ST3Gal1 and suitable donor then.In Figure 46 E, at first the M-antigen of handling yeast expression with mannosidase puts together itself and diphtheria toxin with GnT-I with the N-acetyl-glucosamine donor that is connected with diphtheria toxin to prune the mannose group residue then.In Figure 46 F, by with sialic acid donor suitable terminal residue being added the M-antigen that cap is modified mammalian cell expression, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of peptide to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 46 G, the M-antigen that obtains from mammlian system is by with sialic acid donor and poly-α 2, and the 8-sialyltransferase carries out sialylated and reconstruct.In Figure 46 I, the M-antigen that is expressed in the insect cell is to put together with neisseria protein with the suitable N-acetyl-glucosamine donor that is connected with neisseria (Neisseria) protein with GnT-II.In Figure 46 J, at first handle the M-antigen in yeast, express to prune the glycosyl chain with the inscribe glycanase, with the suitable galactose donor that is connected with neisseria protein itself and neisseria protein are puted together with galactosyltransferase then.Figure 46 K is modified at antigenic another example of the M-that expresses in the yeast.At first handle this polypeptide to prune terminal mannose group residue, add N-acetyl-glucosamine with GnT-I and/or II to it then with mannosidase.Subsequently, make this polypeptide galactosylation, with sialyltransferase and sialic acid donor it is added cap with sialic acid residues then with galactose donor and galactosyltransferase.

In another exemplary embodiment, the invention provides the method that is used to modify human growth hormone (N, V and variant thereof), shown in Figure 47 A～47K.In Figure 47 B, at first handle the human growth hormone that is produced by mammalian cell with sialidase and also with the sialic acid donor of ST3Gal3 and PEGization it carry out PEGization subsequently to prune terminal sialic acid residues, this human growth hormone is to contain the naturally occurring isoform N-connection site or that have the N-connection site (being the placenta enzyme) after the sudden change.In Figure 47 C, be expressed in human growth hormone in the insect cell and be the N-acetyl-glucosamine that adds PEGization by N-acetyl-glucosamine donor and modify with GnT-I and/or II and suitable substance P EGization.In Figure 47 D, human growth hormone is expressed in yeast, handles with the pruning glycosyl group with Endo-H, and further carry out PEGization with the galactose donor of galactosyltransferase and PEGization.In Figure 47 F, be expressed in human growth hormone-Saliva Orthana fused protein in the mammlian system by handling to prune sialic acid residues and with PEG-sialic acid donor and ST3Gal1 it to be carried out PEGization subsequently and modify with sialidase at first.In Figure 47 G, be expressed in human growth hormone-Saliva Orthana fused protein in the insect cell and be by galactose donor and carry out PEGization and reconstruct with galactosyltransferase and PEGization.In Figure 47 H, human growth hormone-Saliva Orthana fused protein produces in bacterium.At first by the effect of N-acetylgalactosamine transferring enzyme N-acetylgalactosamine is added in the fused protein, with PEG-sialic acid donor and sialyltransferase this fused protein carry out PEGization subsequently with the N-acetylgalactosamine donor.Figure 47 I has described another scheme of modifying the human growth hormone-Saliva Orthana fused protein of bacterial expression: the N-acetylgalactosamine donor with PEGization carry out PEGization by the effect of N-acetylgalactosamine transferring enzyme to this fused protein.Figure 47 J provides human growth hormone-further reconfiguration scheme of Saliva Orthana fused protein.This fused protein is contacted with the N-acetylgalactosamine donor with the N-acetylgalactosamine transferring enzyme, this N-acetylgalactosamine donor by linker with reactive sialic acid derivatize, thereby this fused protein can be by linker and N-acetylgalactosamine attached on the reactive sialic acid.This fused protein is contacted with asialylated transferrin with sialyltransferase, therefore it is connected by sialic acid residues with transferrin.Then, with ST3Gal3 and sialic acid donor fused protein is added cap with sialic acid residues.In Figure 47 K, provide the another kind of scheme of modifying the human growth hormone (N) that produces in the bacterium.This polypeptide is contacted and by linker and reactive sialic acid coupling with NHS-CO-linker-SA-CMP.This polypeptide is contacted with asialylated transferrin with ST3Gal3 and this polypeptide is puted together by sialic acid residues and transferrin.Make this polypeptide sialylated with ST3Gal3 and sialic acid donor then.

In another exemplary embodiment, the invention provides and be used to modify TNF acceptor IgG fused protein (TNFR-IgG or Enbrel ^TM) method, shown in Figure 48 A～48G.Figure 48 B has illustrated a kind of modification program, wherein at first makes the TNFR-IgG that expresses in mammlian system sialylated with sialic acid donor and sialyltransferase ST3Gal1; With galactose donor and galactosyltransferase fused protein is carried out galactosylation then; By ST3Gal3 with the sialic acid donor of PEG derivatize fused protein carry out PEGization then.In Figure 48 C, at first handle the TNFR-IgG in mammalian cell, express to prune sialic acid residues with sialidase.By in catalytic reaction, the sialic acid of PEGization being transferred to from donor peg moiety is attached on the TNFR-IgG subsequently by ST3Gal1.In Figure 48 D, TNFR-IgG be expressed in the mammlian system and add PEG by the galactosylation program and modify, this galactosylation program by galactosyltransferase with the mediation of PEG-galactose donor.In Figure 48 E, TNFR-IgG expresses in mammlian system.First step of reconstruct fused protein is to add the sialic acid residues that O-is connected with sialic acid donor with sialyltransferase ST3Gal1.Subsequently, in fused protein, add the semi-lactosi of PEGization with galactosyltransferase and suitable galactose donor with peg moiety.In Figure 48 F, modify the TNFR-IgG that is expressed in the mammalian cell by suitable terminal residue being added cap with sialic acid donor, this sialic acid donor is modified with levulinic acid, thereby has added reactive ketone on sialic acid donor.After on adding the glycosyl residue of fused protein to, this ketone with as hydrazine-or the part of amine-PEG carry out derivatize.In Figure 48 G, the TNFR-IgG that is expressed in the mammalian cell is that the 8-sialyltransferase is reconstructed with α 2, and the reaction of fused protein is transferred to the sialic acid of PEGization in this enzyme catalysis from the sialic acid donor with peg moiety.

In another exemplary embodiment, the invention provides and be used to generate Herceptin ^TMThe method of conjugate is shown in Figure 49 A～49D.In Figure 49 B, Herceptin ^TMBe expressed in the mammlian system, and at first carry out galactosylation with galactose donor and galactosyltransferase.The effect of ST3Gal3 by utilizing reactive sialic acid-toxin complex makes Herceptin then ^TMPut together by sialic acid and toxin.In Figure 49 C, make the Herceptin that produces in mammalian cell or the fungi ^TMPut together by galactosylation process and toxin, this galactosylation process is used galactosyltransferase and reactive semi-lactosi-toxin complex.Figure 49 D contains preparation Herceptin ^TMThe another kind of scheme of conjugate: at first handle the Herceptin that in fungi, produces with Endo-H ^TMTo prune glycosyl group, with galactose donor and galactosyltransferase it is carried out galactosylation then, and put together this sialylated application ST3Gal3 and reactive sialic acid-radio isotope mixture then by sialylated itself and the radio isotope of making.Selectively, reactive sialic acid part can only be adhered to chelating moiety, can load radio isotope in the stage subsequently then.

In another exemplary embodiment, the invention provides and be used to prepare Synagis ^TMThe method of conjugate is shown in Figure 50 A～50D.In Figure 50 B, at first with galactose donor and galactosyltransferase to being expressed in the Synagis in the mammalian cell ^TMCarry out galactosylation, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 50 C, the Synagis that expresses in mammalian cell or the fungi ^TMBe to carry out PEGization with galactosyltransferase and PEG-galactose donor.In Figure 50 D, at first handle the Synagis that is expressing with Endo-H ^TMTo prune glycosyl group, with galactose donor and galactosyltransferase it is carried out galactosylation then, and with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.

In another exemplary embodiment, the invention provides and be used to generate Remicade ^TMThe method of conjugate is shown in Figure 51 A～51D.In Figure 51 B, at first with galactose donor and galactosyltransferase to being expressed in the Remicade in the mammalian cell ^TMCarry out galactosylation, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 51 C, the Remicade that in mammlian system, expresses ^TMBe to modify by the semi-lactosi that adds PEGization with suitable donor and galactosyltransferase.In Figure 51 D, at first handle the Remicade that in fungi, expresses with Endo-H ^TMTo prune glycosyl group, with galactose donor and galactosyltransferase it is carried out galactosylation then, and with ST3Gal3 with the reactive sialic acid of radio isotope derivatize itself and radio isotope are puted together then.

In another exemplary embodiment, the invention provides and be used to modify the method that contains the Reopro of N glycosylation site after the sudden change.Figure 52 A～52L contains this example.In Figure 52 B, at first handle the Reopro in mammlian system, express to prune sialic acid residues with sialidase, with ST3Gal3 and PEG-sialic acid donor it carry out PEGization then.In Figure 52 C, the Reopro that is expressed in the insect cell is by modifying with the N-acetyl-glucosamine of suitable donor and GnT-I and/or II interpolation PEGization.In Figure 52 D, at first handle the Reopro in yeast, express to prune glycosyl group with Endo-H.Subsequently, with galactosyltransferase and PEG-galactose donor this protein carry out PEGization.In Figure 52 F, at first handle the Reopro in mammalian cell, express to prune sialic acid residues with sialidase, with ST3Gal1 and PEG-sialic acid donor it carry out PEGization then.In Figure 52 G, the Reopro that is expressed in the insect cell modifies by the PEGization of carrying out with galactosyltransferase and PEG-galactose donor.In Figure 52 H, at first add N-acetylgalactosamine with N-acetylgalactosamine transferring enzyme and the suitable Reopro of donor in being expressed in bacterium.With sialyltransferase and PEG-sialic acid donor this protein carry out PEGization then.In Figure 52 J, the Reopro that is expressed in the bacterium modifies by different schemes: promptly by the effect of N-acetylgalactosamine transferring enzyme it carry out PEGization with the N-acetylgalactosamine donor of PEGization.In Figure 52 K, the Reopro of bacterial expression modifies with the another one method: at first, this polypeptide is contacted with the N-acetylgalactosamine donor with the N-acetylgalactosamine transferring enzyme, this N-acetylgalactosamine donor by linker with reactive sialic acid derivatize, thereby this polypeptide can be by linker and N-acetylgalactosamine attached on the reactive sialic acid.This polypeptide is contacted with asialylated transferrin with ST3Gal3, thereby it is connected with transferrin by sialic acid residues.With suitable donor and ST3Gal3 this polypeptide is added cap with sialic acid residues then.Figure 52 L provides the extra scheme of modifying the Reopro of bacterial expression.At first make this polypeptide be exposed among NHS-CO-linker-SA-CMP and it is connected with reactive sialic acid by linker.This polypeptide is contacted with asialylated transferrin with ST3Gal3, thereby it is puted together by sialic acid residues and transferrin.With suitable donor and ST3Gal3 this polypeptide is added cap with sialic acid residues then.

In another exemplary embodiment, the invention provides and be used to generate Rituxan ^TMThe method of conjugate.Figure 53 A～53G provides some examples.In Figure 53 B, at first with suitable galactose donor and galactosyltransferase to being expressed in the Rituxan in the various mammlian systems ^TMCarry out galactosylation.Carry out functionalized with sialic acid to this peptide with sialic acid donor and ST3Gal3 then with the toxin moiety derivatize.In Figure 53 C, be expressed in the Rituxan among mammalian cell or the fungal cell ^TMBe to carry out galactosylation with galactosyltransferase and galactose donor, this provides the semi-lactosi that contains drug moiety to peptide.The Rituxan that Figure 53 D provides reconstruct to express in fungal systems ^TMAnother example.At first prune the glycosyl group of this polypeptide with Endo-H.Add semi-lactosi with galactosyltransferase and galactose donor then.Subsequently, by radio isotope compound sialic acid donor and sialyltransferase ST3Gal3 radio isotope and this molecule are puted together.In Figure 53 F, Rituxan ^TMBe expressed in the mammlian system, and at first it carried out galactosylation with galactosyltransferase and suitable galactose donor; Sialic acid donor with ST3Gal3 and PEGization adheres to the sialic acid with peg moiety then.As shown in Figure 53 G, be expressed in the Rituxan in fungi, yeast or the mammalian cell ^TMAlso can modify in the following method: at first, with α-and beta-Mannosidase handle this polypeptide to remove terminal mannose residue; With GnT-I and II and GlcNAc donor GlcNAc is attached on this molecule then, by galactosylation that radio isotope is attached thereto then, this galactosylation use galactosyltransferase and with can the isotopic chelating moiety link coupled of binding radioactivity galactose donor.

In another exemplary embodiment, the invention provides the method that is used to modify Antithrombin III (AT III).Figure 54 A～54O provides some examples.In Figure 54 B, the Antithrombin III that is expressed in the various mammlian systems is reconstructed by adding one or more terminal sialic acid-peg moieties.AT III molecule is contacted with sialidase to remove terminal sialic acid part.Then, molecule and sialyltransferase are contacted with the suitable sialic acid donor with the peg moiety derivatize.In Figure 54 C, the AT III that is expressed in the various mammlian systems is reconstructed by adding sialic acid-peg moiety.AT III molecule is contacted with sialidase to remove terminal sialic acid part.Molecule and ST3Gal3 are contacted with the suitable sialic acid donor with the normal peg moiety derivatize of 1.2mol.Molecule and ST3Gal3 are contacted so that remaining terminal galactose is partly added cap with suitable sialic acid donor.In Figure 54 D, be reconstructed into and have complicated glycan molecule being expressed in AT III in the NSO rat bone marrow tumour cell, this glycan molecule has terminal sialic acid-peg moiety.AT III molecule is contacted with alpha-galactosidase to remove terminal sialic acid and galactose moiety with sialidase.Molecule and galactosyltransferase are contacted with suitable galactose donor.Molecule and ST3Gal3 are contacted with the suitable sialic acid donor with the peg moiety derivatize.In Figure 54 E, be reconstructed into and have terminal substantially completely sialic acid-peg moiety being expressed in AT III in the various mammlian systems.AT III molecule is contacted with sialidase to remove terminal sialic acid part.Molecule and ST3Gal3 are contacted with the suitable sialic acid donor with the normal peg moiety derivatize of 16mol.Molecule and ST3Gal3 are contacted so that remaining terminal galactose is partly added cap with suitable sialic acid donor.In Figure 54 F, the AT III that is expressed in the various mammlian systems is reconstructed by adding one or more terminal sialic acid-peg moieties.AT III molecule and ST3Gal3 are contacted with the suitable sialic acid donor with levulinic acid part derivatize.Molecule is contacted with hydrazine-PEG.In Figure 54 G, the AT III that is expressed in the various mammlian systems is gathered α 2 by adding one or more ends, the sialic acid that 8-connects partly is reconstructed.Make AT III molecule and poly-α 2, the 8-sialyltransferase contacts with suitable sialic acid donor.In Figure 54 I, the AT III that is expressed among insect, yeast or the fungal cell is reconstructed by adding ramose N-N-acetylglucosamine-peg moiety.AT III molecule and GnT-I are contacted with the suitable N-acetyl-glucosamine donor with the PEG derivatize.In Figure 54 J, the AT III that is expressed in the yeast is reconstructed by removing the high mannose glycans structure and adding terminal sialic acid-peg moiety.AT III molecule is contacted with the inscribe glycanase to prune glycosyl group.Molecule and galactosyltransferase are contacted with suitable galactose donor.Molecule and ST3Gal3 are contacted with the suitable sialic acid donor with the peg moiety derivatize.In Figure 54 K, the AT III that is expressed in the various mammlian systems is reconstructed by the transferrin that adds sugar and put together.AT III molecule and ST3Gal3 are contacted with the suitable sialic acid donor with linker-galactose donor part derivatize.The transferrin that molecule and galactosyltransferase and inscribe glycanase are handled contacts.In Figure 54 M, the AT III that is expressed in the yeast is reconstructed by removing the high mannose glycans structure and adding terminal galactose-peg moiety.Molecule is contacted with the inscribe glycanase to prune glycosyl group.Molecule and galactosyltransferase are contacted with the suitable galactose donor with the peg moiety derivatize.In Figure 54 N, the AT III that is expressed in the vegetable cell is reconstructed by glycan structures being changed into the complicated glycan of mammalian type and adding one or more terminal galactose-peg moieties then.AT III molecule is contacted to remove xylose residues with xylosidase (xylosidase).Molecule and galactosyltransferase are contacted with the suitable galactose donor with the peg moiety derivatize.In Figure 54 O, add one or more terminal sialic acid-peg moieties by the terminad galactose moiety and be reconstructed being expressed in ATIII in the various mammlian systems.AT III molecule and ST3Gal3 are contacted with the suitable sialic acid PEG donor with the PEG derivatize.

In another exemplary embodiment, the invention provides the method for the α and the β subunit that are used to modify human chorionic gonadotrophin (hCG).Figure 55 A～55J provides some examples.In Figure 55 B, the hCG that is expressed in various Mammalss and the insect system is reconstructed by adding terminal sialic acid-peg moiety.The hCG molecule is contacted with sialidase to remove terminal sialic acid part.Then, molecule and ST3Gal3 are contacted with the suitable sialic acid donor with the peg moiety derivatize.In Figure 55 C, will be expressed in hCG in insect cell, yeast or the fungal systems by the glycan that makes up N-and connect with add terminal sialic acid-peg moiety and be reconstructed.HCG molecule and GnT-I are contacted with suitable N-acetyl-glucosamine donor with GnT-II.Molecule and galactosyltransferase are contacted with suitable galactose donor.Molecule and ST3Gal3 are contacted with the suitable sialic acid donor with the peg moiety derivatize.In Figure 55 D, the hCG that is expressed in various Mammalss and the insect system is reconstructed by adding one or more terminal sialic acid-peg moieties on the glycan structures that is connected at O-.The hCG molecule is contacted with sialidase to remove terminal sialic acid.Molecule and ST3Gal3 are contacted, glycan structures is added cap with the sialic acid part with suitable sialic acid donor.Molecule and ST3Gal1 are contacted with the suitable sialic acid donor with the peg moiety derivatize.In Figure 55 E, add sialic acid-peg moiety by the glycan structures that is connected to N-and be reconstructed being expressed in hCG in various Mammalss and the insect system.HCG molecule and ST3Gal3 are contacted with the suitable sialic acid donor with the PEG derivatize.In Figure 55 F, the hCG that is expressed in insect cell, yeast or the fungi is reconstructed by adding terminal N-acetyl-glucosamine-PEG molecule.HCG molecule and GnT-I and GnT-II are contacted with the suitable N-acetyl-glucosamine donor with the PEG derivatize.In Figure 55 G, the glycan structures that the hCG that is expressed in insect cell, yeast or the fungi is connected by each N-adds and is no more than a N-acetyl-glucosamine-peg moiety and is reconstructed.HCG molecule and GnT-I are contacted with the suitable N-acetyl-glucosamine donor with the peg moiety derivatize.In Figure 55 H, add one or more terminal sialic acid-peg moieties by the glycan structures that connects to O-and be reconstructed being expressed in hCG in the various mammlian systems.HCG molecule and ST3Gal3 are contacted with the suitable sialic acid donor with the PEG derivatize.In Figure 55 I, the hCG that is expressed in the various mammlian systems is reconstructed by adding terminal sialic acid-peg moiety.Make hCG molecule and α 2,8-SA contacts with the suitable sialic acid donor with the peg moiety derivatize.In Figure 55 J, the hCG that is expressed in the various mammlian systems partly is reconstructed by adding terminal sialic acid.Make hCG molecule and poly-α 2,8-ST contacts with the suitable sialic acid donor with the peg moiety derivatize.

In another exemplary embodiment, the invention provides and be used to modify alpha-galactosidase A (Fabrazyme ^TM) method.Figure 56 A～56J provides some examples.In Figure 56 B, partly be reconstructed by adding one or more terminal galactose-PEG-transferrin being expressed in the alpha-galactosidase A of justacrine in various Mammalss and insect system.The alpha-galactosidase A molecule is contacted with Endo-H to prune glycosyl group.Then, molecule and galactosyltransferase are contacted with the suitable galactose donor with PEG and transferrin derivatize.In Figure 56 C, partly be reconstructed by adding one or more terminal sialic acid-linkers-Man-6-P being expressed in the alpha-galactosidase A of justacrine in various Mammalss and insect cell system.The alpha-galactosidase A molecule is contacted with sialidase to remove terminal sialic acid part.Molecule is contacted with puting together in the suitable sialic acid donor of Man-6-P by linker with ST3Gal3.In Figure 56 D, the alpha-galactosidase A that is expressed in the NSO rat bone marrow tumour cell partly is reconstructed by adding terminal sialic acid-linker-Man-6-P.The alpha-galactosidase A molecule is contacted with alpha-galactosidase to remove terminal sialic acid and galactose moiety with sialidase.Molecule and galactosyltransferase are contacted with suitable galactose donor.Molecule is contacted with puting together in the suitable sialic acid donor of Man-6-P by linker with sialyltransferase.In Figure 56 E, be reconstructed by adding one or more terminal sialic acid-peg moieties being expressed in the alpha-galactosidase A of justacrine in various Mammalss and insect cell system.The alpha-galactosidase A molecule is contacted with sialidase to remove terminal sialic acid part.Molecule and sialyltransferase are contacted with the suitable sialic acid donor with the peg moiety derivatize.In Figure 56 F, the alpha-galactosidase A that is expressed in Mammals, insect, yeast or the fungal systems partly is reconstructed by adding one or more terminal seminose-linkers-ApoE.The alpha-galactosidase A molecule is contacted with puting together in the suitable seminose donor of ApoE by linker with mannose transferase.In Figure 56 G, the alpha-galactosidase A that is expressed in Mammals, insect, yeast or the fungal systems partly is reconstructed by adding semi-lactosi-linker-alpha2-macroglobulin.The alpha-galactosidase A molecule is contacted with Endo-H to prune glycosyl group.Molecule is contacted with puting together in the suitable galactose donor of alpha2-macroglobulin by linker with galactosyltransferase.In Figure 56 H, the alpha-galactosidase A that is expressed in insect, yeast and the fungal systems partly is reconstructed by adding one or more N-acetyl-glucosamines-PEG-Man-6-P.Alpha-galactosidase A molecule and GnT-I are contacted with the suitable N-acetyl-glucosamine donor with PEG and Man-6-P derivatize.In Figure 56 I, the alpha-galactosidase A that is expressed in insect, yeast or the fungal systems partly is reconstructed by adding one or more terminal galactose-PEG-transferrin.Alpha-galactosidase A molecule and GnT-I are contacted with suitable N-acetyl-glucosamine donor.Molecule and galactosyltransferase are contacted with the suitable galactose donor with PEG and transferrin derivatize.In Figure 56 J, the alpha-galactosidase A that is expressed in insect, yeast or the fungal systems is reconstructed by adding one or more terminal sialic acids-PEG-bad luck ferritin (melanotransferrin) part.Alpha-galactosidase A molecule and GnT-I are contacted with suitable N-acetyl-glucosamine donor with GnT-II.Molecule and galactosyltransferase are contacted with suitable galactose donor.Make molecule and sialyltransferase then and contact with the suitable sialic acid donor of bad luck ferritin derivatize with PEG.

In another exemplary embodiment, the invention provides and be used for modified alpha-idose glycosides enzyme (Aldurazyme ^TM) method.Figure 57 A～57J provides some examples.In Figure 57 B, partly be reconstructed by adding one or more terminal galactose-PEG-transferrin being expressed in the α-idose glycosides enzyme of justacrine in various Mammalss and insect system.α-idose glycosides enzyme molecule is contacted with Endo-H to prune glycosyl group.Then, molecule and galactosyltransferase are contacted with the suitable galactose donor with PEG and transferrin derivatize.In Figure 57 C, partly be reconstructed by adding terminal sialic acid-linker-Man-6-P being expressed in the α-idose glycosides enzyme of justacrine in various Mammalss and insect cell system.α-idose glycosides enzyme molecule is contacted with sialidase to remove terminal sialic acid part.Molecule is contacted with puting together in the suitable sialic acid donor of Man-6-P by linker with ST3Gal3.In Figure 57 D, the α-idose glycosides enzyme that is expressed in the NSO rat bone marrow tumour cell partly is reconstructed by adding one or more terminal sialic acid-linkers-Man-6-P.α-idose glycosides enzyme molecule is contacted with alpha-galactosidase to remove terminal sialic acid and galactose moiety with sialidase.Molecule and galactosyltransferase are contacted with suitable galactose donor.Molecule is contacted with puting together in the suitable sialic acid donor of Man-6-P by linker with sialyltransferase.In Figure 57 E, be reconstructed by adding one or more terminal sialic acid-peg moieties being expressed in the α-idose glycosides enzyme of justacrine in various Mammalss and insect cell system.α-idose glycosides enzyme molecule is contacted with sialidase to remove terminal sialic acid part.Molecule and sialyltransferase are contacted with the suitable sialic acid donor with the peg moiety derivatize.In Figure 57 F, the α-idose glycosides enzyme that is expressed in Mammals, insect, yeast or the fungal systems partly is reconstructed by adding one or more terminal seminose-linkers-ApoE.α-idose glycosides enzyme molecule is contacted with puting together in the suitable seminose donor of ApoE by linker with mannose transferase.In Figure 57 G, the α-idose glycosides enzyme that is expressed in Mammals, insect, yeast or the fungal systems partly is reconstructed by adding one or more semi-lactosi-linkers-alpha2-macroglobulin.α-idose glycosides enzyme molecule is contacted with Endo-H to prune glycosyl group.Molecule is contacted with puting together in the suitable galactose donor of alpha2-macroglobulin by linker with galactosyltransferase.In Figure 57 H, the α-idose glycosides enzyme that is expressed in insect, yeast and the fungal systems partly is reconstructed by adding one or more N-acetyl-glucosamines-PEG-Man-6-P.α-idose glycosides enzyme molecule and GnT-I are contacted with the suitable N-acetyl-glucosamine donor with PEG and Man-6-P derivatize.In Figure 57 I, the α-idose glycosides enzyme that is expressed in insect, yeast or the fungal systems partly is reconstructed by adding one or more terminal galactose-PEG-transferrin.α-idose glycosides enzyme molecule and GnT-I are contacted with suitable N-acetyl-glucosamine donor.Molecule and galactosyltransferase are contacted with the suitable galactose donor with PEG and transferrin derivatize.In Figure 57 J, the α-idose glycosides enzyme that is expressed in insect, yeast or the fungal systems partly is reconstructed by adding one or more terminal sialic acids-PEG-bad luck ferritin.α-idose glycosides enzyme molecule and GnT-I are contacted with suitable N-acetyl-glucosamine donor with GnT-II.Molecule and galactosyltransferase are contacted with suitable galactose donor.Further make molecule and sialyltransferase and contact with the suitable sialic acid donor of bad luck ferritin derivatize with PEG.

A. generate or eliminate the glycosylation site that N-connects

The present invention relates to use following peptide, wherein the changing with respect to native peptides of the polysaccharide chains site on this peptide.Usually, the polysaccharide chains that N-connects is that to be connected to the one-level peptide at asparagine residue structural, and wherein be arranged in can be by the aminoacid sequence of the film on the endoplasmic reticulum (ER) in conjunction with glycosyltransferase identification for this asparagine residue.Usually, the structural recognition site of one-level peptide is sequence l-asparagine-X-serine/threonine, and wherein X can be any amino acid except that proline(Pro) and aspartic acid.Although this recognition site is typical, the present invention further is included in other recognition sites and has the peptide that N-connects glycan, and wherein the chain of this N-connection is to add with glycosyltransferase natural or that recombinate.

Because the glycosylated recognition site that N-connects is known, so generating the one-level peptide sequence of sudden change is within those skilled in the art's technical scope, wherein in the sequence of described sudden change, remove natural N-and connected the glycosylation recognition site, perhaps selectively generated one or more extra N-glycosylation recognition sites in addition again.The most simply, asparagine residue can be removed from the primary sequence of peptide, thus the attachment site of removal glycan, thereby can from mature peptide, remove a kind of glycan.For example, available gene engineering method will have L-asparagineThe natural site of identification of-Serine-Serine sequence is processed as to have Leucine-Serine-Serine sequence, thus the glycosylation site that N-connects eliminated in this position.

Further, the glycosylation site that N-connects can be removed by the residue that changes in the recognition site, thereby even there is asparagine residue, one or more other identification residues but do not exist.For example, can make native sequences l-asparagine-Serine- SerineSport l-asparagine-Serine- MethioninThereby, eliminate the N-glycosylation site in this position.The glycosylation site that connects at N-comprises under the situation of the residue except that above-mentioned typical recognition site, the technician can determine that suitable glycosyltransferase discerns necessary sequence and residue, at least one residue that suddenlys change then, thus suitable glycosyltransferase is no longer discerned this site.In other words, handling to generate or to remove or generate and remove glycosylation site simultaneously to the primary sequence of peptide is in technician's technical scope, thereby can generate the peptide of the glycosylation pattern with change.Therefore, any one-level peptide sequence that the present invention should be interpreted as being confined to provide herein is as unique sequence of carrying out glycan reconstruct, and should be interpreted as comprising any or all of peptide sequence that is suitable for carrying out glycan reconstruct.

In order to generate the peptide of sudden change, the nucleotide sequence of encoded peptide primary sequence is changed, thereby the natural codon mutation that makes coding natural amino acid residue is to generate the codon of other amino-acid residues of coding.The technology that changes nucleotide sequence is general in this area, and is described in for example any well-known molecular biology manual.

In addition, the available standards technology is at the nucleic acid of external composite coding one-level peptide structure.For example, available rules synthetic nucleic acid molecule in " gene machine " as the phosphoramidite method.If need the double-stranded DNA of chemosynthesis in as nucleic acid or its segmental application, each complementary strand can synthesize separately so.The production of short nucleic acid (60～80 base pairs) is simple technically, and can its annealing be realized by synthetic complementary strand.For long 300 base pairs of nucleic acid (〉) production, need specific technology, this is because the coupling efficiency in each circulation of chemical dna synthetic seldom is 100%.In order to address this problem, synthetic gene (double-stranded) is to be that the single-chain fragment assembling of 20～100 Nucleotide comes with the pattern of module from length.Summarize for the polynucleotide synthetic, referring to as Glick and Paternak (Molecular Biotechnology, Principles and Applications ofRecombinant DNA, 1994, ASM Press), people such as Itakura (1984, Annu.Rev.Biochem.53:323) and people (1990, Proc.Nat ' 1.Acad.Sci.USA87:633) such as Climie.

In addition, the change in the nucleotide sequence of encoded peptide can form by site-directed mutagenesis.As is understood, this technology is generally used the phage vector that exists with strand and double chain form simultaneously.The general carrier that is used for site-directed mutagenesis comprises the carrier as the M13 phage.These phages are easy to obtain, and it is used, and normally those skilled in the art is well-known.Also use double-stranded plasmid in site-directed mutagenesis routinely, this has eliminated target nucleic acid has been transferred to step the phage from plasmid.

Usually, site-directed mutagenesis is to be undertaken by two chains that at first obtain single-stranded vector or fuse double-stranded carrier, wherein should the two strands carrier comprises the dna sequence dna of the peptide that coding is wanted in its sequence.The normally synthetic preparation of Oligonucleolide primers with mutant nucleotide sequence of wanting.Make the annealing of this primer and single-stranded vector then, handle to finish synthesizing of chain with sudden change with the Klenow fragment of archaeal dna polymerase such as Escherichia coli polymerase I then.Thereby, formed the isodigeranyl serobila, the sequence of the initial nothing sudden change of chain encoding wherein, and another chain has the sudden change of wanting.Then this isodigeranyl serobila is used to transform or cell that transfection is suitable,, and selects to comprise the clone of the recombinant vectors of sequence with sudden change as Bacillus coli cells.People such as Kunkel (1987, people such as Kunkel, Methods Enzymol.154:367-382) have designed a kind of hereditary selection scheme has been integrated the oligonucleotide of mutagenesis with enrichment clone.Selectively, can be with the commercial thermostability enzyme of buying such as Taq polysaccharase and PCR ^TMBe used for the Oligonucleolide primers of mutagenesis is integrated into the dna fragmentation of amplification, this fragment cloning can be gone among the suitable clone or expression vector then.People such as Tomic (1990, Nucl.Acids Res., 12:1656) and people such as Upender (1995, Biotechniques, PCR 18:29-31) ^TM-mediated mutagenesis method provides two examples of this rules.Except that the thermostability polysaccharase, go back the PCR of use heat stability ligase enzyme ^TMAlso can be used for the mutagenic oligonucleotide of phosphorylation is integrated in the dna fragmentation of amplification, this fragment cloning can be gone among the suitable clone or expression vector then.An example of this rules is provided by the mutafacient system of Michael (1994, Biotechniques 16:410-412) description.

Not every Asn-X-Ser/Thr sequence all is that N-is glycosylated, and this points out the residing front and back of this primitive environment is important.In another method, generated and had library that new N-connects consistent site mutation peptide to identify new N-connection site, this new N-connection site is carried out glycosylation in vivo, and is useful to activity, stability or other features of peptide.

As previously noted, the concensus sequence that adds the polysaccharide chains of N-connection in glycoprotein is Asn-X-Ser/Thr, and wherein X can be any amino acid.Coding can suddenly change with coding Ser and/or Thr with the known standard method of those skilled in the art apart from the amino acid whose nucleotide sequence of two positions of C-terminal one side of Asn.Stated that as mentioned not every Asn-X-Ser/Thr site is all modified by adding glycan.Therefore, must make the glycoprotein of each recombination mutation in fungi, yeast or animal or mammalian expression system, express and analyze the interpolation of the polysaccharide chains that N-connects.The technology that characterizes glycosylation site is that those skilled in the art is well-known.Further, the biological function of the recombinant glycoprotein of sudden change can be determined with the assay method that for the specified protein of checking is standard.Thereby, the primary sequence of peptide is handled and identified the new glycosylation site that contains therein, and determine that further this novel site then becomes simple thing to the effect of the biologic activity of peptide.

In a selectable embodiment, coding is that the amino acid whose nucleotide sequence of two positions can suddenly change with coding Asn with the known standard program of those skilled in the art apart from N-terminal one side of Ser and/or Thr.Determined whether to generate new glycosylation site and this site is as indicated above to the method for the effect of the biologic activity of peptide.

B. generate or eliminate the glycosylation site that O-connects

Adding the glycosylation site of O-connection in peptide can realize by the one-level aminoacid sequence that changes peptide easily, contains the glycosylation site that one or more extra O-are connected thereby its peptide one-level aminoacid sequence with beginning is compared.In peptide, add the glycosylation site that O-connects also can comprise-realize in the peptide of OH group (being preferably Serine or threonine residues), thereby the OH group is come-at-able and can be used in the glycosylation that O-connects by one or more amino acid kinds are integrated in its peptide sequence.The change of the glycosylation site that is connected with N-in the peptide is similar, and the one-level aminoacid sequence of peptide preferably changes on nucleotide level.Specific Nucleotide changes in can the dna sequence dna to encoded peptide, thus the amino acid that this sequence encoding is wanted.Sudden change among the DNA preferably realizes with method as known in the art, as the above-mentioned phosphoramidite DNA synthetic method and the technology of site-directed mutagenesis.

Selectively, the coding nucleotide sequence of inferring the site that is used for adding the glycan that O-connects can add this molecule at 5 ' or 3 ' end of dna molecular with one or several copy.Whether can make the dna sequence dna of change express and analyze the sequence of adding and this sequence then in fungi, yeast or animal or mammalian expression system in peptide is the glycosylation site that has the O-of function to connect.In brief, synthetic peptide receptor sequence is 5 ' or 3 ' the terminal introducing at nucleic acid molecule.In principle, the interpolation of such sequence is less to the destructiveness of the glycoprotein of gained as a result when expressing in suitable expression system.The DNA of change is expressed in Chinese hamster ovary celI or other suitable expression systems, and check the glycosylation site of expressed protein to determine whether to exist O-to connect therein.In addition, the existence that can determine polysaccharide chains whether.

In the another one method, the favourable site of new O-connection site can be found in the library of containing the peptide of various new O-connection site by generation in peptide.For example, the consistent aminoacid sequence by N-acetylgalactosamine transferring enzyme interpolation N-acetylgalactosamine depends on used specific transferring enzyme.Can scan the aminoacid sequence of peptide to identify successive amino acid group, this amino acid group can be suddenlyd change to generate the potential site of the polysaccharide chains of adding the O-connection.The available standard program that well known to a person skilled in the art as previously described of these sudden changes generates.For the glycosylation site that determines whether any discovery is actually glycosylated, make the peptide of each recombination mutation in suitable expression system, express and the existence of the interpolation in subsequent analysis site and/or the polysaccharide chains that O-connects whether.

C. the chemosynthesis of peptide

Can in expression system, generate most effectively although be used for the primary structure of the present invention's peptide based on cell, within the scope of the present invention be that peptide can generate synthetically.The chemosynthesis of peptide is well known in the art, and does not restrictedly comprise progressively solid phase synthesis and fragment condensation in solution or solid phase.Typical progressively solid phase synthesis comprises making corresponding to the amino acid whose amino acid of the C-terminal of the peptide chain of wanting and covalently is connected with solid support, then by progressively extending peptide chain in conjunction with the activated amino acid derivative with activatory carboxylic group to N-terminal.After the solid phase streptavidin binding peptide assembling of protection is fully finished,, and remove blocking group to obtain the product peptide by the covalent attachment of suitable chemical reaction cutting peptide-solid phase.Referring to R.Merrifield, Solid Phase Peptide Synthesis:The Synthesis of aTetrapeptide, J.Am.Chem.Soc., 85:2149-2154 (1963).Peptide chain is long more, and it is challenging to obtain highly purified definite product.Because the generation of complicated mixture, progressively solid phase synthesis process has the restriction of size.Usually, have 100 successive or more definite peptide of amino acids residue can not prepare routinely by solid phase synthesis progressively.

The section method of condensing comprise by solid phase progressively method prepare several peptide sections, subsequently from solid phase cutting and these sections of protecting to greatest extent of purifying.The section that makes protection one by one with first section condensation that is attached on the solid phase.

Being used for peptide of the present invention can be by complete solid phase synthesis, part solid phase method, fragment condensation or classical synthetic synthesizing of solution.These synthetic methods are that those skilled in the art are well-known (referring to as Merrifield, J.Am.Chem.Soc., 85:2149 (1963), people such as Stewart, " Solid Phase Peptide Synthesis " (second edition), (PierceChemical Co.1984), Bayer and Rapp, Chem.Pept.Prot.3:3 (1986), people such as Atherton, Solid Phase Peptide Synthesis:A PracticalApproach (IRL Press1989), Fields and Colowick, " Solid-PhasePeptide Synthesis ", people such as Methods in Enzymology 289 volumes (Academic Press1997) and Lloyd-Williams, Chemical Approaches to theSynthesis of Peptides and Peptides (CRC Press, Inc.1997)).The change method of total chemosynthesis strategy as " native chemical connection " and " peptide of expression is connected " also be standard (referring to as people such as Dawson, Science 266:776 (1994), people such as Hackeng, Proc.Nat ' 1.Acad.Sci.USA 94:7845 (1997), Dawson, Methods Enzymol.287:34 (1997), people such as Muir, Proc.Nat ' 1.Acad.Sci.USA 95:6705 (1998) and Severinov and Muir, J.Biol.Chem.273:16205 (1998)).Equally usefully by Gryphon Sciences, South SanFrancisco, the solid-phase peptide synthetic method of CA exploitation.Referring to U.S. Patent No. 6,326,468,6,217,873,6,174,530 and 6,001,364, herein their whole integral body are incorporated herein by reference.

D. posttranslational modification

It should be appreciated by those skilled in the art that peptide and except that the glycan that adds that N-connects and/or O-connection thereon, also can carry out posttranslational modification.The peptide that expection has the posttranslational modification beyond the glycosylation can be used as peptide of the present invention, as long as keep or improved the biologic activity of wanting or the function of this peptide.This posttranslational modification can be the natural modifications of carrying out in vivo usually, perhaps for modifying in the external processing of carrying out to peptide.The known modification of expection is including, but not limited to acetylize; acidylate; the ADP-ribosylation; amidation; the covalent attachment of flavine; the covalent attachment of heme moiety; the covalent attachment of Nucleotide or nucleotide derivative; the covalent attachment of lipid or lipid derivate; the covalent attachment of phosphatidylinositols; crosslinked; cyclisation; disulfide linkage forms; demethylation; the formation of covalent cross-linking; the formation of halfcystine; the formation of Pyrrolidonecarboxylic acid; formylation; the γ carboxylation; glycosylation; the GPI deadman forms; hydroxylation; iodate; methylate; myristoylation; oxidation; proteolysis processing; phosphorylation; isoamyl two acidylates; racemization; selenizing (selenoylation); sulphating; the amino acid of transfer RNA mediation is to the interpolation such as arginylization (and the ubiquitination of peptide.The enzyme that can be used for carrying out many these modifications is well known in the art, and can be from (Indianapolis, IN) (St.Louis, company MO) buys with Sigma Chemical Company as Boehringer Mannheim.

This modification is that those skilled in the art are well-known, and has been described in detail in the scientific literature.Several common especially modifications such as glycosylation, lipid adhere to, γ carboxylation, hydroxylation and the ADP-ribosylation of sulphating, glutaminic acid residue are described in the most basic textbook, as Peptides-Structure and Molecular Properties, 2 ^NdEd., T.E.Creighton, W.H.Freeman and Company, New York (1993).For many detailed summaries are arranged on this theme, as Wold, F., Post-translationalCovalent Modification of Peptides, B.C.Johnson, Ed., AcademicPress, New York 1-12 (1983); People (Ann.N.Y.Acad.Sci.663:48-62 (1992)) such as people such as Seifter (Meth.Enzymol.182:626-646 (1990)) and Rattan.

The covalent modification of peptide also can react and is incorporated in the molecule at external reactive target amino acid residue and organic derivatization reagent by making peptide, this organic derivatization reagent can with side chain of selecting or terminal amino acid residue reaction.The residue of the most frequently used derivatize is cysteinyl, histidyl-, lysyl, arginyl, tyrosyl, glutaminyl, asparaginyl and n terminal residue.The hydroxylation of proline(Pro) and Methionin, the phosphorylation of the oh group of seryl and threonyl residue, the alpha-amino group group of Methionin, Histidine and Histidine side chain methylate the amidation of the acetylize of N-terminal amine and C-terminal carboxyl(group).The part of this derivatize can be improved solvability, absorption, biological half time etc.Any undesired side effect of peptide etc. also can be eliminated or reduce to this part.

In addition, the derivatize with bifunctional reagent can be used for making peptide and water-insoluble support matrix or other macromolecular carrier crosslinked.Linking agent commonly used comprises glutaraldehyde, N-hydroxy-succinamide ester, with difunctional imido-ester, 1, two (diazole ethanoyl)-2-diphenylphosphino ethanes of 1-and difunctional maleimide.Derivatization reagent such as methyl-3-[9-are to the azido-phenyl)] dithio propionyl imidoether produced can be when light exists crosslinked photoactivation type intermediate.Selectively, can will be described in U.S.Pat.No.3, the water reactive insoluble matrix in 969,287 and 3,691,016 such as the sugar of cyanogen bromide-activated and reactive substrate are used for peptide to be fixed.

E. fusogenic peptide/peptide

Be used for peptide of the present invention and can comprise fusogenic peptide.In the time need being combined in the biology of two peptides and/or functional character in the peptide molecule, fusogenic peptide be particularly advantageous.This fusogenic peptide can show that the natural non-existent combination of biologic activity and function is to generate new useful molecule in treatment and the industrial application.Target organism is learned active in enzymic activity, acceptor and/or ligand activity, immunogenicity primitive and structural structural domain.

This fusogenic peptide is well known in the art, and its generation method is that those skilled in the art are well-known.For example, prepared human-human albumin fusogenic peptide, wherein the peptide of gained has treatment benefit with the alpha-interferon of the long cycle life combination of albumin as a result, makes it possible to reduce the frequency and to the therapeutic composition of patient's potential side effect of taking medicine thereby generated.Referring to from Human Genome Sciences, the Albuferon of Inc. and U.S. Patent No. 5,766,883 ^TMOther fusogenic peptides are included in the antibody molecule that other places are described.

The generation of F. less " biologic activity " molecule

Be used for the variant that peptide of the present invention can be native peptides, wherein substitute the native peptides of total length with the fragment of native peptides.In addition, pre-pro-peptide (pre-pro-peptide) and propetide (pre-peptide) are used in expection.The comparable in size native peptides of variant peptides is little, and can comprise one or more structural domains of bigger peptide.When the biologic activity of some structural domain in the peptide be want but in the peptide biologic activity of other structural domains be not want the time, be favourable to the selection of particular peptide structural domain.What comprise equally is that the blocking with inner of peptide that can strengthen the therapeutic action of wanting of peptide lacks.If can keep the biologic activity of wanting of peptide, the peptide of so any this form all can be used among the present invention.

Peptide may have undiscovered distinct advantages in the native peptides than short-form.Under the situation of human albumin, having found to comprise few clipped form to 63% native albumin peptide is favourable as the swelling agent of blood plasma volume.The ovalbumin peptide that it is believed that brachymemma is better than natural peptide on these therapeutic purpose, this is because with respect to natural human serum albumin dosage or recombinant human serum albumin egg dosage, and described peptide is as long as 1/2 to 2/3 dosage can reach colloid osmosis of equal value.Referring to U.S. Patent No. 5,380,712, herein its integral body is incorporated herein by reference.

Find that also less " biologic activity " peptide compares with native peptides and have the enhanced therapeutic activity.The treatment potentiality of IL-2 are subjected to the restriction based on the side effect of vascular leakage syndromes.The less chemosynthesis form that it is found that the peptide of being made up of the residue 1-30 corresponding to whole alpha-helix can correctly fold and contain natural IL-2 biologic activity, and does not have main side effect.

G. the generation of new peptide

Peptide of the present invention can be derived from the primary sequence of native peptides, and the known many methods of perhaps available those skilled in the art are processed.The peptide of this processing can be compared enhanced or new character according to it and design and/or select with natural peptide.For example, can process so that it has in the specificity to substrate, part, acceptor or other binding partners of the enzyme reaction speed of increase, the avidity to substrate or part increase or that reduce, the binding affinity to acceptor increase or that reduce, change, body increase or that reduce and/or the immunogenicity in animal vitro stability or increase or that reduce peptide.

H. sudden change

1. the design of reasoning sudden change

Can the suddenly change undesired character of the biologic activity wanted with enhancing or function, minimizing peptide and/or add new activity or function of the peptide that is used for the inventive method to peptide." reasonably peptide design " can be used for generating the peptide of this change.In case the aminoacid sequence of peptide and structure are known and have planned the sudden change wanted, but most convenient ground suddenlys change at the corresponding nucleic acids codon so, this nucleic acid codon coding is wanted the amino-acid residue that suddenlys change.Those skilled in the art is easy to determine how nucleotide sequence is changed based on the knowledge of the codon-bias of general genetic code and selected expression system.The amino-acid residue that can in codon, suddenly change and polymerization be gone in the peptide to change in translation process.Selectively, can suddenly change, thereby the amino-acid residue of corresponding encoded is constant, but the selection of codon is more suitable in the peptide expression systems of wanting codon.For example, available other amino acid replacement cys-residue to be removing the disulfide linkage in the native peptides, can suddenly change changing biologic activity to the catalytic structural domain, and can process the isotype of peptide usually.This sudden change can be point mutation, disappearance, insertion and brachymemma etc.

The technology that makes amino acid mutation specific in the peptide is well known in the art.Above-mentioned side-directed mutagenesis is highly suitable for the orthomutation of codon.Oligonucleotide mediated mutafacient system also goes through in people such as Sambrook (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York is since 15.51 pages).Can insert mutagenesis, nuclease Bal31 digestion and linker scanning mutagenesis and other methods well known in the art are carried out systematic disappearance, insertion and brachymemma (people such as Sambrook with joint, 2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

The peptide design of reasoning (rational) has been successfully used to increase the stability of enzyme to heat inactivation and oxidation.For example, the stability of enzyme can be by removing asparagine residue in the α-Dian Fenmei (people such as Declerck, 2000, J.Mol.Biol.301:1041-1057), to α-Dian Fenmei (people such as Igarashi, 1999, Biosci.Biotechnol.Bichem.63:1535-1540) and introduce more inflexible structural element such as proline(Pro) in the D-xylose isomerase (people such as Zhu, 1999, Peptide Eng.12:635-638) and improve.Further, the introducing of extra hydrophobic contact can be stablized 3-Isopropylmalate dehydrogenase (people such as Akanuma, 1999, Eur.J.Biochem.260:499-504) hydrogenlyase that obtains and from pseudomonas species (Pseudonomas sp.) (people such as Rojkova, 1999, FEBSLett.445:183-188).The mechanism of the stabilization of these sudden changes can be applicable to many peptides usually.Expect that these and the peptide that similarly suddenlys change for reconstruct in the method for the present invention are useful.

2. random mutagenesis technology

The new peptide that is used for method of the present invention can be used on the technology of introducing random mutation in the encoding sequence of nucleic acid and generates.Nucleic acid is expressed in the expression system of wanting, and to the peptide purpose of appraisals character of gained as a result.The technology of random mutation being introduced dna sequence dna is well known in the art, and comprises PCR mutagenesis, saturation mutagenesis and degenerate oligonucleotide method.Referring to Sambrook and Russell (2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY) and people such as Ausubel (2002, Current Protocols in Molecular Biology, John Wiley﹠amp; Sons, NY).

In PCR mutagenesis, with the fidelity of the Taq polysaccharase that reduces be used for to clone's dna fragmentation introduce random mutation (people such as Leung, 1989, Technique1:11-15).This is that random mutation is incorporated into very strong and relative method fast in the dna sequence dna.The DNA zone of carrying out mutagenesis is to increase under the condition of the fidelity that reduces Taq archaeal dna polymerase synthetic DNA with polymerase chain reaction (PCR), as the dGTP/dATP ratio of application change with by add Mn in the PCR reaction ²⁺The set of dna fragmentation of amplification is inserted in the suitable cloning vector so that the library of sudden change at random to be provided.

Saturation mutagenesis makes it possible to introduce fast a large amount of single bases and substitutes (people such as Mayers, 1985, Science 229:242) in clone's dna fragmentation.This technology comprises that for example being used in external chemical treatment or radiation to single stranded DNA generates sudden change and synthetic complementary DNA chain.Mutation frequency can be regulated by regulating the fierce degree of handling, and it is alternative to obtain all possible base substantially.Segmental heredity is selected because this program does not relate to sudden change, so can obtain substituting of neutral alternation and those change functions.The sequential element guarded is not partial in the distribution of point mutation.

The library of nucleic acid homologue also can generate from a cover degenerate oligonucleotide sequence.The chemosynthesis of degenerate oligonucleotide sequence can be carried out on automatic dna synthesizer, and the synthetic gene can be connected on the suitable expression then.Degenerate oligonucleotide synthetic be as known in the art (referring to as Narang, SA (1983) Tetrahedron 39:3; People such as Itakura (1981) Recombinant DNA, Proc 3 ^RdCleveland Sympos.Macromolecules, ed.AG Walton, Amsterdam:Elsevier pp.273-289; People such as Itakura (1984) Annu.Rev.Biochem.53:323; People such as Itakura (1984) Science198:1056; People such as Ike (1983) Nucleic Acid Res.11:477).This technology has been applied in the orthogenesis of other peptides (referring to as people such as Scott (1990) Science 249:386-390; People such as Roberts (1992) PNAS 89:2429-2433; People such as Devlin (1990) Science 249:404-406; People such as Cwirla (1990) PNAS 87:6378-6382 and U.S.Pat.No.5,223,409,5,198,346 and 5,096,815).

A. orthogenesis

Peptide also available " orthogenesis " technology that is used for the inventive method generates.Opposite with side-directed mutagenesis (wherein the knowledge of peptide structure is essential), exist at present to need not to know the peptide constitutional features and generate the strategy in sudden change library, from this library, can obtain to have the peptide of the character of improvement.These strategies are commonly referred to " orthogenesis " technology and are that with traditional random mutagenesis procedure difference they carry out the sudden change of many wheel multiple, screening and amplification to the nucleotide sequence of coding target peptide.

In some " orthogenesis " technology, the diversity in the nucleic acid of acquisition is to generate by the mutation method that generates point mutation in nucleotide sequence at random.The point mutation technology is including, but not limited to " the PCR of fallibility ^TM" (Caldwell and Joyce, 1994; PCR Methods Appl.2:28-33; With Ke and Madison, 1997, Nucleic Acids Res.25:3371-3372), the mutagenesis instructed of multiple oligonucleotide (people such as Reidhaar-Olson, 1991, MethodsEnzymol.208:564-586) and any aforesaid random mutagenesis methods.

The multifarious method of another kind of generation is to use to increase change (mutator) gene, and orthogenesis can act on this diversity.Target nucleic acid is incubated to increase becomes in the cell strain, the genome of this cell strain generally encode DNA-repair gene (U.S. Patent No. 6,365,410 of defective; People such as Selifonova, 2001, Appl.Environ.Microbiol.67:3645-3649; People such as Long-McGie, 2000, Biotach.Bioeng.68:121-125, referring to GenencorInternational Inc, Palo Alto CA).

Obtain also available degenerated primer of diversity and saturation mutagenesis realization (Gene Site Saturation Mutagenesis with the orthogenesis technology ^TM, Diversa Corp., San Diego, CA).In such saturation mutagenesis, the polysaccharase that the degenerated primer that will carry out diversified nucleotide sequence total length is used for causing the PCR reaction will be designed to cover.In this mode, each codon of amino acid coding can be suddenlyd change so that each of its remaining 19 common amino acids of encoding.This technology also can be used for introducing sudden change, disappearance and insertion to the specific region of nucleic acid coding sequence, and the remainder of nucleic acid molecule is remained unchanged.The program of gene saturation technique is well known in the art, and can be found in United States Patent (USP) 6,171,820.

B.DNA resets (shuffling)

The also available gene rearrangement of new peptide, the primitive that is used for the inventive method reset, exon is reset and/or codon (being referred to as " DNA the rearrangement ") technology of resetting generates.DNA rearrangement technology can be applicable to regulate and is used for the active of peptide of the present invention or can be used for generating the active peptide with change.Usually referring to U.S.Pat.Nos.5,605,793,5,811,238,5,830,721,5,834,252 and 5,837,458 and people such as Stemmer (1994, Nature 370 (6488): 389-391); (1998, Nature 391 (6664): 288-291) for people such as Crameri; People such as Zhang (1997, Proc.Natl.Acad.Sci.USA94 (9): 4504-4509); People such as Stemmer (1994, Proc.Natl.Acad.Sci.USA 91 (22): 10747-10751), and people such as Patten (1997, Curr.Opinion Biotechnol.8:724-33); Harayama (1998, Trends Biotechnol.16 (2): 76-82); People such as Hansson (1999, J.Mol.Biol.287:265-76) and Lorenzo and Blasco (1998, Biotechniques 24 (2): 308-13) (each of these patents all integral body be incorporated herein by reference) herein,

The DNA rearrangement comprises by homology or site-specific nature reorganization assembles two or more dna fragmentations to generate the variation in the polynucleotide sequence.DNA has been reset and be used to generate 1 type human immunodeficiency virus protein (people such as Pekrun, 2002, J.Virol.76 (6): 2924-35), triazine lytic enzyme (people such as Raillard, 2001, Chem Biol 8 (9): 891-898), murine leukemia virus (MLV) protein (people 2000 such as Powell, Nat Biotechnol 18 (12): 1279-1282) and the indoleglycerolphosphoric acid synthetic enzyme (people 2000 such as Merz, Biochemistry 39 (5): 880-889).

The DNA technology of resetting also develops into can be by simulating natural reorganization to generate molecular diversity (Stemmler, 1994, Nature 370:389-391 in the homologous recombination of the external DNA of carrying out; And Stemmler, 1994, PNAS 91:10747-10751).Usually, in the method the colony of genes involved is carried out fragmentation and carry out repeatedly sex change, hybridize once more and be accompanied by of the extension of Taq polysaccharase 5 ' overhang.In each circulation, segmental length increases, and can recombinate when being derived from heterogeneic fragment phase mutual cross.Initial dna fragmentationization can realize by nuclease digestion, generally uses DNase (reference referring to Stemmler sees above), but also can synthesize by the PCR that is interrupted and realize (United States Patent (USP) 5,965,408, integral body is incorporated herein by reference herein; Referring to Diversa Corp., San Diego, CA).The DNA rearrangement is to realize therefore for the peptide phenotype of improving autonomous selection is arranged to taken turns the direct reorganization of the favourable sudden change of resetting generation by each with respect to the advantage of random point mutation method.

DNA rearrangement technology is well known in the art.Detailed explanation to this technology can be found in Stemmler, and 1994, Nature 370:389-391 and Stemmler, 1994, among the PNAS91:10747-10751.DNA resets technology and also is described in United States Patent (USP) 6,180,406,6,165,793,6,132,970,6,117,679,6,096,548,5,837,458,5,834,252, (herein its whole integral body are incorporated herein by reference) in 5,830,721,5,811,238 and 5,605,793.

This area also provides the nearest modification of the basic fundamental that DNA is reset.In an example, utilize the increased combination of exon in ad hoc structure territory of exon rearrangement, exon or encoded peptide of chimeric oligonucleotide.Then the assembling of the PCR by self-priming to the molecule of amplification carried out recombinating (Kolkman and Stemmler, 2001, Nat.Biotech.19:423-428).In another example, utilize in the instantaneous template library construction body mosaic at random to generate the technology of (RACHITT), the single-stranded template of the parental DNA fragment of strand and total length is annealed (people such as Coco, 2001, Nat Biotechnol.19:354-359).Annealing/extension round-robin thermal cycling that (staggered extension process (StEP)) will have a reduction greatly in another example be used for the repeated interruptions DNA polymerization of primer from the side (people such as Zhao, 1998, NatBiotechnol.16:258-261).In being known as the technology of CLERY, with external series reset with yeast in body in homologous recombination make up (people such as Abecassis, 2000, Nucleic Acids Res.28:E88).In order to make between gene the reorganization maximization, will from the single stranded DNA of the complementary strand of each nucleic acid with DNase digestion and anneal (people such as Kikuchi, 2000, Gene243:133-137).The flush end of two brachymemma nucleic acid of the variable-length that is connected by the sequence that can cut is connected generating the gene fusion thing, and need not to carry out homologous recombination (people such as Sieber, 2001, Nat Biotechnol.19:456-460; People such as Lutz, 2001, Nucleic Acids Res.29:E16; People such as Ostermeier, 1999, Nat Biotechnol.17:1205-1209; Lutz and Benkovic, 2000, Curr.Opin.Biotechnol.11:319-324).Linking together to the flush endization of dna fragmentation with fragment of also available exonuclease mediation strengthens reorganization (U.S. Patent No. 6 between the nucleic acid with very little consensus sequence homology so that it is recombinated, 361,974, integral body is incorporated herein by reference herein).The present invention expect use above-mentioned each and all the variant methods be used for the method for the biological property of any peptide of the inventive method and/or enzyme as enhancing.

Except that the rules of detailed description orthogenesis of delivering and gene rearrangement technology, the commerce services that present peptide to selection carries out gene rearrangement and select procedure also is an available.(Redwood City CA) provides the DNA that generates customization to reset the commerce services in library to Maxygen.In addition, the evolution program that the said firm will customize, this program comprises gene rearrangement and the selection to the peptide kind of selecting.

Optigenix, Inc. (Newark, DE) related service that provides plasmid to reset.Optigenix utilizes gene family to obtain wherein to have the sudden change of new property.This target nucleic acid is cloned in the plasmid of Aspergillus (Aspergillus) expression system.The DNA of family of will being correlated with then introduces in the expression system, and the reorganization in the conservative region in the family can take place in the host.Make the mutant DNA expression of gained as a result then, and screening exists character of wanting and the peptide that does not have undesired character from the peptide that wherein produces.

C. screening procedure

After each " evolution " circulation, the peptide of wanting of being expressed by mutator gene is carried out the screening of target signature.The DNA that " candidate " gene is increased and compile to carry out next round resets.Used screening procedure height depends on the peptide and the target signature of carrying out " evolution ".Utilize program well known in the art can select feature as stabilized peptide, biologic activity, antigenicity etc.Single assay method for the biologic activity of the preferred peptide that is used for the inventive method is described elsewhere.

D. the combination of technology

The technician will understand said mutation and the selection technology can make up mutually and make up the most probable peptide molecule that is used for the inventive method with generation with extra program.Thereby the present invention is not limited to any one method that generates peptide, and should be interpreted as being included in any or all of method described herein.For example, begin to carry out in nucleotide sequence, introducing the program of point mutation, carry out the circulation that DNA resets, selects and increase subsequently.The introducing of point mutation of beginning can be used for that diversity is incorporated into shortage should be multifarious in the gene colony, and the circulation of resetting and screening of the DNA subsequently favourable point mutation of will selecting and recombinate.

III. Glycosylase and glycosyltransferase

A. Glycosylase

Glycosylase is the glycosyltransferase of water as acceptor molecule, and generally similarly is glycoside hydrolase.Thermodynamics and kinetics by the control reaction mixture can be used for Glycosylase to become glycosidic link in body profile.Even but under the reaction conditions of modifying, the Glycosylase reaction remains unworkable, and as the result of reaction of reversible transglycosylase and emulative hydrolysis reaction, Glycosylase tends to provide low synthetic result.

Glycosylase can be by keeping its stereochemistry or bring into play function by the key position that ruptures is put upside down its stereochemistry in hydrolytic process to the key position that ruptures in hydrolytic process, thereby respectively Glycosylase is divided into " holding type " Glycosylase or " putting upside down type " Glycosylase.The holding type Glycosylase has two crucial carboxylic moiety that are present in avtive spot, and an one carboxylic acid serves as acid/alkaline catalysts and another serves as nucleophilic reagent, and for putting upside down the type Glycosylase, the function of a carboxylic acid performance acid, and the function of another performance alkali.

The activity of determining any Glycosylase is well known in the art with being connected specific method, comprises the HPLC rules (Jacob and Scudder, 1994, Methods in Enzymol.230:280-300) of simplification.General discussion for Glycosylase and Glycosylase processing can be found in Glycobiology, A Practical Approach, (1993, Fukuda and Kobataeds., Oxford University PressInc., New York).

The Glycosylase that is used for the present invention is including, but not limited to sialidase, tilactase, the inscribe glycanase, mannosidase (is α and β Man I, Man II and Man III), xylosidase, fucosidase, edaphic bacillus species (Agrobacteriumsp.) beta-glucosidase enzyme, muck cellulomonas cartae (Cellulomonas fimi) mannosidase 2A, the Humicolainsolens Glycosylase, sulfolobus solfataricus (Sulfolobus solfataricus) Glycosylase and Bacillus licheniformis (Bacillus licheniformis) Glycosylase.

The selection that is used for the present invention's fucosidase depends on being connected of Fucose and other molecules.Many specificitys that are used for the Alpha-Fucosidase of the inventive method are that those skilled in the art are well-known, and the variant of many fucosidases also is commercial (Glyko, Novato, the CA that buys; PROzyme, San Leandro, CA; Calbiochem-NovabiochemCorp., San Diego, CA etc.).The target Alpha-Fucosidase is including, but not limited to from Turbo cornutus, Charonia lampas, sudden peal of thunder bulging bacterium (Bacillusfulminans), aspergillus niger (Aspergillus niger), perfringens carboxylic bacterium (Clostridium perfringens), ox kidney (Glyko), chicken gizzard (people such as Tyagarajan, 1996, Glycobiology 6:83-93) α-Fucose Portugal enzyme and from cassava pseudomonas (Xanthomonas manihotis) (Glyko, Alpha-Fucosidase II PROzyme).The chicken gizzard fucosidase is useful especially for removing the core Fucose the glycan that connects from N-.

B. glycosyltransferase

Glycosyltransferase is with the interpolation to the non-reduced end of the oligosaccharides of protein, glycopeptide, lipid or glycolipid or growth of the sugar (donor NDP-sugar) of progressively mode catalytic activation.The glycopeptide that N-connects is the oligosaccharides donor Dol-PP-NAG that is connected with lipid by transferring enzyme ₂Glc ₃Man ₉Carrying out overall (enblock) shifts and subsequently core is pruned and synthetic.In this case, some is different for the characteristic and subsequently dirt settling of " core " sugar.A large amount of glycosyltransferases is as known in the art.

Can be any glycosyltransferase that can utilize modification sugar as long as be used for the present invention's glycosyltransferase as saccharide donor.The example of this kind of enzyme comprises the glycosyltransferase of LeloirShi approach, as galactosyltransferase, N-acetyl-glucosamine transferring enzyme, N-acetylgalactosamine transferring enzyme, fucosyltransferase, sialyltransferase, mannose transferase, xylosyltransferase, glucuronyl transferase etc.

Synthetic for the enzymatic sugar that relates to glycosyltransferase reaction, can clone or separate glycosyltransferase from any source.Many clones' glycosyltransferase and polynucleotide sequence thereof are known.Referring to as people such as Taniguchi, 2002, Handbook of glycosyl transferases andrelated genes, Spr inger, Tokyo.

The nucleotide sequence of glycosyltransferase aminoacid sequence and encoding glycosyl transferring enzyme also is found in the various public databases, comprises GenBank, Swiss-Prot, EMBL etc., wherein deducibility aminoacid sequence from this nucleotide sequence.

Can be used for glycosyltransferase in the inventive method including, but not limited to galactosyltransferase, fucosyltransferase, Transglucosylase, N-acetylgalactosamine transferring enzyme, N-acetyl-glucosamine transferring enzyme, glucuronyl transferase, sialyltransferase, mannose transferase, glucuronyl transferase, galacturonic acid transferring enzyme and oligosaccharyl transferase.Suitable glycosyltransferase comprises what those obtained from eukaryote and prokaryotic organism.

The DNA of encoding glycosyl transferring enzyme can by chemosynthesis, by screening from the reverse transcription thing of the mRNA of suitable cell or cloned culture, obtain from the genomic library of suitable cell or by making up these programs by screening.Screening useful source to mRNA or genomic dna carries out from the oligonucleotide probe of glycosyltransferase nucleotide sequence.Probe can carry out mark and be used for conventional hybridization assays according to known program with detectable mark, this mark as but be not limited to fluorophor, radioactive atom or chemiluminescent groups.Selectively, the glycosyltransferase nucleotide sequence can pass through using polymerase chain reaction (PCR) program and obtain, and wherein the Oligonucleolide primers of PCR produces from the glycosyltransferase nucleotide sequence.Referring to people's such as Mullis U.S.Pat.No.4,683,195 and the U.S.Pat.No.4 of Mullis, 683,202.

Glycosyltransferase can be synthetic in the carrier transformed host cells of the DNA that contains the encoding glycosyl transferring enzyme.Carrier is reproducible DNA construct.Carrier can be used for the DNA of amplification coding glycosyltransferase and/or expresses the DNA of encoding glycosyl transferring enzyme.Expression vector is reproducible DNA construct, and wherein the dna sequence dna of encoding glycosyl transferring enzyme is to be operably connected on the suitable control sequence, and this control sequence can realize the expression of glycosyltransferase in appropriate host cell.To depend on the method for transformation of the host of selection and selection to the needs of this control sequence and change.Usually, control sequence comprises that the sequence of selectable operator gene sequence that transcripting promoter, control transcribes, the suitable mRNA ribosome bind site of coding and control transcribes the sequence with translation termination.Amplification vector does not need to express the control texture territory.Required is just common by replication orgin ability of duplicating in the host of giving and the selection gene that promotes transformant identification.

1. fucosyltransferase

In some embodiments, the glycosyltransferase that is used for the inventive method is a fucosyltransferase.Fucosyltransferase is well known to a person skilled in the art.Exemplary fucosyltransferase comprises the L-Fucose from the enzyme of GDP-Fucose to the hydroxy position transfer of acceptor saccharide.The fucosyltransferase that the sugar of non-nucleotide is transferred in the acceptor also can be used among the present invention.

In some embodiments, acceptor saccharide for example is the GlcNAc in Gal β in the oligosaccharides glucosides (1 → 3,4) the GlcNAc beta-yl group.The suitable fucosyltransferase of this reaction comprises the Gal β (1 → 3 that at first characterizes from human milk, 4) GlcNAc β 1-α (1 → 3,4) fucosyltransferase (FTIIIE.C.No.2.4.1.65) (referring to people such as Palcic, Carbohydrate Res.190:1-11 (1989); People such as Prieels, J.Biol.Chem.256:10456-10463 (1981); With people such as Nunez, Can.J.Chem.59:2086-2095 (1981)) and be found in Gal β (1 → 4) GlcNAc β-α fucosyltransferase (FTIV, FTV, FTVI) in the human serum.Also characterized saliva acidic group α (2 → 3) Gal β (1 → 3) GlcNAc β fucosyltransferase FTVII (E.C.No.2.4.1.65).Gal β (1 → 3,4) GlcNAc β 1-α (1 → 3,4) recombinant forms of fucosyltransferase has also carried out characterizing (referring to people such as Dumas, people such as Bioorg.Med.Letters1:425-428 (1991) and Kukowska-Latallo, Genes and Development 4:1288-1303 (1990)).Other exemplary fucosyltransferases comprise as α 1,2 fucosyltransferase (E.C.No.2.4.1.69).The enzymatic fucosylation can be by being described in people such as Mollicone, and the method in Eur.J.Biochem.191:169-176 (1990) or the U.S. Patent No. 5,374,655 is carried out.

2. galactosyltransferase

In another group embodiment, glycosyltransferase is a galactosyltransferase.Exemplary galactosyltransferase comprises α (1,3) galactosyltransferase (E.C.No.2.4.1.151, referring to as people such as Dabkowski, people such as Transpl ant Proc.25:2921 (1993) and Yamamoto, Nature 345:229-233 (1990), (GenBank j04989, the people such as Joziasse of ox, J.Biol.Chem.264:14290-14297 (1989)), (GenBankm26925 of mouse; People such as Larsen, Proc.Nat ' 1.Acad.Sci.USA 86:8227-8231 (1989)), (the GenBank L36152 of pig; People such as Strahan, Immunogenetics41:101-105 (1995)).Another kind of

suitable α

1,3 galactosyltransferase is to participate in (E.C.2.4.1.37, people such as Yamamoto, the J.Biol.Chem.265:1146-1151 (1990) (people's)) that blood B group antigen forms.

Be equally applicable in the inventive method is β (1,4) galactosyltransferase, this enzyme comprise as E.C.2.4.1.90 (LacNAc synthetic enzyme) and E.C.2.4.1.22 (lactose synthetase) (ox (D ' people such as Agostaro, Eur.J.Biochem.183:211-217 (1989)), people's (people such as Masri, Biochem.Biophys.Res.Commun.157:657-663 (1988)), (the people such as Nakazawa of mouse, J.Biochem.104:165-168 (1988)), and E.C.2.4.1.38 and ceramide galactosyltransferase (E.C.2.4.1.45, people such as Stahl, J.Neurosci.Res.38:234-242 (1994).Other suitable galactosyltransferases comprise as

α

1,2 galactosyltransferase (coming grain wine fragmentation sugar yeast (Schizosaccharomyces pombe) freely, people such as Chapell, Mol.Biol.Cell5:519-528 (1994)).For further suitable galactosyltransferase, referring to people such as Taniguchi (2002, Handbook of Glycosyl transferases andRelated Genes, Spr inger, Tokyo), and people such as Guo (2001, Glycobiology, 11 (10): 813-820) and people (1998, J Biochem.123:1000-1009) such as Breton.

From cloned genes, produce T by genetically engineered as enzyme GalNAc _I-XIVProtein be well-known.Referring to as U.S.Pat.No.4,761,371.A method comprises concentrates enough samples, determines the aminoacid sequence of enzyme then by the N-end sequencing.Then this information is used for separating the cDNA clone of coding total length (membrane-bound) transferring enzyme, this cDNA causes the synthetic of complete active enzyme after being cloned in and expressing among the insect cell line Sf9.Then by to amino acid whose semi-quantitative analysis around the known glycosylation site in 16 different proteins and be accompanied by to the external glycosylation research of synthetic peptide receptor-specific with definite enzyme.This research has proved that some amino-acid residue is (overrepresent) that high frequency exists in glycosylated peptide section, and has than other amino acid moieties remarkable influence more for the acceptor effect around the residue of the specific position of glycosylated Serine and threonine residues.

3. sialyltransferase

Sialyltransferase is the another kind of glycosyltransferase that is used for reconstitution cell of the present invention and reaction mixture.The example that is applicable to sialyltransferase of the present invention comprises ST3GalIII (as rat or people's ST3GalIII), ST3GalIV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II and ST6GalNAc III (nomenclature that is used for sialyltransferase herein is described in people such as Tsuji, among the Glycobiology 6:-(1996)).Exemplary α (2, the 3) sialyltransferase that is called α (2,3) sialyltransferase (E.C.2.4.99.6) can be transferred to sialic acid on the non-reduced terminal Gal of Gal β 1 → 3Glc disaccharides or glucosides.Referring to people such as Van den Eijnden, J.Biol.Chem.256:3159 (1981), people such as Weinstein, people such as J.Biol.Chem.257:13845 (1982) and Wen, J.Biol.Chem.267:21011 (1992).Another exemplary α 2,3-sialyltransferase (E.C.2.4.99.4) can be transferred to sialic acid on the non-reduced terminal Gal of disaccharides or glucosides.Referring to people such as Rearick, people such as J.Biol.Chem.254:4444 (1979) and Gillespie, J.Biol.Chem.267:21004 (1992).Further exemplary enzyme comprises Gal-β-1,4-GlcNAc-2,6 sialyltransferases (referring to people such as Kurosawa, Eur.J.Biochem.219:375-381 (1994)).

Preferably, glycosylation for the sugar of glycopeptide, sialyltransferase can be transferred to sialic acid sequence Gal β 1,4GlcNAc-, Gal β 1,3GlcNAc-or Gal β 1, on the 3GalNAc-, terminal sialic modal penult sequence (referring to table 8) on the promptly sialylated fully sugared structure.Sialic acid can be transferred to α 2,3Gal β 1, the α 2 of 4GlcNAc, the 8-sialyltransferase also can be used in the method for the present invention.

Table 8. Gal β 1, the 4GlcNAc sequence is as the sialyltransferase of receptor substrate

1)Goochee?et?al.,Bio/Technology?9：1347-1355(1991)

2)Yamarmoto?et?al.,J.Biochem.120：104-110(1996)

3)Gilbert?et?al.,J.Biol.Chem.271：28171-28276(1996)

An example that is used for the sialyltransferase of the inventive method is ST3Gal III, and it is also referred to as α (2,3) sialyltransferase (E.C.2.4.99.6).This enzyme catalysis sialic acid is to Gal β 1,3GlcNAc or Gal β 1, the transfer of the Gal of 4GlcNAc glucosides (referring to as people such as Wen, J.Biol.Chem.267:21011 (1992); People such as Van den Eijnden, J.Biol.Chem.256:3159 (1991)), and the oligosaccharides that l-asparagine connects in the responsible glycopeptide is sialylated.Sialic acid is connected with Gal, thereby forms α between two sugar-connection.Key between the sugar (connection) is between the 3-position of the 2-position of NeuAc and Gal.This specific enzyme can separate (people such as Weinstein, J.Biol.Chem.257:13845 (1982)) from rats'liver; People's cDNA (people (1993) J.Biol.Chem.268:22782-22787 such as Sasaki; Kitagawa ﹠amp; Paulson (1994) J.Biol.Chem.269:1394-1401) and genome (people (1996) J.Biol.Chem.271:931-938 such as Kitagawa) dna sequence dna be known, thereby promoted by recombinant expressed production to this enzyme.In a preferred embodiment, sialylated method of the present invention is used rat ST3GalIII.

The example of the sialyltransferase of the method that is used to ask for protection be from the CST-I of campylobacter (Campylobacter) (referring to as U.S. Patent No. 6,503,744,6,096; 529 and 6,210,933 and WO99/49051; and the U.S. Patent application of announcing 2002/2,042,369).This enzyme catalysis sialic acid is to Gal β 1,4Glc or Gal β 1, the transfer of 3GalNAc.Be used for other exemplary sialyltransferases of the present invention and comprise that those are isolating from campylobacter jejuni jejunum subspecies (Campylobacter jejuni), comprise α (2,3) sialyltransferase.Referring to as WO99/49051.

Other sialyltransferases (comprise those list in the table 8) also be used for economical and effectively large scale process to carry out sialylated to commercially important glycopeptide.As the simple inspection of the effectiveness of finding these other enzyme, make each enzyme (1-100mU/mg protein) and asialylated-α of various amounts ₁AGP (1-10mg/ml) reacts, and with respect to each or both of ST6Gal I, the ST3Gal III of ox glycopeptide is carried out sialylated ability with the comparison object sialyltransferase.Selectively, the oligosaccharides of other glycopeptides that enzymatic from the peptide main chain can be discharged or glycopeptide or N-connection replaces asialylated-α ₁AGP is used to carry out this estimation.To have sialyltransferase that the oligosaccharides that more effectively N-of glycopeptide is connected than ST6Gal I carries out sialylated ability and be used for the sialylated extensive practice process of peptide (as in this disclosure to as illustrated in the ST3Gal III).

4. other glycosyltransferases

It should be appreciated by those skilled in the art that and other glycosyltransferase can be substituted in the similar transferring enzyme circulation, as sialyltransferase is described in detail.Especially, glycosyltransferase also can be as Transglucosylase, as Alg8 (people such as Stagljov, Proc.Natl.Acad.Sci.USA91:5977 (1994)) or Alg5 (people such as Heesen, Eur.J.Biochem.224:71 (1994)).

The N-acetylgalactosamine transferring enzyme also can be used in the practice of the present invention.Suitable N-acetylgalactosamine transferring enzyme is including, but not limited to α (1,3) N-acetylgalactosamine transferring enzyme, β (1,4) N-acetylgalactosamine transferring enzyme (people such as Nagata, people such as J.Biol.Chem.267:12082-12089 (1992) and Smith, J.Biol.Chem.269:15162 (1994)) and peptide N-acetylgalactosamine transferring enzyme (people such as Homa, J.Biol.Chem.268:12609 (1993)).Suitable N-acetyl-glucosamine transferring enzyme comprises GnT-I (2.4.1.101, people such as Hull, BBRC176:608 (1991)), GnT-II, GnT-III (people such as Ihara, J.Biochem.113:692 (1993)), GnT-IV, GnT-V (people such as Shoreibah, J.Biol.Chem.268:15381 (1993)) and GnT-VI, the N-acetyl-glucosamine transferring enzyme that O-connects (people such as Bierhuizen, Proc.Natl.Acad.Sci.USA 89:9326 (1992)), N-acetyl-glucosamine-1-phosphotransferase (people such as Rajput, Biochem is (1992) J.285:985) and hyaluronic acid synthetase (hyaluronan synthase).

Mannose transferase can be used for shifting the seminose part of modification.Suitable mannose transferase comprises α (1,2) mannose transferase, α (1,3) mannose transferase, α (1,6) mannose transferase, β (1,4) mannose transferase, Dol-P-Man synthetic enzyme, Och1 and Pmt1 (referring to people such as Kornfeld, Annu.Rev.Biochem.54:631-664 (1985)).

Xylosyltransferase also can be used among the present invention.Referring to as people such as Rodgers, Biochem.J., 288:817-822 (1992); With people such as Elbain, U.S. Patent No., 6,168,937.

Other suitable glycosyltransferase looping discriptions are in people such as Ichikawa, people such as JACS114:9283 (1992), Wong, people Carbohydrates and Carbohydrate Polymers such as J.Org.Chem.57:4343 (1992) and Ichikawa, Yaltami, ed. (ATLPress, 1993).

Procaryotic glycosyltransferase also can be used in the practice of the present invention.This glycosyltransferase comprises participation fat oligosaccharides (LOS) synthetic enzyme, and this LOS can be produced by many gram negative bacteriums.LOS generally has simulation on human epithelial cell surface or the terminal glycan sequence of the glycoconjugate of finding (people such as Preston, Critical Reviews in Microbiology 23 (3): 139-180 (1996)) in host's secretory product.This kind of enzyme is including, but not limited to the protein as the rfa operon of intestinal bacteria and Salmonella typhimurium (Salmonella typhimurium) species, this comprises β 1,6 galactosyltransferases and β 1,3 galactosyltransferase are (referring to as EMBL Accession Nos.M80599 and M86935 (intestinal bacteria); EMBL Accession No.S56361 (Salmonella typhimurium)), Transglucosylase (Swiss-Prot Accession No.P25740 (intestinal bacteria), β 1,2-Transglucosylase (rfaJ) (Swiss-Prot Accession No.P27129 (intestinal bacteria) and Swiss-Prot Accession No.P19817 (Salmonella typhimurium)) and β 1,2-N-acetylgucosamine transferase (rfaK) (EMBL Accession No.U00039 (intestinal bacteria)).The known glycosyltransferase of other aminoacid sequences comprises that those are encoded as rfaB by operon, this characterizes in biology below, as the rh1 operon of Klebsiella pneumonia (Klebsiella pneumoniae), intestinal bacteria, Salmonella typhimurium, intestines Salmonellas (Salmonella enterica), yersinia entero-colitica (Yersinia enterocolitica), Mycobacterium leprosum and Pseudomonas aeruginosa (Pseudomonas aeruginosa).

Being equally applicable to of the present invention is to participate in producing containing Lacto-N-neo-tetraose (lacto-N-neotetraose) D-galactosyl-β-1; 4-N-ethanoyl-D-glucose amido-β-1; 3-D-galactosyl-β-1; 4-D-glucose and Pk blood group trisaccharide sequence D-galactosyl-α-1; 4-D-galactosyl-β-1; the glycosyltransferase of the structure of 4-D-glucose; this structure has carried out identifying (people such as Scholten, J.Med.Microbiol.41:236-243 (1994)) in the LOS of mucosal disease substance Diplococcus gonorrhoeae (Neisseria gonnorhoeae) and Neisseria meningitidis (N.meningitidis).Coding from Neisseria meningitidis and Diplococcus gonorrhoeae participates in the gene of the biosynthetic glycosyltransferase of these structures from Neisseria meningitidis immunologic pattern L3 and L1 (people such as Jennings, Mol.Microbiol.18:729-740 (1995)) and among the Diplococcus gonorrhoeae mutant F62 (Gotshlich, J.Exp.Med.180:2181-2190 (1994)) identify.In Neisseria meningitidis, the seat of being made up of 3 gene lgtA, lgtB and lgE is coded in and adds last 3 necessary glycosyltransferases of sugar people such as (, J.Biol.Chem.271:19166-73 (1996)) Wakarchuk in the Lacto-N-neo-tetraose.Proved the enzymic activity of lgtB and lgtA gene product recently, thereby first direct evidence for their glycosyltransferase function (people such as Wakarchuk, J.Biol.Chem.271 (45): 28271-276 (1996)) is provided.In Diplococcus gonorrhoeae, 2 extra genes are arranged, be about to the lgtD in 3 positions of terminal galactose that β-D-GalNAc adds the Lacto-N-neo-tetraose structure to and terminal α-D-Gal added to lgtC in the lactose element of LOS of brachymemma, thereby generated Pk blood group antigenic structure (Gotshlich (1994) sees above).In Neisseria meningitidis, immunologic pattern L1 also expresses P separately ^kBlood group antigen and shown to have the lgtC gene (people (1995) such as Jennings sees above).Neisseria gonorrhoeae glycosyltransferase and genes involved also are described in USPN5, in 545,553 (Gotshlich).Also characterized α 1,2-fucosyltransferase and α 1,3-fucosyltransferase (people such as Martin, J.Biol.Chem.272:21349-21356 (1997)) from helicobacter pylori (Helicobacterpylori).Be used for the present invention equally be campylobacter jejuni jejunum subspecies glycosyltransferase (referring to people such as Taniguchi, 2002, Handbookof glycosyltransferases and related genes, Springer, Tokyo).

B. sulfotransferase

The present invention also provides the method that generates the peptide that comprises sulfating numerator, and this Sulfated molecule comprises as Sulfated polysaccharide such as heparin, heparitin vitriol, carrageenan and related compound.Suitable sulfotransferase comprise as chrondroitin-6-sulfotransferase (by people such as Fukuta, the chicken cDNA that J.Biol.Chem.270:18575-18580 (1995) describes; GenBankAccession No.D49915), glycosaminoglycan N-acetyl-glucosamine N-deacetylase/N-sulfotransferase 1 (people such as Dixon, Genomics 26:239-241 (1995); UL18918) and glycosaminoglycan N-acetyl-glucosamine N-deacetylase/N-sulfotransferase 2 (people such as Orellana, the mouse cDNA that describes among the people such as J.Biol.Chem.269:2270-2276 (1994) and Eriksson, J.Biol.Chem.269:10438-10443 (1994); Be described in the people cDNA that describes among the GenBank Accession No.U2304).

C. cell bonded glycosyltransferase

In another embodiment, the enzyme that is used for the inventive method is a cell bonded glycosyltransferase.Although the glycosyltransferase of many solubilities is known (referring to as U.S.Pat.No.5,032,519), normally film combining form of glycosyltransferase when combining with cell.Yan Jiu many membrane bound enzymes can be thought integral protein so far; Be that they can not be by supersound process discharge from film and need stain remover so that its dissolving.Identified surperficial glycosyltransferase on the surface of vertebrates and invertebral zooblast, and had recognized that these surperficial transferring enzymes can keep catalytic activity under physiological conditions.Yet the function that the cell surface glycosyltransferase is more approved is iuntercellular identification (Roth, 1990, Molecular Approaches toSupracellular Phenomena).

Developed the method that changes by the glycosyltransferase of cell expressing.For example, people such as Larsen, Proc.Natl.Acad.Sci.USA86:8227-8231 (1989) has reported the genetic method of the cDNA sequence of separating clone, this clone's the cDNA sequence decision cell surface oligosaccharide structure and the expression of homologous glycosyltransferase thereof.To be transfected into from the cDNA library that mRNA generates the COS-1 cell, this mRNA is from known expression UDP-semi-lactosi:. β .-D-galactosyl-1,4-N-ethanoyl-D-glucosaminide α-1, the mouse cell line of 3-galactosyltransferase is isolating.Cultivate this cells transfected then and measure α 1-3 galactosyltransferasactivity activity.

People such as Francisco, Proc.Natl.Acad.Sci.USA 89:2713-2717 (1992) discloses the method that β-Nei Xiananmei is anchored to the intestinal bacteria outside surface.Produced treble fusions, the result has caused the active β-Nei Xiananmei molecule of surface bonding, this treble fusions is by the signal sequence of (i) outer membrane protein, (ii) outer membrane protein stride membrane portions and (iii) complete ripe β-Nei Xiananmei sequence is formed.Yet the Francisco method only limits to the prokaryotic cell prokaryocyte system, and admits as the author, needs complete triplen to bring into play correct function.

D. merge enzyme

In another exemplary embodiment, method of the present invention is utilized fusogenic peptide, and this fusogenic peptide has the glycopeptide conjugate synthetic enzymic activity of wanting above a kind of participation.This fused protein can comprise as with the catalytic activity structural domain of the catalytic activity structural domain bonded glycosyltransferase of auxiliary enzymes.A step during but the catalyst structure domain catalysis of this auxiliary enzymes such as nucleotide sugar form, this nucleotide sugar is the donor of glycosyltransferase, perhaps catalysis involved in sugar based transferase round-robin reaction.For example, the polynucleotide of encoding glycosyl transferring enzyme are combined with the polynucleotide that coding participates in nucleotide sugar synthetic enzyme by reading frame (in-frame).But the fusogenic peptide of gained catalysis nucleotide sugar synthetic not only as a result then, and sugar moieties can be transferred on the acceptor molecule.Fusogenic peptide can be the two or more cyclophorases that are connected to an effable nucleotide sequence.In other embodiment, fusogenic peptide comprises the catalytic activity structural domain of two or more glycosyltransferases.Referring to as U.S. Patent No. 5,641,668.The glycopeptide that the present invention modifies can be easy to design and produce (referring to as the PCT patent application PCT delivered as WO 99/31224 on June 24th, 1999/CA98/01180) with various suitable fusogenic peptides.

E. fixed enzyme

Except that cell bonded enzyme, the present invention also provides the application of being fixed in the enzyme on solid and/or the solubility support.In an exemplary embodiment, the glycosyltransferase that provides the method according to this invention to put together by intact glycosyl linker and PEG.This PEG-linker-enzyme conjugate selectively is attached on the solid support.The operation of reaction mixture and the purifying of reaction product have been simplified in the application of the enzyme of solid support in the methods of the invention, and make that also this enzyme is easy to reclaim.This glycosyltransferase conjugate is applied in the method for the present invention.Other combinations of enzyme and support are conspicuous for those skilled in the art.

F. the mutagenesis of glycosyltransferase

The new form that is used for glycosyltransferase, sialyltransferase, sulfotransferase and any other enzyme of method of the present invention can generate with previously described any method and the well-known additive method of those skilled in the art.Interested especially is to have the receptor-specific of change and/or the transferring enzyme of donor specific.Same interested is the enzyme with higher conversion and higher stability etc.

When the sequence of peptide is known, can use the design induced-mutation technique of reasoning.Because it is known being used for sequence and many tertiary structures of transferring enzyme of the present invention and Polyglucosidase, so these enzymes can be used for the sudden change design of reasoning ideally.For example, the catalytic site of enzyme is suddenlyd change to change the donor and/or the receptor-specific of enzyme.

The detailed tertiary structure data of glycosyltransferase and Glycosylase lytic enzyme also make these enzymes can be used to comprise the sudden change of structural domain exchange ideally.Glycosyltransferase and Glycosylase lytic enzyme are modular enzyme (referring to Bourne and Henrissat, 2001, Current Opinion inStructural Biology 11:593-600).Glycosyltransferase can be divided into two classes: GT-A and GT-B based on its structure.The glycosyltransferase of GT-A class comprises 2 different structural domains, and one participates in the Nucleotide combination and another participation receptors bind.Thereby people can be easily will be merged to generate new gene by reading frame from the dna sequence dna in the coding structure territory of a gene and structural domain from second gene, and this new genes encoding has the protein of new acceptor/donor specific.This structural domain exchange can comprise sugared module and other supplementary structure territories extraly.

Aforesaid random mutation and/or orthogenesis technology also can be used for generating glycosyltransferase and the Glycosylase that is used for new form of the present invention.

IV. external and expression in vivo system

A. produce the cell of glycopeptide

The effect of glycosyltransferase is the glycosylated key of peptide, thereby the difference in any given cell type in the expression of a cover glycosyltransferase can influence the glycosylation pattern of any given peptide that produces in this cell.For the glycosylated summary of host cell dependency peptide, referring to Kabata and Takasaki, " Structure and Biosynthesis of CellSurfaceCarbohydrates ", Cell Surface Carbohydrates and CellDevelopment, 1991, pp.1-24, Eds.Minoru Fukuda, CRC Press, Boca Raton, FL.

According to disclosure of the present invention, only to have the necessary reconstruct degree of the glycosylated peptide of wanting relevant with generation for the cell type that produces peptide.For example, having the number of the number of the necessary enzymatic digestion of the glycosylated peptide of wanting reaction and order and enzymatic building-up reactions and order in external generation will depend on the structure of glycan on the peptide that is produced by particular cell types and change.Although the present invention never should be interpreted as being confined to producing peptide from any specific cell type (being included in cell type disclosed herein), but the discussion to several cell systems is provided now, and this discussion has been determined effectiveness of the present invention and has been generated the independence of the cell type of peptide.

Usually, in order to express this peptide from the nucleic acid of encoded peptide, this nucleic acid must be integrated in the expression cassette, this expression cassette comprises the peptide-coding sequence that promoter element, terminator element and between are operably connected.This expression cassette operationally is connected with carrier.For this purpose, connector or linker can be used to connect nucleotide fragments, can comprise that maybe other operations are with the unnecessary Nucleotide of the restriction site, the removal that facilitate, removal restriction site etc.For this purpose, can comprise vitro mutagenesis, primer reparation, restricted cutting, annealing, alternative as conversion and transversion once more.Shuttle vectors has duplicate necessary genetic elements in cell.Some carriers only can duplicate in prokaryotic organism, perhaps can duplicate in prokaryotic organism and eukaryote simultaneously.This plasmid expression vector will be kept in one or more dubbing systems, be preferably two dubbing systems, and this makes it possible to stably keep in yeast host cell and purpose stable maintenance in prokaryotic hosts in order to clone for the purpose of expressing.Many carriers with different characteristics are commercial buying.Carrier is plasmid or phage normally, but also can be clay or minichromosome.Be that many commercial carriers of buying will have the promotor and the terminator of already present expression cassette and can be used for inserting the multi-link position point of the encoding sequence of target peptide easily.The shuttle vectors that contains expression cassette is transformed in intestinal bacteria, and this carrier duplicates in fission process to generate carrier formulation therein, and this carrier formulation is enough to transform the host cell of the expression system with selection.Aforesaid method is well known in the art, and the rules of implementing can be found in people (2001, Molecular Cloning:ALaboratory Manual, Cold Spring Harbor Laboratory, New York) such as Sambrook.

This carrier is in case behind the purifying, then be transformed in the cell of expression system from make its expanded cells.Transform the characteristic that rules depend on cell type and carrier.Transformant is grown on the suitable nutritional medium, and under selection pressure, keep maintenance in due course with source DNA in guaranteeing.When expression is induction type, can make the yeast host growth to obtain highdensity cell, abduction delivering then.The sophisticated heterologous peptides of excretory can be gathered in the crops by any ordinary method, and can wait purifying by chromatography, electrophoresis, dialysis, solvent-solvent extraction.

Molecule clone technology is well known in the art.Further, the technology of molecular cloning program can be found in people such as Sambrook (2001, Molecular Cloning:ALaboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); People such as Glover (1985, DNA Cloning:A PracticalApproach, I and II volume); People such as Gait (1985, OligonucleotideSynthesis); Hames and Higgins (1985, Nucleic Acid Hybridization); Hames and Higgins (1984, Transcription And Translation); People such as Freshney (1986, Animal Cell Culture); Perbal (1986, Immobilized CellsAnd Enzymes, IRL Press); Perbal (1984, A Practical Guide ToMolecular Cloning); People such as Ausubel (2002, Current Protocols inMolecular Biology, John Wiley ﹠amp; Sons, Inc.).

B. fungi and yeast

The peptide that produces in yeast is glycosylated, and the glycan structures that exists thereon mainly is a high mannose structures.Under the situation of N-glycan, the glycan structures that produces in yeast can contain nearly 9 or more mannose residue, and this structure can contain or not contain extra sugar.Example by the type of glycan on the peptide of yeast cell generation shows in Fig. 4 left side.No matter the number of mannose residue and the type and the complicacy of the extra sugar of interpolation thereon, comprise as shown in Figure 4 three seminose core textures as the N-glycan of the composition of the peptide that produces in the yeast cell.When the glycan structures on the peptide that is produced by yeast cell is high mannose structures, removing all seminoses at the suitable mannosidase (except that molecule of the three seminose cores that comprise glycan) from molecule of external use is simple thing for technicians, thereby has generated the peptide that is attached with three basic seminose core textures thereon.At present, utilize in this area the available technology and utilize disclosure of the present invention, add extra sugar moieties to three basic seminose core texture enzymatic ground to have the peptide that is attached with the glycan structures of wanting thereon with generation be simple thing external.Similarly, when the peptide that is produced by yeast cell also comprised high mannose structures thereon except that other complicated sugar that adhere to, it was simple things to obtain three basic seminose core textures that all extra sugar (comprising extra mannose residue) are removed on enzymatic ground.In case produced three basic seminose core textures, then may have the glycosylated peptide of wanting according to the explanation generation that provides herein.

" yeast " meaning refers to ascosporogenous yeast (Endomycetales), basidiosporangium yeast and belongs to the yeast of Fungi Imperfecti (Blastomycetes).Ascosporogenous yeast can be divided into two sections, i.e. Spermophthoraceae and sugar yeast section.The section in back comprises 4 subfamilies, promptly fragmentation sugar yeast subfamily (as the fragmentation saccharomyces), take Xun Shi subfamily, Lipomycetoideae and sugar yeast subfamily (as pichia belong to, Crewe Vickers yeast belong and saccharomyces).The basidiosporangium yeast comprises Leucosporidium, full genus of red winter, locks and throw yeast belong, Filobasidium and Filobasidiella.The yeast that belongs to Fungi Imperfecti can be divided into two sections, i.e. Sporobolomycetaceae (as Sporobolomyces, Bu Shi bullet spore yeast belong) and Cryptococcaceae (as mycocandida).The present invention is interested especially to be the species of saccharomyces, pichia genus, Aspergillus, Trichoderma, Crewe Vickers yeast belong, especially newborn Crewe Vickers yeast (K.lactis) and fruit bat Crewe Vickers yeast (K.drosophilum), mycocandida, Hansenula anomala genus, fragmentation saccharomyces, Yarrowia and ｀ Chrysosporium (Chrysoporium).Because the zymic classification may change in the future, so for the purposes of the present invention, the yeast definition should be as people such as Skinner, and eds. (1980) Biology and Activities of Yeast (Soc.App.Bacteriol.Symp.SeriesNo.9) describes.

Except that previously described, those skilled in the art may be familiar with the operation of zymic biology and yeast genetics.Referring to as people such as Bacila, eds. (1978, Biochemistryand Genetics of Yeast, Academic Press, New York); With Rose and Harrison (1987, The Yeast (2 ^NdEd.) Academic Press, London).The method that foreign DNA is incorporated in the yeast host is well known in the art.There are numerous methods to can be used for transformed yeast.It is that (1978, Nature 275 (5676): 104-109) and people (EPO Publication No.45,573 such as Stinchcomb by people such as Hinnen (1978, Proc.Natl.Acad.Sci.USA 75:1919-1933), Beggs that spheroplast transforms; Be incorporated herein by reference herein) introduce.Electroporation is by Becker and Gaurante (1991, Methods Enzymol.194:182-187) introduces, lithium acetate is to be introduced by people (1996, MethodsMol Biol.53:139-145) such as people such as Gietz (2002, Methods Enzymol.350:87-96) and Mount.For the summary of the conversion system of non--sugar yeast, referring to people such as Wang (Crit Rev Biotechnol.2001; 21 (3): 177-218).For the general procedure of Yeast gene engineering, referring to people such as Barr (1989, Yeast geneticengineering, Butterworths, Boston).

Except that wild-type yeast and fungal cell, the yeast and the fungal bacterial strain that have suddenlyd change and/or selected are also arranged, to strengthen the expression of exogenous gene level, the purity of the peptide of gained, translation post-treatment, and the recovery of mature peptide and purity as a result.The expression of exogenous peptide also can be pointed to the emiocytosis approach, as by (referring to Kjeldsen, 2000, Appl.Microbiol.Biotechnol.54:277-286 and the reference quoted therein) as illustrated in the expression of Regular Insulin.Usually, secrete from yeast cell in order to cause exogenous peptide, but application source is from the secretion signal of yeast genes, kill and wound toxin (Stark and Boyd, 1986, EMBO J.5:1995-2002) or α pheromone (Kurjan and Herskowita as those, 1982, Cell 30:933; The secretion signal of gene people such as Brake, 1988, Yeast 4:S436).

About common filamentous fungus, the method for genetic manipulation can be found in Kinghorn and Turner (1992, Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, New York).Suitably the guide of carrier can be found in Martinelli and Kinghorn (1994, Aspergilus:50 years, Elsevier, Amsterdam).

1. sugar yeast

In sugar yeast, the suitable yeast vector that is used to produce peptide comprises YRp7 (people such as Struhl, Proc.Natl.Acad.Sci.USA 76:1035-1039,1978), YEp13 (people such as Broach, Gene 8:121-133,1979), POT carrier (people such as Kawasaki, U.S.Pat.No.4,931,373, be incorporated herein by reference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108,1978) and derivative thereof.The preferred promotor that is used for yeast comprises Yeast sugar glycolysis genetic expression (people such as Hitzeman, J.Biol.Chem.255:12073-12080,1980; Alber and Kawasaki, J.Mol.Appl.Genet.1:419-434,1982; Kawasaki, U.S.Pat.No.4,599,311) or alcohol dehydrogenase gene (people such as Young, Genetic Engineering of Microorganisms forChemicals, people such as Hollaender, (eds.), and p.355, Plenum, New York, 1982; Ammerer, Meth.Enzymol.101:192-201,1983) promotor and ADH2-4 ^cPromotor (people such as Russell, Nature 304:652-654,1983; Irani and Kilgore, U.S. Patent application Ser.No.07/784,653, CA 1,304, and 020 and EP 284 044, be incorporated herein by reference herein).Ceneme also can comprise transcription terminator.Preferred transcription terminator be the TPI1 terminator (Alber and Kawasaki, as above).

The example of this primary yeast-bacterium shuttle vectors comprises Yep24 (people (1979) Gene 8:17-24 such as Botstein), pC1 (people (1984) Proc.Natl.Acad.Sci.USA 81:4642-4646 such as Brake) and Yrp17 (people (1982) J.Mol.Biol.158:157 such as Stnichomb).In addition, plasmid expression vector can be high or low copy number purpose plasmid, and this copy number range is generally about 1～about 200.Under the situation of high copy number order yeast vector, in single host, will there be at least 10 usually, be preferably at least 20 and be no more than about 150 carriers copy usually.Depend on the heterologous peptides of selection, high or low copy number purpose carrier depends on carrier and recombinant peptide and host's effect all be can be wants.Referring to as people such as Brake (1984) Proc.Natl.Acad.Sci.USA 81:4642-4646.DNA construct of the present invention also can be integrated in the yeast genes group by integrating vector.The example of this carrier is as known in the art.Referring to as people such as Botstein (1979) Gene 8:17-24.

Selecting suitable yeast and other microorganism host is within the technical scope of this area to put into practice the present invention.Interested especially is the sugar yeast species: Saccharomyces cerevisiae (S.cerevisae), Ka Ersibai sugar yeast (S.carlsbergensis), saccharification sugar yeast (S.diastaticus), S.douglasii, S.kluyveri, S.norbensis and avette sugar yeast (S.oviformis).When the peptide of selecting yeast host cell to want with expression, appropriate host cell can comprise that those have good secretion capacity, low proteolytic activity and total vitality of subject etc.Yeast and other microorganisms can obtain from various sources usually, comprise YeastGenetic Stock Center, Department of Biophysics and MedicalPhysics, University of California, Berkeley, Calif.; And American type culture collection (American Type Culture Collection), ManassasVA.As for summary, referring to people such as Strathern, eds. (1981, The MolecularBiology of the Yeast Saccharomyces, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

The method that foreign DNA is incorporated in the yeast host is well known in the art.

2. pichia

Production to recombinant peptide is disclosed among PCT application WO 97/17450, WO 97/17451, WO 98/02536 and the WO 98/02565 as host cell with Pichia methanolica.The dna molecular that is used to transform P.methanolica is normally as double-stranded cyclic plasmid preparation, and this plasmid preferably carried out linearizing before transforming.For the peptide production among the P.methanolica, preferably the promotor of plasmid and terminator are the P.methanolica genes, utilize gene (AUG1 or AUG2) as P.methanolica alcohol.Other useful promotors comprise those Protosol synthetic enzyme (DHAS), hydrogenlyase (FMD) and catalase (CAT) gene and those be disclosed in U.S. Patent No. 5,252, in 726.In order to promote the integration of DNA in host chromosome, the whole expression section of plasmid is joined at two ends and host DNA sequence.The preferred selective marker that is used for Pichiamethanolica is a P.methanolica ADE2 gene, this genes encoding ribose phosphoric acid-5-aminooimidazole carboxylase (phosphoribosy1-5-aminoimidazole carboxylase) (AIRC; EC 4.1.1.21), this makes the ade2 host cell to grow when not having VITAMIN B4.For the minimized large-scale industry process of application of wanting to make methyl alcohol, the host cell that two methyl alcohol utilizes gene (AUG1 and AUG2) all to lack is preferred.For the production of excretory peptide, the host cell of vacuole protein enzyme gene (PEP4 and PRB1) disappearance is preferred.Electroporation can be used for promoting to contain the introducing of plasmid in the P.methanolica cell of DNA of target peptide of encoding.Preferably transform the P.methanolica cell with electroporation, the strength of electric field of this electroporation exponential decay is the pulsed electrical field of 2.5～4.5kV/cm, be preferably about the time constant (t) of 3.75kV/cm and 1～40 millisecond, be most preferably about 20 milliseconds.For using Pichia pastoris (Pichia pastoris) carrying out the summary that extensive antibody fragment is produced, referring to people such as Fischer (1999, Biotechnol Appl Biochem.30 (Pt2): 117-120).

3. Aspergillus

The method of expression of peptides is well known in the art in the Aspergillus species, is described in people such as Carrez including, but not limited to those, 1990, and Gene94:147-154; Contreras, 1991, Bio/Technology 9:378-381; People such as Yelton, 1984, Proc.Natl.Acad.Sci.USA 81:1470-1474; People such as Tilburn, 1983, Gene 26:205-221; Kelly and Hynes, 1985, EMBO is J.4:475-479; People such as Ballance, 1983, Biochem.Biophys.Res.Comm.112:284-289; People such as Buxton, 1985, Gene 37:207-214 and U.S.Pat.No.4, in 935,349, integral body is incorporated herein by reference herein.The example that is used for the promotor of Aspergillus can be found in U.S. Patent No. 5,252,726.The bacterial strain that is used for the Aspergillus of peptide expression can be found in U.S. Patent No. 4,935,349.The commercial product of aspergillus niger and aspergillus oryzae (Aspergillus oryzae) exogenous peptide can be buied from Novoenzymes.

4. wood is mould

For the peptide that expression is wanted, the wooden mould advantage that is better than other recombinant host cell species that has.This biology is easy to raised growth, and has and carry out glycosylation and effectively the recombinant mammalian peptide of high yield is secreted into ability in the substratum, thereby makes relatively easy to the separation of peptide.In addition, the glycosylation pattern on the peptide of expression than the peptide of in many other systems, expressing more to people's peptide on similar.Yet, still variant in the glycan structures on the peptide of in these cells, expressing.For example, terminal sialic acid residues is important for the treatment function of peptide in the mammlian system, and this is because these parts have stoped the removing of peptide from mammalian in the existence of glycan structures end.The mechanism that it is believed that the biological half time that sialylated molecule increases be lectin to their identification reduce (Drickamer, 1988, J.Biol.Chem.263:9557-9560).Yet usually the fungal cell does not add extra terminal sialic acid residues on the glycan on the peptide, and therefore in the fungal cell synthetic peptide be asialylated.According to the present invention, this defective can be used on the of the present invention external glycan reconstructing method of describing in detail in other places and remedies.

Comprise T.reesei as the host with the Trichoderma species of producing the peptide that will be reconstructed, as QM6a, ALKO2442 or CBS383.78 (Centraalbureau voorSchimmelcultures, Oosterstraat 1, PO Box 273,3740 AG Baarn, Holland) or ATCC13631 (American type culture collection, Manassas VA, 10852, the U.S., pattern)); Viride (T.viride) (as CBS189.79 (det.W.Gams)); T.longibrachiatum is as CBS816.68 (pattern); T.pseudokoningii is (as MUCL19358; Mycotheque de l ' Universite Catholique de Louvain); T.saturnisporum CBS330.70 (pattern); T.harzianum CBS316.31 (det.W.Gams); T.virgatum (T.pseudokoningii) ATCC24961.Most preferably, the host is T.reesei, and more preferably be T.reesei bacterial strain QM9414 (ATCC26921), RUT-C-30 (ATCC56765) and mutant such as the VTT-D-79125 (Nevalainen that is derived from the high productivity of QM9414, Technical Research Centre of FinlandPublications 26, (1985), Espoo, Finland).

With DNA to wood mould conversion carry out with any technology as known in the art, be included in European patent No.EP0244234, Harkki (1989, Bio/Technology 7:596-601) and Uusitalo (1991, introduce in J.Biotech.17:35-50).The mould cultivation of wood is supported by a large amount of experience of aforementioned technical scale fermentation technique; For example referring to Finkelstein, 1992, Biotechnology of Filamentous Fungi:Technology and Products, Butterworth-Heinemann, publishers, Stoneham, Mass.

5. Crewe Vickers yeast

The yeast that belongs to Crewe Vickers yeast belong is as the peptide of host living beings with the production reorganization.The peptide that is produced by this genus yeast is rennin (European patent 96 430), thaumatin (European patent 96 910), albumin, interleukin-1 ' beta ', TPA, TIMP (European patent 361 991) and albumin derivant (European patent 413 622) with treatment function especially.Interested especially species comprise newborn Crewe Vickers yeast in the Crewe Vickers yeast belong.

The method of express recombinant peptide is well known in the art in Crewe Vickers yeast belong species.The carrier of expression and secretion people recombinant peptide is (Yeh, J.Cell.Biochem.Suppl.14C:68, Abst.H402 as known in the art in Crewe Vickers yeast belong; Fleer, 1990, Yeast 6 (special issue): S449), the program that recombinant peptide transforms and expresses also is known (people such as Ito, 1983, J.Bacteriol.153:163-168; Van den Berg, 1990, Bio/Technology 8:135-139; U.S. Patent No. 5,633,146, WO8304050A1, EP0096910, EP0241435, EP0301670, EP0361991, all integral body are incorporated herein by reference herein).For the summary of shuttling back and forth by gene targeting and plasmid to the genetic manipulation of newborn Crewe Vickers yeast linear DNA plasmid, referring to people such as Schaffrath (1999, FEMS Microbiol Lett.178 (2): 201-210).

6. golden pityrosporion ovale

The fungi Chrysosporium is used to express the external source recombinant peptide recently.Those skilled in the art utilizes golden pityrosporion ovale can be found in (integral body is incorporated herein by reference herein) among the WO 00/20555 with the description of the program of expression exogenous peptide.The species that are specially adapted to expression system include, but are not limited to C.botryoides, C.carmichaelii, C.crassitunicatum, C.europae, C.evolceannui, F.fastidium, C.filiforme, C.gerogiae, C.globiferum, C.globiferum var.articulatum, C.globiferum var.niveum, C.hirundo, C.hispanicum, C.holmii, C.indicum, C.inops, chrysosporium keratinophilum (C.keratinophilum), C.kreiselii, C.kuzurovianum, C.lignorum, C.lobatum, C.lucknowense, C.lucknowense Garg 27K, C.medium, C.medium var.spissescens, C.mephiticum, C.merdarium, C.merdarium var.roseum, C.minor, C.pannicola, chrysosporium parvum (C.parvum), C.parvum var.crescens, C.pilosum, C.peodomerderium, C.pyriformis, C.queenslandicum, C.sigleri, C.sulfureum, C.synchronum, C.tropicum, C.undulatum, C.vallenarense, C.vespertilium and C.zonatum.

Other

Be used for transforming the method for being permitted Wang Shi yeast belong (Schwanniomyces) and be disclosed in European patent 394538.The method that is used to transform Acremonium chrysogenum is by U.S.Pat.No.5, and 162,228 is open.The method that is used to transform neurospora is by U.S.Pat.No.4, and 486,533 is open.Be known that equally the specific expression system of grain wine fragmentation sugar yeast (European patent 385 391).The ordinary method of expression of peptides can be found in Giga-Hama and Kumagai (1977 in fission yeast grain wine fragmentation sugar yeast, Foreign geneexpression in fission yeast:Schizosaccharomyces pombe, Springer, Berlin).

C. mammlian system

As mentioned above, mammalian cell generally produces the heterogeneous mixture of N-glycan structures, and this N-glycan is changing aspect number that is attached to the extra sugar on the three seminose cores and the arrangement.Usually, mammalian cell produces the peptide with complicated glycan structures, as showing on Fig. 3 right side.Utilize method of the present invention, can be reconstructed the peptide that produces in mammalian cell with generation and have the glycosylated peptide of wanting, this reconstruct is by at first identifying the one-level glycan structures, and then determines to realize for the reconstruct glycan structures must remove which sugar.As discussing herein, the sugar that remove will determine to use which kind of nickase, and thereby the accurate step of restructuring procedure will depend on as the one-level glycan structures of initial substrate and change.Be used for the exemplary arrangement that the glycan structures that produces at mammalian cell usually is reconstructed is shown in Fig. 2.N-glycan biosynthetic pathway in the mammalian cell has carried out sufficient sign (summary is seen Moremen, 1994, Glycobiology 4:113-125).Identified that many glycan synthesize necessary enzyme, and separated defective mutational cell line in this enzymatic pathway, comprised Chinese hamster ovary (CHO) clone Lec23 (alpha-glucosidase I defective) and Lec18 (new GlcNAc-TVIII).The glycosylation pattern of the peptide that is produced by these mutant cells changes with respect to normal Chinese hamster ovary celI.As discussing herein, the glycosylation defect in these and other mutant cells can be applied to produce the purpose of the peptide that lacks complicated glycan structures.For example, the peptide that is produced by the Lec23 cell lacks sialic acid residues, thereby needs less enzyme operation for glycan structures being reduced to basic three seminose cores or Man3GlcNAc4.Thereby the peptide that is produced by these cells can serve as the preferred substrate of glycan reconstruct.Those skilled in the art can separate or identify the clone of other glycosylation-defective based on known method, as is described in people such as Stanley, 1990, Somatic Cell Mol.Genet., the method among the 16:211-223.Purpose for the preferred peptide substrate that generates the restructuring procedure that is used for describing herein can be included in the application of the unidentified glycosylation defect clone of the Buddhist monk of those evaluations among the present invention.

The expression vector that is used at mammalian cell expression exogenous peptide is numerous, and is well known in the art.Many mammalian expression vectors are commercial at present can be buied from company, comprise Novagen, Inc (Madison, WI), Gene Therapy Systems (SanDiego, CA), Promega (Madison, WI), ClonTech Inc. (Palo Alto, CA) and Stratagene (La Jolla, CA) etc.

Several mammal cell lines of expressing exogenous peptide of being good at are especially arranged.General mammal cell line is derived from the tumour cell of the immortalization that extracts from Mammals, promptly they can substantially ad infinitum duplicate in substratum.These clones include, but are not limited to CHO, and (Chinese hamster ovary is as CHO-K1; ATCC No.CCL61) and variant, NS0 (mouse myeloma), BNK, BHK570 (ATCC No.CRL 10314), BHK (ATCC No.CRL 1632), Per.C6 ^TM(people's cell of immortalization, Crucell N.V., Leiden, Holland), COS-1 (ATCC No.CRL 1650), COS-7 (ATCC No.CRL 1651), HEK 293, mouse Lcell, T lymphoid cell line, BW5147 cell and MDCK (Madin-Darby dog kidney), HeLa (people's), A549 (people's lung cancer), 293 (ATCC No.CRL 1573; People such as Graham, 1977, Gen.Virol.36:59-72), BGMK (buffalo grivet kidney (BuffaloGreen Monkey kidney), Hep-2 (people's epiderm-like laryngocarcinoma), LLC-MK ₂(African green monkey kidney, McCoy, NCI-H292 (people's lung mucoepidermoid tumor pipe), RD (rhabdosarcoma), Vero (African green monkey kidney), HEL (human embryonic lung), human fetal lung-Chang, MRC5 (embryo lung), MRHF (people's foreskin) and WI-38 (human embryonic lung).In some cases, the cell of express therapeutic peptide can be the cell that is derived from the patient that will treat, and perhaps can be derived from another kind of relevant or irrelevant Mammals.For example, inoblast can separate from the skin of mammal skin tissue and in vitro culture and conversion.This technology is commercial can be from Transkaryotic Therapies, and (Cambridge MA) obtains Inc..Nearly all at present used clone all can from American type culture collection (ATCC, Manassas, VA) and BioWhittaker (Walkersville, Maryland) acquisition.

Mammalian cell can transform with DNA with any of several technology well-known in the art.This technology transforms (Chen and Okayama, 1988 including, but not limited to calcium phosphate; Graham and van der Eb, 1973; Corsaro and Pearson, 1981, SomaticCell Genetics 7:603), diethylaminoethyl-(DEAE)-dextran transfection (people such as Fujita, 1986; People such as Lopata, 1984; People such as Selden, 1986), electroporation (people such as Neumann, 1982; Potter, 1988; People such as Potter, 1984; Wong and Neuman, 1982), the transfection of cation lipid reagent (Elroy-Stein and Moss, 1990; People such as Feigner, 1987; People such as Rose, 1991; People such as Whitt, 1990; People such as Hawley-Nelson, 1993, Focus 15:73; People such as Ciccarone, 1993, Focus 15:80), retrovirus (people such as Cepko, 1984; Miller and Baltimore, 1986; People such as Pear, 1993; Austin and Cepko, 1990; People such as Bodine, 1991; Fekete and Cepko, 1993; People such as Lemischka, 1986; People such as Turner, 1990; People such as Williams, 1984; Miller and Rosman, 1989, BioTechniques 7:980-90; Wang and Finer, 1996, Nature Med.2:714-6), 1,5-dimethyl-1,5-phenodiazine 11 methylene radical gather Methobromide (polybrene) (people such as Chaney, 1986; Kawai and Nishizawa, 1984), microinjection (Capecchi, 1980) and protoplastis merge (people such as Rassoulzadegan, 1982; People such as Sandri-Goldin, 1981; Schaffer, 1980) etc.Usually, transformation technology is referring to people such as Sambrook (2001, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and people such as Ausubel (2002, Current Protocols in MolecularBiology, John Wiley ﹠amp; Sons, New York).

The rhabdovirus system that insect cell is transformed use is suitable for stable conversion mammalian cell (referring to summary Koat and Condreay, 2002, Trends Biotechnol.20:173-180 reaches the reference of quoting therein).The production of recombinant peptide in the mammalian cell of cultivating is disclosed in as U.S.Pat.No.4, in 713,339,4,784,950,4,579,821 and 4,656,134.Several companies provide the service that transforms and cultivate mammalian cell, comprise Cell Trends, and Inc. (Middletown, MD).The technology of cultivating mammalian cell is well known in the art, and further can be found in people such as Hauser (1997, Mammalian Cell Biotechnology, Walter de Gruyer, Inc., Hawthorne, NY) and people such as Sambrook (2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor) reach in the reference of quoting therein.

D. insect

The insect cell that insect cell is particularly cultivated can be expressed the peptide of the glycan structures with N-connection, and the glycan structures that this N-connects is seldom sialylated, and is generally comprised within the mannose residue that has or do not have the extra Fucose that adheres on it.Being present in this class glycan structures on the peptide that produces in the insect cell of cultivation and the example of seminose glycan thereof shows in Fig. 6.In this case, can have or not exist the core Fucose, if present, this core Fucose can be connected to glycan by several different keys.

Shaft-like virus-mediated expression has become particularly suitable (people such as Altmann, 1999, Glycoconjugate J.16:109-123) to the production of recombinant peptide in the insect cell.Aspect the folding and translation post-treatment, insect cell is only second to mammalian cell at peptide.Yet, noticed as mentioned, the N-glycosylation of peptide and the N-glycosylation in the mammalian cell be variant in many aspects in the insect cell, and particularly insect cell often generates the glycan structures of brachymemma, and this glycan structures comprises and only contains 3 or the oligosaccharides of 2 mannose residues only sometimes.These structures can be substituted by fucosyl residues extraly.

According to the present invention, can be at first be reconstructed with generation at the external peptide that produces in to insect cell and have the glycosylated peptide of wanting by selectively remove any alternate fucosyl residues with suitable fucosidase.Peptide comprises under the situation of three basic seminose core textures after removing fucosyl residues, and what all needed so is to have the glycosylated peptide of wanting external with generation to the suitable sugar of three seminose core textures interpolation.Peptide may only contain under the situation of 2 mannose residues in glycan structures after removing any fucosyl residues, available mannose transferase and suitable donor molecule such as GDP-seminose add the 3rd mannose residue, have the glycosylated peptide of wanting with generation thereby add suitable residue.Randomly, also can from these kinds, generate single feeler glycan.

The rules that transform insect cell with baculovirus are well known in the art.Having published several provides and utilizes the book of rhabdovirus system in the program of expressed in insect cells peptide.These books are including, but not limited to Richardson (Baculovirus Expression Protocols, 1998, Methodsin Molecular Biology, 39 volumes, Humana Pr), people (1994, Baculovirus Expression Vectors:A Laboratory Manual, Oxford Univ Press) and King and Possee (1992 such as O ' Reilly, The BaculovirusExpression System:A Labora ory Guide, Chapman ﹠amp; Hall).In addition, also just like Lucklow (1993, Curr.Opin.Biotechnol.4:564-572) and Miller (1993, publication Curr.Opin.Genet.Dev.3:97-101).

Also authorized baculovirus expression system many and exogenous protein relevant patent.These patents are including, but not limited to U.S. Patent No. 6,210,966 (lack glutamine but contain the insect cell substratum of ammonium salt), U.S. Patent No. 6,090,584 (BVAC (baculovirus artificial chromosome) produces the application of recombinant peptide), U.S. Patent No. 5,871,986 (application of baculovirus express recombinant nucleic acid in mammalian cell), U.S. Patent No. 5,759,809 (in the method for expressed in insect cells peptide and the methods of kill insects), U.S. Patent No. 5,753,220 (cysteine proteinase gene defective type baculoviruss, its production process and produce the process of economic peptide with it), U.S. Patent No. 5,750,383 (baculovirus cloning systems), U.S. Patent No. 5,731,182 (non-mammalian DNA virus of express recombinant nucleic acid in mammalian cell), U.S. Patent No. 5,728,580 (in insect cell line, inducing the method and the substratum of single-cell suspension), U.S. Patent No. 5,583,023 (the baculovirus of modification, its preparation process and as the application of expression vector), U.S. Patent No. 5,571,709 (baculovirus of modification and rhabdovirus expression vectors), U.S. Patent No. 5,521,299 (surveying the oligonucleotide of baculovirus infection), U.S. Patent No. 5,516,657 (baculovirus vectors that are used for expression-secretion type and film mating type peptide), U.S. Patent No. 5,475,090 (coding strengthens the gene to the peptide of the virus infection of host insect), U.S. Patent No. 5,472,858 (productions of recombinant peptide in the insect larvae), U.S. Patent No. 5,348,886 (in bacterium, producing the method for reorganization eukaryotic virus), U.S. Patent No. 5,322,774 (the prokaryotic organism leader sequences in the recombinant baculovirus expression system), U.S. Patent No. 5,278,050 (improving the processing of recombination in the insect system and the method for secernment efficiency), U.S. Patent No. 5,244,805 (rhabdovirus expression vectors), U.S. Patent No. 5,229,293 (recombinant baculovirus), U.S. Patent No. 5,194,376 (can produce the baculovirus expression system of recombinant peptide) with high level, U.S. Patent No. 5,179,007 (method of purification of Recombinant peptide and carrier), U.S. Patent No. 5,169,784 (baculovirus double promoter expression vector), U.S. Patent No. 5,162,222 (application of baculovirus early promoter express recombinant nucleic acid in the insect cell of stable conversion or recombinant baculovirus), U.S. Patent No. 5,155,037 (being used for improving the insect signal sequence of recombinant nucleic acid processing of insect system and secernment efficiency), U.S. Patent No. 5,147,788 (baculovirus vector and application methodes), U.S. Patent No. 5,110,729 (in cultured cells, producing the method for peptide with baculovirus vector), U.S. Patent No. 5,077,214 (the baculovirus early promoter is in the application of the expressed in insect cells recombination of stable conversion), U.S. Patent No. 5,023,328 (lepidopteran (Lepidopteran) AKH signal sequence) and U.S. Patent No.s 4,879,236 and 4,745,051 (producing the method for recombination rhabdovirus expression vector).Above-mentioned patent all herein integral body be incorporated herein by reference.

The insect cell line in present several different plant species source is applied in the expression of peptide, and these clones are well known in the art.Targeted insect clone is including, but not limited to common Diptera and lepidopteran insect cell, Sf9 and variant thereof (mythimna separata in autumn (fall armyworm) fall army worm (Spodoptera frugiperda)), Estigmene acrea, Trichoplusiani, silkworm (Bombyx mori), Malacosoma disstri, drosophila strains Kc1 and SL2 etc. and mosquito.

E. plant

Vegetable cell as the peptide producer provides different situations.Although the N-that produces in the plant connects glycan and comprises three seminose core textures, this pentasaccharides main chain can comprise several different extra sugar as shown in Figure 5.For example, in one case, this three seminoses core texture is to be replaced by the fucosyl residues that the xylose residues of

β

1,2 connection and

α

1,3 connect.In addition, vegetable cell also can produce the Man5GlcNAc2 structure.The peptide that produces in the vegetable cell often is high antigenic owing to have

core α

1,3 wood sugar and Fucose on glycan structures, and will not remove from blood flow apace owing to there is not terminal sialic acid residues after in being incorporated into Mammals.Therefore, these peptides are reconstructed, otherwise it has been generally acknowledged that they are not suitable for as the therapeutical agent in the Mammals unless be used in method provided herein.Although find that some monoclonal antibodies of expressing in the plant are non-immunogenicities in mouse, but this polysaccharide chains that possible is is non-immunogenicity (people such as Chargelegue owing to be imbedded in the Fc zone of these antibody, 2000, Transgenic Res.9 (3): 187-194).

According to the explanation that provides herein, at present possible is to be created on the peptide that produces in the vegetable cell, and wherein the glycan structures of the increase number that exists thereon comprises basic three seminose core textures or Man3GlcNAc4 structure.This is by realizing that up to reaching basic three seminose core textures or Man3GlcNAc4 structure this Glycosylase comprises fucosidase with the extra sugar of the external excision of being combined in of suitable Glycosylase.These cleavage reactions also should comprise from structure removes any Fucose or xylose residues to eliminate the antigenicity of final peptide when in the introducing Mammals.Has the vegetable cell that suppresses the sudden change that Fucose and xylose residues add to three seminose core textures and is (people such as von Schaewen, 1993, PlantPhysiology 102:1109-1118) as known in the art.The present invention has expected that these cells have application in the peptide of the glycan that lacks Fucose and wood sugar in production.After having produced basic three seminose cores or Man3GlcNAc4 structure, extra sugar can be added to then on it to obtain having the glycosylated peptide of wanting, the treatment that therefore this peptide is suitable in the Mammals is used.

Many people think that transgenic plant are the selected expression systems of medicine peptide.Potentially, plant can provide cheap recombinant peptide source.Estimated that the comparable cost of producing identical peptide in intestinal bacteria of the production cost of recombinant peptide hangs down 10～50 times in the plant.Use small difference arranged although compare in the plant codon with animal, this can be compensated by regulating recombinant DNA sequence (referring to people such as Kusnadi, 1997, Biotechnol.Bioeng.56:473-484; People such as Khoudi, 1999, Biotechnol.Bioeng.135-143; People such as Hood, 1999, Adv.Exp.Med.Biol.464:127-147).In addition, peptide is synthetic, secretion is very similar in plant and animal with posttranslational modification, and tiny difference (referring to people such as Fischer, 2000, J.Biol.Regul.Homest.Agents 14:83-92) is only arranged in the plant glycosylation.Then, polluted by animal pathogenic agent, microbial toxin and carcinogenic sequence from the product of transgenic plant is also unlikely.

The expression of recombinant peptide is well known in the art in the vegetable cell.Except that transgenic plant, peptide also can the transgenic plant cells culture (people such as Lee, 1997, Mol.Cell.7:783-787) and in the non-transgenic plant with the recombinant plant virus inoculation produce.Published several the books of describing the genetic transformation process of vegetable cell: Potrykus (1995, Genetransfer to plants, Springer, New York), Nickoloff (1995, Plantcell electroporation and electrofusion protocols, Humana Press, Totowa, New York) and Draper (1988, Plant genetic transformation, Oxford Press, Boston).

Several method has been used for genetic recombination material settling out transformed plant cells at present.These methods are including, but not limited to Agrobacterium-mediated Transformation (Bechtold and Pelletier, 1998; Escudero and Hohn, 1997; Hansen and Chilton, 1999; People such as Touraev, 1997), biological bullet method (micropellet bombardment method) (people such as Finer, 1999; Hansen and Chilton, 1999; Shilito, 1999), protoplastis electroporation (people such as Fromm, 1985; People such as Ou-Lee, 1986; People such as Rhodes, 1988; People such as Saunders, 1989; People such as Trick, 1997), polyoxyethylene glycol is handled (Shilito, 1999; People such as Trick, 1997), in planta microinjection (people such as Leduc, 1996; People such as Zhou, 1983), seed imbibition (people such as Trick, 1997), laser beam (1996) and silicon carbide whiskers (people such as Thompson, 1995; U.S.Patent Appln.No.20020100077, integral body is incorporated herein by reference herein).

The plant of numerous species can carry out the conversion and the expression of exogenous peptide.The interested especially plant that expression is used for the peptide of reconstructing method of the present invention includes, but are not limited to Arabidopis thaliana (Arabidopsis thalliana), Semen Brassicae campestris (Brassica spp.; Ruiz and Blumwald, 2002, Planta 214:965-969)), soybean (Glycine max), Sunflower Receptacle (Helianthus unnuus), oil palm (Elaeis guineeis), Semen arachidis hypogaeae (peanut, Arachis hypogaea; People such as Deng, 2001, Cell.Res.11:156-160), coconut (Cocus nucifera), castor-oil plant (Ricinus communis), safflower (Carthamustinctorius), leaf mustard (Brassica spp. and Sinapis alba), coriandrum (Coriandrum sativum), pumpkin (Cucurbita maxima; Spencer and Snow, 2001, Heredity 86 (Pt6): 694-702), Semen Lini/flax (Linumusitatissimum; People such as Lamblin, 2001, Physiol Plant 112:223-232), Brazilian nut (Bertholletia excelsa), willow material (Simmondsia chinensis), corn (Zea mays; People such as Hood, 1999, Adv.Exp.Med.Biol.464:127-147; People such as Hood, 1997, Mol.Breed.3:291-306; People such as Petolino, 2000, Transgenic Research 9:1-9), alfalfa (people such as Khoudi, 1999, Biotechnol.Bioeng.64:135-143), tobacco (Nicotiana tabacum; People such as Wright, Transgenic Res.10:177-181; People such as Frigerio, 2000, PlantPhysiol.123:1483-1493; People such as Cramer, 1996, Ann.New York Acad.Sci.792:62-8-71; People such as Cabanes-Macheteau, 1999, Glycobiology9:365-372; People such as Ruggiero, 2000, FEBS Lett.469:132-136), canola (people such as Bai, 2001, Biotechnol.Prog.17:168-174; People such as Zhang, 2000, J.Anim.Sci.78:2868-2878)), potato (people such as Tacket, 1998, J.Infect.Dis.182:302-305; People such as Richter, 2000, Nat.Biotechnol.18:1167-1171; People such as Chong, 2000, Transgenic Res.9:71-78), alfalfa (people such as Wigdorovitz, 1999, Virology 255:347-353), pea (Pisum sativum; People such as Perrin, 2000, Mol.Breed.6:345-352), paddy rice (Oryza sativa; People such as Stoger, 2001; Plant Mol.Biol.42:583-590), cotton (Gossypium hirsutum; People such as Kornyeyev, 2001, PhysiolPlant 113:323-331), barley (Hordeum vulgare; People such as Petersen, 2002, Plant Mol Biol 49:45-58); Wheat (Triticumspp.; People such as Pellegrineschi, 2002, Genome 45:421-430) and beans (Vicia spp., people such as Saalbach, 1994, Mol Gen Genet 242:226-236).

If want express recombinant nucleic acid in complete plant rather than cultured cells, can make plant regeneration subsequently at first with the DNA transformed plant cells of encoded peptide so.This comprises general tissue culture program for each plant species optimization.For many species, the plant regeneration program is known in the art.In addition, those skilled in the art can utilize the rules of conventional experiment development to other species.The laboratory manual of many description plant regeneration programs is available, including, but not limited to Smith (2000, Plant tissue culture:techniques and experiments, Academic Press, San Diego), Bhojwani and Razdan (1996, Plant tissue culture:theory and practice, Elsevier Science Pub., Amsterdam), Islam (1996, Plant tissueculture, Oxford ﹠amp; IBH Pub.Co., New Delhi, India), Dodds and Roberts (1995, Experiments in plant tissue culture, New York:CambridgeUniversity Press, Cambridge Britain), Bhojwani (Plant tissue culture:applications and limitations, Elsevier, Amsterdam, 1990), Trigiano and Gray (2000, Plant tissue culture concepts andlaboratory exercises, CRC Press, Boca Raton, Fla) and Lindsey (1991, Plant tissue culture manual:fundamentals andapplications, Kluwer Academic, Boston).

Although the peptide of purification of Recombinant may be expensive from plant, several systems have been developed so that this minimizing costs.A kind of method is with the endosperm of synthetic peptide guiding seed, this peptide is easy to extract (people such as Wright in this endosperm, 2001, Transgenic Res.10:177-181, people such as Guda, 2000, Plant Cell Res.19:257-262 and U.S. Patent No. 5,767,379, integral body is incorporated herein by reference herein).A kind of selectable method is to make recombinant peptide and conventional plant product such as the extraction altogether of starch, meal or oil.In the oil grain rape, oleosin-hurudin fusogenic peptide is attached on the oil body of seed when expressing in plant, and can extract (Parmenter, 1995, Plant Mol.Biol.29:1167-1180 with oil from plant seed; U.S. Patent No. 5,650,554,5,792,922,5,948,682 and 6,288,304 and U. S. application 2002/0037303, herein their whole integral body are incorporated herein by reference).In a variant of this method, the peptide fusion (U.S. Patent No. 5,856,452, integral body is incorporated herein by reference herein) that makes oleosin and target peptide have avidity to the external source coexpression.

The expression of recombinant peptide in plant plastid such as chloroplast(id) generated the peptide that does not have glycan structures thereon, and the situation in this and the prokaryotic organism is similar.Yet the output of this peptide is extremely big when expressing in this vegetable cell device, thereby this class expression system can have the advantage that is better than other system.For the general summary of exogenous peptide expression technology in the higher plant plastid, referring to Hager and Beck (2000, Appl.Microbiol.Biotechnol.54:302-310 and the reference of quoting therein).Plastid be expressed in the tobacco be successful especially (referring to as people such as Staub, 2000, Nat.Biotechnol.18:333-338).

F. transgenic animal

The standard technique that recombinant DNA is incorporated into any number in the available transgenic animal technology in the zygote of animal (as Mammals) realizes.Referring to as people such as Hogan, Manipulating the Mouse Embryo:A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986; And U.S.Pat.No.5,811,634, integral body is incorporated herein by reference herein.More generally, recombinant DNA can be incorporated into (people such as Gordon, 1980, PNAS77:7380-7384 among the embryo by the pronucleus microinjection; Gordon and Ruddle, 1981, Science 214:1244-1246; People such as Brinster, 1981, Cell 27:223-231; Costantini and Lacy, 1981, Nature 294:92-94).Microinjection has the advantage that can be applicable in a large amount of species.The also available retrovirus of pre-implantation embryos transforms (Jaenisch and Mintz, 1974, Proc.Natl.Acad.Sci.U.S.A.71:1250-1254; People such as Jaenisch, 1976, Hamatol Bluttransfus.19:341-356; People such as Stuhlmann, 1984, Proc.Natl.Acad.Sci.U.S.A.81:7151-7155).The conversion of retrovirus mediation has the advantage that the recombinant nucleic acid of single copy can be added in the cell, but it has produced high-caliber mosaicism.Recently, used the technology (people such as Gossler of embryonic stem cell mediation, 1986, Proc.Natl.Acad.Sci.U.S.A.83:9065-9069), the transfer of complete chromosome segment (people such as Lavitrano, 1989, Cell 57:717-723), and also used and bonded gamete in vitro fertilization transfection (people such as Lavitrano, 1989, Cell 57:717-723).The book of the laboratory procedure of a few these technology of the disclosure: Cid-Arregui and Garc í a-Carranc á (1998 have been published, Microinjection andTransgenesis:Strategies and Protocols, Springer, Berlin), Clarke (2002, Transgenesis Techniques:Principles and Protocols, Humana Press, Totowa, NJ) and Pinkert (1994, Transgenic AnimalTechnology:A Laboratory Handbook, Academic Press, San Diego).

In case recombinant DNA is incorporated in the ovum, then that the ovum incubation is the short time period, transfers to then in the false pregnancy animal of the same species that obtains this ovum people such as (, see above) Hogan.Under mammiferous situation, 125 ovum of general each experiment injection, wherein about 2/3rds will survive in program.20 viable ovum are transferred in the Mammals of false pregnancy, wherein 4～10 offsprings that will grow for living.Usually, offspring's 10-30% (under the situation of mouse) carries recombinant DNA.

Although can be with whole animal as the expression system of peptide of the present invention, in a preferred embodiment, exogenous peptide gathers in the product of animal, and this exogenous peptide can be gathered in the crops from this product and need not to injure this animal.In preferred embodiments, exogenous peptide accumulates in breast, ovum, hair, blood and the urine.

If recombinant peptide accumulates in the Ruzhong of animal, so suitable Mammals is the Mammals and the milcher of ruminating animal, ungulate, domestication.Particularly preferred animal is goat, sheep, camel, cow, pig, horse, bull and yamma (llamas).Being created on its Ruzhong, to gather the technology of the immunocow of recombinant peptide be well-known: referring to people such as Newton (1999, J.Immunol.Methods 231:159-167), Ebert (1991, Biotechnology9:835-838) and U.S. Patent No. 6,210,736,5,849,992,5,843,705,5,827,690,6,222,094, herein their whole integral body are incorporated herein by reference.The generation that can produce the transgenic animal of the recombinant peptide of wanting is commercial from GTCBiotherapeutics, Framingham, and MA is obtainable.

If recombinant peptide is wanted to accumulate in the ovum, suitable bird is including, but not limited to chicken, goose and turkey.Other target animals are including, but not limited to other species of birds, fish, Reptilia and Amphibians.It is well known in the art introducing recombinant DNA by the retrovirus conversion in chicken: people such as Thoraval (1995, Transgenic Research 4:369-376), people such as Bosselman, (1989, Science 243:533-535), people (1992 such as Petropoulos, J.Virol.66:3391-3397), U.S. Patent No. 5,162,215, integral body is incorporated herein by reference herein.With recombinant DNA to the successful conversion of chicken also by DNA being introduced blastoderm cells and the blastoderm cells of such transfection being introduced the embryo realize: people such as Brazolot (1991, Mol.Reprod.Dev.30:304-312), people (1993 such as Fraster, Int.J.Dev.Biol.37:381-385) and people such as Petitte (1990, Development108:185-189).Developed high-throughout technology and estimated whether transgenic chicken expresses the peptide wanted (people such as Harvey, 2002, Poult.Sci.81:202-212, U.S. Patent No. 6,423,488, integral body is incorporated herein by reference herein).Utilize recombinant DNA that the retrovirus of chicken is transformed, the external source β-Nei Xiananmei accumulates in the egg white of chicken (people such as Harvey, 2002, Nat.Biotechnol.20 (4): 396-399).The production of the chicken of generation exogenous peptide is commercial in ovum can be from AviGenics, Inc., and Athens GA buys.

G. bacterium

The recombinant expressed peptide that produces in bacterium is not glycosylated usually.Yet it is conspicuous that the glycosylated bacterial system of peptide has been become, therefore in the future may produce glycosylated recombinant peptide in bacterium.

Many bacterial expression systems are as known in the art.Preferred bacterial species is including, but not limited to intestinal bacteria and bacillus (Bacillus) species.

The expression of recombinant peptide in intestinal bacteria is well known in the art.Rules based on colibacillary expression system can be found in U.S.Appln No.20020064835, U.S. Patent No. 6,245,539,5,606,031,5,420,027,5,151,511 and RE33,653 etc.The method of transform bacteria is including, but not limited to calcium chloride (people such as Cohen, 1972, Proc.Natl.Acad.Sci.U.S.A.69:2110-2114; Hanahan, 1983, J.Mol.Biol.166:557-580; Mandel and Higa, 1970, J.Mol.Biol.53:159-162) and electroporation (Shigekawa and Dower, 1988, Biotechniques 6:742-751), and those are described in people such as Sambrook, in 2001 (the seeing above).Summary for the laboratory rules of microbial transformation and expression system, referring to Saunders and Saunders (1987, Microbial Genetics Applied to Biotechnology:Principles andTechniques of Gene Transfer and Manipulation, Croom Helm, London), P ü hler (1993, Genetic Engineering of Microorganisms, Weinheim, New York), people such as Lee (1999, Metabolic Engineering, Marcel Dekker, New York), Adolph (1996, Microbial Genome Methods, CRC Press, Boca Raton) and Birren and Lai (1996, Nonmammalian GenomicAnalysis:A Practica lGuide, Academic Press, San Diego).

The general summary of the document of expressing for peptide in the intestinal bacteria, referring to Balbas (2001, Mol.Biotechnol.19:251-267).Several companies provide the bacterial isolates of selecting to be used to express mammalian-derived peptides at present, as colibacillary Rosetta ^TMBacterial strain (Novagen, inc., Madison, WI; Enhancing expression and enhanced disulfide linkage with the eukaryote codon that is not used in usually in the bacterial cell form).

H. cell engineering

See from disclosure of the present invention it is evident that, get over homogeneous by the parent material that cell produces, it is then effective more to have the glycosylated peptide of wanting in a large number in external generation.Thereby, disclosedly herein host cell is carried out genetically engineered operation provide the significant advantage that is better than with peptide parent material thereon as the parent material of external enzymatic reaction with the heterogeneous glycan structures that adheres to produce the glycosylated peptide of homogeneous.It is a kind of that to be used for preferred peptide parent material of the present invention be the peptide that mainly has unique glycan molecule of being made up of three basic seminose core textures.Another kind of preferred parent material is Man3GlcNAc4.Behind restructuring procedure, this preferred peptide will produce the glycosylated peptide that having of maximum wanted, thereby improve clinical efficacy.Yet other glycan parent materials also are applicable to the method for describing herein, for example can be easy to be cut to three basic seminose core textures with a series of mannosidases at external high mannose.As describing elsewhere, also can use other glycan parent materials, as long as thereby can excise all irrelevant sugar moieties generates three basic seminose core texture or Man3GlcNAc4.Thereby the cell of using gene engineering processing is to have the peptide of the glycan structures that adheres to of homogeneous as far as possible thereon in order to generate with the purpose of producing peptide of the present invention, and wherein glycan structures can have the glycosylated peptide of wanting external being reconstructed with generation.This will cause the rapid reduction of these peptide production costs.Because the glycopeptide that is produced by this method mainly has identical N-and connects glycan structures, produce the back and modify norm standardization and optimization to produce consistence between bigger end product batch so can make.As a result, heterogeneous comparable those present available of the chain product of finally finishing are lower.This product is compared with the product in prior art field and will be had the biological half time and the biological activity of improvement.Selectively, if desired, the present invention can be used for introducing limited and specific heterogeneity, as the reaction conditions that adds by the difference of selecting to cause sugar moieties.

Preferably (be not strict prerequisite), the cell of genetically engineered processing are the cells that can produce the peptide that has mainly the glycan structures of being made up of basic three seminose core textures or Man3GlcNAc4.MIN requirement is: the ratio of these preferred construction that produced by the cell of genetically engineered processing must be enough to produce after the reconstruct rules and has the glycosylated peptide of wanting.

Usually, can modify so that it becomes host cell of the present invention any eukaryotic cell.At first, determine endogenous and by the glycosylation pattern of biogenic reorganization glycopeptide to identify the interpolation/deletion of enzymic activity, wherein this enzymic activity can cause producing basic three seminose core glycopeptides or Man3GlcNAc4 glycopeptide.This generally need deletion with three seminose glycopeptides as the activity of the glycosyltransferase of substrate with increase the more complicated N-that degrades and be connected glycan with the enzymic activity of generation than short chain.In addition, the cell of genetically engineered processing can produce high mannose glycans, and this glycan can cut the initial glycan structures of wanting with generation with mannosidase.That this mannosidase can be is activated in cell paste (promptly this cell being carried out genetically engineered processing to produce this enzyme), and perhaps they can be used in the reaction after the produced in vitro.

It is well-known with the technology of the glycosylation character of the peptide of change expressing that host cell is carried out genetic modification.Referring to as people such as Altmann (1999, Glycoconjugate J.16:109-123), people such as Ailor (2000, Glycobiology 10 (8): 837-847), people such as Jarvis (In vitrogen Conference, March, 1999, summary), Hollister and Jarvis (2001, Glycobiology 11 (1): 1-9) and people (1999 such as Palacpac, PNAS USA 96:4697), people such as Jarvis (1998, Curr.Opin.Biotechnol.9:528-533), Gerngross (U.S.Patent Publication No.20020137134), they all disclose by make the technology of insect or vegetable cell expression system " Mammalsization (mammalianize) " with glycosyltransferase gene transfection insect or vegetable cell.

The technology of glycosylation character that changes the peptide of expression in escherichia coli hereditarily also exists.Intestinal bacteria used the various glycosyltransferases from bacterium Neisseria meningitidis and nitrogen-fixing root nodule Pseudomonas carried out processing with produce in vivo oligosaccharides (people such as Bettler, 1999, Glycoconj.J.16:205-212).Carried out the β 1 of genetically engineered processing with the overexpression Neisseria meningitidis, the intestinal bacteria of 3N acetylgucosamine transferase lgtA gene will make external source lactose glycosylation (people such as Priem, 2002, Glycobiology 12:235-240) effectively.

Also the fungal cell has been carried out genetic modification so that its produce external source glycosyltransferase (people such as Yoshida, 1999, Glycobiology 9 (1): 53-58; People such as Kalsner, 1995, Glycoconj.J.12:360-370; Schwientek and Ernst, 1994, Gene145 (2): 299-303; People such as Chiba, 1995, Biochem is J.308:405-409).

Thereby, on the one hand, the invention provides and make glycopeptide colony glycosylated cells, thereby a part of glycopeptide that produces has basic three seminose cores or Man3GlcNAc4 structure.Preferably, cell produces the peptide with unique glycan structures of being made up of basic three seminose cores.MIN requirement is: the ratio with peptide of three basic seminose cores or Man3GlcNAc4 structure must be enough to produce behind restructuring procedure and has the glycosylated peptide of wanting.Introduced one or more heterologous nucleic acids cenemes in this cell, each of these cenemes can comprise the nucleotide sequence of one or more one or more target peptide of encoding.The natural form of target glycopeptide can comprise one or more complicated N-and connect glycan or may simply be high mannose glycans.

This cell can be the cell of any kind, and is preferably eukaryotic cell.This cell can be mammalian cell, as the mammalian cell people, mouse, rat, rabbit, hamster or other types.When this cell is mammalian cell, this mammalian cell can be derived from or be contained in the inhuman transgene mammal, wherein the glycopeptide wanted of the cell coding in the Mammals and produce necessary various glycosylations of glycopeptide molecule and the Glycosylase enzyme of wanting.In addition, this cell can be the fungal cell, is preferably yeast cell, and perhaps this cell can be insect or vegetable cell.Similarly, when this cell was vegetable cell, this vegetable cell can be derived from or be contained in the transgenic plant, wherein the glycopeptide wanted of this plant code and produce necessary various glycosylations of glycopeptide molecule and the Glycosylase enzyme of wanting.

In some embodiments, host cell can be the eukaryotic cell of expressing one or more allos glycosyltransferases and/or one or more allos Glycosylases, and the expression of glycopeptide in host cell of wherein recombinating causes having the generation of basic three seminose cores as the reorganization glycopeptide of the main glycan structures that adheres to thereon.

In some embodiments, the allos glycosyltransferase that is used for cell can be selected from any known glycosyltransferase, this glycosyltransferase is included in for example people (2002 such as Taniguchi, Handbook of Glycosyltransferases and Related Genes, Springer, New York) in the glycosyltransferase family tabulation.

In other embodiment, allogenic glycosylase can be selected from mannosidase 1, mannosidase 2, mannosidase 3 and other mannosidases, including, but not limited to the microorganism mannosidase.Extra disclosure about the enzyme that is used for the present invention provides elsewhere.

In other other embodiment, host cell can be eukaryotic cell, wherein made one or more endogenous glycosyltransferases and/or one or more endogenous Glycosylase inactivations, thereby the expression of reorganization glycopeptide causes having the generation of basic three seminose cores as the reorganization glycopeptide of the main glycan structures that adheres to thereon in the host cell.

In extra embodiment, but host cell expressing heterologous glycosyltransferase and/or Glycosylase, and one or more endogenous glycosyltransferases of while and/or Glycosylase are inactivations.Endogenous glycosyltransferase and/or Glycosylase can be with well known to a person skilled in the art that any technology carries out inactivation, and this technology is including, but not limited to antisense technology and relate to the technology that nucleic acid is inserted in the host cell gene group.In some embodiments, endogenous enzyme can be selected from GnT-I, mannosidase, xylosyltransferase,

core α

1,3 fucosyltransferase, serine/threonine O-mannose transferase etc.

Selectively, can use and make the glycosylated natively expression system of peptide, thus the glycan that N-connects mainly be three seminose core types or the Man3GlcNAc4 type.An example that produces the cell type of three seminose cores is the Sf9 cell.Other this systems can be by analyzing natively or the glycopeptide that expresses in cell on reorganization ground and select the system of the glycosylation feature that those displayings want to identify.The present invention should be interpreted as comprising any or all these be used to produce the cell of peptide of the present invention.

V. purifying glycan reconstruct and/or the peptide that sugar is puted together

If the sugar-protein of modifying be produce in the cell or excretory, so the first step can by as centrifugal or ultrafiltration remove particulate residue or host cell cracked fragment; Selectively, protein can concentrate filter with the commercial protein of buying and concentrate, subsequently by one or more steps isolated peptides variant from other impurity, this step is selected from immunoaffinity chromatography, ion exchange column fractional separation (as diethylaminoethyl-(DEAE) or contain carboxymethyl or the matrix of sulfopropyl), at the Blue-agarose, CM Blue-agarose, MONO-Q, MONO-S, lens lectin-agarose, the WGA-agarose, Con A-agarose, ether Toyopearl, butyl Toyopearl, chromatography on phenyl Toyopearl or the a-protein agarose, the SDS-PAGE chromatography, silicon layer is analysed, chromatofocusing, reversed-phase HPLC (RP-HPLC), utilize gel-filtration as sephadex molecular sieve or size exclusion chromatography, chromatography on the pillar of selective binding peptide, and ethanol, pH or ammonium sulfate precipitation, membrane filtration and various technology.

The peptide of the modification that in culture, produces normally by from the initial extraction of cell, enzyme etc. and subsequently by one or more concentrate, saltout, water ion-exchange or size exclusion chromatography step and isolating.In addition, the glycoprotein of modification can come purifying by affinity chromatography.Can use HPLC then and carry out last purification step.

Proteinase inhibitor such as phenylmethylsulfonyl fluoride (PMSF) can be included in aforesaid any step with the arrestin hydrolysis, and can comprise that microbiotic is to prevent the growth of external contaminant.

In another embodiment, at first concentrate the concentrated supernatant liquor of filter, this concentrated filter such as Amicon or Millipore Pellicon ultrafiltration unit from the system that produces modified peptides of the present invention with the commercial protein of buying.After enrichment step, enriched material can be applied in the suitable purifying matrix.For example, suitable affinity matrix can comprise peptide part, with suitable support bonded lectin or antibody molecule.Selectively, can use anionite-exchange resin, as have the matrix or the substrate of the DEAE group that stretches out.Suitable matrix comprises other types commonly used in acrylamide, agarose, dextran, Mierocrystalline cellulose or the protein purification.Selectively, can use cation-exchange step.Suitable cationite comprises various insoluble matrix, and this matrix comprises sulfopropyl or carboxymethyl group.The sulfopropyl group is particularly preferred.

Then, can use one or more RP-HPLC steps being further purified the peptide variant compositions, the hydrophobic RP-HPLC medium of this RP-HPLC step application is as having the methyl that stretches out or the silica gel of other aliphatic groups.The various combinations of some or all of aforementioned purification step also can be used for providing the glycoprotein of the modification of homogeneity.

The peptide that the present invention of obtaining from large scale fermentation modifies can by with by people such as Urdal, the disclosed similar method of J.Chromatog.296:171 (1984) is carried out purifying.This reference has been described two the successive RP-HPLC steps of purification of Recombinant human IL-2 on the preparation HPLC post.Selectively, can use the glycoprotein of modifying with purifying as the technology of affinity chromatography.

The nucleic acid of VI. preferred peptide and the preferred peptide of coding

The present invention includes coding various peptides and proteinic isolating nucleic acid and similar molecule or fragment.The present invention should not be understood that only to be confined to the application in the methods of the invention of these peptides by any way, and should be understood to include any and all to those skilled in the art at present available peptides maybe will become the available peptide.In addition, the present invention should not be understood that only to comprise the specific nucleic acid or the aminoacid sequence of listed peptide herein, and should be understood to include any of each peptide and all variants, homologue, mutant etc.Should be noted that when peptide to be accredited as the sudden change that has in this peptide sequence or other when changing, unless explanation is arranged herein in addition, the amino acid whose numbering of following definite change or sudden change, promptly first amino acid in the mature peptide sequence is the 1st amino acids like this.

Preferred peptide is including, but not limited to Filgrastim (G-CSF), human interferon-alpha (IFN-α), human interferon beta (IFN-β), human blood coagulation factor VII (FactorVII), human blood coagulation IX (FactorIX), Human Fallicle-Stimulating Hormone (FSH), human erythropoietin (EPO), human granulocyte/macrophage colony stimulating factor (GM-CSF), human interferon gamma (IFN-γ), people α-1-proteinase inhibitor (also is known as α-1-antitrypsin or α-1-trypsin inhibitor; A-1-PI), glucocerebrosidase, human histiotype activation factor (TPA), Human Inter Leukin-2 (IL-2), human blood coagulation factor VII I (FactorVIII), with the Tumor Necrosis Factor Receptors of the 75kDa of human IgG immunoglobulin Fc meromixis, the commercial ENBREL that is known as ^TMOr ETANERCEPT ^TM(chimeric TNFR), human urokinase (urokinase), specificity are in conjunction with glycoprotein iib/iiia and vitronectin α _vβ ₃The Fab fragment of the monoclonal antibody that the people/mouse of acceptor is chimeric, the commercial REOPRO that is known as ^TMOr ABCIXIMAB (chimeric anti-glycoprotein iib/iiia), specificity be in conjunction with the chimeric monoclonal antibody of people/mouse of people HER2, the commercial HERCEPTIN that is known as ^TM(chimeric anti--HER2), specificity is in conjunction with the chimeric antibody of people/mouse of respiratory syncystial virus F protein matter or A antigenic site, the commercial SYNAGIS that is known as ^TMOr PALIVIZUMAB (chimeric anti--RSV), specificity is in conjunction with the chimeric people/mouse monoclonal antibody of the CD20 on the people B-cell, the commercial RITUXAN that is known as ^TMOr RITUXAMAB (chimeric anti-CD 20), people recombinate DNase (DNase), specificity in conjunction with the chimeric people/mouse monoclonal antibody of human tumor necrosis factor, the commercial REMICADE that is known as ^TMOr surface antigen (the adw hypotype of INFLIXIMAB (chimeric anti-TNF), insulin human, hepatitis B virus; HBsAg) and human growth hormone (HGH), alpha-galactosidase A (Fabryzyme ^TM), α-idose glycosides enzyme (Aldurazyme ^TM), antithrombin (Antithrombin III, AT-III), human chorionic gonadotrophin (hCG), Interferon, rabbit ω etc.

The isolating nucleic acid of the present invention should be interpreted as comprising RNA or the dna sequence dna and the modified forms thereof of any above-mentioned peptide of the present invention of coding, and this modified forms comprises the chemically modified of DNA or RNA so that this nucleotide sequence is more stable when combining when acellular or with cell.As nonrestrictive example, known this oligonucleotide of giving of oligonucleotide that contains at least a thiophosphoric acid modification is to nuclease enhanced resistance.The specific example of the oligonucleotide of modifying comprise those contain connect between thiophosphoric acid, phosphotriester, methyl phosphorodithioate, short-chain alkyl or cycloalkyl sugar or short chain heteroatoms or heterocycle sugar between (" main chain ") connect.5,034,506) or the oligonucleotide of polyamide skeleton structure (people such as Nielsen, 1991, Science 254:1497) in addition, also can use and have the morpholine backbone structure (US Patent No:.

Efficient and this nucleotides sequence of the chemically modified of Nucleotide being can be used for strengthening the cellular uptake nucleotide sequence are listed in the efficient of expressing in the cell.The present invention relates to any and all combinations that nucleotide sequence is modified.

The present invention should not be construed as and only is confined to disclosed herein nucleic acid and aminoacid sequence.As elsewhere in greater detail, in case read the present invention, then those skilled in the art it is evident that other nucleic acid of code book invention peptide can obtain by program (being site-directed mutagenesis, phase shift mutation etc.) and the technology of describing herein well known in the art.

What comprise equally is the nucleic acid of separated coding peptide fragment, and wherein this peptide fragment has kept the biologic activity that peptide is wanted.In addition, although disclose the exemplary nucleic acid of encoded peptide herein with specific SEQ ID NOS, the present invention never should be interpreted as being confined to disclosed herein any specific nucleic acid.On the contrary, the present invention should be interpreted as comprising with disclosed sequence herein to have conforming any or all nucleic acid molecule of enough per-cent, thereby these nucleic acid are also encoded and had the peptide of the disclosed herein biologic activity of wanting.What relate to equally is the isolating nucleic acid shorter than total length nucleic acid, and it has kept the biologic activity of the peptide of its coding.Determining the mensuration of nucleic acid and the conforming method of the per-cent between another and the biologic activity of determining any specific preferred peptide, the same in the text other is local open.

Same as other place is disclosed in the text, any other program can be used for generating with recombinant DNA method well known in the art derivative, mutant or the variant form of peptide of the present invention, this recombinant DNA method is as being described in people (1989 such as Sambrook, MolecularCloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York) and people (1997 such as Ausubel, Current Protocols inMolecular Biology, Green ﹠amp; Wiley, New York).The program of introducing amino acid change in peptide or the polypeptide by the dna sequence dna that changes encoded peptide is well known in the art and is described in people (1989, see above) such as Sambrook equally; Among the people such as Ausubel (1997, see above).

The present invention includes coding G-CSF, IFN-α, IFN-β, proconvertin, plasma thromboplastin component, FSH, EPO, GM-CSF, IFN-γ, A-1-PI, glucocerebrosidase, TPA, IL-2, blood coagulation factor VIII, chimeric TNFR, urokinase, chimeric anti--glycoprotein iib/iiia, chimeric anti--HER2, chimeric anti--RSV, chimeric anti-CD 20, DNase, chimeric anti-TNF, insulin human, HBsAg and HGH, nucleic acid, the wherein covalently bound thereon nucleic acid of coded markings peptide.Be that the present invention comprises chimeric nucleic acid, wherein the nucleotide sequence of coded markings peptide covalently is connected with the nucleic acid of coding peptide of the present invention.This mark peptide is well known in the art, and comprises as green fluorescent protein (GFP), myc, myc-pyruvate kinase (myc-PK), His ₆, maltose binding protein matter (MBP), influenza virus hemagglutinin labeling polypeptide, flag form (flag) labeling polypeptide (FLAG) and glutathione-S-transferase (GST) labeling polypeptide.Yet, the present invention's never should be interpreted as being confined to encoding nucleic acid of top listed mark peptide.On the contrary, coding can all should be interpreted as being included among the present invention to these mark peptides are brought into play the peptide of function in similar substantially mode any nucleotide sequence.

The nucleic acid that comprises the nucleic acid of coded markings peptide can be used for peptide of the present invention is positioned in cell, tissue and/or the whole biology (as mammal embryo), surveys peptide of the present invention and the effect of this peptide of research cell from emiocytosis.Further, the interpolation of mark peptide has promoted the separation and the purifying of " mark " peptide, thereby peptide of the present invention can be easy to produce and purifying.

Preferred isolated peptides: G-CSF below the present invention includes, IFN-α, IFN-β, proconvertin, plasma thromboplastin component, FSH, EPO, GM-CSF, IFN-γ, A-1-PI, glucocerebrosidase, TPA, IL-2, blood coagulation factor VIII, chimeric TNFR, urokinase, chimeric anti--glycoprotein iib/iiia, chimeric anti--HER2, chimeric anti--RSV, chimeric anti-CD 20, DNase, chimeric anti-TNF, the insulin human, HBsAg, HGH, alpha-galactosidase A, α-idose glycosides enzyme, Antithrombin III, hCG and Interferon, rabbit ω etc.

The present invention also should be interpreted as comprising " derivative " of peptide of the present invention (or DNA of this peptide of encoding), " mutant " and " variant ", this derivative, mutant and variant have carried out change (perhaps when referring to the nucleotide sequence of this peptide of coding at one or more amino acid, carried out change at one or more base pairs), thereby the peptide of gained (or DNA) is different with the sequence of enumerating herein as a result, but have and the identical biological property of disclosed peptide herein, promptly this peptide has G-CSF, IFN-α, IFN-β, proconvertin, plasma thromboplastin component, FSH, EPO, GM-CSF, IFN-γ, A-1-PI, glucocerebrosidase, TPA, IL-2, blood coagulation factor VIII, chimeric TNFR, urokinase, chimeric anti--glycoprotein iib/iiia, chimeric anti--HER2, chimeric anti--RSV, chimeric anti-CD 20, DNase, chimeric anti-TNF, the insulin human, biology/biochemical property of HBsAg and HGH.

What further comprise is the peptide fragment that has kept the biologic activity of wanting of peptide, and no matter its length.Within technician's technical scope, be to separate the peptide shorter fully, and be used in mensuration provided herein and determine which isolating fragment has kept the biologic activity wanted also thereby be to can be used for peptide of the present invention than the total length form of any peptide that is used for the present invention.

Proteinic biological property of the present invention should be interpreted as including, but not limited to the ability of this peptide performance function in the environment that biology is measured and described herein, this function as reduce inflammation, eliminate immune response, blood aggegation, increase hematopoiesis output, proteolytic enzyme inhibition, immune system, conjugated antigen, grow, alleviate or treat disease, DNA and cut etc.

A.G-CSF

The present invention comprises the method that G-CSF goes up glycan structures of modifying.G-CSF is the cytokine that is produced by activated T-cell, scavenger cell, endotheliocyte and matrix inoblast well known in the art.G-CSF mainly plays a role increasing the leukocytic generation of inflammation at marrow, and the function of further bringing into play endocrine hormone is with initial replenishing the neutrophilic granulocyte that consumes in the inflammation function course.The marrow of G-CSF after chemotherapy also has clinical application in substituting.

The G-CSF peptide of reconstruct can be applied to following patient, this patient is selected from and accepts the chemotherapeutic non-bone marrow cancer patient of bone marrow depression, accept to induce or strengthen chemotherapeutic acute myeloid leukaemia (AML) patient, accept bone marrow transplantation non-bone marrow cancer patient, carry out patient that the peripheral blood ancester cell collects, suffer from the neutropenic patient of severe chronic and suffer from the persistence neutrocytopenia and also suffer from the patient that late period, HIV infected.Preferably, the patient is people patient.

Although G-CSF for Mammals particularly people's therapeutic to use be important in useful, but the existing method of producing G-CSF from reconstitution cell causes following product, the forfeiture that this product has the short relatively biology life-span, can cause immunogenic incorrect glycosylation pattern, function potentially with to greater amount with more frequent take medicine need be to reach identical effect etc.

Separated and cloned G-CSF, and its nucleic acid and aminoacid sequence are respectively shown in SEQ ID NO:1 and SEQ ID NO:2 (being respectively Figure 58 A and 58B).The present invention comprises the method for modifying G-CSF, relates to that G-CSF serves as effectively and when the ability of biological molecule of function is arranged when it especially.When utilizing disclosure of the present invention and introducing herein, technician's easy to understand the invention provides the composition and the method for modifying G-CSF.

The present invention further comprises the G-CSF variant, as is well known in the art.As an example, but never mean restriction the present invention, in U.S. Patent No. 6,166, described the G-CSF variant in 183, wherein described the natural complement (complement) that comprises lysine residue and further be connected to G-CSF on one or two peg molecule.In addition, U.S. Patent No. 6,004,548,5,580,755,5,582,823 and 5,676,941 have described the G-CSF variant, and wherein the one or more cysteine residues on the

position

17,36,42,64 and 74 are substituted by L-Ala or Serine.U.S. Patent No. 5,416,195 have described the G-CSF molecule, and wherein the halfcystine on the position 17, the aspartic acid on the position 27 and the Serine on position 65 and 66 are substituted by Serine, Serine, proline(Pro) and proline(Pro) respectively.Other variants are well known in the art, and are described in as U.S. Patent No. 5,399, in 345.

The expression of the G-CSF molecule that the present invention modifies and active available well known in the art and be described in as U.S. Patent No. 4,810, the method in 643 is measured.As an example, the available radiolabeled thymus pyrimidine picked-up assay method of activity is measured.In brief, make people's marrow Ficoll-Hypaque (1.077g/ml from healthy donors, Pharmacia, Piscataway, NJ) carry out density separation (cut), and low-density cell suspension in containing 10% foetal calf serum, glutamine and antibiotic IscoveShi substratum (GIBCO, LaJolla, CA) in.Make about 2 X 10 ⁴The human bone marrow cell and control medium or G-CSF of the present invention on the flat flat board in 96-hole in air 5% CO ₂In about 2 days of about 37 ℃ of incubations.Use 0.5 μ Ci/ hole then ³The H-thymus pyrimidine (New England Nuclear, Boston, Mass.) about 4 hours to the culture burst process, and to be described in (1983, Blood61:781) method in is measured picked-up as people such as Ventua.Compare with the medullary cell of handling with control compound ³The increase that the H-thymus pyrimidine is integrated in the human bone marrow cell is the indication of active and viable G-CSF compound.

B.IFN α, IFN β and IFN ω

The present invention further comprises reconstruct and modifies IFN α, IFN β With IFN ωMethod.IFN α is that weight is the part of family of about 20 peptides of about 18kDa.IFN ω is very similar to IFN α on 26S Proteasome Structure and Function.When thereby the host increases when making that this is failed to respond to any medical treatment the immune response of IFN α, IFN ω can be used for treating infection with hepatitis C virus.The antibody of anti-IFN α can not with IFN ω cross reaction.Thereby, when IFN α therapy is no longer possible, can uses IFN ω and continue the treatment hepatitis C.

IFN α, the ω that is known as I type Interferon, rabbit jointly is attached on the identical cell receptor with IFN β and causes similar reaction.I type IFN suppresses virus replication, increases the cracking potentiality of NK cell, regulates the expression of MHC molecule and suppresses cell proliferation etc.I type IFN is with doing virus infection hepatites virus infections and to the therapeutical agent of multiple sclerosis particularly.

The existing composition of I type IFN is for the dysregulation immunological response with making the compound useful to the therapeutical agent of various diseases as mentioned above.Yet, compare the obstruction of transformation period in ability that they are reduced and function and the limited body with the n cell factor that comprises glycosylated natural complement.

The IFN α peptide of reconstruct can be applied to following patient, and this patient is selected from the patient who suffers from hairy cell leukemia, the patient who suffers from malignant melanoma, the patient who suffers from follicular lymphoma, the patient who suffers from pointed condyloma, the patient who suffers from the relevant Kaposi sarcoma of AIDS-, the patient who suffers from hepatitis C, the patient who suffers from hepatitis B, the patient who suffers from human papilloma virus infection, the patient who suffers from chronic myelogenous leukemia (CML), the patient who suffers from chronic phase Philadelphia chromosome (Ph) positive chronic myelocytic leukemia, the patient who suffers from non_hodgkin lymphoma (NHL), suffers from lymphadenomatous patient, the patient and the patient who suffers from kidney that suffer from bladder cancer.Preferably, the patient is people patient.

The IFN β peptide of reconstruct can be applied to following patient, this patient be selected from suffer from multiple sclerosis (MS) the patient, suffer from hepatitis B the patient, suffer from hepatitis C the patient, suffer from human papilloma virus infection the patient, suffer from mammary cancer the patient, suffer from the cancer of the brain the patient, suffer from colorectal carcinoma the patient, suffer from the patient of pulmonary fibrosis and suffer from the patient of rheumatoid arthritis.Preferably, the patient is people patient.

The IFN of reconstruct ωPeptide can be applied to following patient, and this patient is selected from the patient who suffers from hairy cell leukemia, the patient who suffers from malignant melanoma, the patient who suffers from follicular lymphoma, the patient who suffers from pointed condyloma, the patient who suffers from the relevant Kaposi sarcoma of AIDS-, the patient who suffers from hepatitis C, the patient who suffers from hepatitis B, the patient who suffers from human papilloma virus infection, the patient who suffers from chronic myelogenous leukemia (CML), the patient who suffers from chronic phase Philadelphia chromosome (Ph) positive chronic myelocytic leukemia, the patient who suffers from non_hodgkin lymphoma (NHL), suffers from lymphadenomatous patient, the patient and the patient who suffers from kidney that suffer from bladder cancer.Preferably, the patient is people patient.

The prototype Nucleotide of IFN α and aminoacid sequence propose (being respectively Figure 59 A and 59B) with SEQ ID NO:3 and SEQ ID NO:4 respectively herein.The prototype Nucleotide of IFN ω and aminoacid sequence propose (being respectively Figure 84 A and 84B) with SEQ ID NO:74 and SEQ ID NO:75 respectively herein.IFN β comprises the single-gene product of about 20kDa, and its nucleic acid and aminoacid sequence propose (being respectively Figure 60 A and 60B) with SEQ ID NO:5 and SEQ ID NO:6 respectively herein.The present invention is not limited to Nucleotide and aminoacid sequence herein.There is the variant of many IFN in those skilled in the art natively or as the derivative of processing with easy to understand.Similarly, IFN β is modified to realize more favourable therapeutic property.The example of the I type IFN that modifies is (referring to table 9) well known in the art, and is described in as United States Patent (USP) 6,323, in 006, wherein halfcystine-60 is substituted by tyrosine, also is described in as U.S. Patent No. 4,737,462,4,588,585,4,545,723 and 6,127, in 332, wherein described and had multiple amino acids alternate IFN β.In addition, U.S. Patent No. 4,966,843,5,376,567,5,795,779 have described IFN α-61 and IFN-76.U.S. Patent No. 4,748,233 and 4,695,543 have described IFN α gx-1, and U.S. Patent No. 4,975,276 has been described IFN α-54.In addition, U.S. Patent No. 4,695,623,4,897,471,5,661,009 and 5,541,293 have all described the consistent IFN α sequence of representative at known all variants of submission date.Although this tabulation of I type IFN and variant thereof anything but exhaustively, the present invention of those skilled in the art's easy to understand comprises IFN β and IFN alpha molecule, derivative and variant known or that find in the future.

Table 9. interferon-' alpha ' isotype

The method of expressing IFN in reconstitution cell is well known in the art, and be easy to realize with following technology, this technical description is in as U.S. Patent No. 4,966, and 843 and people (2001 such as Sambrook, Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York) and people (1997 such as Ausubel, CurrentProtocols in Molecular Biology, Green ﹠amp; Wiley, New York).

The assay method of determining the biologic activity of the I type IFN that modified by the present invention is that those skilled in the art is well-known.For example, being described in assay method among the people such as Rubinstein (1981, Journalof Virology 37:755-758) is generally used for determining the effect of I type IFN by measuring virus infection cytopathic effect in the cell colony.This method only is one of method of many biological functions that become known for measuring I type IFN in this area.

C. proconvertin a

The method that the present invention further comprises reconstruct and modifies proconvertin.The coagulation of blood approach is the complex reaction that comprises many incidents.An intermediate event in this approach is a proconvertin, its be by when tissue factor and calcium ion exist, factor X changed into the proenzyme that Xa participates in the external approach of coagulation of blood (the activation back for proconvertin a).Factor Xa changes thrombogen into zymoplasm then when prothrombinase, calcium ion and phosphatide exist.Factor X is inherent and the common incident of external coagulation of blood approach to the activation of factor Xa, so proconvertin a can be used for treating the patient with blood coagulation factor VIII defective or inhibition.Evidence suggests that also proconvertin a also can participate in inherent approach, therefore increased high-lighting and importance that proconvertin acts in coagulation of blood.

Proconvertin is the strand glycoprotein of the about 50kDa of molecular weight.When this form, this factor proenzyme with non-activity in blood circulates.Proconvertin can be by several different plasma proteins enzyme catalysiss, as Hageman factor a to the activation of proconvertin a.The activation of proconvertin causes heavy chain and light chain by at least one disulfide bonds.Further, described the proconvertin molecule of the modification that can not change proconvertin a into, and this molecule can be used as the resist coagulation medicine, as under situations such as blood clot, thrombus.In view of proconvertin in the coagulation of blood approach importance and can be used as treatment to the condensate level that increases and reduce, the molecule that has following character so will be favourable and can be used for the coagulation of blood treatment of conditions, this molecule has than long biological half time, the ability of increase, and has usually and the synthetic and more similar therapeutic property of excretory wild-type proconvertin in healthy human body.

The proconvertin peptide of reconstruct can be applied to following patient, and this patient is selected from the haemophiliac with the outbreak of bleeding; The patient who suffers from haemophilia A; The patient who suffers from haemophilia B; The patient who suffers from haemophilia A, wherein this patient also has the antibody of anticoagulin VIII; The patient who suffers from haemophilia B, wherein this patient also has the antibody of anticoagulin IX; The patient who suffers from liver cirrhosis; Sclerosis patient with coordination liver transplantation; Has the sclerosis patient that last stomach and intestine are bled; Patient and patient with bone marrow transplantation with hepatectomy.Preferably, the patient is people patient.

Proconvertin is cloned and is checked order, and its nucleic acid and aminoacid sequence provide (being respectively Figure 61 A and 61B) with SEQ ID NO:7 and SEQ ID NO:8 herein.The present invention never should be interpreted as the proconvertin nucleic acid and the aminoacid sequence that are confined to propose herein.The variant of proconvertin is described in as U.S. Patent No. 4,784,950 and 5,580, in 560, wherein Methionin-38, Methionin-32, arginine-290, arginine-341, Isoleucine-42, tyrosine-278 and tyrosine-332 are by various amino acid replacements.Further, U.S. Patent No. 5,861,374,6,039,944,5,833,982,5,788,965,6,183,743,5,997,864 and 5,817,788 have described the coagulation factor VII variants that can not cut with formation proconvertin a.The technician can recognize the coagulation of blood approach and wherein the effect of proconvertin be well-known, therefore comprise the variant of many aforesaid naturally occurring and processing in the present invention.

Express and the active method of definite proconvertin is well known in the art, and be described in, in 950 as U.S. Patent No. 4,784.In brief, the expression of proconvertin or its variant can realize in various prokaryotic organism and eukaryote system, the insect cell that comprises intestinal bacteria, Chinese hamster ovary celI, bhk cell, application baculovirus expression system, they all are well known in the art.

The active mensuration of the proconvertin that prepared according to the methods of the invention is modified is well known in the art.As nonrestrictive example, people such as Quick (HemorragicDisease and Thrombosis, 2 ^NdEd., Leat Febiger, Philadelphia, 1966) step of having described the biologic activity that is used for determining prepared according to the methods of the invention proconvertin molecule assay method (one-stage clotting assay) of condensing.

D. plasma thromboplastin component

The method that the present invention further comprises reconstruct and/or modifies plasma thromboplastin component.As mentioned above, plasma thromboplastin component is vital in the coagulation of blood cascade.The defective of plasma thromboplastin component is the feature of a hemophilioid disease (Type B) in the health.This treatment of diseases is confined to import usually the human plasma protein fraction matter enriched material of plasma thromboplastin component.Yet except that the shortcoming of operating time and cost, the input of blood enriched material relates to the danger of transmitting viral hepatitis, acquired immune deficiency syndrome (AIDS) or thromboembolic disorders to acceptor.

Although proved that it is important and useful compound that plasma thromboplastin component itself is used for treatment, but produce the existing method (U.S. Patent No. 4 of plasma thromboplastin component from reconstitution cell, 770,999) cause following product, the forfeiture that this product has the quite short biology life-span, can potentially cause immunogenic incorrect glycosylation pattern, function with to greater amount with more frequent take medicine need be to reach identical effect etc.

The plasma thromboplastin component peptide of reconstruct can be applied to following patient, this patient be selected from having bleed outbreak and also suffer from haemophilia B the haemophiliac, suffer from haemophilia B the patient, suffer from haemophilia B and also have the antibody of anticoagulin IX the patient, suffer from liver cirrhosis the patient, have the coordination liver transplantation the sclerosis patient, have sclerosis patient that last stomach and intestine bleed, have the patient of bone marrow transplantation and have the patient of hepatectomy.The plasma thromboplastin component peptide that also can use reconstruct is with the bleeding episodes among the patient who controls and/or prevent to suffer from haemophilia B, congenital plasma thromboplastin component deficiency or christmas disease.Also can use the plasma thromboplastin component peptide of reconstruct with the bleeding episodes among the patient in control and/or the prevention of surgical surgical procedure to the patient.Preferably, the patient is people patient.

The nucleic acid of plasma thromboplastin component and aminoacid sequence propose (being respectively Figure 62 A and 62B) with SEQ ID NO:9 and SEQ IDNO:10 herein.The sequence that the present invention is in no way limited to propose herein.The plasma thromboplastin component variant is well known in the art, as is described in U.S. Patent No. 4,770,999, wherein the tyrosine of first position is by L-Ala alternate 5,521,070, wherein plasma thromboplastin antecedent is connected to the U.S. Patent No. 6 on the alkylene oxide group, 037,452, and the DNA of the plasma thromboplastin component of wherein the encoding U.S. Patent No. 6 of modifying at least one splice site, in 046,380.As proof herein, the variant of plasma thromboplastin component is well known in the art, and disclosure of the present invention comprises those known or in the future can develop or find variants.

The method of determining the plasma thromboplastin component that prepared according to the methods of the invention is modified can realize with above-mentioned method, perhaps this external application method well known in the art is implemented, as step activatory local organization thromboplastin timing, as be described in Biggs (1972, HumanBlood Coagulation Haemostasis and Thrombosis (Ed.1), Oxford, Blackwell, Scientific, in pg.614).In brief, for the biologic activity of the plasma thromboplastin component molecule of measuring the method according to this invention development, available isopyknic activatory local organization thromboplastin reagent, with aseptic venotomy technology well known in the art isolating plasma thromboplastin component defective type blood plasma and measure from the haemophilia B patient as the blood plasma of the normal collection of standard or sample.In this was measured, the activity of 1 unit was defined as this amount that exists in 1 milliliter of normal blood plasma of collecting.Further, can carry out reducing from the assay method of the ability of blood plasma time of coagulation to normal time of plasma thromboplastin component defective type patient based on plasma thromboplastin component, as be described in Proctor and Rapaport (1961, in Amer.J.Clin.Path.36:212).

E.FSH

The present invention further comprises reconstruct and/or the method for modifying FSH.People's reproductive function partly is that this family has 92 amino acid whose glycoprotein α subunits of common, but variant on the specific β subunit of its hormone by the control of the human glycoprotein hormone family of heterodimer.This family comprises follicle stimulating hormone (FSH), lutropin (LH), thyrotropin or thyrotropic hormone (TSH) and human chorionic gonadotrophin (hCG).People FSH can be used for regulating metabolic all respects relevant with reproduction among the human women with LH in treatment.For example, part FSH of purifying from urine can be used for stimulating the maturation of the no ovum women ovarian follicle of no ovum syndromes or luteal phase defect clinically.Lutropin (LH) and FSH are used to stimulate the growth of ovarian follicle in vitro fertilization to carry out capable of being combinedly.The effect of FSH in reproduction circulation is fully known, thereby allows it to treat application, but part is owing to heterogeneity and the impure difficulty that run into from the preparation of natural origin.This heterogeneity is owing to the variation in the glycosylation pattern.

FSH in vitro fertilization and to the stimulation of internal fertilization in all are valuable instruments, but as mentioned above, its clinical efficacy is subjected to the obstruction of the discordance in the Protein Glycosylation Overview.Therefore be apparent that the method for reconstruct FSH will have huge benefit for reproductive science.

The FSH peptide of reconstruct can be applied to following patient, and this patient is selected from the patient who carries out intrauterine insemination (IUI), the patient who carries out (IVF) in vitro fertilization and sterility patient.The FSH peptide that also can use reconstruct is to induce or to increase ovulation among the patient; Stimulate the growth of ovarian follicle among the patient; Induce the gametogenic ovarian follicular growth among the patient; Stimulate, induce or increase sterile among follicular development among the patient and ovulation subsequently or the treatment patient.Preferably, the patient is people's female patient.Also the FSH peptide of reconstruct can be applied to the patient that suffers from the hypophysis defective or the patient in pubescence.Preferably, the patient is the people male patient.

FSH clones and checks order, and its nucleic acid and aminoacid sequence are herein respectively with SEQIDNO:11, SEQ ID NO:12 (α subunit) with respectively with SEQ ID NO:13 and SEQID NO:14 (β subunit) proposition (being respectively Figure 63 A, 63B, 63C and 63D).The technician is not limited to the sequence of describing herein with easy to understand the present invention, and this is because the variant of FSH is well known in the art.As nonrestrictive example, U.S. Patent No. 5,639,640 have described the β subunit that comprises two different aminoacids sequences, and U.S. Patent No. 5,338,835 have described the β subunit that comprises extra aminoacid sequence, and this extra aminoacid sequence is derived from human chorion gonadotrophic hormone beta subunit and is about 27 amino acid.Therefore, the present invention comprises natural and by the FSH variant of the manual processing of the mankind, they all are well known in the art.

The method of expressing FSH in protokaryon and eukaryotic cell is well known in the art, and is described in (U.S. Patent No. 4,840,896,4,923,805,5,156,957) in the document in a large number.Further, the method of the biologic activity of the FSH molecule of estimation reconstruct of the present invention is well known in the art, and be described in as U.S. Patent No. 4,589, in 402, the method for FSH to the influence of the generation of fertility, ovum and pregnancy rate of determining described wherein among inhuman primates and the people subject.

F.EPO

The method that the present invention further comprises reconstruct and/or modifies EPO.EPO is the acid glycoprotein of about 34kDa, and can exist with 3 kinds of natural forms: α, β and asialylated.α and beta form only have nuance on sugared composition, but have identical ability, biologic activity and molecular weight.Asialylated form is to have removed terminal sialic α or beta form.EPO is present in the blood plasma with low-down concentration when state of health, wherein organizes and accept enough oxidations from the red corpuscle of existing number.This normal concentration is enough to stimulate substituting by the red blood cell of usual aging loss.The amount of erythropoietin increases under the hypoxemia situation when the oxygen of being transported by hemocyte in the circulation reduces in the circulation.Hypoxemia can by by the hemorrhage a large amount of blood loss that cause, red blood cell because the destruction of over-exposure in radiation, since minimizing or various forms of anaemia that high height above sea level or the secular athymia oxygen that causes are taken in cause.Therefore EPO is the useful compound that replenishes red blood cell after the situation of radiotherapy, anaemia and other life threatenings.

The EPO peptide of reconstruct can be applied to following patient, this patient is selected from the patient that suffers from anaemia, have the incompetent anaemia patient of chronic renal, have the anaemia patient of latter stage nephropathy, the anaemia patient who dialyses, anaemia patient, anaemia azidothymidine in treating with chronic renal failure the HIV infected patient, have non-bone marrow cancer and carry out chemotherapeutic anaemia patient and the anaemia patient of the non-vascular operation of non-heart is carried out in arrangement.Also the EPO peptide of reconstruct can be applied to and carry out operating patient to reduce the needs of allogeneic blood transfusion.The EPO peptide of reconstruct can be applied to also that expection has significant blood loss and patient with intra-operative blood transfusion risk of increase.Preferably, the patient is people patient.

In auxiliary importance from various diseases and illness are recovered, the present invention can be used for producing and has natural and the therefore EPO of more effective sugared composition according to EPO.At present the synthetic EPO of institute lacks glycosylation completely, therefore since its in vivo short life and must give more continually and with higher dosage.The present invention also provides the production of PEGization EPO molecule, and this EPO molecule has the improved greatly transformation period with can comparing by the EPO molecule that makes required sugar form maximization gained.

EPO clones and checks order, and its Nucleotide and aminoacid sequence propose (being respectively Figure 64 A and 64B) with SEQ ID NO:15 and SEQ ID NO:16 respectively herein.The sequence that those skilled in the art's easy to understand proposes herein only is coding and the example that comprises the sequence of EPO.As an example, U.S. Patent No. 6,187,564 have described the fused protein of the aminoacid sequence that comprises two or more EPO peptides, U.S. Patent No. 6,048,971 and 5,614,184 have described in the position 101,103, the 104 and 108 sudden change EPO molecules with amino acid replacement.U.S. Patent No. 5,106,954 have described the EPO molecule of brachymemma, and U.S. Patent No. 5,888,772 has been described in the position 33,139 and 166 and had alternate EPO analogue.Therefore, the technician will recognize that the present invention comprises EPO and EPO derivative and variant, and these integral body in document and this area fully prove.

In addition, the method for expression EPO is well known in the art in cell.As in U.S. Patent No. 4,703,008,5,688,679 and 6,376, example in 218 grades, EPO can express in prokaryotic organism and eukaryotic expression system.The method of measuring the biologic activity of EPO is well known in the art equally.As an example, Krystal measure (Krystal, 1983, Exp.Hematol.11:649-660) can be used for determining the activity of prepared according to the methods of the invention EPO.In brief, this measures the effect of erythropoietin to complete mouse boosting cell of measuring.Handle mouse to stimulate the generation of the reactive red blood cell ancester cell of erythropoietin with phenylhydrazine.After processing, obtain spleen, complete splenocyte is separated and make its erythropoietin protein carry out incubation with the wild-type erythropoietin of various amounts or description herein.Behind the incubation that spends the night, add ³The H-thymus pyrimidine is also measured its integration in cell DNA. ³The amount that the H-thymus pyrimidine is integrated has been indicated the production of the red blood cell of the erythropoietin stimulating that the interaction by erythropoietin and its cell receptor causes.The concentration of proteinic concentration of erythropoietin of the present invention and wild-type erythropoietin has been undertaken quantitatively by competitive radioimmunoassay method well known in the art.Specific activity is calculated divided by the proteinic micrograms of being measured by radioimmunoassay of immunoprecipitation with the international unit of measuring in Krystal measures.

Several different sudden change EPO have been reported with different glycosylation pattern.Many have the active stimulation of improved reticulosis, and do not influence the transformation period of this peptide in the animal blood flow.The EPO peptide of expection sudden change can substitute natural EPO peptide and be used for any glycan reconstruct described herein, the Glycopegylated and/or sugared embodiment of puting together.During preferred EPO sudden change is listed in the table below, but be not limited in being listed in the table below those (referring to as people such as Chern, 1991, Eur.J.Biochem.202:225-229; People such as Grodberg, 1993, Eur.J.Biochem.218:597-601; People such as Burns, 2002, Blood 99:4400-4405; U.S. Patent No. 5,614,184; GenBank Accession No.AAN76993; People such as O ' Connell, 1992, J.Biol.Chem.267:25010-25018; People such as Elliott, 1984, Proc.Natl.Acad.Sci.U.S.A.81:2708-2712; People such as Biossel, 1993, J.Biol.Chem.268:15983-15993).Most preferred EPO sudden change is Arg ¹³⁹To Ala ¹³⁹, Arg ¹⁴³To Ala ¹⁴³And Lys ¹⁵⁴To Ala ¹⁵⁴The form that the preferred natural EPO form for preparing these sudden changes is 165aa, it is described among Figure 65; Yet also can use the natural form of other EPO.At last, the sudden change that is described in the table 10 can be made up mutually or with other sudden changes, is used for the present invention's EPO peptide with preparation.

Table 10.EPO sudden change

G.GM-CSF

The present invention comprises the method for modifying GM-CSF.GM-CSF is the cytokine that is produced by activated T-cell, scavenger cell, endotheliocyte and matrix inoblast well known in the art.GM-CSF mainly plays a role increasing the leukocytic generation of inflammation at marrow, and the function of further bringing into play endocrine hormone is with initial replenishing the neutrophilic granulocyte that consumes in the inflammation function course.GM-CSF further is macrophage stimulation factor and promotes the differentiation of Lagerhans cell to dendritic cell.Similar with G-CSF, the marrow of GM-CSF after chemotherapy also has clinical application in substituting.

Although having proved G-CSF itself is the important and useful compound that therapeutic is used, but the existing method of producing G-CSF from reconstitution cell causes following product, the forfeiture that this product has the short relatively biology life-span, can cause immunogenic incorrect glycosylation pattern, function potentially with to greater amount with more frequent take medicine need be to reach identical effect etc.

The GM-CSF peptide of reconstruct can be applied to following patient, this patient be selected from suffer from acute myelocytic leukemia (AML) or acute nonlymphocytic leukemia (ANLL) the patient, carry out the white corpuscle extraction method with the patient that from peripheral blood, collects the hematopoiesis ancester cell, carry out patient, non_hodgkin lymphoma (NHL) patient who carries out autologous bone marrow transplantation that the autologous peripheral blood ancester cell transplants, carry out the Hokdkin disease patient of autologous bone marrow transplantation and carry out acute lymphoblastic leukemia (ALL) patient of autologous bone marrow transplantation.Also the GM-CSF peptide of reconstruct can be applied to the patient to accelerate marrow and implant, shorten neutrophilic granulocyte recovers after the chemotherapy time, to transfer the hematopoiesis ancester cell and enter peripheral blood to collect by the white corpuscle extraction method or to promote marrow-reconstitution from body or allogeneic bone marrow transplantation (BMT) back.Also the GM-CSF peptide of reconstruct can be applied to bone marrow transplantation failure or marrow and implant the patient who postpones.Preferably, the patient is people patient.

Separated and cloned GM-CSF, and its nucleic acid and aminoacid sequence (being respectively Figure 66 A and 66B) that proposed as SEQ IDNO:17 and SEQ ID NO:18 respectively.The present invention comprises the method for modifying GM-CSF, especially when relating to GM-CSF and serve as effectively ability with the biological molecule that function is arranged.When utilizing disclosure of the present invention and introducing herein, technician's easy to understand the invention provides the composition and the method for modifying GM-CSF.

The present invention further comprises the GM-CSF variant, as is well known in the art.As an example, but never mean restriction the present invention, described the GM-CSF variant in WO 86/06358, protein modification wherein is a kind of alternate quaternary structure.Further, U.S. Patent No. 6,287,557 have described the GM-CSF nucleotide sequence that the application for gene therapy is connected with the genome of simplexvirus.In addition, European patent publication No.0288809 (corresponding to PCT patent publications No.WO 87/02060) has reported the fused protein that comprises IL-2 and GM-CSF.The IL-2 sequence can be at N-or the C-end of GM-CSF, thereby after the acidity cutting to fused protein, can generate and have the GM-CSF that N-or C-end sequence are modified.Therefore, GM-CSF derivative, mutant and variant are well known in the art, and are contained within the method for the present invention.

The expression of the GM-CSF molecule that the present invention modifies and active available well known in the art and be described in as U.S. Patent No. 4,810, the method in 643 is measured.As an example, the available radiolabeled thymus pyrimidine picked-up assay method of activity is measured.In brief, make people's marrow Ficoll-Hypaque (1.077g/ml from healthy donors, Pharmacia, Piscataway, NJ) carry out density separation, and low-density cell suspension in containing 10% foetal calf serum, glutamine and antibiotic IscoveShi substratum (GIBCO, La Jolla, CA) in.Make about 2 X 10 ⁴The human bone marrow cell and control medium or G-CSF of the present invention on the flat flat board in 96-hole in air 5% CO ₂In about 2 days of about 37 ℃ of incubations.Use 0.5 μ Ci/ hole then ³The H-thymus pyrimidine (New England Nuclear, Boston, Mass.) about 4 hours to the culture burst process, and as the method that is described among the people (1983, Blood 61:781) such as Ventua picked-up is measured.Compare with the medullary cell of handling with control compound ³The increase that the H-thymus pyrimidine is integrated in the human bone marrow cell is the indication of active and viable GM-CSF compound.

H.IFN-γ

An object of the present invention is to comprise the method for modification and/or reconstruct IFN-γ.The IFN-γ that is also referred to as II type Interferon, rabbit is opposite with IFN β with IFN α, and it is the homodimer glycoprotein that comprises two subunits of about 21-24kDa.The variation of this size is owing to the glycosylation pattern that changes, and this pattern is when not reproducing usually during recombinant production in the various expression systems known in the art.IFN-γ is the strong activator of scavenger cell, can increase the expression of I type MHC molecule, and is the stimulant of II type MHC molecule on lower degree.Further, isotype conversion in the differentiation of IFN-γ promotion T-cell and the B-cell.Prove fully that also IFN-γ is neutrophilic granulocyte, NK cell and the stimulant that causes the antibody response of macrophage-mediated removing.IFN-γ has been intended to be the treatment medicine and has been used for the infection of intracellular pathogen, and as tuberculosis and leishmaniasis, can be used as perhaps that the antiproliferative therapeutical agent is used for the abnormal cell proliferation is the illness of characteristics, as various cancers and other tumorigenesis.

IFN-γ has proved to have strong immunologic competence, but because from the glycosylated difference of the system that is used to express IFN-γ at present, the ability of its therapeutical agent, effect, biological half time and other important factors are variable.The present invention comprises the method for correcting this critical defect.

The IFN-γ peptide of reconstruct can be applied to following patient, and this patient is selected from patient, the patient who suffers from pernicious osteopetrosis, the patient who suffers from pulmonary fibrosis, the patient who suffers from tuberculosis that suffer from chronic granulomatous disease, suffers from the patient of cryptococcal meningitis and suffers from the patient that lung mycobacterium avium (Mycobacterium avium) compound (MAC) infects.Preferably, the patient is people patient.

The Nucleotide of IFN-γ and aminoacid sequence propose (being respectively Figure 67 A and 67B) with SEQ ID NO:19 and SEQ IDNO:20 respectively herein.The sequence that easy to understand proposes herein never limits the present invention.On the contrary, the variant of IFN-γ, derivative and mutant are that the technician is well-known.As an example, U.S. Patent No. 6,083,724 have described the birds IFN-γ of reorganization, and U.S. Patent No. 5,770,191 has been described the terminal variant of the C-of people IFN-γ.In addition, U.S. Patent No. 4,758,656 methods of having described new IFN-γ derivative and in various expression systems, having synthesized this derivative.Therefore the present invention is not limited to the disclosed IFN-γ in other places sequence in the text, but comprises all derivatives well known in the art, variant, mutant etc.

The expression system of IFN-γ is well known in the art equally, and comprises prokaryotic organism and eukaryote system and plant and insect preparation, and its method is that the technician is known.As an example, U.S. Patent No. 4,758,656 have described the system at expression in escherichia coli IFN-γ derivative, and U.S. Patent No. 4,889,803 has been described the expression system of using Chinese hamster ovary cell and SV40 promotor.

To the mensuration according to the biologic activity of the IFN-γ of the reconstruct of the preparation of disclosed method herein is that those skilled in the art are well-known.The biology that IFN-γ expresses is measured and can be found in as U.S. Patent No. 5,807, in 744.In brief, add IFN-γ to CD34 ⁺⁺CD38 ^-In cell (100 cells in the every hole) culture, this culture has carried out stimulating to induce CD34 with combination of cytokines ⁺⁺CD38 ^-The propagation of cell, wherein cytokine is as IL-3, c-kit part and IL-1, IL-6 or G-CSF.The generation that the cell proliferation and second colony form sexual cell will be suppressed greatly in the mode of dose-dependently, and approaching inhibition fully when 5000U/ milliliter IFN-γ.As to the inhibiting confirmation check of IFN-γ, can add the IFN-gamma antibodies in contrast.

I. α-proteinase inhibitor (alpha antitrypsin)

The present invention further comprises the method for reconstruct α-proteinase inhibitor (A-1-PI, α-1-antitrypsin or α-1-trypsin inhibitor), and this α-proteinase inhibitor also is known as alpha antitrypsin.A-1-PI is that molecular weight is the glycoprotein of 53kDa.A-1-PI works in the disorganization that control is caused by endogenous serine protease, and is the most significant serpin in the blood plasma.Especially, A-1-PI suppresses various elastoser, comprises the neutrophilic granulocyte elastoser.Elastoser is the proteolytic enzyme of degraded tissue, and can be problematic especially when its activity is not regulated in lung tissue.This proteolytic enzyme is functionating by the degraded foreign protein.Yet, when API does not exist with the amount that is enough to regulate elastase activity, elastoser degradable lung tissue.Therefore, this imbalance can cause chronic pulmonary tissue injury and pulmonary emphysema.In fact, the hereditary defect of A-1-PI has shown relevant with emophysematous too early development.A-1-PI replenishes and has been successfully used in such emophysematous treatment.Further, the defective of A-1-PI also will cause the deterioration of other diseases, as cystic fibrosis and sacroiliitis, wherein white corpuscle move in the lung or in the joint with to anti-infective.

Therefore, A-1-PI can be used for the treatment of disease convincingly, in this disease inhibitor and proteolytic enzyme especially the imbalance between the neutrophilic granulocyte elastoser be the disease condition reasons of development.A-1-PI also has antiviral activity.According to these, can infer logically that the present invention can be used for producing A-1-PI, this A-1-PI is safe, effective and strong in the environment of the eternal variation of lung.

The A-1-PI peptide of reconstruct can be applied to following patient, this patient be selected from suffer from congenital α-the 1-antitrypsin is not enough and emophysematous patient, suffer from the patient of cystic fibrosis and suffer from the patient of pulmonary fibrosis.Preferably, the patient is people patient.

A-1-PI clones and checks order, and proposes (being respectively Figure 68 A and 68B) in SEQ ID NO:21 and SEQ ID NO:22.As skilled in the art to understand, the variant natural and processing of A-1-PI exists, and is contained among the present invention.As an example, U.S. Patent No. 5,723,316 have described at position 356-361 and have amino acid replacement and further comprise the terminal A-1-PI derivatives that extend of about 3 amino acid whose N-.U.S. Patent No. 5,674,708 have described the A-1-PI analogue that has amino acid replacement in the position 358 of one-level aminoacid sequence.The technician will readily recognize that the present invention comprises known and A-1-PI variant, derivative and mutant that will find.

The expression of the A-1-PI of the reconstruct that the method according to this invention produces and actively determine that method is well known in the art, and being described in as U.S. Patent No. 5,674,708 and U.S. Patent No. 5,723,316 in.In brief, biologic activity can be by measuring the enzymatic substrate N-suc-Ala-Ala-Pro-Phe-p-nitroanilide of chymotrypsin protein (the 10mM solution among the 90%DMSO of 0.1ml) the inhibition of hydrolysis use the assay method of chymotrypsin activity determined, as be described in people such as DelMar (1979, in Anal.Biochem.99:316).1.0 milliliters of general chymotrypsin protein enzymatic determination things comprise: 100mM Tris-Cl damping fluid, pH8.3,0.005% (v/v) Triton X-100, BCTR (18kmmol) and A-1-PI of the present invention.Make this mensuration mixture at room temperature about 5 minutes of incubation in advance, add substrate (the 10mM solution among the 90%DMSO of 0.01ml), and remaining chymotrypsin activity is by determining by p-nitroanilide being discharged the change scope that absorbs at 410nm that causes.The measurement of photoabsorption is to carry out with the spectrophotometer that the temperature control sample chamber has been installed at 25 ℃.

J. glucocerebrosidase

The present invention of Miao Shuing further comprises the method for modifying glucocerebrosidase herein.Glucocerebrosidase is the lysosome glucoproteinase that catalysis glycolipid glucocerebroside is hydrolyzed to glucose and ceramide.The variant of glucocerebrosidase is with Cerezyme ^TMAnd Ceredase ^TMIn commercial sale, and be the therapeutical agent of the treatment gaucher's disease of approved.Ceredase ^TMIt is placenta derivatize form with glucocerebrosidase of the structure that complete N-connects.Cerezyme ^TMBe that length is the reorganization variant of 497 amino acid and the glucocerebrosidase of expressing in Chinese hamster ovary celI.4 N-connection glycan of Cerezyme are modified to end at three seminose cores.

Glucocerebrosidase produces in the recombinant mammalian cells culture at present, therefore the glycosylation pattern that has reflected those cells, be generally rodent cells such as Chinese hamster ovary cell or baby hamster kidney cell, this glycosylation pattern and people's glycosylation pattern have huge different, thereby cause immunogenicity and usefulness forfeiture etc.

The glucocerebrosidase peptide of reconstruct can be applied to following patient, and this patient is selected from the patient that suffers from lysosomal storage disease, suffer from the insufficient patient of glucocerebrosidase and suffer from the patient of gaucher's disease.Preferably, the patient is people patient.

The nucleic acid of glucocerebrosidase and aminoacid sequence propose (being respectively Figure 69 A and 69B) with SEQ ID NO:23 and 24 herein.Yet as understood as technical staff, Dai Biao sequence is the prototype sequence herein, and does not limit the present invention.In fact, the variant of glucocerebrosidase is well-known and is described in as U.S. Patent No. 6,015,703 that the enhanced that this patent has been described glucocerebrosidase analogue and variant thereof produces.Further, U.S. Patent No. 6,087,131 have described the clone and the order-checking of another one glucocerebroside enzyme variants.The present invention will comprise that these and other are known or in the future findable derivative, variant and mutant.

The method of expressing glucocerebrosidase with standard technique is well known in the art, and is described in detail in as U.S. Patent No. 6,015, in 703.Mensuration to the biological effectiveness of prepared according to the methods of the invention glucocerebrosidase molecule also is well known in the art, and the mouse gaucher's disease model description that is used to estimate and uses the glucocerebrosidase therapeutical agent in as people such as Marshall (2002, Mol.Ther.6:179) in.

K.TPA

The present invention further comprises the method for reconstruct tissue-type activation factor (TPA).TPA activation Profibrinolysin is to form the plasmin of dissolving fibrin, and this fibrin is the main component of thrombus protein substrate.The TPA preparation has developed into the thrombolytics in the thrombolytics treatment, and this thrombolytics has the selectivity of height to the thrombotic thrombus that causes myocardial infarction and cerebrum block.

Further, in order to obtain purpose, produced the TPA of various modifications by genetically engineered to the more high-affinity of fibrin and the blood halflife longer than natural TPA.TPA is the protein that the common utmost point is insoluble in water.Especially, the TPA of modification more is insoluble in water than natural TPA, thereby the preparation of the feasible TPA that modifies is very difficult.Thereby the TPA that modifies when giving the patient is difficult to water-soluble.Yet the TPA of modification has various advantages, as avidity that fibrin is increased and the longer transformation period in the blood.The objective of the invention is to increase the solvability of the TPA of modification.

The TPA peptide of reconstruct can be applied to following patient, and this patient is selected from the patient who suffers from acute myocardial infarction and suffers from the patient of acute ischemic outbreak.The TPA peptide of reconstruct also can be applied to the patient to improve ventricular function behind the acute myocardial infarction, to reduce the sickness rate or the minimizing mortality ratio relevant with acute myocardial infarction of congestive heart failure behind the acute myocardial infarction.The TPA peptide of reconstruct also can be applied to the patient and recover or reduce the sickness rate of postictal deformity of acute ischemic and paralysis to improve the postictal neuroscience of acute ischemic.Preferably, the patient is people patient.

The nucleic acid of TPA and aminoacid sequence propose (being respectively Figure 70 A and 70B) with SEQ ID NO:25 and SEQ ID NO:26 respectively herein.As mentioned above, made up TPA variant and being used for the treatment of in the application.For example, U.S. Patent No. 5,770,425 have described some of them or the deleted TPA variant of all fibrin structural domains.Further, U.S. Patent No. 5,736,134 have described the TPA that the amino acid of position 276 has been modified.The technician utilizes disclosure of the present invention and introduction herein that easy to understand the present invention is included in the TPA sequence that proposes and well-known those variants of technician of being proficient in document herein.

The expression of TPA from the nucleotide sequence of coding TPA is well-known, and is described in detail in as U.S. Patent No. 5,753, in 486.The mensuration that is used for the biological property of definite prepared according to the methods of the invention TPA molecule is well known in the art.In brief, other places disclosed method synthetic TPA molecule can (1988, method Anal.Biochem.168:428) be measured the ability of its cracking fibrin when the Profibrinolysin of saturation concentration exists according to people such as Carlsen with in the literary composition.External grumeleuse cracking assay method is measured the activity of tissue-type activation factor by turbidity measurement with the Eppendorf centrifuge analyser.Make in the centrifugal mixture of going into fibrinogen and Profibrinolysin of the mixture of zymoplasm and TPA and form and clot dissolution subsequently with initial grumeleuse.Absorption-the timing relationship of gained is as a result analyzed to determine to measure terminal point.The activity of TPA variant and the typical curve of TPA are compared.Used damping fluid is the 0.06M sodium phosphate that contains 0.01% (v/v) TWEEN80 and 0.01% (w/v) sodiumazide in the mensuration process, pH7.4.The concentration of people's zymoplasm is about 33 units/ml.Fibrinogen (2.0mg/ml can caked protein) wet freezing with the precipitation fibronectin, is carried out gravity filtration on ice then.Glu-Profibrinolysin concentration is 1mg/ml.Analyser compartment temperature is made as 37 ℃.Hopper loader is set at and distributes the sample of 20 microlitre TPA (about 500 nanograms/milliliter～about 1.5 mcg/ml) as typical curve, perhaps can cause in standard curve range cracked concentration to distribute 20 microlitre TPA variants.With 20 microlitre zymoplasms as second class grade chemical, and with the fibrinogen of 200 microlitre 50:1 (v/v): the Profibrinolysin mixture is as one-level reagent.Absorption/time-program(me) is used incubation time, 340-nanometer-wave filter and 90 seconds readings at interval of 5 minutes.

L.IL-2

The method that the present invention further comprises reconstruct and modifies IL-2.IL-2 is the lymphocytic main somatomedin of T and can increases body fluid and cell immune response by irritation cell toxicity cd8 t cell and NK cell.Therefore IL-2 is crucial in the defense mechanism to antitumor and virus infection.IL-2 also is used for resisting the treatment of metastatic melanoma and renal adenocarcinoma, and has been used for the clinical trial of many form cancers.Further, IL-2 also has been used for the patient that HIV infects, and it causes the remarkable increase of CD4 number therein.

Because IL-2 is handling and the disease of treatment life threatening such as the success among various cancer and the AIDS, method so of the present invention is useful for the following IL-2 molecule of development, and this IL-2 molecule has the ability and the more similar therapeutic property of wild-type IL-2 common and synthesis secretion in healthy people of longer biological half time, increase.

The IL-2 peptide of reconstruct can be applied to following patient, and this patient is selected from patient, the patient who suffers from metastatic melanoma, the patient who suffers from ovarian cancer, the patient who suffers from acute myelocytic leukemia (AML), the patient who suffers from non_hodgkin lymphoma (NHL) that suffer from metastatic renal cell cancer, has infected the patient of HIV and infected the patient of hepatitis C.The IL-2 peptide of reconstruct also can be applied to the patient as the Theratope adjuvant.Preferably, the patient is people patient.

IL-2 clones and checks order, and nucleic acid and aminoacid sequence propose (being respectively Figure 71 A and 71B) with SEQ ID NO:27 and SEQ ID NO:28 herein.The present invention never should be interpreted as the IL-2 nucleic acid and the aminoacid sequence that are confined to propose herein.The variant of IL-2 is described in as U.S. Patent No. 6,348, and in 193, wherein the l-asparagine of position 88 is substituted by arginine, and in the U.S. Patent No. 5,206,344, has wherein described the polymer that comprises the IL-2 variant with various amino acid replacements.The present invention comprises these and other IL-2 variants well known in the art.

The expression of IL-2 and the active method of determining are well known in the art, and are described in as U.S. Patent No. 5,417, in 970.In brief, the expression of IL-2 or its variant can realize in various prokaryotic organism and eukaryote system that comprise the insect cell of intestinal bacteria, Chinese hamster ovary celI, bhk cell, application baculovirus expression system, they all are well known in the art.

The active mensuration of the IL-2 that prepared according to the methods of the invention is modified can be carried out as follows.By being described in (Methods in Enzymology, 1984,108 volumes as people such as A.Boyum, 88 pages, Academic Press, method Inc.) is at Ficoll-Hypaque (Pharmacia, Piscataway is NJ) centrifugal to separate peripheral blood lymphocyte from red corpuscle with granulocyte on the gradient.In substratum, lymphocyte is washed about 3 times subsequently, this substratum is by RPMI 1640 (Gibco-BRL, La Jolla, CA) and the heating (carrying out 1 hour) inactivation in 56 ℃ 10%AB human serum (CTS Purpan, Toulouse, France), 2mM Sodium.alpha.-ketopropionate, 5mM HEPES, 4mM L-glutaminate, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates and 0.25 μ g/ml amphotericin are formed (perfect medium).By attaching on the plastics and adhesive cell (monocyte and scavenger cell) is removed, with remaining cell with every milliliter of about 5～10 X 10 ⁵The concentration of cell is resuspended in the perfect medium and with every square centimeter of about 1-2 X 10 ⁵The density of cell is inoculated in the culturing bottle.Make bottle at 5% CO then ₂In in about 1 hour of 37 ℃ of incubations, after stirring culturing bottle gently, reclaim the non-lymphocyte that adheres to thereafter by suction.

The non-lymphocyte that adheres to is washed once, and with every milliliter about 10 ⁵The concentration of cell was being cultivated about 48 hours in aforesaid incubator on the perfect medium when IL-2 of the present invention exists.Then once with cell washing.

The cellular cytoxicity activity of cell be with estimate after the target cell of the people T lymphoid cell line C8166-45/C63 (HT1 cell) of anti-NK cell cytotoxicity contacts about 4 hours, as by people such as Salahuddin (1983, Virology 129:51-64; 1984, Science 223:703-707) described.Make 6 X 10 ⁵Individual HT1 cell in 37 ℃ of perfect mediums at serum-free with about 200 μ Ci's ⁵¹Cr (Sodium chromate, Amersham, Arlington Heights, IL) radio-labeling is about 1 hour, and washing is several times then.Target cell is distributed on the microtiter plate of round bottom the ratio (50:1,10:1,1:1) of target cell with different effective cells with effective cell.Microtiter plate is centrifugal, and after aforesaid incubation, reclaim supernatant liquor from each hole and measure radioactivity with gamma counter.Cytotoxicity is from being discharged by dead target cell ⁵¹The Cr amount is determined.Nonspecific cytotoxicity be from when not having effective cell from target cell the radioactivity amount of spontaneous release determine.

Method of the present invention only is of Cytotoxic many methods who is used for measuring effect cell well known in the art, and should not be construed as limiting the invention.

M. blood coagulation factor VIII

The method that the present invention further comprises reconstruct and modifies blood coagulation factor VIII.As described to proconvertin and plasma thromboplastin component at preamble, blood coagulation factor VIII is a key component in the coagulation of blood approach.Human blood coagulation factor VII I (antihemophilic factor; FVIII:C) the human plasma protein fraction matter of forming by two peptides (molecular weight is that light chain and the molecular weight of 80kDa depends on the heavy chain of glycosylation state at 90～220kDa).It is to condense in the approach basic cofactor and be that factor X changes necessary to its activity form (factor Xa).Blood coagulation factor VIII circulates the peptide that this Feng's von willebrand's factor is 2050 aa (referring to U.S. Patent No. 6,307,032) as the non-covalent complex (akaVIII:RP) with Feng's von Willebrand (Von willibrand) factor in blood plasma.20% the blood coagulation factor VIII haemoconcentration that is lower than normal value causes being called the bleeding property disease of hemophilia A.Be lower than 1% blood coagulation factor VIII blood levels and cause the serious venereal disease disease of bleeding, bleeding in wherein spontaneous joint is modal symptom.

With other coagulation of blood factor types seemingly, blood coagulation factor VIII is the therapeutical agent that various treatments of bleeding venereal disease disease such as hemophilia A and hemophilia B are had great potential.Because the glycosylation of heavy chain, the existing method of preparation blood coagulation factor VIII causes not as the effective product of natural blood coagulation factor VIII from reconstitution cell.Cause thick composition more effective and the more difficult preparation of ratio reorganization blood coagulation factor VIII from the purification process of human plasma.The present invention seeks to improve this situation.

The blood coagulation factor VIII peptide of reconstruct can be applied to following patient, and this patient is selected from the patient, the patient who suffers from haemophilia A that suffer from von Willebrand's disease, suffers from blood coagulation factor VIII: the insufficient patient of C, suffer from the insufficient patient of fibrinogen, suffer from the insufficient patient of Hageman factor I and have the patient of the blood coagulation factor VIII inhibitor (acquired hemophilia) of acquisition.The blood coagulation factor VIII peptide of reconstruct also can be applied to the patient and bleed or bleeding episodes with prevention, treatment or control.Preferably, the patient is people patient.

The nucleic acid of blood coagulation factor VIII and aminoacid sequence propose (being respectively Figure 72 A and 72B) with SEQ ID NO:29 and SEQ ID NO:30 respectively herein.Various blood coagulation factor VIIIs are general in the art, as are described in U.S. Patent No. 5,668, in 108, wherein the aspartic acid of position 1241 is substituted by L-glutamic acid, and is accompanied by the same variation of nucleic acid.U.S. Patent No. 5,149,637 have described the blood coagulation factor VIII variant that comprises glycosylated or nonglycosylated C-terminal fragment, and U.S. Patent No. 5,661,008 have described the blood coagulation factor VIII variant that comprises the amino acid/11-740 that is connected by at least 3 amino-acid residues with amino acid/11 649～2332.Therefore, the variant of blood coagulation factor VIII, derivative, modifier and mixture are well known in the art and are contained among the present invention.

The expression system that produces blood coagulation factor VIII is well known in the art, and comprises protokaryon and eukaryotic cell, as in U.S. Patent No. 5,633, and 150,5,804,420 and 5,422, example shown in 250.

In order to determine the biologic activity of the method according to this invention synthetic blood coagulation factor VIII molecule, the technician will recognize that being used to of describing herein estimate that the mensuration of proconvertin and plasma thromboplastin component can be applicable to blood coagulation factor VIII.

N. urokinase

The present invention also comprises reconstruct and/or the method for modifying urokinase.Urokinase is that the Profibrinolysin activation is the serine protease of plasmin.This protein is synthetic in various tissues, comprises endothelium and kidney, and is excreted in the urine with trace.The urokinase of purifying exists with two kinds of activity forms, i.e. high-molecular weight form (HUK; About 50kDa) and low-molecular-weight form (LUK; About 30kDa).Shown that LUK is derived from HUK, it passes through the proteolysis behind Methionin 135, thereby discharges preceding 135 amino acid from HUK.It is generally acknowledged that HUK or LUK must be by carrying out proteolysis changes proteolytic activity into the activity form (this form continues corresponding to HUK or LUK) that generates two chains form to the strand precursor that is called uPA between Methionin 158 and Isoleucine 159.The urokinase kind of the proteolytic activity that obtains from this proteolysis is pruned contains two amino acid chains that link together by single disulfide linkage.Prune these two chains that form by activation and be called A or A ₁Chain (being respectively HUK or LUK) and comprise the B chain of the proteolytic enzyme structural domain of molecule.

Shown that urokinase is effective thrombolytics.Yet, because it is natively with the trace generation, so the cost of the enzyme of effective dose is high.Urokinase is produced in the reconstitution cell culture, and the coding DNA of urokinase and appropriate carriers and host microorganism all are known.The method of existing composition that comprises urokinase and recombinant production urokinase is had the obstruction of product of the glycosylation pattern of defective, and owing to there is complicated proteolysis to cut incident when urokinase activate, this unusual glycosylation causes poor efficiency and product low ability.

The urokinase peptide of reconstruct can be applied to following patient, and this patient is selected from the patient that suffers from embolism, suffer from the patient of acute massive pulmonary embolism and suffer from the patient of Coronary thrombosis.Preferably, the patient is people patient.The urokinase peptide of reconstruct also can be used for recovering the opening of intravenous catheter, comprises the central vein conduit that is stopped up by agglomerative blood or fibrin.

The nucleotide sequence of coding urokinase one-level amino acid chain is described among SEQ ID NO:33 and the SEQID NO:34 and (is respectively Figure 73 A and 73B).The variant of urokinase is well known in the art, so the present invention is not limited to the sequence that proposes herein.In fact, the technician is easy to recognize in U.S. Patent No. 5,219, and the urokinase variant of describing in 569,5,648,253 and 4,892,826 exists as the part that function is arranged, and therefore is contained among the present invention.

Expression and estimation to prepared according to the methods of the invention urokinase molecule also are well known in the art.As nonrestrictive example, the expression of urokinase in various systems is described in detail in U.S. Patent No. 5,219, in 569.Activity and functional assay method of the urokinase of the method preparation that definite basis provides herein have description in the document, and the mensuration relevant with fibrin with other Profibrinolysins of describing elsewhere is similar.An active assay method of determining described herein method synthetic urokinase molecule is described in as people such as Ploug (1957, Biochim.Biophys.Acta 24:278-282) in, this assay method is used the fibrin flat board that comprises 1.25% agarose, 4.1mg/ml human plasminogen, 0.3 unit/ml zymoplasm and 0.5 μ g/ml Trypsin inhibitor SBTI.

O. people DNase

The present invention further comprises the method for reconstruct and/or modified and recombined human DNase.People DNase I has been verified as therapeutical agent and has shown can be in the mucous viscosity of external minimizing cystic fibrosis.Fixed is the DNA that pyogenic mucus contains the 10-13mg/ml that has an appointment, and promptly influences the ionic polymer of respiratory tract fluidic rheological property.Therefore, the enzyme ox pancreas DNase I of degradable DNA promptly is verified as mucolytic agent many years ago, but does not enter clinical practice, and this is the side effect that causes because of the proteolytic enzyme by antigenicity and/or pollution.Recombinant human DNase useful as therapeutics is to alleviate the symptom as the cystic fibrosis disease at present.

The rDNase peptide of reconstruct can be applied to the patient who suffers from cystic fibrosis.The rDNase peptide of reconstruct also can be applied to cystic fibrosis patient to improve pulmonary function.Preferably, the patient is people patient.

Similar with the DNase that is derived from Niu Laiyuan, recombinant human DNase has also caused some problems, and great majority are owing to the effect that reduces, and this is owing to the unsuitable glycosylation that is caused by present used mammalian expression system.The invention describes the method for reconstruct DNase, this method can cause the effect that increases and better treatment result.

The Nucleotide of people DNase and aminoacid sequence propose (being respectively Figure 74 A and 74B) with SEQ ID NO:39 and SEQ ID NO:40 herein.The peptide variant that comprises DNase is well known in the art.As an example, U.S. Patent No. 6,348,343 have described the people DNase that has a plurality of amino acid replacements in whole primary structure.In addition, U.S. Patent No. 6,391,607 have described the DNase variant that 9,14,74,75 and 205 extremes with a plurality of amino acid replacements are enlivened in the position.Present embodiment and other are well known in the art or in the future findablely all be contained among the present invention.

The expression system that produces the DNase peptide is that the technician is well-known, and has been described in prokaryotic organism and the eukaryote system.For example, PCT patent publications No.WO 90/07572 has described in detail these methods.

The assay method of determining the biologic activity of the DNase molecule that the method according to this invention develops is well known in the art.As an example, but never mean restriction the present invention, the assay method of the DNA-hydrolytic activity of definite people DNase I is provided herein.In brief, two different plasmid digestion assay methods have been used.First assay method (" super coiled DNA digestion assay method ") is measured the double-stranded plasmid DNA of superhelix to transformation lax (the tool breach), linearity and form degraded.The double-stranded plasmid DNA of second assay method (" linear DNA digestion assay method ") measure linear is to the transformation of the form of degraded.Specifically, prepared according to the methods of the invention DNase is joined in the 160 microlitre solution, and this solution comprises the super spirial plasmid DNA in 25mM HEPES (pH7.1) of every milliliter 25 microgram or linearizing plasmid DNA, 100 μ g/ml bovine serum albumins, the 1mM MgCl of EcoR I-digestion ₂, 2.5mM CaCl ₂, 150mM NaCl, and sample carried out incubation in room temperature.Obtain the reaction mixture of sample aliquot and termination reaction in each time by adding 25mM EDTA and dimethylbenzene nitrile indigo plant, tetrabromophenol sulfonphthalein and glycerine.The integrity of plasmid DNA can be analyzed by sample is carried out electrophoresis on sepharose in the terminated sample.Behind electrophoresis, gel is dyeed with bromination second key, and the DNA in the gel is manifested by ultraviolet ray.Supercoiled in the plasmid DNA, the lax relative quantity with linear forms is by scanning gel with fluorescent image instrument (as MolecularDynamics Model 575 FluorImager) and to carrying out quantitatively determining corresponding to the DNA in the multi-form band on the gel.

P. Regular Insulin

The present invention further comprises the method for reconstruct Regular Insulin.Regular Insulin is well-known the most effective treatment medicine to type i diabetes, and the β islet cells of pancreas does not produce the Regular Insulin that is used to regulate blood glucose levels in type i diabetes.The consequence of diabetes and blood-glucose out of control comprises circulation and foot problems and blind, and various other complication that caused by diabetes or that worsened by diabetes.

Before the insulin human being cloned and check order, pork insulin is used as the treatment of diabetes medicine.Regular Insulin is recombinant production at present, but the short sequence of 51 amino acid of ripe molecule is the complex construction that comprises a plurality of disulfide linkage.The method of existing recombinant production pancreas islet element causes lacking with the natural protein that produces the product of similarity in the non-insulin subject of health.The present invention seeks to remedy this shortcoming.

The insulin peptide of reconstruct can be applied to following patient, this patient be selected from suffer from type i diabetes (diabetes) the patient and suffer from diabetes B and need basis (long term) Regular Insulin with control hyperglycemia the patient.The insulin peptide of reconstruct also can be applied to the diabetic subject with the control hyperglycemia.Preferably, the patient is people patient.

Insulin human's Nucleotide and aminoacid sequence are described (being respectively Figure 75 A and 75B) respectively in SEQ ID NO:43 and SEQ ID NO:44.The Regular Insulin variant is a large amount of in the art.U.S. Patent No. 6,337,194 have described Regular Insulin fused protein analogue, U.S. Patent No. 6,323,311 have described the insulin derivates of the cyclic anhydride that comprises dicarboxylic acid, and U.S. Patent No. 6,251,856 have described the insulin derivates that comprises a plurality of amino acid replacements and lipophilic group.Example that the technician will recognize following insulin derivates is exclusiveness anything but, has only represented those small portions well known in the art.Therefore, the present invention comprises known maybe with the insulin derivates of finding.

The expression system that is used to produce Regular Insulin is well known in the art, and the useful molecules biology techniques is realized, as be described in people (1989 such as Sambrook, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) in.

Be used for determining that functional assay method of prepared according to the methods of the invention insulin molecule also is well known in the art.For example, the external model that suppresses of glucose can be used for estimating the biologic activity with method synthetic Regular Insulin of the present invention.What can be used for this purpose is rat model.Before experiment, make animal overnight fasting (16 hours), give vetanarcol or other appropriate anaesthetization agent such as ketamine with intraperitoneal then and anaesthetize.The intravenous injection (tail vein) of each animals received particular insulin derivative (20 μ g/ml/kg).15 and 5 minutes and injection back obtained blood sample from jugular vein in 15,30,60,90,120,180 and 240 minutes before injection.Blood glucose levels is measured with the blood-glucose monitor, and this monitor can be available from each commercial supplier.

Q. hepatitis B vaccine (HBsAg)

The present invention further comprises the method for the antigen (HbsAg or hepatitis B surface antigen(HBsAg)) that reconstruct is used for hepatitis B vaccine.HBsAg is the proteinic surface antigen of the hepatitis B S-of recombinant production, and be used to cause immune response to hepatitis B virus, this virus is the virus of increasingly dangerous, can cause hepatopathy such as liver cirrhosis and cancer etc., and causes that the whole world is annual to surpass 100 ten thousand death.The HBsAg vaccine gives 3 times to cause the immune response of protectiveness and neutrality 6 months the timed interval at present.

HBsAg produces in the yeast strain at present, has therefore reflected the glycosylation pattern that fungi is natural.The invention provides the method for reconstruct HBsAg, the immunogenicity that this method also causes improving, have improvement to antibody of the avidity of virus etc.

The HBsAg peptide of reconstruct can be applied to the patient, the patient is resisted the immunization by hepatitis B virus-induced diseases.The HBsAg peptide of reconstruct also can be applied to dialysis preceding (predialysis) or dialysis patients, the patient is resisted the immunization by hepatitis B virus-induced diseases.Preferably, the patient is people patient.

With SEQ ID NO:45 and SEQ ID NO:46 (being respectively Figure 76 A and 76B) proposed herein from the nucleic acid of hepatitis B virus S-protein (HBsAg) and the sequence of one-level amino acid chain.Length of nucleotides is 1203 bases.Amino acid is that 400 residues are long.Last 226 amino-acid residues are little S-antigen, and this antigen can be used for GlaxoSmithKline vaccine and Merck vaccine.55 amino acid of little S-antigen upstream are the Pre-S initiator codons.It is middle S-antigen that this Pre-S adds the S zone, and this antigen can be used for Aventis Pasteur vaccine.Comprise the proteinic rest part of S-the initiator codon from first codon to Pre-S, and be called big S-protein.This only is an example that is used for the HBsAg of vaccine, and other hypotypes are well-known, as by GenBank Acc No.:AF415222, AF415221, AF415220 and AF415219 institute example.The sequence of Ti Chuing only is the example of HBsAg as known in the art herein.Similarly antigen separates from other strains of hepatitis B virus, and estimation has been done or do not done to its antigenicity and potentiality as vaccine candidate object.Therefore the present invention comprises known maybe with the Hepatitis B virus vaccine S-protein surface antigen of finding.

The expression of HBsAg in expression system is conventional procedure for a person skilled in the art, and is described in as U.S. Patent No. 5,851, in 823.Mensuration to vaccine immunogenicity is well known in the art, and comprises the various mensuration that produce neutralizing antibody, and uses as technology such as ELISA, neutralization mensuration, western blotting, immunoprecipitations.In brief, the sandwich ELISA that is used to survey effective resisting-HBsAg antibody has been described.(AventisBehring, King of Prussia PA) can be used in this method Enzygnost HBsAg mensuration.The hole is covered with anti--HB.Add to the protein of serum blood plasma or purifying and suitable contrast in the hole and carry out incubation.After the washing, make the antibody and the reaction of remaining antigenic determinant of the peroxidase labelling of anti-HBsAg.Remove unconjugated enzyme len antibody by washing, and the enzymic activity on the solid phase is determined by method well known in the art.Enzymatic reaction to hydrogen peroxide and chromogen is terminated by the sulfuric acid that adds dilution.The concentration of HBsAg is proportional in colour intensity and the sample, and is by relatively obtaining to the colour intensity of unknown sample and the negative contrast of following with over against the luminosity according to the colour intensity of serum.

R. human growth hormone

The present invention further comprises the method for reconstruct human growth hormone (HGH).Excretory HGH isotype is made up of 191 amino acid and is had a molecular weight of about 21,500 in people's hypophysis.The HGH isotype for preparing in placenta is glycosylated form.HGH participates in many adjustings of normal people's g and D, comprises linear growth (physique generation), lactation, macrophage activation and Insulin-Like and causes glycosuria effect etc.

HGH is complicated hormone, and it is used as with the results of interaction of various cell receptors and changes.Be used under the clinical setting although comprise the composition of HGH, especially treated in the nanism, its effect is subjected to recombinating and does not have glycosylated restriction among the HGH that produces.

The HGH peptide of reconstruct can be applied to following patient, this patient is selected from the patient, the patient who suffers from Turner syndrome that suffer from growth hormone deficiency, suffer from owing to lack enough endogenous growth hormone secretions grow insufficient patient, suffer from because Pu-Wei syndrome (PWS) is grown insufficient patient, suffer from and suffer from becoming thin or cachectic patient that AIDS is correlated with the insufficient relevant insufficient patient of growth of chronic renal.The HGH peptide of reconstruct also can be applied to the little patient of stature.Preferably, the patient is people patient.

The nucleic acid of HGH and aminoacid sequence propose (being respectively Figure 77 A and 77B) with SEQ ID NO:47 and SEQ ID NO:48 herein.The technician will recognize that variant, derivative and the mutant of HGH are well-known.Example can be found in U.S. Patent No. 6,143, in 523, wherein

position

10,14,18,21,167,171,174,176 and 179 amino-acid residue are alternate, and are found in U.S. Patent No. 5,962, in 411, this patent has been described the splice variant of HGH.The present invention comprises these HGH variants as known in the art and with the variant of finding.

The method of expressing HGH in reconstitution cell is described in as U.S. Patent No. 5,795, in 745.Inter alia, the method for expression HGH is well known in the art in prokaryotic organism, eukaryote, insect cell system, plant and external translating system.

The known the whole bag of tricks of HGH molecule available techniques personnel that produces with method of the present invention carries out determination of activity.For example, U.S. Patent No. 5,734,024 has described the method for biological function of the HGH of definite expression.

S. Antithrombin III

(Antithrombin III AT-III) is the strong inhibitor of cascade that condenses in the blood to antithrombin.It is the non-vitamin k-dependent protein enzyme of Trombin inhibiting and other procoagulant factors (procoagulant factor) (as factor Xa).Congenital Antithrombin III deficiency is the autosomal dominant symptom, wherein idiogenetics the dcc gene of a copy.This situation causes the vein that increases and the risk of artery thrombosis, and the outbreak of clinical manifestation generally comes across the Young Adults phase.The severe congenital Antithrombin III deficiency of two dcc genes of idiogenetics is and the relevant autosomal recessive situation of thrombosis that increases, generally remarkable in petticoats.Acquired Antithrombin III deficiency is most commonly in the incorrect activatory situation of condensed system.Cause the insufficient general situation of acquired Antithrombin III comprise disseminated inravascular coagulation, because the microangiopathic hemolytic anemia (being hemolytic uremic syndrome) of endothelial injury and see veno-occlusive disease (VOD) among the patient who carries out bone marrow transplantation.AT-III can not carry out short-term by infusion AT-III enriched material completely and correct.

The AT-III peptide of reconstruct can be applied to following patient, and this patient is selected from the not enough patient of the heredity AT-III that suffers from the heredity AT-III insufficient patient relevant with surgery or obstetric operation and suffer from thromboembolism.Preferably, the patient is people patient.

Antithrombin III (AT-III) is that molecular weight is 58,000 α 2-glycoprotein.It is with Thrombate III ^TMCarry out commercial distribution (Bayer Corp., West Haven, CT).The nucleic acid of people's Antithrombin III and aminoacid sequence are shown in respectively among Figure 78 A (SEQ ID NO:63) and the 78B (SEQ ID NO:64).

The method for preparing Antithrombin III is that those skilled in the art are well-known.For example, the nucleic acid and the aminoacid sequence of the announcement of the mutant of people's Antithrombin III (referring to U.S. Patent No. 4,517,294) and people's Antithrombin III (referring to U.S. Patent No. 5,420,252,5,618,713,5,700,663) are obtainable.Method of the present invention can be used any of these aminoacid sequence and any its encoding sequence, but is not limited to these sequences.The illustrative methods that produces the reorganization Antithrombin III is well known in the art, and several method is described in U.S. Patent No. 5,420, in 252,5,843,705,6,441,145 and 5,994,628.The illustrative methods of purification of Recombinant Antithrombin III is described in U.S. Patent No. 5,989, in 593,6,268,487,6,395,888,6,395,881,6,451,978 and 6,518,406.

The reorganization Antithrombin III has many known purposes.Antithrombin III can be used as the antithrombotics (U.S. Patent No. 5 in the surgical operation, 252,557,5,182,259), as the part (U.S. Patent No. 5 that suppresses thrombotic pharmaceutical preparation or method, 565,471,6,001,820) and the harmful side effect (U.S. Patent No. 6 that reduces Transplanted cells, 387,366).In addition, the Antithrombin III preparation can be used for increasing placental blood flow (U.S. Patent No. 5,888,964), suppresses fertilization (U.S. Patent No. 5,545,615), treatment asthma (U.S. Patent No. 6,355,626) and treatment of arthritis (U.S. Patent No. 5,252,557) and other inflammation (U.S. Patent No. 6,399,572).Antithrombin III also can be used for making to be replaced blood plasma (U.S. Patent No. 4,900,720) or prepares the stabilized cell blood products (U.S. Patent No. 6,139,878) that is used to transfuse blood.Antithrombin III can be used as that pharmaceutical preparation is used (U.S. Patent No. 5,084,273,5,866,122,6,399,572,6,156,731 and 6,514,940) or the applying gene methods of treatment is used (U.S. Patent No. 6,410,015).The composition that comprises Antithrombin III can be used as tissue adhesive (U.S. Patent No. 6,500,427) or is incorporated into the lubricant (U.S. Patent No. 6,391,832) of the medical facilities among the patient.Antithrombin III also can be used for bag usually by stent (U.S. Patent No. 6,355,055,6,240,616,5,985,307,5,685,847 and 5,222,971), ocular implant (U.S. Patent No. 5,944,753) and false organ (U.S. Patent No. 6,503,556,6,491,965 and 6,451,373).Antithrombin III also can be used for inside in the position patient and bleeds and stop blooding among site (U.S. Patent No. 6,314,314) and the definite patient in the method for dysfunction (U.S. Patent No. 6,429,017).

T. human chorionic gonadotrophin

The glycoprotein that human chorionic gonadotrophin (hCG) is made up of α subunit and β subunit.HCG and two other gonad-stimulating hormone lutropin (LH) and follicle stimulating hormone (FSH) and thyrotropic hormone (TSH) are closely related, and all these three kinds of hormones all are glycoprotein hormoneses.The α subunit of these different sugar proteohormonees is structurally closely similar, but the β subunit is different on aminoacid sequence.

The nucleic acid of human chorionic gonadotrophin α subunit and aminoacid sequence are shown in respectively among Figure 79 A (SEQ ID NO:69) and the 79B (SEQ ID NO:70).The nucleic acid of human chorion gonadotrophic hormone beta subunit and aminoacid sequence are shown in respectively among Figure 79 C (SEQ ID NO:71) and the 79D (SEQ IDNO:72).

Human chorionic gonadotrophin be used for infertility treatment with promote can not own ovulation the women ovulate or from ovary, discharge ovum.Human chorionic gonadotrophin also is applied to the young male sex and does not drop to scrotum or undergrown testis with treatment.It is used for the generation of male sex's moderate stimulation testosterone.Some doctors also give the patient who suffers from the erectile dysfunction that lacks sexual desire and human chorionic gonadotrophin are opened in male sex's's " climacteric " treatment write out a prescription.

The hCG peptide of reconstruct can be applied to following patient, and this patient is selected from the patient who carries out auxiliary procreation technology (ART), the patient who carries out (IVF) in vitro fertilization, the patient who carries out embryo transfer, infertile patients, suffer from and be not because the male patient of the preadolescence cryptorchidism that anatomy is blocked and suffer from the male patient of low gonadotropin hypofunction.The hCG peptide that also can use reconstruct in sterile female patient is to induce final follicle maturity, and wherein this sterile female patient has carried out the hypophysis desensitization and carried out pretreat with follicle stimulating hormone.Also can in the infertile patients of not ovulating, use the hCG peptide of reconstruct with induced ovulation and pregnancy.Preferably, the patient is people patient.

The method for preparing human chorionic gonadotrophin is that those skilled in the art are well-known.The heterodimer hCG preparation of can in the many expression systems that are used for the industrial production recombinant protein at present any, recombinating.A kind of method for preparing the hCG that recombinates is described in U.S. Patent No. 5,639, in 639.Normally well known in the art by in same cell, expressing two subunit preparation reorganization heterodimer method of protein, and several method is described in U.S. Patent No. 5,643,745 (in filamentous fungus, expressing), 5,985,611 and 6,087,129 (in secretory cell, expressing).Selectively, each subunit can be expressed in cell separately, and external two subunits is placed together to be assembled into heterodimer later on.

It is very many and be well known in the art to use the method for human chorionic gonadotrophin.Usually, hCG is used to induce the Mammals ovulation or makes Mammals ovulation synchronization (referring to U.S. Patent No. 6,489,288,5,589,457,5,532,155,4,196,123,4,062,942 and 4,845,077).In addition, hCG can be used in the conceived test, particularly based in the agglutinative test (referring to U.S. Patent No. 3,991,175,4,003,988,4,071,314 and 4,088,749).HCG also can be used for (referring to U.S. Patent No. 4,161,519 and 4,966,888) in the contraceptive bian vaccine.In addition, hCG can be used for treating situation such as the benign prostatauxe (referring to U.S. Patent No. 5,610,136) relevant with hormonal equilibrium old and that change and is common in central nervous system disease (referring to U.S. Patent No. 4,791,099) among the elderly.

Selectively, hCG can be used for surveying and treating the cancer of expressing a hCG or an one subunit.The tumour of expressing hCG includes, but are not limited to mammary cancer, prostate cancer, ovarian cancer and cancer of the stomach, and neuroblastoma such as Kaposi sarcoma.Can produce the antibody of anti-hCG, thereby this hCG has carried out sugared reconstruct to have and the similar glycan structures of finding on the hCG of tumour expression of glycan, and these antibody can be used for surveying the tumour of expression hCG (referring to U.S. Patent No. 4 in the patient according to method well known in the art, 311,688,4,478,815 and 4,323,546).In addition, the hCG of reconstruct can be used for causing the immune response (referring to U.S. Patent No. 5,677,275,5,762,931,5,877,148,4,970,071 and 4,966,753) to the tumour of expressing hCG.

HCG also can be used for usually animal being carried out in the immunomodulatory method, as is described in U.S. Patent No. 5,554, the method in 595,5,851,997 and 5,700,781.In addition, hCG can be used as the inhibitor of matrix metalloproteinase in the situation that benefits from this treatment, the disease (referring to U.S. Patent No. 6,444,639) that this situation such as chronic inflammatory diseases, multiple sclerosis and dependence blood vessel take place.

U. α-idose glycosides enzyme

α-idose glycosides enzyme is with Aldurazyme ^TMCarry out commercial distribution (BioMarin andGenzyme).It is used for the replacement therapy of MPS I (a kind of lysosomal storage disease).MPS I (being also referred to as dysostosis multiplex) is by the genetic diseases that α-L-idose glycosides enzyme deficiency causes, and this enzyme normally some compounding sugar that is called glycosaminoglycan (GAG) decomposes necessary.If enzyme does not exist with enough amounts, the normal decomposition of GAG is incomplete or is blocked so.Thereby cell can not be discharged saccharide residue, so they are accumulated in the lysosome of cell and cause MPS I.

The α of reconstruct-idose glycosides enzyme peptide can be applied to following patient, and this patient is selected from the patient that suffers from lysosomal storage disease, suffer from α-insufficient patient of L-idose glycosides enzyme, suffer from the patient of mucopolysaccharidosis I (MPS I) and suffer from the patient of dysostosis multiplex.Preferably, the patient is people patient.

Produce and the method for purifying α-idose glycosides enzyme and the method for the treatment of some hereditary symptom are described in U.S. Patent No. 6,426, in 208, this heredity symptom comprises α-L-idose glycosides enzyme deficiency and mucopolysaccharidosis I (MPS I).The nucleic acid and the aminoacid sequence of human α-idose glycosides enzyme see respectively among Figure 80 A (SEQ ID NO:65) and the 80B (SEQ ID NO:66).

V. alpha-galactosidase A

Alpha-galactosidase A (being also referred to as agalsidase β) is with Fabrazyme ^TMCarry out commercial distribution (Genzyme).Alpha-galactosidase A is used for the treatment of Fabry disease.Fabry disease is the rare hereditary symptom that the alpha-galactosidase A deficiency by key causes.During this enzyme, the Fabry patient can not decompose the fatty acid material that is called globotriasylceramide (GL-3) in its body, accumulates in the cell of this material in the heart, kidney, brain and other critical organ blood vessels.Building up of this material makes the patient have the risk of apoplexy, heart attack, injury of the kidney and debilitating pain.Many patients develop into renal failure in adulthood, and serious organ complication caused death in the time of about 40 years old.

The alpha-galactosidase A peptide of reconstruct can be applied to following patient, and this patient is selected from the patient that suffers from lysosomal storage disease, suffer from the insufficient patient of alpha-galactosidase A and suffer from the patient of Fabry disease.Preferably, the patient is people patient.

Alpha-galactosidase A is hydrolysis globotriaosylceramide and relevant lysosomal enzyme with glycolipid of terminal alpha-galactosidase key.It is the N-glycosylated protein of the 45kDa of coding on X chromosome is long-armed.The initial glycosylation form of synthetic (Mr=55,000～58,000) is processed to ripe glycosylation form (Mr=50,000) in human fibroblasts or Chang liver cell.From people tissue and blood plasma the ripe organized enzyme of purifying be homodimer (people such as Bishop, 1986, Proc.Natl.Acad.Sci.USA83:4859-4863).The nucleic acid of alpha-galactosidase A and aminoacid sequence see respectively among Figure 81 A (SEQ ID NO:67) and the 81B (SEQ ID NO:68).The nucleic acid of the alpha-galactosidase A that other are useful and aminoacid sequence see U.S. Patent No. 6,329, in 191.

The reference how instruction prepares alpha-galactosidase A sees U.S. Patent No. 5,179,023 and 5,658,567 (in expressed in insect cells), U.S. Patent No. 5,356,804 (express in mammalian cell and secretion, comprise Chinese hamster ovary celI), U.S. Patent No. 5,401,451 (in mammalian cell, expressing), U.S. Patent No. 5,580,757 (in mammalian cell, expressing) and U.S. Patent No.s 5,929,304 (in vegetable cell, expressing) as fused protein.The method that is used for the purification of Recombinant alpha-galactosidase A sees U.S. Patent No. 6,395, in 884.

How instruction is used alpha-galactosidase A and is included, but are not limited to U.S. Patent No. 6,066,626 (gene therapies) and U.S. Patent No. 6,461,609 (treating with protein) with the reference for the treatment of the patient.The alpha-galactosidase A mutant that is used for the inventive method includes, but are not limited to those and is described in U.S. Patent No. 6,210, the mutant in 666.

W. antibody

The present invention further comprises the various methods that comprise the antibody preparation of chimeric antibody preparation of reconstruct, comprise chimeric TNFR, chimeric anti--glycoprotein iib/iiia, chimeric anti--HER2, chimeric anti--RSV, chimeric anti-CD 20 and chimeric anti-TNF.The chimeric antibody preparation comprises from the people Fc part of IgG antibody with from the variable region to the specific monoclonal antibody of antigen.Other preparations comprise the acceptor with human IgG Fc meromixis, as the TNF acceptor of 75kDa.These molecules further comprise the Fab fragment that comprises from light chain and the heavy chain of people and mouse.Chimeric TNFR can be used for treating inflammatory disease, as rheumatoid arthritis.Chimeric anti--glycoprotein iib/iiia can be used for treating heart abnormality, coagulation of blood and platelet function disorder.Chimeric anti--HER2 can be used as the medicine of mammary cancer, and chimeric anti--RSV can be used for treating respiratory syncytial virus, and chimeric anti-CD 20 can be used for treating non_hodgkin lymphoma, and chimeric anti-TNF can be used for treating the CrohnShi disease.

Although having proved, these chimeric antibodies can be used for handling various diseases, owing to short relatively transformation period of the recombinant protein that produces needs quite frequently and with quite high dosage to give in the rodents cell.Although most of chimeric antibodies are people, therefore be " self " by immune system recognition, they will be degraded and destroy owing to non-natural glycosylation pattern.The present invention will address this problem, thereby increases the effect of these novel drugs greatly.

Antibody and generation method thereof

As used herein, term " antibody " refer to can be specifically with antigen on specific antigen decision position bonded immunoglobulin molecules.Antibody can be the complete immunoglobulin (Ig) that is derived from natural origin or is derived from recombinant sources, perhaps the immunoreactivity part of complete immunoglobulin (Ig).Antibody generally is the immunoglobulin molecules tetramer.Antibody among the present invention can exist with various forms, and this form comprises as polyclonal antibody, monoclonal antibody, Fv, Fab and F (ab) ₂And single-chain antibody and humanized antibody (people such as Harlow, 1999, Using Antibodies:ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; People such as Harlow, 1989, Antibodies:A Laboratory Manual, Cold Spring Harbor, New York; People such as Houston, 1988, Proc.Natl.Acad.Sci.USA 85:5879-5883; People such as Bird, 1988, Science 242:423-426).

As used herein, term " synthetic antibody " refers to the antibody that generates with recombinant DNA technology, as by the antibody of described phage expression herein.This term also should be interpreted as the antibody that the aminoacid sequence by the DNA of composite coding and expressing antibodies or encoding antibody generates, and wherein DNA or aminoacid sequence are to obtain with available in this area and well-known synthetic DNA or aminoacid sequence technology.

Monoclonal antibody at full-length peptide or peptide fragment can prepare with any well-known Monoclonal Antibody program, be described in people such as Harlow (1988 as those, Antibodies, ALaboratory Manual, Cold Spring Harbor, NY) and people such as Tuszynski (1988, Blood is in 72:109-115).The also available chemical synthesising technology of the peptide of wanting is synthetic.Selectively, the DNA of the peptide wanted of coding can clone and express from suitable promoter sequence in being suitable for generating the cell of a large amount of peptides.At the monoclonal antibody of peptide be with as herein the standard program of institute's reference from mouse, generate with this peptide immunization.

Coding is used in the nucleic acid of the monoclonal antibody that program described herein obtains and can clones and check order with available technology in this area, and be described in as people such as Wright (1992, Critical Rev.in Immunol.12 (3,4): 125-168) reach in the reference of quoting therein.Further, antibody of the present invention can (1997, Thrombosis and Hematocyst 77 (4): the technology 755-759) be carried out " humanization " with being described in people's (seeing above) such as Wright and people such as reference of quoting therein and Gu.

In order to generate phage antibody library, at first from the mRNA of cell (as hybridoma), obtain the cDNA library from separating, this mRNA expresses the peptide of wanting that will be expressed on the phage surface, as the antibody of wanting.The cDNA copy of mRNA produces with ThermoScript II.The cDNA of coding immunoglobulin fragment obtains by PCR, and the dna clone of gained is as a result gone in the suitable phage vector to generate the phage DNA library, and this library comprises the immunoglobulin gene that DNA limits.The program of phage library that preparation comprises allogeneic dna sequence DNA is well known in the art, and be described in as Sambrook and Russell (2001, Molecular Cloning:A LaboratoryManual, Cold Spring Harbor, NY).

The phage of the antibody that can want coding is processed, thereby peptide shows in its surface that in such a way promptly these peptides can its corresponding binding peptide combination, as antibody at antigen.Therefore, when the phage of expressing specific antibodies carries out incubation when the cell of expressing corresponding antigens exists, phage will be attached on the cell.The phage of expressing antibodies will not be not joined on the cell.This panning technique is well known in the art and is described in as among the people such as Wright (seeing above).

Developed the method for producing people's antibody with the M13 phage display, as those are described in the above (people such as Burton, 1994, Adv.Immunol.57:191-280).Basically, the cDNA library generates from mRNA, and this mRNA obtains from the cell colony that produces antibody.The immunoglobulin gene that this mRNA coding is reset, thereby the immunoglobulin gene of the same rearrangement of cDNA coding.The cDNA of amplification is cloned in the M13 expression vector, thereby is created on the phage library of its surface expression people antibody fragment.Show the phage of target antibody by antigen in conjunction with selection, and it is bred in bacterium to produce the human normal immunoglobulin of solubility.Thereby synthetic opposite with conventional monoclonal antibody, this program makes the DNA of coding human normal immunoglobulin rather than the cell immortalityization of expressing human immunoglobulin (Ig).

The reconstruct of antagonist molecule glycan

One class peptide is the specific glycosylation of immunoglobulin (Ig) has particularly important to the biologic activity of these peptides effect.The present invention should not be construed as the immunoglobulin (Ig) that only is confined to the IgG class, and should be interpreted as comprising the immunoglobulin (Ig) of IgA, IgE and IgM antibody-like.

Further, the present invention should not be construed as the conventional antibody structure that only is confined to any kind.On the contrary, the present invention should be interpreted as comprising all types of antibody molecules, comprises as antibody fragment, chimeric antibody, people's antibody, humanized antibody etc.

General immunoglobulin molecules comprises effect part and antigen-binding portion thereof.For the summary of immunoglobulin (Ig), referring to people such as Harlow, 1988, Antibodies:A Laboratory Manual, Cold Spring Harbor, New York; With people such as Harlow, 1999, Using Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY.The effect of immunoglobulin (Ig) partly is positioned at the Fc part and the part of molecule and is responsible for the effective combination of immunoglobulin (Ig) to its relevant cell receptor.The immunoglobulin molecules particularly incorrect glycosylation in this molecule Fc partial C H2 structural domain can influence the biologic activity of immunoglobulin (Ig).

About immunoglobulin IgG, more specific is, whether IgG effector function major part contains the decision of N-acetyl-glucosamine (GlcNAc) residue by IgG, and this residue is attached to the 4-0 position of branch's seminose of three seminose cores of the connection of the N-on the l-asparagine (Asn) 297 glycan in the IgG molecule CH2 structural domain.This residue is known as " GlcNAc of five equilibrium ".The purpose of adding the GlcNAc of five equilibrium to natural or reorganization IgG molecule or containing in the N-polysaccharide chains of chimeric construct body of IgG-Fc is the Fc immunological effect function optimization that makes this molecule Fc part.This effector function can comprise cytotoxicity (ADCC) and any other the biological action that relies on antibody, this biological action need to effective combination of Fc γ R acceptor and with the combining of the C1 composition of complement.The GlcNAc that has described five equilibrium is for the importance of the maximum immunological effect function that realizes the IgG molecule (people such as Lifely, 1995, Glycobiology5 (8): 813-822; People such as Jeffris, 1990, Biochem.J.268 (3): 529-537).

The glycan of finding at the glycosylation site of the Asn297 of IgG molecule CH2 structural domain is structurally for the IgG molecule of finding to be circulated in the humans and animals blood plasma, and the IgG that is produced by the immortalization Mammals and the insect cell line of myeloma cell, hybridoma and various transfections is distinctive.In all cases, the N-glycan is (Man3, GlcNAc4, Gal2, NeuAc2, the Fuc1) completely of high mannose chain or the GlcNAc that has or do not have five equilibrium or incomplete pair of variable feeler chain (people such as Raju, 2000, Glycobiology 10 (5): 477-486; People such as Jeffris, 1998, Immunological.Rev.163L59-76; People such as Lerouge, 1998, Plant Mol.Biol.38:41-48; People such as James, 1995, Biotechnology 13:592-596).

The invention provides glycosylation immunoglobulin molecules in external customization.This immunoglobulin molecules can be any immunoglobulin molecules, including, but not limited to monoclonal antibody, synthetic antibody, chimeric antibody, humanized antibody etc.Generation antibody reaches to be described elsewhere to its ad hoc approach that characterizes.Preferably, immunoglobulin (Ig) is IgG, and more preferably IgG is humanized or human IgG, is most preferably IgG1.

The present invention specifically relates to use β 1,4-mannose group-glycopeptide β 1,4-N-acetylgucosamine transferase GnT-III:EC2.4.1.144 is as external reagent, so that N-acetyl-glucosamine (GlcNAc) is connected to by glycosidic link on the 4-0 position of branch's seminose of three seminose cores of the N-glycan that is positioned at Asn 297 in the IgG molecule CH2 structural domain.Yet as understandable from the disclosure that provides herein, the present invention should not be construed as and only comprises that this enzyme of application is to provide the GlcNAc of five equilibrium to immunoglobulin molecules.On the contrary, having found possible is that the glycosylation pattern of antagonist molecule is regulated, thereby this antibody molecule has the enhanced biologic activity except that the potential enhancing of other character such as stability etc., i.e. effector function.

Provide in the present invention for strengthen to the combination of Fc-γ RIIIA and enhanced rely on antibody Cytotoxic purpose and from the glycan that Asn (297) N-is connected the general method of removal Fucose molecule (referring to people such as Shields, 2002, J.Biol.Chem.277:26733-26740).The fucosidase that is connected of Fucose molecule contact on this method need make antibody molecule and be suitable for the antibody glycan.Selectively, recombinant antibodies can be expressed in the cell of expressing fucosyltransferase, as the Lec13 variant of Chinese hamster ovary celI.The removal of Fucose from the antibody glycan can be carried out separately, or combines as the GlcNAc that adds five equilibrium with the method for other reconstruct glycan and to carry out.The expression of antibody in the cell that lacks GnT-I also produces the Fc glycan that lacks the core Fucose, and this glycan can further be modified by the present invention.

The general method of introducing the GlcNAc of five equilibrium for the purpose that strengthens Fc immunological effect function in the IgG molecular preparation is provided in the present invention, and wherein this IgG molecule particularly contains the oligosaccharides that N-connects at Asn 297 in the CH2 structural domain.This method need make IgG molecule colony reach certain glycosylation state, thereby polysaccharide chains is the acceptor of GnT-III.This is by any one realization of following 3 approach: 1) by selection or genetic manipulation to host expression system, this host expression system secretion is as the IgG with N-polysaccharide chains of the substrate of GnT-III; 2) by IgG sugar form colony being handled, be the acceptor of GnT-III thereby handle the remaining glycan structures in back at exoglycosidase with exoglycosidase; 3) as mentioned 1) and 2) in the host selects and exoglycosidase is handled some combinations, add subsequently and add the acceptor of GlcNAc with generation GnT-III by GnT-I and GnT-II.

For example, the IgG that obtains from chicken plasma mainly contains the high mannose chain, and need digest with the generation substrate with one or more alpha-Mannosidases, thereby by GnT-I GlcNAc is added in α 1, the 3 seminose branch of seminose core.This substrate can be three basic seminose core, i.e. Man3GlcNAc2.As saccharide donor the processing of this core texture is generated as shown in Figure 1 Man3GlcNAc5 with UDP-GlcNAc with the combination of GnT-I, GnT-II and GnT-III.The sequence of operation of these glycosyltransferases can change so that the production optimization of required product.Selectively, available then

β

1,4 galactosyltransferase of this structure is handled and is extended.If desired, the oligosaccharides of galactosylation can further use α 2,3-or α 2,6-sialyltransferase to extend to obtain complete two feeler structures.Utilize present method, can be reconstructed (Fig. 3) to two feeler polysaccharide chains as the suitableeest Fc immunological effect function of the therapeutic IgG in any exploitation is necessary.

Selectively, be found in the IgG molecule in the blood plasma of most of animals or generally comprise a series of pairs of feeler sugar forms by most of zooblasts or by transgenic animal excretory IgG as recombinant products, this sugar form comprise the GlcNAc that has or do not have five equilibrium complete form (NeuAc2, Gal2, GlcNAc4, Man3, ± Fuc1) (Fig. 3) or variable incomplete form (people such as Raju, 2000, Glycobiology 10 (5): 477-486; People such as Jeffris, 1998, Immunological Rev.163:59-76).Be present in order to ensure the GlcNAc of five equilibrium in the whole immunoglobulin (Ig) colony of such generation, can in turn or in mixture handle molecule mixture: neuraminidase, beta-galactosidase enzymes, Hex, Alpha-Fucosidase with following exoglycosidase.Three seminose cores of gained can be reconstructed with aforesaid glycosyltransferase as a result then.

In some cases, need from existing antibody molecule, eliminate effector function.The present invention also comprises with suitable Glycosylase and glycosyltransferase modification Fc glycan, to eliminate effector function.Be contemplated that interpolation equally with PEG or other polymer-modified sugar, this PEG or other polymkeric substance are used to hinder or eliminate combining of Fc acceptor or complement and antibody.

In addition, to characterizing by the transgenic animal excretory with by the IgG of transgenic plant as " plant materials (plantibodies) " storage.What produce in transgenic plant has a β of containing 1,2 wood sugar and/or the α 1 that connect, the also available exoglycosidase of cutting except above-mentioned points outside the Glycosylase of the IgG molecule of the N-glycan of 3 Fucoses that connect is handled to remove these residues, thereby generate three seminose cores or Man3GlcNAc4 structure, handle with the above-mentioned N-glycan of reconstruct with glycosyltransferase then.

Main innovation of the present invention aspect is the application of suitable glycosyltransferase, and the exoglycosidase that this application has or do not have is in advance handled, and uses so that the effector function optimization of antibody with correct order.In an exemplary embodiment, the GlcNAc of five equilibrium is incorporated in the glycan of the IgG molecule of the GlcNAc that needs this five equilibrium or other IgG-Fc-chimeric construct bodies.In another exemplary embodiment, the core Fucose is removed from the glycan of IgG molecule or other IgG-Fc-chimeric construct bodies.

X.TNF acceptor-IgG Fc fused protein

The Nucleotide of the people TNF acceptor of 75kDa and aminoacid sequence propose (being respectively Figure 82 A and 82B) with SEQ ID NO:31 and SEQ ID NO:32 respectively herein.Chimeric anti--the light chain of HER2 and the aminoacid sequence of variable region of heavy chain propose (being respectively Figure 83 A and 83B) with SEQ ID NO:35 and SEQ ID NO:36 respectively.Chimeric anti--the heavy chain of RSV and the aminoacid sequence of variable region of light chain propose (being respectively Figure 84 A and 84B) with SEQ ID NO:38 and SEQ ID NO:37 respectively.The inhuman amino acid sequences of anti-TNF proposes (being respectively Figure 85 A and 85B) with SEQ ID NO:41 and SEQ ID NO:42 respectively herein.The Nucleotide and the aminoacid sequence of the Fc part of human IgG propose (being respectively Figure 86 A and 86B) with SEQ ID NO:49 and SEQ ID NO:50 respectively.

The chimeric ENBREL of reconstruct ^TMCan be applied to following patient, this patient is selected from the patient who suffers from rheumatoid arthritis and suffers from the patient of the juvenile arthritis,juvenile chronic arthritis,juvenile rheumatoid arthritis of multi-joint process.The chimeric ENBREL of reconstruct ^TMAlso can be applied to the arthritic to alleviate symptom, symptom or the structural impairment among the patient.Preferably, the patient is people patient.

The Synagis of reconstruct ^TMAntibody can be applied to the patient the patient is carried out the immunization that anti respiratory syncytial virus (RSV) infects.The Synagis of reconstruct ^TMAntibody also can be applied to the patient to prevent or to alleviate the seriousness of the lower respiratory illness that is caused by RSV.Preferably, the patient is people patient.

Y.MAb resists-glycoprotein iib/iiia

The aminoacid sequence of mouse-anti-glycoprotein iib/iiia antibody variable region respectively with SEQ ID NO:52 (the sophisticated variable light chain of mouse, Figure 87) and SEQ ID NO:54 propose (the sophisticated variable heavy chain of mouse, Figure 88).The sequence of these mouse can be according to U.S. Patent No. 5,777, program in 085 and human IgG aminoacid sequence SEQ ID NO:51 (the sophisticated variable light chain of people, Figure 89), SEQ ID NO:53 (the sophisticated variable heavy chain of people, Figure 90), SEQ ID NO:55 (people's light chain, Figure 91) (people's heavy chain Figure 92) makes up to generate chimeric humanization mouse-anti-glycoprotein iib/iiia antibody with SEQ ID NO:56.Other anti--humanized antibody of glycoprotein iib/iiia can be found in U.S. Patent No. 5,877, in 006.Express the clone of anti--glycoprotein iib/iiia MAb7E3 can be commercial (Manassas VA) buys with catalog number (Cat.No.) no.HB-8832 from ATCC.

The indication of selected antibody

The Reopro of reconstruct ^TMCan be applied to following patient, this patient is selected from the patient who carries out endermic crown intervention, and (wherein this patient plans to carry out endermic crown intervention in 24 hours with the patient who suffers from unstable angina.The Reopro of reconstruct ^TMAlso can be applied to the patient that carries out endermic crown intervention to alleviate or to prevent heart ischemia complication among the patient.Preferably, the patient is people patient.

The Herceptin of reconstruct ^TMCan be applied to the patient who suffers from the proteinic metastatic breast cancer of overexpression HER2.Preferably, the patient is people patient.

The Remicade of reconstruct ^TMCan be applied to following patient, this patient is selected from the patient that suffers from rheumatoid arthritis, suffer from the patient of CrohnShi disease and suffer from the patient of the CrohnShi disease of fistulization.The Remicade of reconstruct ^TMAlso can be applied to symptom and the symptom of rheumatoid arthritis patients to alleviate rheumatoid arthritis among the patient.Remicade ^TMAlso can be applied to symptom and the symptom of CrohnShi patient to alleviate CrohnShi disease among the patient.Preferably, the patient is people patient.

The Z.MAb anti-CD 20

The nucleic acid of chimeric anti-CD 20 antibodies and aminoacid sequence are at the SEQ ID NO:59 (nucleotide sequence of mouse variable region light chain, Figure 93 A), the SEQ ID NO:60 (aminoacid sequence of mouse variable region light chain, Figure 93 B), the SEQ ID NO:61 (nucleotide sequence of mouse variable region heavy chain, Figure 94 A) and among the SEQID NO:62 (aminoacid sequence of mouse variable region heavy chain, Figure 94 B) propose.In order to make the murine antibody humanization, can use the TCAE 8 (SEQID NO:57, Figure 95 A-95E) that contains human IgG heavy chain and light chain constant domain easily.According to U.S. Patent No. 5,736,137 explanations that provide are cloned into above-mentioned mouse variable region coding DNA and have made up a carrier (SEQ ID NO:58, Figure 96 A-96E) in TCAE 8 carriers, and this carrier can be expressed chimeric anti-CD 20 antibodies in being transformed into mammal cell line the time.Other humanized anti-CD 20 antibodies can be found in U.S. Patent No. 6,120, in 767.Express the clone of anti-CD 20 antibodies MAb C273 can be commercial (Manassas VA) buys with catalog number (Cat.No.) no.HB-9303 from ATCC.

The sequence that the technician proposes easy to understand herein is not an exclusiveness, but the example of the variable region of chimeric antibody, acceptor and other bound fractions.Further, it is well known in the art making up method chimeric or " humanized " antibody, and is described in as U.S. Patent No. 6,329,511 and U.S. Patent No. 6,210,671 in.In conjunction with present disclosure and method well known in the art, the technician will recognize that the present invention is not limited to disclosed herein sequence.

The expression of chimeric antibody is well known in the art, and is described in detail in as U.S. Patent No. 6,329, in 511.It is procaryotic, Eukaryotic that expression system can be etc.Further, chimeric antibody is in being described in people such as Putlitz (1990, Bio/Technology 8:651-654) with the expression in the insect cell of baculovirus expression system.In addition, the method of expressing the nucleic acid of coding fusion or chimeric protein is well known in the art, and be described in as people such as Sambrook (2001, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, New York) and people (1997 such as Ausubel, Current Protocolsin Molecular Biology, Green ﹠amp; Wiley, New York) in.

The function of the chimeric antibody that the method according to this invention is produced and biologic activity determine it also is basic operation for a person skilled in the art.Determine that by competition assay the method for affinity of antibody is described in detail among the Berzofsky (J.A.Berzofsky and I.J.Berkower, 1984, Fundamental Immunology (ed.W.E.Paul), Raven Press (NewYork), 595).In brief, use radioiodinated monoclonal antibody with the avidity of chimeric antibody and comparing of monoclonal antibody, wherein this chimeric antibody is a deutero-from this monoclonal antibody.

The anti-CD 20 antibodies of reconstruct can be applied to following patient, and this patient suffers from rudimentary or ovarian follicle recurrence or refractory, the CD20-positive, B-cell non-Hodgkin's.Preferably, the patient is people patient.

VII. pharmaceutical composition

On the other hand, the invention provides pharmaceutical composition.This pharmaceutical composition comprises the covalent conjugates between medicine acceptable diluent and the water-soluble polymers that exists at non-natural, treatment part or biomolecules and glycosylation or the nonglycosylated peptide.This polymkeric substance, treatment part or biomolecules are puted together by intact glycosyl linking group and peptide, and this glycosyl linking group inserts between peptide and polymkeric substance, treatment part or the biomolecules and covalently is connected with both.

Pharmaceutical composition of the present invention is applicable to various drug delivery system.Be used for appropriate formulations of the present invention and can be found in Remington ' s Pharmaceutical Sciences, MacePublishing Company, Philadelphia, PA, 17 ^ThEd. (1985).Send the summary of the method for passing for medicine, referring to Langer, Science 249:1527-1533 (1990).

This pharmaceutical composition can be prepared as any suitable mode that gives, and comprises giving as partial, oral, nose, intravenous, encephalic, endoperitoneal, subcutaneous or intramuscular.Give for parenteral, as subcutaneous injection, carrier preferably comprises water, salt solution, alcohol, fat, wax or damping fluid.For orally give, can use any above-mentioned carrier or solid carrier, as N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, talcum, Mierocrystalline cellulose, glucose, sucrose and magnesiumcarbonate.Biodegradable microsphere (as polylactate polyglycolate) also can be used as the carrier of pharmaceutical composition of the present invention.Suitable biodegradable microsphere is disclosed in as U.S. Patent No. 4,897, in 268 and 5,075,109.

Usually, pharmaceutical composition is that parenteral gives, and gives as intravenously.Thereby, the invention provides and be used for the composition that parenteral gives, said composition comprises dissolving or is suspended in compound in the appropriate carriers, is preferably aqueous phase carriers, as water, buffered water, salt solution, PBS etc.Said composition can contain near the acceptable auxiliary substance of the required medicine of physiology situation, as regulate pH with buffered reagent, tension regulator, wetting agent, stain remover etc.

These compositions can sterilize by the sterilising technology of routine, or can be filtration sterilization.But the aqueous solution former state of gained is packed for use as a result, perhaps can freeze-drying, and freeze dried preparation was with the combination of aseptic aqueous phase carriers before giving.The pH of preparation is generally 3～11, is 5～9 more preferably, is most preferably 7～8.

In some embodiments, peptide of the present invention can be integrated into from the liposome that vesicle formation property lipid of standard forms.The whole bag of tricks of preparation liposome is an available, as is described in people such as Szoka, Ann.Rev.Biophys.Bioeng.9:467 (1980), U.S. Patent No. 4,235,871,4,501,728 and 4,837,028.Using various directed agents (as sialic acid galactoside of the present invention) is (referring to U.S. Patent No. 4,957,773 and 4,603,044) well known in the art to the guiding of liposome.

Can use and make directed agents and the standard method of liposome link coupled.These methods generally comprise lipid composition are integrated in the liposome, and as activating to adhere to the phosphatidylethanolamine of directed agents, the perhaps lipophilic compound of derivatize is as lipid deutero-peptide of the present invention.

The machine-processed surface that generally needs directed agents to be positioned liposome by this way of guiding, thus targeting part can interact with target such as cell surface receptor.The available those skilled in the art's known method of sugar of the present invention (as alkylation or the acylations to being present in the oh group on the sugar with chain alkyl halogenide or lipid acid respectively) is attached on the lipid molecule before forming liposome.Selectively, can form liposome by this way, thereby the linker part when forming, film can be integrated in the film at first.This linker part must have lipophilic part, and this part can firmly embed and be anchored in the film.It also must have reactive moieties, and this part is can to utilize by chemistry on the water surface of liposome.Reactive moieties is selected, thereby it chemically is being suitable for forming stable chemical bond with directed agents of adding afterwards or sugar.In some cases, possible is that directed agents and linker molecule are directly adhered to, but in most of the cases, be more suitable in the 3rd molecule as chemical bridge joint, thereby the linker molecule in the junctional membrane and directed agents or sugar, this directed agents or sugar extend from the vesicle surface three dimension.The dosage range that peptide of the present invention gives is the dosage of the effect wanted even as big as generation, and wherein immunoreactive symptom has shown the inhibition of some degree.This dosage should not cause side effect too greatly.Usually, this dosage will change according to the degree of disease in age, situation, sex and the animal, and can be determined by those skilled in the art.This dosage can be regulated under the situation that any contraindication occurs by single doctor.

Extra pharmaceutical methods can be applicable to the time length of control action kou.Controlled release preparation can put together by using polymer, compound or absorption peptide is realized.Controlled release send to be passed and can put into practice with sustained release by selecting suitable macromole (as polyester, polyamino carboxymethyl cellulose and Protamine sulfates) and macromolecular concentration and integration method.Another possible method of control controlled release preparation action time is that peptide is integrated in the particle of polymeric material, as polyester, amino acids, hydrogel, poly-(lactic acid) or ethylene vinyl acetate copolymer.

In order to prevent that peptide from combining with plasma proteins, preferably peptide is trapped in the micro-capsule by condensation technique or interfacial polymerization preparation, as be respectively Walocel MT 20.000PV or gelatin-micro-capsule and gather (methyl methacrylate) micro-capsule, perhaps peptide is trapped in the colloid drug delivery system, in liposome, albumin microsphere spheroid, micro emulsion, nano particle (nanoparticle) and nanocapsule (nanocapsule) or big emulsion.This introduction is disclosed in Remington ' s Pharmaceutical Sciences (16 ^ThEd., A.Oslo, ed., Mark, Easton, Pa., 1980).

Peptide of the present invention is highly suitable for the drug delivery system that can lead, synthetic or natural polymkeric substance as macromolecular complex, nanocapsule, microsphere or pearl form, and be applicable to the system based on lipid, this system comprises oil-in-water emulsion, micelle, blended micelle, liposome and heavily seals red corpuscle.These systems are known as the colloid drug delivery system jointly.Usually, this colloidal particle diameter that contains the dispersive peptide is about 50nm-2 μ m.The size of colloidal solid makes it to give or to give as aerosol through intravenously by injection.The material that is used for the colloid system preparation generally by filtration sterilization sterilize, avirulent and biodegradable, for example albumin, ethyl cellulose, casein, gelatin, Yelkin TTS, phosphatide and soya-bean oil.The polymeric colloid system is by preparing with the similar method of cohesion microencapsulation.

In an exemplary embodiment, peptide is the composition that send the liposome of delivery system as guiding.When being dispersed in phosphatide in the aqueous media gently, they expand, hydration also spontaneously forms multi-concentric double-deck vesicle, and in this vesicle, the aqueous media layer is separated lipid bilayer.This system is commonly referred to multilamellar liposome or multilayer vesicle (MLV), and diameter range is about 100nm～about 4 μ m.When MLV was carried out supersound process, then forming diameter range was the little monolayer vesicle (SUVS) of about 20～about 50nm, and this little monolayer vesicle contains the aqueous solution in the SUV core.

The example that is used for the lipid of liposome production comprises the phosphatidyl compound, as phosphatidyl glycerol, phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine.Useful especially is the diacyl phosphatidyl glycerol, and wherein lipid part contains 14-18 carbon atom, is 16-18 carbon atom especially, and is saturated.Exemplary phosphatide comprises Yelkin TTS phatidylcholine, dipalmitoyl phosphatidylcholine and distearoyl phosphatidylcholine.

Contain in the liposome of peptide of the present invention in preparation, should consider these variablees, as the unstable of the efficient of peptide tunicaization, peptide, the medicine acceptability of the qualitative and preparation of the osmotic transient of the homogeneity of the liposome colony of gained and size, peptide-lipid ratio, preparation as a result.People such as Szoka, Annua lReview of Biophysics and Bioengineering, 9:467 (1980); People such as Deamer, Liposomes, Marcel Dekker, New York, 1983,27; People such as Hope, Chem.Phys.Lipids, 40:89 (1986)).

The guiding that contains peptide of the present invention send delivery system can give host, particularly mammalian hosts in every way, as in intravenous, intramuscular, subcutaneous, endoperitoneal, endovascular, partial, the chamber, in endermic, the nose or suck.The concentration of peptide will depend on the characteristic of specific application, disease, the frequency that gives etc. and change.The peptide that send the delivery system tunicaization of guiding can provide this medium such as salt solution, phosphate buffered saline (PBS) etc. in the preparation that comprises other suitable compounds and water physiology acceptable medium.

Compound by method preparation of the present invention also can be used as diagnostic reagent.For example, the compound of mark is used in and suspects the zone of locating inflammation or metastases among the patient with inflammation.Use, this compound can be used for this reason ¹²⁵I, ¹⁴C or tritium carry out mark.

EXPERIMENTAL EXAMPLE

The present invention now will be described in conjunction with the following examples.These embodiment provide just to the purpose of illustrating, and the present invention never should be interpreted as being confined to these embodiment, and should be interpreted as comprising owing to the introduction that provides herein becomes conspicuous any and all variations.

The material and the method for the experiment that is used for proposing have in the present embodiment been described now.

A. general method

1.CMP-SA-PEG preparation

Present embodiment has proposed the preparation to CMP-SA-PEG.

The preparation of 2-(benzyloxy acid amides)-glycyl acid amides-2-deoxidation-D-mannopyranose.(3.125g 10.2mmol) adds to contain and is dissolved in MeOH (10mL) and H with N-benzyloxycarbonyl-glycyl-N-hydroxy-succinamide ester ₂D-mannosamine-HCl among the O (6mL) (2g, 9.3mmol) and triethylamine (1.42mL is in solution 10.2mmol).Reactant was stirred 16 hours and concentrated with the evaporation (rotoevaporation) of circling round in room temperature.Chromatography (silicon, 10%MeOH/CH ₂Cl ₂) obtained 1.71g (50% output) white solid product: R _f=0.62 (silicon, CHCl ₃: MeOH:H ₂O, 6/4/1); ¹H NMR (CD ₃OD, 500MHz) δ 3.24-3.27 (m, 2H), 3.44 (t, 1H), 3.55 (t, 1H), 3.63-3.66 (m, 1H), 3.76-3.90 (m, 6H), 3.91 (s, 2H), 4.0 (dd, 2H), 4.28 (d, 1H, J=4.4), 4.41 (d, 1H, J=3.2), 5.03 (s, 1H), 5.10 (m, 3H), 7.29-7.38 (m, 10H).

5-(N-benzyloxy acid amides) glycyl acid amides-3, the two deoxidations of 5--D-glycerine-D-semi-lactosi The preparation of-2-nonulopyranosuronate.With 2-(N-benzyloxy acid amides) glycyl acid amides-2-deoxidation-D-mannopyranose (1.59g, 4.3mmol) be dissolved in 0.1M HEPES (12mL, pH7.5) and Sodium.alpha.-ketopropionate (4.73g is in solution 43mmol).Make neuraminic acid zymohexase (containing the enzyme of 540U in the 10mM phosphate buffer soln of pH6.9 of 0.1M NaCl) and reaction mixture be heated to 37 ℃ and continue 24 hours at 45mL.Then that reaction mixture is centrifugal and supernatant liquor carried out chromatography (C18 silicon, the gradient from H20 (100%) to 30%MeoH/ water).Collect suitable fraction, concentrate, resistates is carried out chromatography, and (C18 silicon is from 10%MeOH/CH ₂Cl ₂To CH ₂Cl ₂/ MeOH/H ₂The gradient of O 6/4/1).Collect suitable fraction, it is concentrated and residuum is resuspended in the water.After the freeze-drying, obtained white solid product (1.67g, 87% output): R _f=0.26 (silicon, CHCl ₃: MeOH:H ₂O is 6/4/1); ¹H NMR (D ₂O, 500MHz) δ 1.82 (t, 1H), 2.20 (m, 1H), 3.49 (d, 1H), 3.59 (dd, 1H), 3.67-3.86 (m, 2H), 3.87 (s, 2H), 8.89-4.05 (m, 3H), 5.16 (s, 2H), 7.45 (m, 5H).

5-glycyl acid amides-3, the two deoxidations of 5--D-glycerine-D-semi-lactosi The preparation of-2-nonulopyranosuronate.With 5-(N-benzyloxy acid amides) glycyl acid amides-3,5-is two, and deoxidation-(1.66g 3.6mmol) is dissolved in water/methyl alcohol of 20mL 50% D-glycerine-D-semi-lactosi-nonulopyranosuronate.Flask is repeatedly vacuumized and place argon, add 10% Pd/C (0.225g) then.After repeating to vacuumize, (about 1atm) joins in the flask with hydrogen, and reaction mixture was stirred 18 hours.Make reaction mixture pass through diatomite filtration, concentrate also freeze-drying to obtain the white solid product of 1.24g (100% output): R with the vaporizer that rotates _f=0.25 (silicon, IPA/H ₂O/NH ₄OH 7/2/1); ¹H NMR (D ₂O, 500MHz) δ 1.83 (t, 1H, J=9.9), 2.23 (dd, 1H, J=12.9,4.69), 3.51-3.70 (m, 2H), 3.61 (s, 2H), 3.75-3.84 (m, 2H), 3.95-4.06 (m, 3H).

Cytidine-5 '-single phosphinylidyne-[5-(N-fluorenes methoxyl group-acid amides) glycyl acid amides-3, two deoxidations of 5- -β-D-glycerine-D-semi-lactosi-2-nonulopyranosuronate] preparation.To contain and be dissolved in 20mL H ₂5-glycyl acid amides-3 among the O, the two deoxidations of 5--D-glycerine-D-semi-lactosi-2-nonulopyranosuronate (0.55g, solution 1.70mmol) add to Tris (1.38g, 11.4mmol), 1M MgCl ₂(1.1mL) and in the solution of BSA (55mg).With 1MNaOH (2mL) with the pH regulator to 8.8 of solution and add CTP-2Na ⁺(2.23g, 4.2mmol).The pH of reaction mixture is by the control of pH controller, and this controller can discharge needed 1MNaOH to keep pH8.8.Fused protein (sialyltransferase/CMP-neuraminic acid synthetic enzyme) is added in the solution and stir reaction mixture in room temperature.After 2 days, add the fused protein of additional quantity and reactant was stirred extra 40 hours.Reaction mixture is precipitated in EtOH, and will precipitate with cold EtOH washing 5 times to obtain 2.3 gram white solids.About 1.0g crude product is dissolved in 1,4 diox (4mL), H ₂O (4mL) and saturated NaHCO ₃(3mL), and dropwise add FMOC-Cl (308mg, 1.2mmol) solution that is dissolved in the 2ml diox.After room temperature was stirred 16 hours, reaction mixture is concentrated to about 6mL by rotary evaporation, and also (C18 silicon was from 100%H with chromatography ₂O is to 30%MeOH/H ₂The gradient of O) carries out purifying.Combination also concentrates suitable fraction.Residuum is dissolved in the water also freeze-drying to obtain 253mg white solid: R _f=0.50 (silicon, IPA/H ₂O/NH ₄OH 7/2/1); ¹H NMR (D ₂O, 500MHz) δ 1.64 (dt, 1H, J=12.0,6.0), 2.50 (dd, 1H, J=13.2,4.9), 3.38 (d, J=9.67,1H), 3.60 (dd, J=11.65,6.64,1H), 3.79 (d, J=4.11,1H), 3.87 (dd, J=12.24,1.0,1H), 3.97 (m, 2H), 4.07 (td, J=10.75,4.84,1H), 4.17 (dd, J=10.68,1.0,1H), 4.25 (s, 2H), 4.32 (t, J=4.4,1H), 4.37 (t, J=5.8,1H), (4.6-4.7 m is caused bluring by the peak of solvent), 5.95 (d, J=4,1H), 6.03 (d, J=7.4,1H), 7.43-7.53 (m, 3H), 7.74 (m, 2H), 7.94 (q, J=7,3H).MS (ES); To C ₃₅H ₄₂N ₅O ₁₈P ([M-H] ^-) calculated value be 851.7; Measured value 850.0.

Cytidine-5 '-single phosphinylidyne-(5-glycyl acid amides-3, two deoxidation-β of 5--D-glycerine-D-gala The preparation of sugar-2-nonulopyranosuronate).With diisopropylamine (83 μ L, 0.587 μ mol) add the Cytidine-5 that is dissolved in water (3mL) and the methyl alcohol (1mL) to '-single phosphinylidyne-[5-(N-fluorenes methoxyl group-acid amides) glycyl acid amides-3, two deoxidation-the β of 5--D-glycerine-D-semi-lactosi-2-nonulopyranosuronate] (100mg, 0.117mmol) in.Reaction mixture was stirred 16 hours and reactant methanol is removed from reaction mixture by rotary evaporation in room temperature.Thick reaction mixture is filtered by the C18 silicagel column of answering water, and elutriant collected and freeze-drying to obtain white solid product (87mg, 100%): R _f=0.21 (silicon, IPA/H ₂O/NH ₄OH 7/2/1); ¹H NMR (D ₂O, 500MHz) δ 1.66 (td, 1H, J=5.3), 2.50 (dd, 1H, J=13.2,4.6), 3.43 (d, J=9.58,1H), 3.63 (dd, J=11.9,6.44,1H), 3.88 (dd, J=11.8,1.0,1H), 3.95 (td, J=9.0,2.3,1H), 4.10 (t, J=10.42,1H), 4.12 (td, J=10.34,4.66,1H), 4.18 (d, J=10.36,1H), 4.24 (m, 2H), 4.31 (t, J=4.64,1H), 4.35 (t, 1H), 6.00 (d, J=4.37,1H), 6.13 (d, J=7.71,1H), 7.98 (d, J=7.64,1H).MS (ES); To C ₂₁H ₃₂N ₅O ₁₁P ([M-H] ^-) calculated value be 629.47; Measured value 627.9.

Cytidine-5 '-single phosphinylidyne-[5-(N-methoxyl group-polyoxyethylene-(1kDa)-3-oxo propionyl Amine)-and glycyl acid amides-3, the two deoxidation-β of 5--D-glycerine-D-semi-lactosi -2-nonulopyranosuronate] preparation.With benzyl triazol-1-yl oxygen-three (dimethylamino)-Phosphonium hexafluorophosphate (BOP, 21mg, 48 μ mol) add to and be dissolved in dry DMF (700 μ L) and triethylamine (13 μ L, 95 μ mol) in the methoxy polyoxyethylene in-(1kDa molecular-weight average)-3-oxo propionic acid (48mg, the 48 μ mol) solution.After 30 minutes, add and contain Cytidine-5 '-single phosphinylidyne-(5-glycyl acid amides-3, the solution of (30mg, 48 μ mol), water (400 μ L) and triethylamine (13 μ L, 95 μ mol) of two deoxidation-β of 5--D-glycerine-D-semi-lactosi-2-nonulopyranosuronate).This solution was stirred 20 minutes in room temperature, carry out chromatography (C18 silicon, methanol gradient) then.Collect suitable fraction, it is concentrated and residuum is resuspended in the water, and freeze-drying is to obtain 40mg (50% output) white solid: R _f=0.36 (silicon, IPA/H ₂O/NH ₄OH 7/2/1); ¹H NMR (D ₂O, 500MHz) δ 1.66 (td, 1H, J=5.3), 2.50 (dd, 1H, J=13.2,4.6), 2.64 (t, J=5.99,3H), 3.43 (d, J=9.58,1H), 3.63 (m, 1H), 3.71 (s, 70H), (3.79 m, the peak by 3.71 causes bluring), 3.82 (t, J=6.19,1H), 3.88 (dd, J=11.8,1.0,1H), 3.95 (td, J=9.0,2.3,1H), 3.98 (t, J=5.06,1H), 4.12 (td, J=10.34,4.66,1H), 4.18 (d, J=10.36,1H), 4.23 (d, J=4.85,2H), 4.31 (t, J=4.64,1H), 4.35 (t, 1H), 6.00 (d, J=4.55,1H), 6.13 (d, J=7.56,1H), 7.98 (d, J=7.54,1H).MS (MALDI), [M-H] of observation; 1594.5,1638.5,1682.4,1726.4,1770.3,1814.4,1858.2,1881.5,1903.5,1947.3.

Cytidine-5 '-single phosphinylidyne-[5-(N-methoxyl group-polyoxyethylene-(10kDa)-the oxo acid amides) -glycyl acid amides-3, the two deoxidation-β of 5--D-glycerine-D-semi-lactosi -2-nonulopyranosuronate] preparation.With Cytidine-5 '-single phosphinylidyne-(5-glycyl acid amides-3, (the 2.5mg of two deoxidation-β of 5--D-glycerine-D-semi-lactosi-2-nonulopyranosuronate), 4 μ mol) and water (180 μ L) add to and contain triethylamine (1.1 μ L, 8 μ mol) (the methoxy polyoxyethylene-(10kDa in the dry DMF (800 μ L), molecular-weight average)-oxo carbonyl-(N-oxo benzotriazole) ester (40mg, 4 μ mol) in the solution, and reaction mixture stirred 1 hour in room temperature.Water (8mL) diluted reaction mixture also carries out purifying by anti-phase flash chromatography (C18 silicon, methanol gradient) then.Merge suitable fraction, it is concentrated and residuum is dissolved in the water, and freeze-drying is to obtain 20mg (46% output) white solid product: R _f=0.35 (silicon, IPA/H ₂O/NH ₄OH 7/2/1); ¹H NMR (D ₂O, 500MHz) δ 1.66 (td, 1H), 2.50 (dd, 1H), 2.64 (t, 3H), 3.55-3.7 (m, the peak by 3.71 causes bluring), 3.71 (s, 488H), 3.72-4.0 (m, the peak by 3.71 causes bluring), 4.23 (m, 3H), 4.31 (t, 1H), 4.35 (t, 1H), 6.00 (d, J=4.77,1H), 6.12 (d, J=7.52,1H), 7.98 (d, J=7.89,1H).MS (MALDI), [M-CMP+Na] of observation; 10780.

2. The preparation of CMP-SA-PEG II

Present embodiment has proposed the general method of preparation CMP-SA-PEG, and specifically is the method for preparing CMP-SA-PEG (1kDa) and CMP-SA-PEG (20kDa).

The preparation Cytidine-5 '-single phosphinylidyne-(5-glycyl acid amides-3, two deoxidation-β of 5--D-glycerine-D- The general method of semi-lactosi-2-nonulopyranosuronate).Will Cytidine-5 '-single phosphinylidyne- (5-glycyl acid amides-3, the two deoxidation-β of 5--D-glycerine-D-semi-lactosi -2-nonulopyranosuronate) (870mg 1.02mmol) is dissolved in the 25mL water, And the dimethylamine agueous solution of interpolation 5.5mL 40wt%.Reactant was stirred 1 hour, remove excessive dimethylamine by rotary evaporation then.The aqueous solution is filtered by the C-18 silicagel column, and wash this post with water.Elutriant merged and freeze-drying to obtain 638mg (93%) white solid product: R _f=0.10 (silicon, IPA/H ₂O/NH ₄OH 7/2/1); ¹H NMR (D ₂O, 500MHz) δ 1.66 (td, 1H, J=5.3), 2.50 (dd, 1H, J=13.2,4.6), 3.43 (d, J=9.58,1H), 3.63 (dd, J=11.9,6.44,1H), 3.88 (dd, J=11.8,1.0,1H), 3.95 (td, J=9.0,2.3,1H), 4.10 (t, J=10.42,1H), 4.12 (td, J=10.34,4.66,1H), 4.18 (d, J=10.36,1H), 4.24 (m, 2H), 4.31 (t, J=4.64,1H), 4.35 (t, 1H), 6.00 (d, J=4.37,1H), 6.13 (d, J=7.71,1H), 7.98 (d, J=7.64,1H).MS (ES); To C ₂₁H ₃₂N ₅O ₁₁P ([M-H] ^-) calculated value be 629.47; Measured value 627.9.

Use the general method that mPEG-(p-NP) carbonic ether prepares CMP-SA-PEG.

With Cytidine-5 '-single phosphinylidyne-(5-glycyl acid amides-3,5-is two, and deoxidation-β-D-glycerine-D-semi-lactosi-2-nonulopyranosuronate) (175mg 0.259mMol) is dissolved in the water of pH8.5 and the mixture of DMF or THF (ratio is 1:2).In 8 hours, (0.519mMole) divide several parts to add mPEG-nitrophenols carbonic ether (2～20kDa mPEG) in room temperature, and reaction mixture was stirred 3 in room temperature.When finishing, add entry (40ml) and 1.5ml NH ₄OH (29% the aqueous solution).The xanchromatic reaction mixture was stirred 2 hours again, concentrate by rotary evaporation then.Water (pH8.5) is diluted to about 500ml volume with reaction mixture then, and carries out purifying by anti-phase flash chromatography method (Biotage 40M, C18 silicon post) with the methanol gradient.Merge suitable fraction, and concentrate to obtain white solid product.R _f(silicon; 1-propanol/water/29%NH ₄OH; 7/2/1); (2kDa PEG)=0.31; (5kDa PEG)=0.33; (10kDaPEG)=0.36; (20kDa PEG)=0.38 (TLC silicon, IPA/H ₂O/NH ₄OH 7/2/1); MS (MALDI), [M-CMP+Na] observed value; (2kDa)=2460; (5kDa)=5250; (10kDa)=10700; (20kDa)=22500.

The preparation Cytidine-5 '-single phosphinylidyne-[5-(N-fluorenes methoxyl group-methane amide)-glycyl acid amides-3,5- Two deoxidation-β-D-glycerine-D-semi-lactosi-2-nonulopyranosuronate] general method.

With Sodium.alpha.-ketopropionate (2.4g, 218mmol), the HEPES damping fluid (0.25M, pH7.34) and 1.0g (22mmol) Fmoc-glycyl seminose acid amides in the polycarbonate bottles of 150mL, mix.Add then neuraminic acid zymohexase solution (19mL ,～600U), and reaction mixture carried out incubation in 30 ℃ on orbital shaker.After 23 hours, about 75% transformation to product has taken place in thin-layer chromatography (TLC) demonstration.(1.72g is 33mmol) with 0.1M MnCl to add CTP then in reaction mixture ₂(6mL).With 1M NaOH (5.5mL) with pH regulator to 7.5, and add the solution contain CMP-neuraminic acid synthetic enzyme (Neisseria) (25mL, 386U).Finish after being reflected at 24 hours, and reaction mixture is carried out chromatography (C-18 silicon, H ₂O (100%) is to 10%MeOH/H ₂The gradient of O).Merge suitable fraction, concentrate and freeze-drying to obtain white solid.R _f(IPA/H ₂O/NH ₄OH, 7/2/1)=0.52. ¹H NMR (D ₂O, 500MHz) δ 1.64 (dt, 1H, J=12.0,6.0), 2.50 (dd, 1H, J=13.2,4.9), 3.38 (d, J=9.67,1H), 3.60 (dd, J=11.65,6.64,1H), 3.79 (d, J=4.11,1H), 3.87 (dd, J=12.24,1.0,1H), 3.97 (m, 2H), 4.07 (td, J=10.75,4.84,1H), 4.17 (dd, J=10.68,1.0,1H), 4.25 (s, 2H), 4.32 (t, J=4.4,1H), 4.37 (t, J=5.81H), (4.6-4.7 m is caused bluring by the peak of solvent), 5.95 (d, J=4,1H), 6.03 (d, J=7.4,1H), 7.43-7.53 (m, 3H), 7.74 (m, 2H), 7.94 (q, J=7,3H) .MS (ES); To C ₃₅H ₄₂N ₅O ₁₈P ([M-H] ^-) calculated value be 850.7; Measured value 850.8.

The preparation Cytidine-5 '-single phosphinylidyne-[5-(N-methoxyl group-polyoxyethylene-(1kDa)-the 3-oxo Propionic acid amide)-and glycyl acid amides-3, the two deoxidation-β of 5--D-glycerine-D-semi-lactosi -2-nonulopyranosuronate] general method.Methoxyl group-polyoxyethylene-(1kDa molecular-weight average)-3-oxo propionic acid-N-succinimide ester (52mg, 52 μ mol) is dissolved in dry DMF (450 μ L) and the triethylamine (33 μ L, 238 μ mol).Add the solid Cytidine-5 '-single phosphinylidyne-(5-glycyl acid amides-3, two deoxidation-β of 5--D-glycerine-D-semi-lactosi-2-nonulopyranosuronate) (30mg, 48 μ mol).Add the water (330 μ L) of pH8, and after 30 minutes, add other 28mg NHS-activatory PEG.After 5 minutes, reaction mixture is carried out chromatography (C-18 silicon, methanol gradient), and concentrate the white solid that suitable fraction obtains 32mg (40% output).R _f=0.31 (silicon, IPA/H ₂O/NH ₄OH 7/2/1); ¹H NMR (D ₂O, 500MHz) δ 1.66 (td, 1H, J=5.3), 2.50 (dd, 1H, J=13.2,4.6), 2.64 (t, J=5.99,3H) 3.43 (d, J=9.58,1H), 3.63 (m, 1H), 3.71 (s, 70H), 3.79 (m, peak and cause bluring), 3.82 (t by 3.71, J=6.19, and 1H) 3.88 (dd, J=11.8,1.0,1H), 3.95 (td, J=9.0,2.3,1H), 3.98 (t, J=5.06,1H), 4.12 (td, J=10.34,4.66,1H), 4.18 (d, J=10.36,1H), 4.23 (d, J=4.85,2H), 4.31 (t, J=4.64,1H), 4.35 (t, 1H), 6.00 (d, J=4.55,1H), 6.13 (d, J=7.56,1H), 7.98 (d, J=7.54,1H) .MS (MALDI), [(M-CMP)-and H] observed value; 1506.4,1550.4,1594.5,1638.5,1682.4,1726.4,1770.3,1814.4,1858.2.

The preparation Cytidine-5 '-single phosphinylidyne-{ 5-[N-(2,6-dimethoxy-polyoxyethylene-(20kDa)-3-oxo propionic acid amide-lysyl acid amides-glycyl acid amides-3, the general method of the two deoxidation-β of 5--D-glycerine-D-semi-lactosi-2-nonulopyranosuronate}. with 2,6-two [methoxyl group-polyoxyethylene-(20kDa molecular-weight average)-3-oxo propionic acid amide]-lysyl acid amides-N-succinimide ester (367mg, 9 μ mol) be dissolved in anhydrous THF (7mL) and the triethylamine (5 μ L, 36 μ mol).With Cytidine-5 '-single phosphinylidyne-(5-glycyl acid amides-3, two deoxidation-β of 5--D-glycerine-D-semi-lactosi-2-nonulopyranosuronate) (30mg, 48 μ mol) are dissolved in the 1.0mL water, and join in the reaction mixture.Reactant was stirred 4 hours in room temperature, carry out chromatography (HPLC, Waters Xterra RP8, water/NH then ₄OH, 100% to 20% methanol/NH ₄The gradient of OH, 1mL/ minute) to obtain white solid, its R _t=22.8 minutes.MS (MALDI), [(M-CMP)-and H] observed value; (43027.01 40,000-45,500).

3.UDP-Gal-PEG preparation

Present embodiment has proposed the general method of preparation UDP-Gal-PEG.

348mg methoxyl group among the THF (0.5ml)-polyoxyethylene propionic acid N-hydroxy-succinamide ester (mPEG-SPA, MW1,000) is joined 25mg GalN-1-phosphoric acid in the solution of 1ml water, add 67 μ L triethylamines subsequently.The mixture of gained was as a result stirred 17 hours in room temperature.Under reduced pressure concentrate thick reaction mixture is provided, this thick reaction mixture is by chromatography (C-18 silicon, use the stepwise gradient of 10%, 20%, 30%, 40% moisture MeOH) carry out purifying, after merging suitable fraction and being concentrated into drying, to obtain the product of 90mg (74%).R _f=0.5 (silicon, propyl alcohol/H ₂O/NH ₄OH 30/20/2); MS (MALDI), observed value 1356,1400,1444,1488,1532,1576,1620.

[α-1-(uridine-5 '-two phosphinylidyne)]-2-deoxidation-2-(methoxyl group-polyoxyethylene-propionyl Amine-1kDa)-α-D-galactosamine.(the single phosphoric acid-D-galactosamine of methoxyl group-polyoxyethylene-propionic acid amide-1kDa)-α-1-(58mg) is dissolved in 6mL DMF and the 1.2mL pyridine with 2-deoxidation-2-.Add UMP-morpholidate (60mg) then, and stirred 48 hours in 70 ℃ of mixtures with gained as a result.After concentrating, resistates is carried out chromatography (C-18 silicon, the stepwise gradient of application 10%, 20%, 30%, 40%, 50%, 80%MeOH) to obtain the 50mg product after concentrating suitable fraction.R _f=0.54 (silicon, propyl alcohol/H ₂O/NH ₄OH 30/20/2).MS (MALDI); Observed value 1485,1529,1618,1706.

[α-1-(uridine-5 '-two phosphinylidyne)]-6-deoxidation-6-(methoxyl group-polyoxyethylene-amino -2kDa)-α-D-semi-lactosi.[α-1-(uridine-5 '-two phosphinylidyne)]-6-carbonyl aldehyde (carboxaldehyde)-α-D-semi-lactosi (10mg) is dissolved in the sodium phosphate buffer (pH6.0) of 2mL 25mM, and with methoxyl group-polyoxamide (MW 2,000,70mg) handle, use 25uL 1M NaBH then ₃CN solution is handled in 0 ℃.The mixture of gained as a result is freezing 3 days in-20 ℃.Reaction mixture is carried out chromatography (HPLC, Water Xterra P8), use 0.015M NH ₄OH is as mobile phase A, and MeOH is as Mobile phase B, and flow velocity is 1.0mL/ minute.Collect product, concentrate and obtain solid: R _t=9.4 minutes.R _f=0.27 (silicon, EtOH/H ₂O7/3).

[α-1-(uridine-5 '-two phosphinylidyne)]-6-amino-6-deoxidation-α-D-semi-lactosi.The 15mg ammonium acetate is joined in [α-1-(uridine-5 '-two phosphinylidyne)]-6-carbonyl aldehyde (carboxaldehyde)-α-solution of D-galactopyranose (10mg) in sodium phosphate buffer (pH6.0).Add 1M NaBH then ₃CN solution (25 μ L), and with mixture stirring 24 hours.Solution is concentrated and resistates is carried out chromatography (Sephadex G ₁₀) to obtain the 10mg white solid.R _f=0.62 (silicon, EtOH/0.1M NH ₄Ac).

[α-1-(uridine-5 '-two phosphinylidyne)]-6-deoxidation-6-(methoxyl group-polyoxyethylene-propionyl Amine ,～2kDa)-α-D-galactopyranose.[α-1-(uridine-5 '-two phosphinylidyne)]-6-amino-6-deoxidation-α-D-galactopyranose (5mg) is dissolved in 1mL H ₂Among the O.(MW～2,000 66mg), add 4.6 μ L triethylamines then to add methoxyl group-polyoxyethylene glycol propionyl-NHS ester then.The mixture of gained as a result stirred in room temperature spend the night, go up purifying to obtain product, R at HPLC (C-8 silicon) then _t=9.0 minutes.

[α-1-(uridine-5 '-two phosphinylidyne)]-6-deoxidation-6-(methoxyl group-polyoxyethylene-formyl Amine ,～2kDa)-α-D-galactopyranose.With [α-1-(uridine-5 '-two phosphinylidyne)]-6-amino-6-deoxidation-α-D-galactopyranose (10mg) and methoxyl group-polyoxyethylene glycol carboxyl-HOBT (MW2,000,67mg) at 1mL H ₂Mix among the O, add 6.4mg EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloric acid and 4.6 μ L triethylamines subsequently.The mixture of gained was as a result stirred 24 hours in room temperature.With mixture chromatography (C-8 silicon) to obtain product.

4.UDP-GlcNAc-PEG preparation

Present embodiment has proposed the general method of preparation UDP-GlcNAc-PEG.In the left side of scheme 17, aminosugar bisphosphate-Nucleotide of protecting is carried out oxidation form aldehyde with 6-position at sugar.Formation and reduction by schiff bases change this aldehyde into corresponding primary amines.Make as a result that the adducts of gained contacts with the p-NP carbonic ether of m-PEG, this ester and amine reaction, thus m-PEG is attached on the sugar nuclear by amido linkage.Right side on scheme 17 tops, aminosugar bisphosphate-Nucleotide of handling protection with chemical oxidizing agent is to form carboxyl on 6 carbon of sugar nuclear.Make activated carboxylic and with m-PEG amine reaction, thereby m-PEG is attached on the sugar nuclear by amido linkage.Right side in scheme 17 bottoms, reaction be identical with right upper portion substantially, initial sugar nucleotide contacted with oxidisability enzyme such as desaturase, rather than contact with chemical oxidizing agent.

Scheme 17.

5.UDP-GalNAc-PEG preparation

Present embodiment (scheme 18) has proposed the general method of preparation UDP-GalNAc-PEG.The reaction that proposes above starts from sugared bisphosphate-Nucleotide, and wherein R is the amine 2 of hydroxyl 1 or protection.In step a,, thereby change 6 of sugar into aldehyde part (3 and 4) with oxydase and the initial sugar of catalatic mixture process.In step c, formation by schiff bases and reduction change this aldehyde into corresponding amine (7 and 8).In step e, optional handle this amine with activatory m-PEG derivative, thereby with the amine acidylate to produce corresponding m-PEG acid amides (11 and 13).Selectively, in step f, amine is contacted with activatory m-PEG kind such as m-PEG active ester, thereby form corresponding m-PEG acid amides (12 and 14).In step b, also handle parent material with catalase and oxydase, complete oxidation methylol part, thereby at 6-position formation carboxyl.In steps d,, and change m-PEG adducts (9 and 10) into by reacting subsequently with m-PEG amine intermediate with the carboxy moiety activation.This is shown in the scheme 18.

Scheme 18.

A and b): galactose oxidase and catalase are dissolved in the 25mM sodium phosphate buffer (pH6.0); C) NH ₄Ac, NaBH ₃CN is dissolved in the 25mM sodium phosphate buffer (pH6.0);

d)CH ₃(OCH ₂CH ₂)NH ₂，EDC，H ₂O；e).CH ₃(OCH ₂CH ₂) _nNH ₂，NaBH ₃CN，H ₂O

For 15 and 16; F) CH ₃O (CH ₂CH ₂O) _nCH ₂CH ₂CONHS, H ₂O, Et ₃N

Amino-sugared phosphoric acid is contacted with m-PEG N-hydroxy-succinamide active ester, thereby form corresponding sugar-PEG-acid amides.This acid amides is contacted with UMP-morpholidate to form the sugared bisphosphate-Nucleotide of corresponding activity.

Scheme 19.

6.CMP-SA-levulinic acid is synthetic

Present embodiment has proposed the program of synthetic CMP-SA-levulinic acid.

The preparation of 2-leyulamide-2-deoxidation-D-mannopyranose.With isobutyl chlorocarbonate (100 μ L, 0.77mmol) dropwise join levulinic acid (86 μ L, 0.84mmol), (127 μ L are in solution 0.91mmol) for anhydrous THF (3mL) and triethylamine.This solution was stirred 3 hours in room temperature, dropwise join then contain D-mannosamine hydrochloric acid (151mg, 0.7mmol), triethylamine (127 μ L, 0.91mmol), in the solution of THF (2mL) and water (2mL).Reaction mixture was stirred 15 hours, be concentrated into drying by rotary evaporation then.With chromatography (silicon, 5-15%MeOH/CH ₂Cl ₂Gradient step by step) is used for separated product, thereby obtained 0.156g (73% output) white solid: R _f=0.41 (silicon, CHCl ₃/ MeOH/ water 6/4/1); ¹H NMR (D ₂O, 500MHz) δ 2.23 (s, 3H), 2.24 (s, 3H), 2.57 (td, J=6.54,3.68,2H), 2.63 (t, J=6.71,2H), 2.86-2.90 (m, 4H), 3.42 (m, 1H), 3.53 (t, J=9.76,1H), 3.64 (t, J=9.43,1H), 3.80-3.91 (m, 4H), 4.04 (dd, J=9.79,4,71,1H), 4.31 (dd, J=4.63,1.14,1H), 4.45 (dd, J=4.16,1.13,1H), 5.02 (d, J=1.29,1H), 5.11 (s, J=1.30,1H), MS (ES); C ₁₁H ₁₉NO ₇Calculated value, 277.27; Measured value [M+1] 277.9.

5-leyulamide-3, the two deoxidations of 5--D-glycerine-D-semi-lactosi The preparation of-2-nonulopyranosuronate.With Sodium.alpha.-ketopropionate (0.616g, 5.6mmol) and N-acetylneuraminate aldolase (50U) add in 2-leyulamide-2-deoxidation-D-mannopyranose solution among the 0.1M HEPES (pH7.5) (0.156g, 0,56mmol).Reaction mixture is heated to 37 ℃ continues 20 hours, and freezing subsequently.Then reaction mixture is passed through the filtration of C18 silicon, freezing and freeze-drying.(silicon is at first used 10-40%MeOH/CH with flash chromatography with thick solid ₂Cl ₂Be CH then ₂Cl ₂/ MeOH/H ₂O6/4/0.5) carry out purifying.Merge and concentrated suitable fraction, thereby obtain 45mg (80% output) white solid: R _f=0.15 (silicon, CHCl ₃/ MeOH/ water 6/4/1); ¹H NMR (D ₂O, 500MHz) δ 1.82 (t, J=11.9,1H), 2.21 (dd, J=13.76,4.84,1H), 2.23 (s, 3H), 2.57 (appq, J=6.6,2H), 2.86-2.95 (m, 2H), and 3.15-3.18 (m, 1H), 3.28-3.61 (compound, 1H), 3.60 (dd, J=11.91,6.66,1H), 3.75 (td, J=6.65,2.62,1H), 3.84 (dd, J=11.89,2.65,1H), 3.88-4.01 (compound, 2H), 4.04 (td, J=11.18,4.67,1H), MS (ES); To C ₁₄H ₂₃NO ₁₀Calculated value, 365.33; Measured value ([M-1] ^-), 363.97.

Cytidine-5 '-single phosphinylidyne-(5-leyulamide-3, two deoxidation-β of 5--D-glycerine-D-gala The preparation of sugar-2-nonulopyranosuronate).With 5-leyulamide-3, the two deoxidations of 5--D-glycerine-D-semi-lactosi-2-nonulopyranosuronate (50mg, 137 μ mol) is dissolved in the damping fluid of 100mM HEPES (pH7.5) of 2mL, and adds 1M MnCl ₂(300 μ L, 300 μ mol).With CTP-2Na ⁺(79mg, 1.5 μ mol) are dissolved in the 5mL HEPES damping fluid and add in the sugar.Interpolation sialyltransferase/CMP-neuraminic acid synthetic enzyme merges enzyme (11U) and reaction mixture was stirred 45 hours in room temperature.By 10, the 000MWCO filter filters and the filtrate that will contain reaction product is directly used without being further purified: R with reaction mixture _f=0.35 (silicon, IPA/ water/NH ₄OH7/2/1).

B. the sugar of peptide is puted together with Glycopegylated

α-proteinase inhibitor (alpha antitrypsin)

7. recombinant glycoprotein Antithrombin III, Pp63 glycophosphoproteins and alpha1-antitrypsin is sialylated

Present embodiment has proposed the preparation to the sialylated form of several recombinant peptides.

With ST3Gal III sialylated to recombinant glycoprotein.Checked that several glycoprotein are by the sialylated ability of recombinant rat ST3Gal III.For in these glycoprotein each, sialylated will be valuable procedure of processing at each glycoprotein in as the exploitation of commercial product.

Reaction conditions.Reaction conditions is summarized in the table 11.Sialyltransferase is reflected between room temperature and 37 ℃ and carried out 24 hours.Sialylated degree is by determining to be integrated in the oligosaccharides that glycoprotein connects ¹⁴The amount of C-NeuAc and definite.For each proteinic reaction conditions referring to table 11.

Table 11. reaction conditions

¹" circulation " refers to generate CMP-NeuAc with standard conditions (20mM NeuAc and 2mM CMP) " original position " enzymatic that is described in the specification sheets.Damping fluid is 0.1M HEPES, pH7.5.

Although the result who proposes in the table 12 proof has been used low-level enzyme, under each situation, all realized the sialylated of significance degree.Basically, obtained sialylated completely based on estimation to the terminal galactose that can get.Table 12 shows the result of sialylated reaction.In each research, enzyme amount (mU/mg) benchmark as a comparison that every mg protein is used.Among the embodiment shown in several, every mg protein only needs the ST3Gal III of 7-13mU just can obtain sialylated substantially completely after 24 hours.

Table 12. analytical results

Protein	The source	Terminal Gal ¹Moles/mole	NeuAc integrates ²Moles/mole	％Rxn ³	Other features
Protein	The source	Terminal Gal ¹Moles/mole	NeuAc integrates ²Moles/mole	％Rxn ³	Other features	AT?III ⁴	GenzymeTransgenics	102	104	117	Do not have
AT?III ⁴	GenzymeTransgenics	102	1.3	108	SDS-gel: lipidated protein FACs: carbohydrate sugar form	AT?III ⁴	GenzymeTransgenics	102	104	117	Do not have
AT?III ⁴	GenzymeTransgenics	102	1.3	108	SDS-gel: lipidated protein FACs: carbohydrate sugar form	Asialylated-Pp63 glycophosphoproteins	Sigma	802	905	116	Do not have
Asialylated-AAAT ⁵	PPL	7	7.0	100	SDS-gel: lipidated protein	Asialylated-Pp63 glycophosphoproteins	Sigma	802	905	116	Do not have

¹Terminal (exposure) Gal amount on the oligosaccharides that determine by supplier or that connect from the N-of literature value (Pp63 glycophosphoproteins, asialylated-AAAT).

²By after separating from the precursor of free radioactivity mark through gel-filtration by the NeuAc of the determined integration of integration of 14C-NeuAc.

³%Rxn refers to the reacting finisheding degree per-cent based on the terminal Gal amount of theoretical maximum.

⁴Antithrombin III.

⁵Alpha1 Anti-trypsin.

These results significantly with those opposite with report in the studying in great detail of ox ST6Gal I, in this research, in 24 hours, be less than 50mU/mg protein can cause be less than 50% sialylated, and 1070mU/mg protein can cause that about 85-90%'s is sialylated.People such as Paulson (1977), J.Biol.Chem.252:2363-2371; People such as Paulson (1978), J.Biol.Chem.253:5617-5624.By another study group to rat

α

2,3 and the research of α 2, the 6 sialyltransferases complete sialylated proteinic enzyme concn of 150-250mU/mg people (1982) J.Biol.Chem.257:13845-13853 such as () Weinstein that needs that discloses asialylated-AGP.These early stage researchs show that together ST6Gal I sialyltransferase need surpass 50mU/mg and sialylated fully to realize up to 150mU/mg.

The present embodiment proof need be than expection enzyme still less to the sialylated of recombinant glycoprotein with ST3Gal III sialyltransferase.For the reaction of a kilogram levels, need the ST3GalIII sialyltransferase of about 7,000 units, rather than shown 100 in the early stage research, 000-150,000 unit.These enzymes are unpractical from the purifying of natural origin, only obtain the output of 1-10 unit in mass preparation after 1-2 month.Suppose that ST6Gal I and ST3Gal III sialyltransferase all are to produce as the reorganization sialyltransferase, and realized the identical expression level of two enzymes, with respect to ST3Gal III sialyltransferase, ST6Gal I sialyltransferase needs the fermentation scale of 14-21 doubly high (or higher) so.For ST6Gal I sialyltransferase, reported the expression level of 0.3U/1 in the yeast (people (1995) Biochem.Biophys.Res.Commun.210:14-20 such as Borsig).In aspergillus niger, realized the expression level that ST3Gal III sialyltransferase 1000U/ rises.On existing expression level, will need 300-450,000 liter yeast fermentation thing carries out sialylated needed enough enzyme with ST6Gal I sialyltransferase to 1kg glycoprotein to produce.On the contrary, with ST3Gal III sialyltransferase 1kg glycoprotein is carried out sialylated needs and be less than 10 liters of fermentation of Aspergillus niger things.Thereby, produce the required fermentation capacity ratio of ST3Gal III sialyltransferase that is used for extensive sialylated reaction and produce the required low 10-100 of ST6Gal I doubly; The cost of producing sialyltransferase will reduce pro rata.

Cri-IgG antibody

8.Cri-IgG1 the sugared reconstruct of antibody

Present embodiment has proposed Cri-IgG1 antibody is carried out the method for external reconstruct.

The N-glycosylation in the conservative site of Asn297 can be regulated its pharmacokinetic performance and effector function (people such as Dwek, 1995, J.Anat.187:279-292 in monoclonal antibody Fc structural domain; People such as Boyd, 1995, Mol.Immunol.32:1311-1318; People such as Lund, 1995, FASEB J.1995,9:115-119; People such as Lund, 1996, J.Immunol.157:4963-4969; Wright ﹠amp; Morrison, 1998, J.Immunol.160:3393-3402; Flynn ﹠amp; Byrd, 2000, Curr.Opin.Oncol.12:574-581).In cell cultures fermentation or some pathological conditions process, in the glycosylation pattern in this site, produced significant heterogeneity.The different glycosylation pattern feature of gained is the complexity two feeler structures (being respectively G0, G1 and G2, referring to table 13) with 0,1 and 2 terminal galactose residues as a result on the Fc structural domain.Observed sugar form variation, shown the therapeutic property that can influence antibody as the variation in the modification of the N-sugar form of galactosylation, brachymemma endways and five equilibrium, especially its by complement combination and activation regulate the ability that target cell kills and wounds (people such as Boyd, 1995, the same; Wrght﹠amp; Morrison, 1998, the same; People such as Mimura, 2000, Molec.Immunol.37:697-706; People such as Davies, 2001, Biotechnol.Bioeng.74:288-294).

For the different sugar form that obtains Cri-IgG1 antibody and check its Fc effector function, use exoglycosidase and progressively prune Cri-IgG1 antibody to generate the sialic sugar form (G2 of shortage, G1), lack the sugar form (G0) of sialic acid and semi-lactosi and the sugar form (M3N2F) that lacks sialic acid, semi-lactosi and N-acetyl-glucosamine, as shown in table 13.These molecules are modified with different glycosyltransferases and suitable sugar subsequently.The modification condition is developed into the condition that causes the initial antibodies glycan structures to change the different sugar form into: M3N2, GnT-I-M3N2 (using the M3M2 form that GnT-I has added the GlcNAc part), G0, five equilibrium-G0 (having added the G0 part of the GlcNAc of five equilibrium with GnT-III), five equilibrium-the G0 of galactosylation (having added the five equilibrium-G0 sugar form of terminal galactose part), G2, (α 2 for the S1 of mono-sialylated, 6)-G2 (has one with α 2, the G2 sugar form of the terminal sialic acid part that the 6-sialyltransferase adds), (α 2 for S1,3)-G2 (has one with α 2, the G2 sugar form of the terminal sialic acid part that the 3-sialyltransferase adds) and asialylated S2 (α 2,3)-G2 (G2 sugar form).After each sugared reconstruction step, glycan structures enzymatic from the antibody protein discharges, and ins all sorts of ways and analyze, and comprises characterizing by capillary electrophoresis, 2-AA HPLC separating with the MALDI-TOF mass spectrometry.

The abbreviation of table 13. sugar form structure

◇=Fucose,=GlcNAc, zero=seminose, ●=semi-lactosi

Be described in material and the method used in these experiments now.

The Cri-IgG1 monoclonal antibody.Cri-IgG1 antibody is available from R.Jefferies (MRCCenter for Immune Regulation, The Medical School, University ofBirmingham, Britain).Antibody is non-recombinant antibodies, and separates from human myeloma.Antibody is used three kinds of method preparations.In first method, this method is called " DEAE ", and antibody is used the DEAE ion exchange column and separated under gentle relatively condition.In the second approach, this method is called " SPA ", and antibody carries out purifying on A albumen post (streptococcus aureus (Staphylococcus aureus) A albumen), wherein have the elution step of low pH.In the third method, this method is called " Fc ", uses protease treatment antibody, thereby only keeps the Fc part of antibody, and has removed the antibodies structural domain.These antibody purification methods are that those skilled in the art are well-known, and do not repeat in detail herein.

The affinity purification of reshaped antibody.The antibody that to modify by exoglycosidase or glycosyltransferase is at ProA-agarose 4-post (the Amersham Bioscience that flows fast, Arlington Heights, IL) carry out purifying on, with 0.1M glycine-HCl damping fluid (pH2.7) wash-out, and neutralize with the Tris of 1M pH9.5 immediately.Eluate is used the NAP-10 post, and (Amersham Bioscience, Arlington Heights IL) carry out buffer exchange, are replaced by to carry out next step glycosylated suitable damping fluid, as the MES of 100mM pH6.5 or the Tris-HCl of 50mMpH7.2.In 4 ℃ at Tube-O-Dialyzers ^TM(ChemiconInternational, Temecula CA) upward fully dialyse the end product of reconstruct to PBS, and MWCO is 8kDa.

The external Glycosylase of Cri-antibody is handled.(IL) antagonist carries out buffer exchange for AmershamBioscience, Arlington Heights, is replaced by sodium phosphate/Trisodium Citrate of 50mM pH6.0 to use the NAP-10 post.By making antibody (5mg/mL) contact spend the night (to remove terminal sialic acid part) in 37 ℃ with 20mU/mg protein neuraminidase, contact and spend the night (to remove the terminal galactose part in 37 ℃ with 20mU/mg protein beta-galactosidase enzymes, thereby cause the G0 sugar form), and/or with the 2mU/mg beta-N-acetylhexosaminidase (from sword bean, Seikagaku, Tokyo, Japan) spend the night (to remove terminal N-acetyl-glucosamine in 37 ℃ of contacts, thereby cause the M3N2 sugar form), with progressively at external pruning sugar moieties.With the sample affinity purification that carries out as indicated above.

The external glycosylation of Cri-antibody.Utilize following condition to carry out external GnT1 and modify, promptly use 1mg/mlM3N2 sugar form antibody as substrate, use 25mU/mg recombinant human β 1,2-mannose group-UDP-N-acetylglucosamine based transferase is at MES, the 5mM MnCl of 100mM pH6.5 ₂, 5mM UDP-GlcNAc and 0.02% NaN ₃Damping fluid in carried out 24 hours in 32 ℃.Obtain aliquots containig and carry out glycan analysis, and the product affinity purification that carries out as indicated above of gained as a result.

Utilize following condition to carry out the external modification of five equilibrium-sugar form, promptly use 1mg/ml M3N2 sugar form antibody as substrate, use 25mU/mg β 1,2-recombinant human mannose group-UDP-N-acetylglucosamine based transferase I, use 25mU/mg β 1,2-recombinant human mannose group-UDP-N-acetylglucosamine based transferase II and application 3.5mU/mg β 1,4-recombined small-mouse mannose group-UDP-N-acetylglucosamine based transferase III is at MES, the 10mM MnCl of 100mM pH6.5 ₂, 5mMUDP-GlcNAc and 0.02% NaN ₃Damping fluid in carried out 24 hours in 32 ℃.Obtain aliquots containig and carry out glycan analysis, and the remaining product affinity purification that carries out as indicated above.

Utilize following condition application G0 sugar form antibody or five equilibrium sugar form antibody to carry out external galactosylation, promptly by making antibody and 0.6U/mg reorganization milk β 1, the 4-galactosyltransferase is at Tris-HCl, 150mM NaCl, 5mM UDP-semi-lactosi, the 5mM MnCl of 50mMpH7.4 ₂Damping fluid in 32 ℃ the contact 24 hours.Obtain aliquots containig and carry out glycan analysis, and the remaining product affinity purification that carries out as indicated above.

Utilize following condition use G2 sugar form antibody (1mg/ml) carry out external sialylated, promptly by making antibody and 0.1U/mg ST3Gal3 or 0.1U/mg ST6Gal1,5mM cmp sialic acid in the damping fluid of Tris, the 150mM NaCl of 50mM pH7.4 and 3mM CMP-SA, contact 24 hours in 32 ℃.Obtain aliquots containig and carry out glycan analysis, and the remaining product affinity purification that carries out as indicated above.

Glycan analysis:

Capillary electrophoresis with fluorescence detection of induced with laser.By dilution with at Microcon ^TM(Millipore, Bedford concentrate on MA) from the aliquots containig of the antibody of sugared reconstruct and remove buffer reagent composition and nucleotide sugar the YM-30 microconcentrator.The method that provides by application manufacturer makes protein and PNGase F (Prozyme, San Leandro, CA) contact and discharge the oligosaccharides that N-connects from this protein.In brief, make sample sex change 10 minutes in the damping fluid of 100 ℃ of sodium phosphates, 0.1%SDS and 50mM beta-mercaptoethanol at 50mM pH7.5.Add TX100 to 0.75% (v/v) and 10U PNGase F/200 μ g protein then.After 3 hours, protein is carried out ethanol sedimentation in 37 ℃ of incubations, and make the supernatant liquor drying.The free oligosaccharides that will discharge the then amino pyrene-1 of 8-, 3, the 6-trisulfonic acid carries out mark, and use available from Beckman-Coulter, Inc. (Fullerton, CA) carbohydrate mark and assay kit are analyzed by capillary electrophoresis, as shown in the manufacturers (also referring to Ma and Nashabeh, 1999, Anal.Chem.71:5185-5192).

Capillary electrophoresis (CE) is at eCAP ^TM(50 μ m I.D. are 40cm to the length of detector to the kapillary of N-CHO bag quilt; Beckman-Coulter, Inc., Fullerton carries out on CA), wherein uses fluorescent probe (Beckman-Coulter, Inc.Fullerton, P/ACE CA) with induced with laser ^TMMDQ glycoprotein system (Beckman-Coulter, Inc.Fullerton, CA).By 20 pounds/inch ²The pressure effect 10 seconds sample was incorporated in the cylinder, under 25kV, separated 20 minutes then with reversed polarity.The cylinder temperature is controlled at 20 ℃.Electrophorogram is to be generated in the excitation wavelength of 488nm and the emission wavelength of 520nm by the fluorescent penetrant method of induced with laser.

The carbohydrate standard substance (

EMD Biosciences, Inc., San Diego, CA) comprise M3N2 (the three seminose cores that the N-of coreless Fucose connects), G0 (takes off sialic acid, take off semi-lactosi, two feelers have the oligosaccharides of the N-connection of core Fucose), G2 (takes off sialic acid, two feelers have the oligosaccharides that the N-of core Fucose connects) and the G2 of no Fucose, S1-G2 (the mono-sialylated of coreless Fucose, the two feeler oligosaccharides of galactosylation) and S2-G2 (coreless Fucose two sialylated, the two feeler oligosaccharides of galactosylation) (available from Glyko, referring to Prozyme, San Leandro, CA), M3N2F (three seminose cores with N-connection of core Fucose) and NGA2F (take off sialic acid, take off semi-lactosi, two feelers have the oligosaccharides that the core Fucose is connected with the N-with five equilibrium GlcNAc), with these carbohydrate standard substances amino pyrene-3 of 1-, 6,8-trisulfonate (APTS, Beckman-Coulter, Inc., Fullerton CA) carries out the distribution that mark and being used to is identified the glycan that discharges from antibody.

2-AA HPLC.According to the method (1998, Glycobiology 8:685-694) by Anumula and Dhume description of slightly modified, the glycan that PNGase F is discharged carries out mark with 2-AA (2-anthranilic acid).The N-glycan of reductive amination is used ShodexAsahipak NH2P-50 4D nh 2 column (4.6mm x 150mm), and (Showa Denko K.K., Tokyo Japan) analyzes.Being used for isolating two kinds of solvents is A) 2% acetate and 1% tetrahydrofuran (THF), be dissolved in the acetonitrile, and B) 5% acetate, 3% triethylamine and 1% tetrahydrofuran (THF), soluble in water.

In order to separate the neutral glycan of 2-AA mark, pillar is used 70%A isocratic elution 5 minutes, be subsequently in 60 minute time period from 70% to 50%B linear gradient, be from 50% to 5%B abrupt slope gradient, at last for 5%B isocratic elution 10 minutes subsequently in 5 minute time period.The peak of wash-out is surveyed with fluorescence detection, wherein excites at 230nm, and the detection wavelength is 420nm.Under this gradient condition, the G0 sugar form will be at about 30.5 minutes wash-outs, and the G1 sugar form will be at about 34.0 minutes wash-outs, and the G2 sugar form will be at about 37.0 minutes wash-outs.Under these conditions, the existence of Fucose does not change elution time.

In order to separate the negatively charged ion glycan of 2-AA mark, with pillar with 70%A isocratic elution 2.5 minutes, be subsequently in 97.5 minute time period from 70% to 5%A linear gradient, be usefulness 5%A isocratic elution 15 minutes at last.The peak of wash-out is surveyed with fluorescence detection, wherein excites at 230nm, surveys at 420nm.Under this gradient condition, neutral glycan is expected at wash-out between 18.00-29.00 minute, glycan with an electric charge is expected at wash-out between 30.00-40.00 minute, glycan with two electric charges is expected at wash-out between 43.00-52.00 minute, glycan with three electric charges is expected at wash-out between 54.00-63.00 minute, and the glycan with four electric charges is expected at wash-out between 65.00-74.00 minute.

Maldi analysis to the N-glycan of reductive amination.The little aliquots containig of the N-glycan that will discharge with the PNGase-of 2-anthranilic acid (2AA) mark goes up dialysis 45 minutes at MF-Millipore membrane filter (hole of 0.025 μ m, diameter 47mm), and this membrane filter swims in waterborne.With the dialysis aliquots containig at Speedvac ^TM(ThermoSavant, Holbrook NY) go up dryly, be dissolved in the small amount of water again, and with water-soluble/acetonitrile (50:50) in 2,5-resorcylic acid (10g/L) solution mixes.

On the MALDI target, and (FosterCity CA) analyzes for Applied Biosystems, Inc. to be applied in the Biosystems DE-Pro mass spectrometer of linearity/negative ion mode operation with the mixture drying.Determine the structure of oligosaccharides based on observed mass-to-charge ratio and former document.Not attempting fully, sign waits laminated structure.

SDS-PAGE.For the stability of the antibody of determining sugared reconstruct, all samples is analyzed by SDS-PAGE.The end product of sample is used 8-16% Tris-glycine gels, and (Invitrogen, Carlsbad CA) carry out electrophoresis under non-reduced condition.Bovine serum albumin carries out electrophoresis with as quantitation standard under reductive condition.(Pierce Chemical Co., Rockford IL) dyes, to develop the color with GelCode BlueStain Reagent with gel.

Result of experiment is described now.

Be expressed in the natural sugar form of the Cri in the human myeloma cell.The Cri-IgG1 antibody of purifying contains indefinite sugar form from the patients serum who suffers from multiple myeloma.Figure 97 A-97C has shown the HPLC curve of the glycan that enzymatic discharges from Cri-IgG1 antibody.Figure 98 A-98C has shown the MALDI curve of the glycan that enzymatic discharges from the Cri-IgG1 antibody that is expressed in human myeloma cell.Main form is the insufficient G0 of galactosylation, G1, and G2 is relative few (table 14 and Figure 97 C) with sialylated structure.For the influence to mab treatment character of the glycan of checking modification, external exoglycosidase is pruned and external glycosylation reconstruct is modified Cri-IgG1 antibody by carrying out, to generate the different sugar form of this antibody.

The relative quantity of the Cri-IgG1 different sugar form that table 14. is expressed by the isolating human myeloma cell of HPLC is that the area from separately peak calculates and gets.

At first, (each 100 μ g) are carried out in the optimization of each step in exoglycosidase pruning and the glycosylation on small-scale.

Three seminose core sugar forms (M3N2) of Cri-IgG1 antibody.Progressively processing by Glycosylase generates M3N2, and this Glycosylase comprises neuraminidase, β 1,4-tilactase and β 1-2,3,4,6N-acetyl hexosaminidase.For the removal of terminal galactose and GlcNAc on the Cri-IgG1 antibody sample of estimating sugared reconstruct, used quantitative capillary electrophoresis (CE) method.Glycan is to discharge with PNGase F enzymatic from the antibody of sugared reconstruct, and with 8-amino pyrene-1,3,6-trisulfonic acid (APTS) carries out derivatize at reducing end under neutral.The CE of the fluorescence detection (LIF) of the product of gained by having induced with laser on the post analyzes (Ma ﹠amp as a result; Nashabeh, 1999, the same).Because the separation of glycan is based on the difference of hydromeehanics size, so the glycan of APTS mark moves (M3N2＜M3N2F＜G0＜G1＜G2) with the order that size increases gradually.

Figure 99 A-99D shows the glycan that discharges from the Cri-IgG1 antibody of sugared reconstruct and with the electrophorogram of the glycan standard substance (Figure 99 A) of APTS derivatize.Sugar form is identified by its electrophoretic mobility and standard substance are compared.The relative quantity of each glycan kind is that the relative area percentage calculation from the peak shown in each gets, and the results are shown in the table 15.The M3N2F sugar form is represented 91% DEAE-Cri glycan, 80% SPA-Cri glycan and 100% Fc-Cri glycan.In glycan structures, observe the incomplete removal (referring to table 15) of the GlcNAc part that causes the GnT-I-M3N2F sugar form from DEAE-Cri (8.6%) and SPA-Cri (～20%).Sugar form GnT-I-M3N2F is the M3N2F sugar form with an extra GlcNAc, as being added by GnT-I.

Table 15. has calculated the area at the independent peak of CE curve among Figure 99, and has determined the relative quantity of M3N2F and GnT-I-M3N2F sugar form.

Take off the sugar form (G0) of galactosylation.Cri-IgG1 antibody with G0 sugar form is that the 4-tilactase is progressively handled natural Cri-IgG1 antibody and obtained by usefulness neuraminidase and β 1, and each reaction was carried out 24 hours.Analyze by CE, HPLC and MALDI from the glycan that the antibody of sugared reconstruct discharges.Figure 100 A has shown the CE curve of the glycan that discharges.In all three samples, only observed a peak, based on standard substance relatively with this peak called after G0 sugar form (Figure 100 A and table 16).

The relative quantity of the Cri-IgG1 G0 sugar form that table 16. is determined by CE and HPLC.

Except the glycan analysis that is provided by CE, quantitatively the HPLC method also is used for determining the per-cent by the G0 sugar form of the reconstruct glycan representative of Cri-IgG1 antibody.It is by discharging glycan with PNGase F enzymatic and monitoring at the product that the reducing end under neutral derivatize discharges with 2-anthranilic acid (2-AA) that glycan on the sugar reshaped antibody distributes.The mixture of derivatize separates by HPLC having on the Shodex Asahipak NH2P-50 4D post of fluorescence detection.Figure 101 A-101C shows the chromatogram that obtains from the glycan that discharges.Owing in all three samples, only found a main peak, so HPLC result has confirmed the CE analysis.With CE and HPLC data consistent, maldi analysis shows that also almost completely sugar is reconstructed into G0 sugar form (Figure 102 A-102C).

The G2 sugar form (G2) of complete galactosylation.Handle Cri-IgG antibody to obtain the taking off sialic acid sugar form with neuraminidase, this sugar form also is that galactosylation is insufficient.Use 0.6U/

ml ox β

1,4 galactosyltransferase and galactose donor molecule to handle these then and take off the sialic acid sugar form, have the G2 sugar form so that antibody sugar is reconstructed into.

The degree of terminal galactose baseization is determined by glycan analysis.In CE and HPLC curve, only observe a main peak (Figure 103 A-103C and Figure 104 A-104C).This peak is corresponding to the G2 sugar form in each situation.The calculating of per-cent total peak area shows that almost completely (～90%) changes G2 (referring to table 14) into from the not enough sugar form of galactosylation of original sample.These the results are summarized in the table 17.The maldi analysis of glycan further is supported in all samples almost completely, and sugar is reconstructed into G2 sugar form (Figure 105 A-105C).

The relative quantity of the reconstruct Cri-IgI1 antibody G2 sugar form that the per-cent total peak area was determined during table 17. was analyzed by CE and HPLC.

GnT-I-sugar form (GnT-I-M3N2).By add a GlcNAc part to molecule M3N2 sugar form Cri-IgG antibody sugar is reconstructed into the GnT-I-M3N2 sugar form.This molecule and 25mU GnT-I/mg antibody are contacted with suitable GlcNAc donor molecule.CE, the HPLC of the glycan that discharges and maldi analysis (being respectively Figure 106 A-106D, Figure 107 A-107C and Figure 108 A-108C) show that original M 3N2F sugar form is by reconstruct fully.Yet the modification structure that 40-60% is only arranged is the GnT-I-M3N2 sugar form, and about 30% is the G0 sugar form.The existence of G0 sugar form may be the result that incomplete GlcNAc prunes when preparation original M 3N2 form.

Five equilibrium sugar form (NGA2F).Combination by making M3N2 sugar form Cri-IgG antibody and three kinds of transferring enzyme GnT-I, GnT-II and GnT-III and suitable N-acetyl-glucosamine donor molecule contact and its sugar are reconstructed into the NGA2F sugar form.Be reflected in 24 hours and finish.In order to determine that five equilibrium-GlcNAc partly adds the degree in the glycan to, use CE and analyze the sugar form of determining to be present on the sugared reshaped antibody.

Figure 109 A-109D shows the electrophorogram of analyzing acquisition from the CB of the glycan of the Cri-IgG1 antibody release of sugared reconstruct.4 peaks have appearred after reconstruct.A main peak is to move with the identic retention time of NGA2F standard sugar.3 other secondary peaks may be the glycan of incomplete reconstruct.In order to compare, also used quantitative HPLC method, wherein the sequentially eluting that increases gradually with size of the glycan of 2-AA mark (Gn1＜G0＜NGA2F).Shown in Figure 110 A-110C, from the CE analysis of glycan, obtained similar result.Use CE or HPLC analysis and all do not find M3N2F.The NGA2F glycan is the main peaks that CE and HPLC analyze.Still the Gn1 and the G0 glycan that are retained in the sample may be the results who not exclusively modifies.Most of original M 3N2F sugar forms partly are reconstructed into NGA2F sugar form (60～70%) by 3 GlcNAc, about 15～18% partly are reconstructed into the G0 sugar form by adding 2 GlcNAc, and only have a small amount of (～7%) partly to be reconstructed by only adding a GlcNAc.The maldi analysis (Figure 111 A-111C) of the glycan that discharges shown have 1, the peak of the sugar form of 2 or 3 terminal GlcNAc parts, it is consistent with CE and HPLC analysis (Figure 109 and 110).The relative quantity of each glycan kind is that the relative area percentage calculation from peak shown in each gets, and is summarized in the table 18.

Table 18. is determined by CE and HPLC from the relative quantity of the different sugar form of the Cri-IgG1 of GnT-I, II and III reconstruct.

The five equilibrium of galactosylation (Gal-NGA2F) sugar form.The NGA2F sugar form of Cri-IgG1 antibody is with ox β 1, and 4-galactosyltransferase and suitable galactose donor carry out sugared reconstruct.Use 0.6U/ml β 1, the 4-galactosyltransferase adds the terminal galactose part.Figure 112 A-112D shows the electrophorogram that obtains with 2-AA HPLC method.In brief, terminal for the sugar form of GalNAc 100% be galactosylation almost.Compare Figure 112 A～112B of DEAE Cri-IgG1 and Figure 112 C～112D of Fc Cri-IgG1, the 2-AA HPLC curve (Figure 112 A and 112C) of the glycan that GnT-I, II and III modify is modified by GalT1, thereby the peak of all glycan is all owing to size after adding galactose moiety increases the later wash-out (Figure 112 B and 112D) that becomes.These results have further obtained the confirmation that MALDI-MS analyzes.

Sialylated (S2G2) sugar form of Cri-IgG1.The sugared reconstruct G2 sugar form of Cri-IgG1 antibody further uses ST3Gal3 and ST6Gal1 is reconstructed.Figure 113 A-113C has shown the HPLC curve with the G2 sugar form of ST3Gal3 reconstruct.Most of G2 sugar forms change S2G2 sugar form (the G2 sugar form with 2 extra terminal sialic acid parts into;～70%, referring to table 19), a small amount of S1G2 sugar form (G2 sugar form with 1 extra terminal sialic acid part that is is only arranged;＜25%, referring to table 19).These results have further obtained being shown in the confirmation of the maldi analysis among Figure 114 A-114C.The MALDI data also show all G2 sugar forms all by sialylated for S2G2 or S1G2 sugar form.

The relative quantity that table 19. is determined by HPLC from the different sugar form of the Cri-IgG1 of ST3Gal3 reconstruct.

By comparing, the ST6Gal1 reconstruct of G0 sugar form can not reach the complete level of finding in ST3Gal3 reconstruct.Figure 115 A-115D and Figure 116 A-116C have shown respectively from the result of CE and HPLC analysis acquisition.In any one sugared reconstruct sample, all do not find the S2G2 sugar form.Yet all G2 sugar forms all change S1-G2 into.MALDI-MS analyzes and also supports these data (Figure 117 A-117C).

The stability of the reconstruct glycan of Cri-IgG1.At last, studied the stability of the Cri-IgG1 glycan that is reconstructed by exoglycosidase processing and glycosylation.The Cri-IgG1 antibody of each sugared reconstruct is stored in 4 ℃, and after reconstruct, 2 weeks checked degraded by SDS-PAGE.Shown in Figure 118 A-118E, the DEAE of reconstruct and SPA antibody all keep the molecular weight of about 150kDa, show seldom to not degraded, and no matter the sugared reconstruct type of carrying out how.Fc Cri-IgG1 antibody keeps the molecular weight of about 38kDa, also shows seldom to not degraded, and regardless of the reconstruct type of carrying out how.

The effector function biological assay of the Cri-IgG1 antibody of reconstruct.The effector function biological assay is derived from the method for people such as Mimura (2000, Molecular Immunology 37:697-706).The IC of the sugar form of Cri-IgG1 antibody ₅₀Be to determine by the super-oxide reaction of the U937 cell that suppresses to cause by red corpuscle, wherein red corpuscle by natural anti--NIP antibody carried out sensitization.

In 1000 units/when the mL interferon-gamma exists with single celled U937 cell cultures 2 days, with the inducing cell differentiation and produce the ability of super-oxide.Then with cell washing and with 2 x 10 ⁶Cell/mL is resuspended in the Hanks balanced salt solution, does not have phenol redly in this salts solution, and contains HEPES and the 0.15mM BSA of 20mM pH7.4.When not existing or exist, the various sugar forms of Cri-IgG1 antibody with anti--NIP (5-iodo-4-hydroxyl-3-oil of mirbane acetyl) antibody red corpuscle is carried out sensitization in 37 ℃ of incubations 30 minutes.Use the PBS washed cell then 3 times, and with it with 2.5 x10 ⁷Cell/mL is resuspended among the HBSS-BSA.With U937 cell (100 μ l, 2 x 10 ⁶Cell/mL) join in the plastic test tube, and with nitric acid two-(20 μ l 2.5mM) join in the test tube N-methylacridine.Test tube was heated 5 minutes in 37 ℃ of water-baths.Then with red corpuscle (80 μ l, 2.5 x 10 of sensitization ⁷Cell/mL) join in the test tube.Superoxide anion production is by using Berthold LV953 luminometer (Berthold Australia Pty Ltd, Bundoora, Australia) in 30 minute time period in the time of 37 ℃ nitric acid two-chemoluminescence of N-methylacridine enhanced measures.

Compare with natural antibody, G0 and M3N2 sugar form Cri-IgG1 antibody have respectively 92% with 85% relative inhibiting value.Yet, natural Cri-IgG1 antibody deficiency core Fucose.(2002, J.Biol.Chem.277:26733-26740) prompting lacks the core Fucose with 10 times of inhibiting value improvement to people such as Shields.Based on these results, the inhibiting value of expection galactosylation-five equilibrium-G0 sugar form will be higher than five equilibrium-G0 sugar form, five equilibrium-G0 sugar form will be much higher than the G2 sugar form, the G2 sugar form will approximate two sialylated-G2 sugar forms and mono-sialylated-G2 sugar form greatly, two sialylated-G2 sugar forms and mono-sialylated-G2 sugar form will be higher than the natural antibody sugar form, the natural antibody sugar form will be higher than the G0 sugar form, and the G0 sugar form will be higher than the M3N2 sugar form.

Complement receptor-1

9. Sialylated and the fucosylation of TP10

Present embodiment has proposed to the preparation of the TP10 with sialic acid Lewis X part with to the analysis of enhanced biologic activity.

Also can trigger inflammatory episode in the brain microvessel structure even interrupt blood flow in the brain in the short period of time, this will aggravate the damage of cerebral tissue.This tissue injury that produces can increase by the activation of the inflammation and the cascade of condensing.In the mouse model of apoplexy, P-selects the expression of the increase of albumen and ICAM-1 can promote leukocyte recruitment.SCR-1 is the recombinant forms of the extracellular domain of complement receptor-1 (CR-1).SCR-1 is the strong inhibitor of complement activation.SCR1sLe ^X(CD20) be the substituting glycosylation form of sCR1, it has been carried out substituting glycosylation to show sialylated Lewis ^XAntigen.In the past, find in the Lec11 of through engineering approaches Chinese hamster ovary celI, to express in vivo and glycosylated sCR-1sLeX correctly is positioned in the big cerebral microvascular of ischemic and in the neurone of expression C1q, thereby suppressing neutrophilic granulocyte and thrombocyte gathers and reduces infarction of brain volume (people such as Huang, 1999, Science 285:595-599).In the present embodiment, at external sCR1sLe by glycan reconstruct preparation ^XShow and the interior glycosylated sCRsLe of body ^XSimilar enhanced biologic activity.

The TP10 peptide is expressed in BUK B11 Chinese hamster ovary celI.This Chinese hamster ovary celI system produces has the glycosylated TP10 peptide of typical Chinese hamster ovary celI, and this peptide has many but is not the glycan that all adds cap with sialic acid.

66mg TP10's is sialylated.With TP10 (2.5mg/mL), CMPSA (5mM) and ST3Gal3 (0.1U/mL) at 50mM Tris, 0.15M NaCl, 0.05% sodiumazide, among the pH7.2 in 32 ℃ of incubations 48 hours.Radiolabeled CMP sialic acid is added in the little sample aliquot with the monitoring integration.TP10 is isolating from nucleotide sugar with SEC HPLC.The sample proved response of analyzing 24 hours and 48 hours finished after 24 hours.Make reaction mixture freezing then.With reaction product carry out the auxiliary sugared electrophoresis of fluorophore (Fluorophore AssistedCarbohydrate Electrophoresis) (

Glyko, Inc, Novato CA) analysis (Figure 119).

Pharmacokinetics research.Purchase has the rat of jugular vein intubate.With 10mg/kg sialylated before or sialylated after the TP10 peptide give each 3 rat (n=3) that group is handled by tail vein injection.Obtained 14 blood samples from 0～50 hour.At each time point, the concentration of the TP10 peptide in the blood after sialylated is all than the height (Figure 120) of the TP10 before sialylated after 0 hour.Sialic interpolation is compared with parent material, makes the area under the plasma concentration-time curve (AUC) of pharmacokinetics curve double (Figure 121).

The fucosylation of sialylated TP10.The above-mentioned sialylated mixture of 10mL (25mgTP10) is thawed, and add the GDP-Fucose to 5mM, MnCl ₂To 5mM, and FTVI (fucosyltransferase VI) is to 0.05U/mL.Make reactant 32 ℃ of incubations 48 hours.With reaction product carry out the auxiliary sugared electrophoresis of fluorophore (Fluorophore Assisted CarbohydrateElectrophoresis) (

Glyko, Inc, Novato CA) analysis (Figure 122).Adding radiolabeled GDP-Fucose in little sample aliquot integrates with monitoring.TP10 is isolating from nucleotide sugar with SEC HPLC.The sample proved response of analyzing 24 hours and 48 hours finished at 24 hours.Measurement selects proteic bonded external test method to show to E-: add the E-selection protein ligands (Figure 123) that Fucose can produce biologic activity.

Enbrel ^TM

10. AntibodyEnbrel ^TM Glycopegylated

Present embodiment has proposed the PEGization program of the O-connection glycan of antagonist molecule.At this, with Enbrel ^TMAs example, yet, it should be appreciated by those skilled in the art that this program can be applicable to many antibody molecules.

Enbrel ^TM The preparation of-SA-PEG (10KDa).To before PEGization, make O-connect the sialylated or not sialylated Enbrel of glycan ^TM(TNF-acceptor-IgG ₁-mosaic) is dissolved in 50mM Tris-HCl, 0.15M NaCl, 5mM MnCl with 2.5mg/mL ₂, 0.05% NaN ₃, among the pH7.2.Make this solution and 5mM UDP-semi-lactosi and 0.1U/mL galactosyltransferase in 32 ℃ of incubations 2 days so that the insufficient glycan of galactosylation is added cap with semi-lactosi.In order to monitor the integration of semi-lactosi, added to the little sample aliquot of reactant ¹⁴C-semi-lactosi-UDP part; The mark that is integrated in the peptide is isolating with free label by gel-filtration with Toso Haas G2000SW analysis mode post in the first alcohol and water.The radioactivity seeker that is integrated into radio-labeling in the peptide and is with built-in (in-line) carries out quantitative.

When reaction finished, the ST3Gal3 that makes this solution and 1mM cmp sialic acid-linker-PEG (10kDa) and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-linker-PEG, peptide is separated by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).When reaction finishes, reaction mixture is prepared the type post with the Toso Haas TSK-Gel-3000 that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.The fraction that will contain product merges, concentrated, exchange buffering liquid, freeze-drying then.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

Erythropoietin (EPO)

11. GlcNAc is to the interpolation of EPO

Present embodiment has provided the interpolation of GlcNAc residue to three seminose cores.

GlcNAc is to the interpolation of EPO.EPO SF-9 expressed in insect cells and purifying (Protein Sciences, Meriden, CT).Change to 100% of " three seminose cores+2 GlcNAc " (peak 1, P1 among Figure 124) from the three seminose sugar forms of Epo and to be in 32 ℃ of incubations 24 hours during with below end reaction concentration of 100mU/ml GlcNAcT-I and 100mU/ml GlcNAcT-II and realization:

100mM MES pH6.5, or 100mM Tris pH7.5

5mM?UDP-GlcNAc

20mM?MnCl ₂

100mU/ml?GlcNAcT-I

100mU/ml?GlcNAcT-II

1mg/ml EPO (purifying, be expressed in the Sf9 cell) available from Protein Sciences.

The analysis of sugar form.This mensuration is K-R Anumula and ST Dhume, the trickle modification of Glycobiolgy8 (1998) 685-69.The N-glycan that N-glycanase (PNGase) discharges reduces mark with anthranilic acid.The N-glycan of reductive amination is injected on the ShodexAsahipak NH2P-50 4D nh 2 column (4.6mm x 150mm).Two kinds of solvents are used for separating: A) acetate of 5% (v/v), 1% tetrahydrofuran (THF) and 3% triethylamine, soluble in water; And B) 2% acetate and 1% tetrahydrofuran (THF) are dissolved in acetonitrile.Make pillar with 70%B isocratic elution 2.5 minutes then, be subsequently on 97.5 minutes time period from 70% to 5%B linear gradient, used the 5%B isocratic elution at last 15 minutes.The peak of wash-out is surveyed with fluorescent probe, and excitation wavelength is that 230nm and emission wavelength are 420nm.

Under these conditions, three seminose cores have 22.3 minutes retention time, and the product of GnT reaction has 30 minutes retention time.Parent material all is three seminose cores (Figure 124) with core GlcNAc.

12. have the preparation of the EPO of the complicated glycan of many feelers

Present embodiment has proposed to prepare PEGization, the EPO of two feelers and the sialylated EPO of three feelers from the EPO of insect cell expression.

To recombinant human erythropoietin (rhEPO) (Protein Sciences Corp. from baculovirus/Sf9 expression system, Meriden, CT) carry out glycan analysis, and the glycan of gained demonstration as a result is mainly three seminose cores with core Fucose, has only the glycan of little per-cent also to have single GlcNAc.

Add N-acetyl-glucosamine with GnT-I and GnT-II.Make two crowdes of rhEPO (1mg/ml) and GnT-I and GnT-II, 5mM UDP-GlcNAc, 20mM MnCl ₂With 0.02% sodiumazide in the MES of 100mM pH6.5 in 32 ℃ of incubations 24 hours.A criticizes and contains 20mg EPO and 100mU/mL GnT-I and 60mU/mL GnT-II.B criticizes and contains 41mg EPO and 41mU/mL GnT-I+50mU/mL GnT-II.After reaction, with sample by the gel-filtration desalination (the PD10 pillar, Pharmacia LKB Biotechnology Inc., Piscataway, NJ).

EPO glycan by 2-AA HPLC atlas analysis.This assay method is to Anumula and Dhume, the trickle modification of Glycobiology 8 (1998) 685-69.The N-glycan of reductive amination is injected on the Shodex Asahipak NH2P-50 4D nh 2 column (4.6mm x 150mm).Two kinds of solvents are used for separating: A) acetate of 5% (v/v), 1% tetrahydrofuran (THF) and 3% triethylamine, soluble in water; And B) 2% acetate and 1% tetrahydrofuran (THF) are dissolved in the acetonitrile.Make pillar with 70%B isocratic elution 2.5 minutes then, be subsequently on 100 minutes time period from 70% to 5%B linear gradient, used the 5%B isocratic elution at last 20 minutes.The peak of wash-out is that the emission wavelength with the excitation wavelength of 230nm and 420nm carries out fluorescence detection.Non-sialylated N-connects in the LC scope that glycan is in 23-34 minute, mono-sialylated be in 34-42 minute, two sialylated are in 42-52 minute, three sialylated are in 55-65 minute, and four sialylated be in 68-78 minute.

Glycan by 2AA HPLC characterizes and discloses A and have 92% to change the two feeler structures (all the other (balance) have single GlcNAc) with two GlcNAc in criticizing.B criticizes and shows 97% and change the product of wanting (Figure 125 A and 125B) into.

Introduce the 3rd tentillum with GnT-V.Make product EPO (1mg/mL that B criticizes) from GnT-I and GnT-II reaction in desalination on the PD-10 pillar and concentrate subsequently back and 10mU/mLGnT-V and 5mM UDP-GlcNAc in the MES of 100mM pH6.5 in 32 ℃ of incubations 24 hours, this MES contains 5mM MnCl ₂With 0.02% sodiumazide.It is (Figure 126) that takes place with 92% efficient that 2AA HPLC analytical proof changes.

After desalination (PD-10) and concentrating, add semi-lactosi: be about to EPO (1mg/ml) and 0.1U/ml GalT1,5mM UDP-semi-lactosi, 5mM MnCl with rGalTI ₂In 32 ℃ of incubations 24 hours.

To maldi analysis from the N-glycan of the reductive amination of EPO.The little sample aliquot of carrying out the N-glycan that discharges from EPO with PNGase of reductibility mark with anthranilic acid is gone up dialysis 45 minutes floating on MF-Millipore membrane filter waterborne (hole of 0.025 μ m, diameter 47mm).The sample aliquot of dialysis is dry in SpeedVac, be dissolved in once more in the water in a small amount, and be dissolved in 2 of water/acetonitrile (50:50), 5-resorcylic acid (10g/L) solution mixes.Make this mixture dry and analyze on target in order to the Applied Biosystems DE-Pro MALDI-TOF mass spectrograph of linearity/negative ion mode operation.Oligosaccharides is based on observed mass-to-charge ratio and former document and is definite.

By MALDI the analysis of the glycan that discharges is shown that semi-lactosi is (Figure 127) that quantitatively adds all available sites to.Then will from the EPO of above-mentioned galactosylation by on the Superdex1.6/60 pillar in 50mM Tris, 0.15M NaCl, carry out gel-filtration and purifying among the pH6.

It is sialylated.After concentrated and desalination (PD-10), make the EPO (1mg/mL) and ST3Gal3 (0.05U/mL) and CMP-SA (3mM) incubation in the 50mM of the pH7.2 that contains 0.02% sodiumazide Tris, 150mM NaCl of 10mg galactosylation.Independent sample aliquot contains radiolabeled CMP-SA.With the integration mark of gained as a result and the free mark size exclusion chromatography/HPLC by the degree such as grade in 45%MeOH, 0.1%TFA to separate (pillar of 7.8mm x 30cm, granular size 5 μ m, TSK G2000SW in 0.5mL/ minute _XL, Toso Haas, Ansys Technologies, Lake Forest, CA).Utilize this program, integrated 12% number (360 micromoles are in 33 micromole EPO, or about 10.9 moles/mole).Theoretical value (site that 3N-connects, three feelers) is the integration of about 9 moles/mole.These meet the limited field of present method.Replacing in the same reaction of ST3Gal3 with ST6Gal1,5.7% radio-labeling is integrated among the EPO of galactosylation, or compares about 48% with ST3Gal3.

13. the EPO's that produces in the insect cell is Glycopegylated

Present embodiment has proposed to prepare from the EPO of insect cell expression two feeler EPO of PEGization.

Will be from recombinant human erythropoietin (rhEPO) (the Protein Sciences Corp. of baculovirus/Sf9 expression system, Meriden, CT) carry out glycan analysis, and show that glycan is mainly three seminose cores with core Fucose, and the glycan of little per-cent also has one GlcNAc (Figure 128).

Add N-acetyl-glucosamine with GnT-I and GnT-II.Make two crowdes of rhEPO (1mg/ml) and GnT-I and GnT-II, 5mM UDP-GlcNAc, 20mM MnCl ₂With 0.02% sodiumazide in the MES of 100mM pH6.5 in 32 ℃ of incubations 24 hours.A criticizes and contains 20mg EPO and 100mU/mL GnT-I and 60mU/mL GnT-II.B criticizes and contains 41mg EPO and 41mU/mL GnT-I+50mU/mL GnT-II.After reaction, with sample by the gel-filtration desalination (the PD10 pillar, Pharmacia LKB Biotechnology Inc., Pi scataway, NJ).

Glycan by 2AA HPLC characterizes and discloses A and criticize 92% and change the two feeler structures (all the other have single GlcNAc) with two GlcNAc into.B criticizes and shows that 97% changes the product of wanting (Figure 125 A and 125B) into.

A criticizes the galactosylation of EPO.EPO (A of～16mg criticizes) is handled to finish the interpolation of GlcNAc with GnT-II.This is reflected at and contains 150mM NaCl, EPO mg/mL, 1mMUDP-GlcNAc, 5mM MnCl ₂, 0.02% sodiumazide and 0.02U/ml GnT-II the 50mM Tris of pH7.2 in carried out 4 hours in 32 ℃.Then by the UDP-semi-lactosi being added into 3mM and GalT1 being added into 0.5U/ml and carrying out the galactosylation of EPO in 32 ℃ of incubations in 48 hours.

The EPO of galactosylation is then at 50mM Tris, 0.15M NaCl, among the pH6 on the Superdex751.6/60 pillar by gel-filtration purifying.The peak that contains EPO is analyzed by 2AAHPLC then.Based on the data of HPLC, contain two semi-lactosis at the glycan of galactosylation reaction back～85%, and～15% glycan do not have any semi-lactosi.

The EPO's of galactosylation is sialylated.Galactosyl EPO sialylated is to contain among the Tris of 150mM NaCl, 0.5mg/ml EPO, 200U/ml ST3Gal3 with 0.5mM CMP-SA or CMP-SA-PEG (1kDa) or CMP-SA-PEG (10kPa) at 100mM to carry out 48 hours in 32 ℃.At the nearly all glycan with two galactose residues in sialylated reaction back with CMP-SA all are complete sialylated (2 sialic acid/glycan).MALDI-TOF analyzes and has confirmed the HPLC data.

The PEGization of the EPO of galactosylation.For the PEGization reaction of using CMP-SA-PEG (1kDa) and CMP-SA-PEG (10kDa), the sample aliquot of reaction mixture is analyzed (Figure 129) with SDS-PAGE.The molecular weight of EPO peptide increases along with adding each sugar, and molecular weight has rapider increase after the PEGization reaction.

The vitro bioassay of EPO.External EPO mensuration (from people such as Hammerling, 1996, the J.Pharm.Biomed.Anal.14:1455-1469 reorganization) based on the reactivity of TF-1 clone to multiple concentration EPO.The TF-1 cell provides the better systems of research marrow sample ancester cell propagation and differentiation.This clone is to be drawn the sample by the marrow of the heparinization of people such as T.Kitamura in October, 1987 from 35 years old Japanese male sex suffering from serious pancytopenia from one to set up.These cells place one's entire reliance upon interleukin or rHuGM-CSF (GM-CSF).

TF-1 clone (ATCC, Cat.No.CRL-2003) grow among the RPMI+FBS10%+GM-CSF (12ng/ml) and in 37 ℃ at 5%CO ₂Middle incubation.This cell is present in the suspension with the concentration of 5000 cells/ml substratum, and 200 μ l are positioned in the 96 hole flat boards.Make EPO (the 0.1 μ g/ml-10 μ g/ml) incubation 48 hours of this cell and various concentration.Carrying out the MTT viability then measures, this mensuration is as follows: add 25 μ l 5mg/ml MTT (SIGMA M5655), make dull and stereotyped in 37 ℃ of incubations 20 minutes～4 hours, add 100 μ l Virahol/HCl solution (100ml Virahol+333 μ l HCl6N), read OD at 570nm and 630nm or 690nm, and from the reading of 570nm, deduct the reading of 630nm or 690nm.

Figure 130 contains when with sialylated EPO with carry out the result of Glycopegylated EPO gained when carrying out the check of external EPO biological activity with 1kDa or 10kDa PEG.When equal concentration, carry out Glycopegylated EPO and do not carry out Glycopegylated EPO and have much at one activity with 1kDa PEG at about 5 μ g/ml.When equal concentration, carry out Glycopegylated EPO with 10kDa PEG and have and do not carry out the Glycopegylated EPO activity of half approximately at about 5 μ g/ml.

14.CHO the O-of the EPO that produces in the cell connects the Glycopegylated of glycan

The preparation of the EPO-SA-PEG (10kDa) that O-connects.Asialylated-the EPO that is produced by Chinese hamster ovary celI at first is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The ST3Gal3 that makes this solution and 5mM CMP-SA and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid in N-connection glycan, added CMP-SA-to the little sample aliquot of reactant ¹⁴C; Peptide is isolating by gel-filtration on the Toso Haas G3000SW analysis mode post of using the first alcohol and water, and product is surveyed with radiation monitor.When reaction finishes, solution is concentrated with the Centricon-20 filter.With rest solution and 0.05MTris (pH7.2), 0.15M NaCl, 0.05% NaN ₃Exchange buffering liquid is to the final volume of 7.2mL, up to no longer detecting CMP-SA.Then retentate is resuspended in 0.05M Tris (pH7.2), 0.15M NaCl, 0.05% NaN with 2.5mg/mL protein ₃In.In order to make the glycosylation of O-connection site, make this solution and 1mM CMP-SA-PEG (10kDa) and ST3Gal1 in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-PEG, the little sample aliquot of reactant is analyzed using on the Toso Haas TSK-gel-3000 analysis mode post that PBS pH7.0 carries out wash-out to separate by gel-filtration and to survey by UV.When reaction finishes, reaction mixture is carried out purifying and absorbs the collection fraction based on UV with the Toso Haas TSK-gel-3000 preparation type post of using PBS damping fluid (pH7.0).Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

15.EPO-fortune iron egg

Present embodiment has proposed to make protein to be connected glycan with O-to carry out the program that sugar is puted together, and particularly transferrin and EPO is carried out sugar and puts together.Sialic acid residues is removed from the O-connection glycan of EPO, and preparation EPO-SA-linker-SA-CMP.Making EPO-SA-linker-SA-CMP and asialylated transferrin carry out sugar with ST3Gal3 puts together.

The preparation of asialylated-EPO that O-connects.The EPo (erythropoietin) that produces in the Chinese hamster ovary celI is dissolved among 50mM Tris-HCl pH7.4, the 0.15M NaCl with 2.5mg/mL, and makes itself and 300mU/mL sialidase (vibrio cholerae (Vibrio cholera))-agarose conjugate in 32 ℃ of incubations 16 hours.In order to monitor reaction, with the little sample aliquot of reactant with suitable damping fluid dilution and according to the program run IEF gel of Invitrogen.10,000rpm is centrifugal and collect supernatant liquor with mixture.With supernatant concentration to 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN ₃, the EPO concentration of about 2.5mg/mL among the pH7.2.The ST3Gal3 that makes this solution and 5mM cmp sialic acid and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor sialic integration, added the CMP-SA-fluorescent ligand to the little sample aliquot of reactant; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).When reaction finishes, reaction mixture is prepared the type post with the Toso Haas G3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Sample is dialysed to water and analyze by MALDI-TOF MS.

The preparation of EPO-SA-linker-SA-CMP.Asialylated-EPO that O-is connected is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The ST3Gal1 that makes this solution and 1mM cmp sialic acid-linker-SA-CMP and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-linker-SA-CMP, peptide is separated by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).

After 2 days, reaction mixture is prepared the type post with the Toso HaasG3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

The preparation of transferrin-SA-linker-SA-EPO.Above-mentioned EPO-SA-linker-SA-CMP is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The ST3Gal3 that makes asialylated transferrin of this solution and 2.5mg/mL and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of transferrin, peptide is separated by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1), and product is surveyed by the UV absorption.When reaction finished, the ST3Gal3 (to add cap to any unreacted transferrin glycan) that makes this solution and 5mM CMP-SA and 0.1U/mL was in 32 ℃ of incubations 2 days.Reaction mixture is prepared the type post with the Toso HaasG3000SW that uses PBS damping fluid (pH7.1) to carry out purifying and absorbs the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

16.EPO-GDNF

Present embodiment has proposed protein is carried out the program that sugar is puted together, particularly the preparation of EPO-SA-linker-SA-GDNF.

The preparation of EPO-SA-linker-SA-GDNF.Above-mentioned EPO-SA-linker-SA-CMP is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The ST3Gal3 that makes this solution and 2.5mg/mL GDNF (producing) and 0.1U/mL in the NSO cell was in 32 ℃ of incubations 2 days.In order to monitor the integration of GDNF, peptide is separated by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1), and product is surveyed by the UV absorption.When reaction finished, the ST3Gal3 (to add cap to any unreacted GDNF glycan) that makes this solution and 5mM CMP-SA and 0.1U/mL was in 32 ℃ of incubations 2 days.Reaction mixture is prepared the type post with the Toso HaasG3000SW that uses PBS damping fluid (pH7.1) to carry out purifying and absorbs the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

17.EPO single feeler Glycopegylated

Present embodiment has proposed to prepare the method for Glycopegylated single feeler erythropoietin (EPO), and biological activity in external and the body.

As EPO (GenBank Accession No.P01588) when being expressed in the Chinese hamster ovary celI, form the glycan that N-are connected at amino-

acid residue

24,38 with 83, and form glycan (Figure 131 of O-connections at amino-acid residue 126; People such as Lai, 1986, J.Biol.Chem.261:3116-3121).The biological activity of this glycoprotein is directly relevant with NeuAc content.The sialic acid that increases has reduced EPO combining at external and its acceptor; Yet the sialic acid that increases has increased the interior biological activity of the body of EPO.The glycan that O-connects is to the not influence of pharmacokinetics of external or activity in vivo or this molecule of EPO (people such as Wasley, 1991, Blood 77:2624-2632).

When EPO is expressed in the insect cell, as use that baculovirus/Sf9 expression system realized (also referring to people such as Wojchowshi, 1987, Biochem.Biophys.Acta910:224-232; People such as Quelle, 1989, Blood 74:652-657), form the glycan that N-is connected at amino-acid residue 24,38 with 83, and do not form the glycan (Figure 132) that O-connects at amino-acid residue 126.This is because insect cell does not have the glycosyltransferase of amino-acid residue 126 aminoacid sequence on every side of identification EPO.The glycan that most of N-connect is by GlcNAc ₂Man ₃Fuc forms.In the present embodiment, the EPO that is expressed in the insect cell is carried out efficient reconstruction to generate complex plycan SA by protein is contacted with ST with GnT1,2, GalT-1 continuously when suitable donor molecule exists ₂Gal ₂GlcNAc ₂Man ₃FucGlcNAc ₂These enzymatic reactions are to use reaction conditions described herein to carry out on the EPO of insect cell expression, thereby 92% total efficiency produces complex plycan (table 21) herein.Randomly, also can add the glycan that O-connects (O ' Connell and Tabak, 1993, J.Dent.Res.72:1554-1558; People such as Wang, 1993, J.Biol.Chem.268:22979-22983).

Table 21. is at the EPO of insect cell expression (" parent material ") per-cent last and each glycan structures kind in the glycan structures colony on EPO after each successive enzymatic reconstruction step.

◇=Fucose,=GlcNAc, zero=seminose,

=semi-lactosi, ▲=the N-n acetylneuraminic acid n

Equally in the present embodiment, be reconstructed forming single feeler, two feelers and glycan three feelers being expressed in EPO in the insect cell, this glycan carries out Glycopegylated with the method that other places are herein described with 1kDa, 10kDa and 20kDa PEG molecule subsequently.Determine the molecular weight of these EPO forms, then with Epoetin with glycan that 3 N-are connected ^TMWith NESP (Aranesp with glycan that 5 N-are connected ^TM) compare (Figure 133).Provide among the example embodiment 7 herein of two feelers of preparation and three feeler glycan structures.

EPO with single feeler PEGization glycan structures prepares by the following method, promptly by at expressed in insect cells EPO peptide, the EPO peptide is only contacted with GnTI (perhaps selectively only with GnTII).The EPO peptide is contacted with GalT-I.When existing, the SA-PEG donor molecule make EPO peptide and ST contact (Figure 134 A) have the EPO peptide (Figure 134 B) of single feeler PEGization glycan structures that 3 N-are connected with generation then.

EPO-SA that generates from the EPO of insect cell expression and the external biological activity of EPO-SA-PEG are to stimulate the propagation of TF-1 erythroleukemia cell to estimate by measuring this molecule.The biological activity that three feeler EPO-SA-PEG1kDa show nearly all three feeler EPO-SA, and two feeler EPO-SA-PEG10kDa shows the biological activity (Figure 135) of nearly all couple of feeler EPO-SA in some EPO concentration range.Be created on reconstruct in the insect cell and Glycopegylated EPO and show Epogen up to 94% ^TMThe external biological activity, this Epogen ^TMBe the EPO (table 22) among the CHO of being expressed in further glycan reconstruct or PEGization.

Table 22. is when 2 μ g/ml protein and 48 hours and Epogen ^TMThe external activity of the EPO construct of comparing.

¹Three feelers-SA2,3 constructs have with 2,3 key bonded SA molecules.

Determined the drug disposition dynamic metabolism of Glycopegylated and non-Glycopegylated EPO.With Glycopegylated and non-Glycopegylated [I ¹²⁵]-mark EPO injects in the rat, and the pharmacokinetics of definite molecule.Compare with the EPO of two feelers, the AUC of the EPO-PEG1kDa of two feelers is high 1.8 times, and the AUC of the EPO-PEG 10kDa of two feelers high 11 times (Figure 136).Compare with the EPO of two feelers, the AUC of the EPO-PEG 1kDa of two feelers is high 1.6 times, and the AUC of the EPO-PEG 10kDa of two feelers high 46 times (Figure 136).Therefore, the Glycopegylated pharmacokinetics of improving EPO greatly.

Also determine biological activity in the body of Glycopegylated and non-Glycopegylated EPO by the degree of measuring the EPO construct and stimulate reticulosis.Reticulosis is the measurement of red corpuscle precursor cell maturation for the speed of mature erythrocyte (erythrocyte).8 mouse of each treatment group are carried out the single subcutaneous injection of 10 μ g protein/Kg, and measured skein cell per-cent (Figure 137) at 96 hours.The EPO of three feelers and two feeler PEGization (comprises Epogen than the EPO form of non-PEGization ^TM) the higher interior biological activity of body of displaying.

Bioactively in the EPO construct body further determine to estimate by the following method, i.e. hematocrit by measurement CD-1 female mice on the 15th behind the peritoneal injection that carries out at EPO construct on every Wendesdays time (per-cent of the whole blood of forming by red corpuscle) with 2.5 μ g peptide/Kg body weight.The hematocrit increment increases with the size of EPO form, and single feeler EPO-PEG20kDa of 82.7kDa has the NESP (Aranesp that is slightly larger than 35.6kDa ^TM) activity, and its activity is the Epogen of 28.5kDa ^TMBioactive about 2 times (Figure 138).

The Glycopegylated EPO that present embodiment is illustrated generation longer action time is feasible.The pharmacokinetics feature of Glycopegylated EPO can customize at the PEG of the transformation period of blood flow molecular size by being used for changing peptide after changing Glycopegylated number of loci and adding.At last, Glycopegylated EPO has kept external and the interior biological activity of body.

18. the list of sialylated and PEGization-, two-and preparation and the biological activity of three-feeler EPO

Present embodiment has been illustrated the production of Glycopegylated EPO, particularly has the EPO of the PEGization of single feeler of having connected PEG thereon and two feeler glycan.Produced following EPO variant: single feeler PEG (1kDa) and PEG (20kDa); Two feelers 2,3-sialic acid (SA), two feeler SA-PEG (1kDa), two feeler SA-PEG (10kDa); With 2,3-SA adds three feelers 2 of cap, 3-SA and three feelers 2,6-SA.

The recombinant erythropoietin (rEPO) that is expressed in insect cell is available from ProteinSciences (Lot #060302, Meridan CT).The glycan composition of this batch EPO has about 98% three seminose core textures.Figure 139 A has described from the HPLC of the glycan of this EPO release and has analyzed, wherein peak " P2 " expression three seminose cores.Figure 139 B shows the maldi analysis of the glycan that discharges, and wherein the structure of the glycan of Shi Fanging is shown in the next door, peak of their representatives.

Single tentillum

Carried out several steps to produce the structure of single tentillum.In brief, the first step is the GnT-I/GalT-1 reaction, carries out purifying with the Superdex-75 chromatography subsequently.This reaction is added the GlcNAc part to a branch of three seminose cores, partly adds galactose moiety to GlcNAc.Partly upward interpolation SA-PEG (10kDa) part or SA-PEG (20kDa) part are extended by be reflected at terminal galactose with ST3Gal3 in branch.Last purifying with the Superdex-200 chromatography realize (Amersham Biosciences, Arlington Heights, IL).

The GnT-I/GalT-1 reaction. combination GnT-I and GalT-1 reactant, and in 32 ℃ of incubations 36 hours.Reactant contains 1mg/mL EPO, 100mM Tris-Cl pH7.2,150mM NaCl, 5mM MnCl ₂, 0.02% NaN ₃, 3mM UDP-GlcNAc, 50mU/mg GnT-I, 3mM UDP-Gal and 200mU/mg GalT-1.Figure 140 has described the maldi analysis from the glycan of EPO release in GnT-I/GalT-1 reaction back.Glycan analysis shows that about 90% glycan has single tentillum structure of expection, and this structure has the terminal galactose part.

The Superdex-75 purifying. after the GnT-I/GalT-1 reaction, use Superdex-75 gel permeation chromatography (the Amersham Biosciences of 1.6cm x 60cm, ArlingtonHeights, IL) at the PBS that contains 0.02% Tween 20 (Sigma-Aldrich Corp., St.Louis, MO) in from zymoprotein pollutent and nucleotide sugar purifying EPO.

The ST3Gal3 reaction. with the ST3Gal3PEGization reactant in 32 ℃ of incubations 24 hours.Reactant contains 1mg/mL EPO, 100mM Tris-Cl pH7.2,150mM NaCl, 0.02% NaN ₃, 200mU/mg ST3Gal3 and 0.5mM CMP-SA-PEG (10kDa) or 0.5mM CMP-SA-PEG (20kDa).The SDS-PAGE that Figure 141 has described this reacted EPO analyzes.The corresponding molecular weight of protein band shows that the EPO glycan that forms by the GnT-I/GalT-1 reaction is sialylated by the PEG derivative fully.

The Superdex-200 purifying. use then 1.6cm x 60cm the Superdex-200 gel permeation chromatography (Amersham Biosciences, Arlington Heights, IL) in the PBS that contains 0.02% Tween 20 from ST3Gal3 reaction contaminant purifying EPO.

The TF-1 cells in vitro of single feeler PEGization EPO is biological to be measured. TF-1 clone is used for the activity of the outer EPO of estimated body.TF-1 clone is can be from American type culture collection (Catalogue No.CRL-2003, Rockyille, the marrow ancester cell that MD) obtains system.The viability of this clone place one's entire reliance upon interleukin 3 or granulocyte-macrophage colony stimutaing factor.The TF-1 cell provides the better systems of research EPO to the effect of propagation and differentiation.

The TF-1 cell in 37 ℃ at 5%CO ₂In grow in the RPMI substratum with 10%FBS and 12ng/ml GM-CSF.Cell suspends with the concentration of 10,000 cells/ml substratum.200 μ l cell aliquots containigs are scattered in the flat board of 96-hole.Make cell and 0.1～10 μ g/ml EPO incubation 48 hours.

Then the MTT by at first adding 25 μ l, 5 μ g/ml (bromination 3-[4,5-dimethylthiazole-2-yl]-2,5-phenylbenzene tetrazole or thiazolyl indigo plant (thiazolyl blue); SigmaChemical Co., St.Louis, Mo., Catalogue No.M5655) carry out the MTT viability and measure.With flat board in 37 ℃ of incubations 4 hours.Add 100 μ l Virahol/HCl solution (100ml Virahol and 333 μ l HCl 6N).570nm and 630 or 690nm read dull and stereotyped light absorption ratio, and from the reading of 570nm, deduct 630 or the reading of 690nm.

Figure 142 has described at its single feeler glycan and has carried out the PEGization back to the active bioassay results of EPO.In this biological assay, the EPO (Epogen) of the non-PEGization of specific activity of the EPO of single feeler PEGization is much lower.

Two tentillums

Several reactions have been carried out to realize two tentillums of EPO.In brief, first reaction is the combination of GnT-I and GnT-II reaction, to add the GlcNAc part to two three seminose core branches.Second reaction is that the GalT-1 reaction is partly added galactose moiety to each GlcNAc.Before ST3Gal3 reaction, carry out the Superdex-75 chromatography (Amersham Biosciences, Arlington Heights, IL).Two tentillums further react with ST3Gal3 and extend, to add 2,3-SA or SA-PEG (1kDa), SA-PEG (10kDa).Use the Superdex-200 chromatography realize last purifying (Amersham Biosciences, Arlington Heights, IL).

The GnT-I/GnT-II reaction. combination GnT-I and GnT-II reactant, and in 32 ℃ of incubations 48 hours.Reactant contains 1mg/mL EPO, 100mM MES pH6.5,150mM NaCl, 20mM MnCl ₂, 0.02% NaN ₃, 5mM UDP-GlcNAc, 100mU/mg GnT-I, 60mU/mgGnT-II.Reaction has realized adding 92% performance level of two feeler GlcNAc parts, and 8% be single feeler GlcNAc part.Figure 143 A shows that the HPLC of the glycan that discharges analyzes, wherein the two feeler GlcNAc glycan of peak " P3 " expression.Figure 143 B shows the maldi analysis of the glycan that discharges, and wherein the structure of glycan is shown in the next door, peak of their representatives.

In order further to react, together with 1mM UDP-GlcNAc, 5mM MnCl ₂, 0.02% NaN ₃Add other 20mU/mg GnT-II together, make mixture in 32 ℃ of incubations 4 hours.This reaction realized finishing of two feeler GlcNAc glycan greater than 99%.

The GalT-1 reaction. after finishing second GnT-II reaction, begin the GalT-1 reaction immediately.Concentration with 0.5mU/mg GalT-1 and 3mM UDP-Gal is added enzyme and nucleotide sugar in the GnT-II reactant of finishing.

When GalT-1 was reflected at small-scale that each reaction has about 100 μ g EPO and carries out, about 95% reaction produces had two feeler terminal galactose EPO partly.The HPLC of the glycan that Figure 144 A show to discharge analyzes, and wherein peak " P2 " is two feeler glycan with terminal galactose part (glycan 85%), and peak " P1 " do not have partly two feeler glycan of terminal galactose (glycan 15%).

GalT-1 is reflected at each reaction and has carrying out on a large scale of about 16mg EPO.Figure 144 B shows that wherein peak " P2 " is the two feeler glycan with terminal galactose part from the HPLC analysis of the glycan of extensive GalT-1 reaction release, and peak " P1 " is the two feeler glycan with terminal galactose part.

The Superdex-75 purifying. after the GnT-1/GalT1 reaction, use Superdex-75 gel permeation chromatography (the Amersham Biosciences of 1.6cm x 60cm, ArlingtonHeights, IL) in the PBS that contains 0.02%Tween 20 from zymoprotein pollutent and nucleotide sugar purifying EPO.Figure 145 has described the chromatogram of Superdex 75 gel-filtrations, and wherein peak 2 is the two feeler glycan with terminal galactose part.The SDS-PAGE that Figure 146 shows each reconstruction step product analyzes, and has shown the increase of EPO molecular weight along with each reconstruction step.

The ST3Gal3 reaction. with the ST3Gal3 reactant in 32 ℃ of incubations 24 hours.Reactant contains 0.5mg/mL EPO, 100mM Tris-Cl pH7.2,150mM NaCl, 0.02% NaN ₃, 100mU/mg ST3Gal3 and 0.5mM CMP-SA, 0.5mM CMP-SA-PEG (1kDa) or 0.5mM CMP-SA-PEG (10kDa).Figure 147 has described before the ST3Gal3 reaction and the SDS-PAGE of reacted EPO analyzes.Analyze based on this SDS-PAGE, after each ST3Gal3 reaction, the two feeler EPO that contain terminal Gal no longer can be detected visibly.Non-sialylated EPO compared and shows that size increases when all sialylated EPO variants were initial with reaction.

The Superdex-200 purifying. use then 1.6cm x 60cm the Superdex-200 gel permeation chromatography (Amersham Biosciences, Arlington Heights, IL) in the PBS that contains 0.02% Tween 20 from ST3Ga l3 reaction contaminant purifying EPO.The distribution of glycan structures when table 23 has been summed up each reconstruction step.

EPO goes up the summary of glycan structures after each reconstruction step of table 23..

Rhombus is represented Fucose, and square expression GlcNAc, and circle is represented seminose, and empty circles is represented semi-lactosi.

Three tentillums

Several reactions have been carried out to realize three tentillumizations of EPO.In brief, first reaction is the combination of GnT-I and GnT-II reaction, to add the GlcNAc part to two outsides of glycan, three seminose core branches.Second reaction is that the GnT-V reaction is added second GlcNAc part to one of two outsides, three seminose core branches, thereby 3 GlcNAc parts are arranged now.The 3rd reaction is that the GalT-1 reaction is partly added galactose moiety to each terminal GlcNAc.The EPO product separates by the Superdex-75 chromatography then.Three tentillums further react with ST3Gal3 and extend, adding 2, and 3-SA or 2, the 6-SA part, and with 2,3-SA partly adds cap.Use the Superdex-75 chromatography and realize last purifying.

The GnT-I/GnT-II reaction. combination GnT-I and GnT-II reactant, and in 32 ℃ of incubations 24 hours.Reactant contains 1mg/mL EPO, 100mM MES pH6.5,150mM NaCl, 20mM MnCl ₂, 0.02% NaN ₃, 5mM UDP-GlcNAc, 50mU/mg GnT-I and 41mU/mgGnT-II.Reaction has realized adding 97% performance level of two feeler GlcNAc parts, and remains 3% 3 seminose core.Figure 148 has described the HPLC analysis from the glycan of EPO release in GnT-I/GnT-II reaction back.

The GnT-V reaction.GnT-V reactant contains 100mM MES pH6.5,5mM UDP-GlcNAc, 5mM MnCl ₂, 0.02% NaN ₃, 10mU/mg GnT-V and 1mg/mL EPO, and in 32 ℃ of incubations 24 hours.This reaction is partly added the GlcNAc part to containing GlcNAc outside seminose partly.Figure 149 has described the HPLC analysis from the glycan of EPO release in GnT-V reaction back.Based on glycan and maldi analysis, about 92% the glycan that discharges from EPO is the expection product, promptly has the EPO of three tentillums of terminal GlcNAc part.The residue of glycan 8% is the structure that contains two tentillums of terminal GlcNAc part.

The GalT-1 reaction.GalT-1 reactant contains 100mM Tris pH7.2,150mM NaCl, 5mM UDP-Gal, 100mU/mg GalT-1,5mM MnCl ₂, 0.02% NaN ₃With 1mg/mL EPO, and in 32 ℃ of incubations 24 hours.Figure 150 has described in this reaction back from the HPLC analysis of the glycan of EPO release.97% of the glycan that glycan and maldi analysis demonstration discharge has the terminal galactose part on three tentillum structures.Remaining 3% for containing two feeler structures of terminal galactose.

The Superdex-75 purifying. after the GnT-I/GalTl reaction, use Superdex-75 gel permeation chromatography (the Amersham Biosciences of 1.6cm x 60cm, ArlingtonHeights, IL) in the PBS that contains 0.02% Tween 20 from zymoprotein pollutent and nucleotide sugar purifying EPO.The material of purifying is divided into two batches, have with generation terminal 2, three feeler glycan of 6-SA part and having with 2,6-SA partly adds the end 2 of cap, 6-SA three feeler glycan partly.

The ST3Gal3 reaction. with the ST3Gal3 reactant in 32 ℃ of incubations 24 hours.Reactant contains BPO, 100mM Tris-Cl pH7.2,150mM NaCl, the 0.02%NaN of 1mg/mL galactosylation ₃, 50mU/mg ST3Gal3 and 3mM CMP-SA.The HPLC that Figure 151 has described after this step the glycan that discharges from EPO analyzes.Based on glycan and maldi analysis, the glycan of about 80% release is to have end 2, the structure of three tentillums of 3-SA part.The residue 20% of the glycan that discharges is to contain end 2, two feeler structures of 3-SA part.

The sialylated reaction of the reacted ST6Gal1 of ST3Gal3. with the ST6Gal1 reactant in 32 ℃ of incubations 24 hours.Reactant contains EPO, 100mM Tris-Cl pH7.2,150mM NaCl, 0.02% NaN of the sialylated and galactosylation of 1mg/mL ₃, 50mU/mg ST6Gal1 and 3mM CMP-SA.Figure 152 has described the HPLC analysis of ST6Gal1 reaction back from the glycan of EPO release.Based on glycan and maldi analysis, the glycan of about 80% three tentillums contains end 2,3-SA part.The residue of glycan 20% is to contain end 2, two feelers of 3-SA part.

The Superdex-75 purifying. the Superdex-75 that uses 1.6cm x 60cm coagulate the amine filtration chromatography (Amersham Biosciences, Arlington Heights, IL) in containing the PBS of 0.02%Tween20 from ST3Gal3 reaction contaminant purifying EPO.

The biological assay of the sialylated or PEGization EPO of three feelers and two feelers. as indicated above, the activity of application TF-1 clone and MTT viability test determination three feelers and two sialylated EPO sugar forms of feeler and PEG 10kDa and the two feeler sugar forms of 1kDa.Figure 153 has described the result of MTT cell proliferating determining.When 2 μ g/ml EDP, the sialylated EPO of two feelers almost has the activity of contrast Epogen, and the sialylated EPO of three feelers has significantly low activity.

Plasma thromboplastin component

19. The plasma thromboplastin component that produces by Chinese hamster ovary celI Glycopegylated

Present embodiment has proposed the preparation of asialylated plasma thromboplastin component and sialylated to its with cmp sialic acid-PEG.

RFactor IX's is asialylated.The plasma thromboplastin component that condenses of recombinant forms (r FacorIX) produces in Chinese hamster ovary celI.The rFactor IX of 6000IU is dissolved in the USP H of 12mL altogether ₂Among the O.With this solution and other 6mL USP H ₂O transfers to Centricon Plus 20, in the PL-10 centrifugal filter.This solution concentration to 2mL, is used 50M Tris-HClpH7.4,0.15M NaCl, the 5mM CaCl of 15mL then ₂, 0.05% NaN ₃Dilute and concentrate once more.Make dilution/concentrate to repeat 4 times effectively damping fluid is changed into the final volume of 3.0mL.The 2.9mL (about 29mg rFactor IX) of this solution transferred in the little plastic test tube and to wherein adding 530 mU α 2-3,6,8-neuraminidase-agarose conjugate (vibrio cholerae, Calbiochem, 450 μ L).Reaction mixture was rotated 26.5 hours gently in 32 ℃.In 10, centrifugal 2 minutes of 000rpm also collects supernatant liquor with mixture.With 50M Tris-HCl pH7.12,1M NaCl, 0.05% NaN of agarose pearl (containing neuraminidase) with 0.5mL ₃Wash 6 times.Once more in 10, centrifugal 2 minutes of 000rpm is to remove any residual agarose resin with the washings collected and supernatant liquor.The asialylated protein soln of collecting is diluted to 19mL with identical damping fluid and in Centricon Plus 20 PL-10 centrifugal filters, is concentrated into～2mL.With 50M Tris-HCl pH7.4,0.15M NaCl, the 0.05%NaN of this solution with 15mL ₃The dilution twice also is concentrated into 2mL.With final asialylated rFactor IX solution with the Tris damping fluid be diluted to 3mL final volume (～10mg/mL).Natural and asialylated rFactor IX sample is analyzed with the IEF-electrophoresis.Isoelectric Fousing Gels (pH3-7) moves with 1.5 μ L (15 μ g) sample, and this sample at first mixes with the dilution of 10 μ L Tris damping fluids and with 12 μ L sample pipetting volume damping fluids.Gel is with standard program application of sample, operation and fixed.Gel dyes (Figure 154) with Colloidal Blue Stain, shows the band of asialylated plasma thromboplastin component.

The preparation of PEG (1kDa and 10kDa)-SA-plasma thromboplastin component.(29mg 3mL) is divided into the sample of two 1.5mL (14.5mg) in the centrifuge tube of two 1.5mL with asialylated rFactor-IX.Each solution is all used 50mM Tris-HCl pH7.4,0.15M NaCl, 0.05% NaN of 12.67mL ₃Dilution, and add CMP-SA-PEG-1k or 10k (7.25 μ mol).Test tube is put upside down gently with mixing, and added 2.9U ST3Gal3 (326 μ L) (14.5mL cumulative volume).Test tube is put upside down once more, and gently in 32 ℃ of rotations 65 hours.This reaction is by in-20 ℃ of freezing stopping.10 μ g response samples are analyzed by SDS-PAGE.The protein of PEGization is that (21.5 x 30cm 13um) carry out purifying on the HPLC pillar, this pillar is used the DulbeccoShi phosphate buffered saline (PBS) (Gibco) of pH7.1,6mL/ minute at Toso Haas Biosep G3000SW.Reaction and purifying are monitored with SDS Page and IEF gel.With 50mM Tris-HCl pH7.4,150mM NaCl, 0.05% NaN of sample with 2 μ L ₃The damping fluid dilution mixes with the 0.5M DTT of 12 μ L sample pipetting volume damping fluids and 1 μ L, and after 6 minutes, loads 10 μ Ls (10 μ g) this sample to Novex Tris-glycine 4-20%1mm gel in 85 ℃ of heating.Gel is with Colloidal Blue Stain painted (Figure 155), shows the band of PEG (1kDa and 10kDa)-SA-plasma thromboplastin component.

20. the direct sialic acid of plasma thromboplastin component-Glycopegylated

Present embodiment has proposed without sialidase processing in advance and plasma thromboplastin component has been carried out sialic acid-PEGization.

Use the sialic acid-PEGization of CMP-SA-PEG (10kDa) to plasma thromboplastin component.Plasma thromboplastin component (1100 IU) is dissolved in 20mM Histidine, 520mM glycine, 2% sucrose, 0.05% NaN of 5mL ₃With 0.01% polysorbate 80, among the pH5.0, described plasma thromboplastin component is expressed in the Chinese hamster ovary celI, and is fully sialylated.Be dissolved in CMP-SA-PEG-(10kDa) (27mg, 2.5 μ mol) in this solution then and add 1U ST3Gal3.Mixing 28 hours afterreactions gently in 32 ℃ finishes.Reactant is to analyze with the SDS-pAGE that Invitrogen describes.Product albumen matter is carried out purifying on Amersham Superdex 200 (10 x 300nm, 13 μ m) HPLC pillar, this pillar is used the phosphate buffered saline (PBS) (PBS) of pH7.0,1mL/ minute.R _t=9.5 minutes.

Use the sialic acid-PEGization of CMP-SA-PEG (20kDa) to plasma thromboplastin component.Plasma thromboplastin component (1100 IU) is dissolved in 20mM Histidine, 520mM glycine, 2% sucrose, 0.05% NaN of 5mL ₃With 0.01% polysorbate 80, among the pH5.0, described plasma thromboplastin component is expressed in the Chinese hamster ovary celI, and is fully sialylated.Be dissolved in CMP-SA-PEG-(20kDa) (50mg, 2.3 μ mol) in this solution then and add CST-II.Mix 42 hours afterreactions gently in 32 ℃ and mix end.Reactant is to analyze with the SDS-PAGE that Invitrogen describes.

Product albumen matter is carried out purifying on Amersham Superdex 200 (10 x 300nm, 13 μ m) HPLC pillar, this pillar is used the phosphate buffered saline (PBS) (Fisher) of pH7.0,1mL/ minute.R _t=8.6 minutes.

21. the sialic acid of Glycopegylated plasma thromboplastin component adds cap

Present embodiment has proposed sialic acid-Glycopegylated peptide is carried out the program that sialic acid adds cap.At this, thrombin-IX is exemplary peptide.

The N-of thrombin-IX-SA-PEG (10kDa) connects the sialic acid that is connected glycan with O-and adds Cap. the rFactor-IX-PEG (10kDa) of purifying (2.4mg) is existed

(Millipore Corp., Bedford MA) concentrates in the centrifugal filter Plus20PL-10, and damping fluid is changed into 50mM Tris-HCl pH7.2,0.15M NaCl, 0.05% NaN of 1.85mL final volume ₃Protein soln is diluted and interpolation 7.4mg CMP-SA (12 μ mol) solid with the identical Tris damping fluid of 372 μ L.This solution is put upside down gently to mix and interpolation 0.1U ST3Gal1 and 0.1U ST3Gal3.Reaction mixture was rotated 42 hours gently in 32 ℃.

10 μ g response samples are analyzed with SDS-PAGE.Novex Tris-glycine 4-12%1mm gel is operation and painted with Colloidd Blue as described in Invitrogen.In brief, 10 μ L (10 μ g) sample is mixed with the 0.5M DTT of 12 μ L sample pipetting volume damping fluids and 1 μ L, and in 6 minutes (Figure 156, swimming lane 4) of 85 ℃ of heating.

Proconvertin a

22. The recombinant blood coagulation factor VIIa's that produces in the bhk cell is Glycopegylated

Present embodiment has proposed the PEGization to the recombinant blood coagulation factor VIIa that produces in the bhk cell.

The preparation of asialylated proconvertin a.Recombinant blood coagulation factor VIIa produces in bhk cell (baby hamster kidney cell).Proconvertin a (14.2mg) is dissolved in damping fluid (pH7.4,0.05M Tris, 0.15M NaCl, 0.001M CaCl with 1mg/ml ₂, 0.05% NaN ₃) in, and with 300mU/mL sialidase (vibrio cholerae)-agarose conjugate in 32 ℃ of incubations 3 days.In order to monitor reaction, the little sample aliquot of reactant is diluted with suitable damping fluid, and carried out IEF gel (Figure 157) analysis according to the program of Invitrogen.In 3,500rpm carries out centrifugal and collects supernatant liquor with mixture.With this resin with above-mentioned damping fluid (pH7.4,0.05M Tris, 0.15M NaCl, 0.05% NaN ₃) wash 3 times (3 * 2mL) and the washings that merges is concentrated in Centricon-Plus-20.With rest solution and 0.05MTris (pH7.4), 0.15M NaCl, 0.05% NaN ₃Carry out buffer-exchanged to obtain the final volume of 14.4mL.

The preparation of proconvertin a-SA-PEG (1kDa and 10kDa).Asialylated rFactorVIIa solution is divided into two parts of equal 7.2ml samples.In each sample, add CMP-SA-5-PEG (1kDa) (7.4mg) or CMP-SA-5-PEG (10kDa) (7.4mg).ST3Gal3 (1.58U) is added in two test tubes, and make reaction mixture in 32 ℃ of incubations 96 hours.This reaction is to monitor by the SDS-PAGE gel with reagent and condition that Invitrogen describes.When reaction finishes, reaction mixture is prepared the type pillar with Toso Haas TSK-Gel-3000 carry out purifying, this pillar is used PBS damping fluid (pH7.1), and absorbs the collection fraction based on UV.With the fraction that contains product that merges in 4 ℃ at Centricon-Plus-20 centrifugal filter (Millipore, Bedford, MA) concentrate in, and spissated solution prepared again to obtain 1.97mg (bicinchoninic acid protein determination, BCA measures, Sigma-Aldrich, St.LouisMO) proconvertin a-PEG.Reaction product is to analyze with SDS-PAGE and IEF analytical method according to the program and the reagent that are provided by Invitrogen.Sample be water is dialysed and analyze by MALDI-TOF.Figure 158 shows the MALDI result who obtains from natural proconvertin a.Figure 159 contains for the MALDI result who carries out the proconvertin a of PEGization with 1kDa PEG, and wherein the peak that carries out the proconvertin a of PEGization with 1kDa PEG is tangible.Figure 160 contains for the MALDI result who carries out the proconvertin a of PEGization with 10kDa PEG, and wherein the peak that carries out the proconvertin a of PEGization with 10kDa PEG is tangible.The SDS-PAGE that Figure 161 has described all reaction product analyzes, and wherein the band of proconvertin a-SA-PEG (10-kDa) is tangible.

Follicle stimulating hormone (FSH)

23. People's hypophysis deutero-FSH's is Glycopegylated

Present embodiment has been illustrated the assembling of conjugate of the present invention.Put together with CMP-(sialic acid)-PEG then follicle stimulating hormone (FSH) is asialylated.

Follicle stimulating hormone asialylated.1mg follicle stimulating hormone (FSH) (HumanPituitary, Calbiochem Cat No.869001) is dissolved in 50mM Tris-HCl, 0.15M NaCl, the 5mM CaCl of the pH7.4 of 500 μ L ₂In.375 these solution of μ L are transferred in the little plastic test tube, and to wherein adding 263mU neuraminidase II (vibrio cholerae).Reaction mixture was shaken 15 hours gently in 32 ℃.Reaction mixture is joined in 600 μ L N-(p-aminophenyl) oxaminic acid-agarose conjugates, and this conjugate is used Tris-HCl, 150mM NaCl and 0.05% NaN of 50mM pH7.4 in advance ₃Carry out balance, and rotated gently 6.5 hours in 4 ℃.In 14, centrifugal 2 minutes of 000rpm also collects supernatant liquor with suspension.With pearl with 0.5mL damping fluid washing 5 times and collect all supernatant liquors.Enzyme solution is contained 50mMTris-HCl pH7.4,1M NaCl, 0.05% NaN with 2L ₃Solution in 4 ℃ of dialysis (7000MWCO) 15 hours, then in 4 ℃ in 4 hours to 50mM Tris-HCl pH7.4,1M NaCl, 0.05% NaN ₃Twice of middle dialysis.With Speed Vac with this solution concentration to 2 μ g/ μ L and be stored in-20 ℃.Response sample (Invitrogen) is analyzed (Figure 162) by IEF gel (pH3-7).

The preparation of people's hypophysis deutero-SA-FSH and PEG-SA-follicle stimulating hormone.Asialylated FSH (100 μ g, 50 μ L) and cmp sialic acid or CMP-SA-PEG (1kDa or 10kDa) (0.05 μ mol) are dissolved in 13.5 μ L H in the 0.5mL plastic test tube ₂O (being adjusted to pH8) with NaOH.With test tube vortex and add 40mU ST3Gal3 (36.5 μ L) (100 μ L cumulative volume) simply.Test tube is carried out vortex once more, and shook 24 hours in 32 ℃ gently.Should react by in-80 ℃ of freezing stopping.15 μ g response samples are analyzed with SDS-pAGE (Figure 163), IEF gel (Figure 164) and MALDI-TOF.Natural FSH also analyzes (Figure 165) with SDS-PAGE.

SDS PAGE and IEF gel analysis to reaction product.The Novex Tris-glycine 8-16%1mm gel that is used for SDS PAGE analysis is available from Invitrogen.With 50mM Tris-HCl pH7.4,150mM NaCl, 0.05% NaN of 7.5 μ L (15 μ g) FSH response sample with 5 μ L ₃The damping fluid dilution is with 15 μ L sample pipetting volume damping fluids and mixed 85 ℃ of heating 6 minutes that are incorporated in of 1 μ L 9M μ-mercaptoethanol.Gel is the also usefulness ColloidalBlue Stain dyeing (Invitrogen) as the indicated operation of Invit rogen.

FSH sample (15 μ g) is mixed (Figure 162) with the dilution of 5 μ L Tris damping fluids and with 15 μ L sample pipetting volume damping fluids.Then with sample application (pH3-7) (Invitrogen) (Figure 165) in Isoelectric Focusing Gels.Gel is as Invitrogen indicated operation and fixed, then with Colloidal Blue Stain dyeing.

24 .CHO the Recombinant FSH that reorganization produces in the cell is Glycopegylated

Present embodiment has been illustrated the assembling to conjugate of the present invention.Asialylated FSH and CMP-(sialic acid)-PEG put together.

The recombinate preparation of asialylated-follicle stimulating hormone.The Puregon that will produce from CHO (rFSH) is used for these research.With 7, the rFSH of 500IU is dissolved in the 8mL water.Make FSH solution at 50mM Tris-HCl pH7.4,0.15M NaCl, 5mM CaCl ₂Middle dialysis also is concentrated into 500 μ L on Centricon Plus 20 centrifugal filters.Transfer to the part (400 μ L) (～0.8mg FSH) of this solution in the little plastic test tube and to wherein adding 275mU neuraminidase II (vibrio cholerae).Reaction mixture was mixed 16 hours in 32 ℃.Reaction mixture is made an addition in advance in N-(p-aminophenyl) oxaminic acid-agarose conjugate (800 μ L) of washing and and rotated gently 24 hours in 4 ℃.In 10,000rpm is centrifugal and collect supernatant liquor with mixture.Pearl with 0.6mL Tris-EDTA damping fluid washing 3 times, is washed 1 time also with 0.2mL Tris-EDTA damping fluid washing 1 time with 0.4mL Tris-EDTA damping fluid, and collects all supernatant liquors.With supernatant liquor in 4 ℃ of 50mM Tris-HCl pH7.4,1M NaCl, 0.05%NaN to 2L ₃Dialyse, and then to 50mM Tris-HCl pH7.4,1M NaCl, 0.05% NaN ₃Dialyse twice.Solution with dialysis is concentrated into 420 μ L and is stored in-20 ℃ in Centricon Plus 20 centrifugal filters then.

Natural and asialylated rFSH sample is analyzed (Figure 166) with SDS-PAGE and IEF.Novex Tris-glycine 8-16% 1mm gel is available from Invitrogen.With 50mM Tris-HCl pH7.4,150mM NaCl, 0.05% NaN of sample (7.5 μ L, 15 μ g) with 5 μ L ₃The damping fluid dilution is with 15 μ L sample pipetting volume damping fluids and mixed 85 ℃ of heating 6 minutes that are incorporated in of 1 μ L 9M beta-mercaptoethanol.Gel is also dyeing with ColloidalBlue Stain (Invitrogen) as the indicated operation of Invitrogen.Isoelectric Focusing Gels (pH3-7) is available from Invitrogen.Sample (7.5 μ L, 15 μ g) is mixed with the dilution of 5 μ L Tris damping fluids and with 15 μ L sample pipetting volume damping fluids.Gel is as the indicated application of sample of Invitrogen, operation and fixed.Gel dyes with Colloidal Blue Stain.Natural and asialylated FSH sample is also dialysed to water and is analyzed with MALDI-TOF.

Sialic acid-the PEGization of Puregon.Asialylated FSH (100 μ g, 54 μ L) and CMP-SA-PEG (1kDa or 10kDa) (0.05 μ mol) are dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN of 28 μ L in the 0.5mL plastic test tube ₃Among the pH7.2.With test tube vortex and add 20mU ST3Gal3 (100 μ L cumulative volume) simply.Test tube is carried out vortex once more, and mixed 24 hours in 32 ℃ gently, and this reaction is by in-80 ℃ of freezing stopping.Sample to this reaction is analyzed with aforesaid SDS-PAGE (Figure 167), IEF gel (Figure 168) and MALDI-TOF MS.

Also the rFSH to PEGization has carried out MALDI.In ionization process, SA-PEG is removed from the N-glycan structures of glycoprotein.Natural FSH has provided the peak 13928; AS-rFSH (13282); Sialylated once more r-FSH (13332); PEG1000-rFSH (13515; 14960 (1); 16455 (2); 17796 (3); 19321 (4)); And PEG10000 (23560 (1); 34790 (2); 45670 (3) and 56760 (4)).

25. the pharmacokinetics research of Glycopegylated FSH

Present embodiment has proposed to check in the body of the pharmacokinetics character that prepared according to the methods of the invention Glycopegylated follicle stimulating hormone (FSH) compares with the FSH of non-PEGization.

(Amersham Biosciences, Arlington Heights IL) carry out radioiodination and preparing in containing the phosphate buffered saline (PBS) of 0.1%BSA with standard conditions with FSH, FSH-SA-PEG (1kDa) and FSH-SA-PEG (10kDa).After in phosphate buffered saline buffer, being diluted to suitable concentration, FSH protein (the 0.4 μ g separately) intravenous injection of each check gone into (body weight is 250-300g) in the female Sprague Dawley rat and 0～80 hour time point blood drawing.Radioactivity in the blood sample is analyzed with gamma counter, and pharmacokinetics is analyzed (Figure 169) with standard method.FSH removes from blood quickly than FSH-PEG (1kDa), and FSH-PEG (1kDa) is removed than FSH-PEG (10kDa) a little soon.

26. the sustenticular cell biological assay of Glycopegylated FSH external activity

Present embodiment has proposed based on the biological assay of the sustenticular cell of cultivating to follicle stimulating hormone (FSH).This mensuration can be used for determining the biological activity of glycan reconstruct (comprise sugar put together) back FSH.

This biological assay is based on the dose-response relationship that exists between the estradiol amount, and this estradiol is to produce when FSH rather than lutropin (LH) add in the sustenticular cell of the cultivation that obtains from jejune old rats.The testosterone of external source can change 17 beta estradiols into when FSH exists.

The old Sprague-Dawley rat of 7～10 ages in days is used to obtain sustenticular cell.After execution, by at collagenase (1mg/ml), trypsin 1mg/ml), incubation made testis take off tunicle in 5～10 minutes and tissue is disperseed among Unidasa (1mg/ml) and the DNase (5 μ g/ml).The tubule fragment is placed drag and wash at PBS (1x).Make the tubule fragment once more with the medium incubation that contains same enzyme 20 minutes: collagenase (1mg/ml), trypsin 1mg/ml), Unidasa (1mg/ml) and DNase (5 μ g/ml).

With the homogenate of tubule fragment and place the substratum of serum-free on the 24 hole flat boards.5x10 is placed in every hole ⁵Individual cell.Be 37 ℃ and 5% CO ₂Middle incubation added fresh substratum to cell after 48 hours.The composition of serum free medium: DMEM (1 volume), Ham ' s F10 nutritional blend (1 volume), Regular Insulin 1 μ g/ml, transferrin 5 μ g/ml, EGF10ng/ml, T420pg/ml, hydrocortisone 10 ^-8M, vitamin A acid 10 ^-6M.

Stimulation test is: with standard FSH or sample in 37 ℃ and 5% CO ₂Middle incubation 24 hours.Average coefficient of variation in measuring is 9%, and the average coefficient of variation between measuring is 11%.

With 17B-estradiol Elisa test kit DE2000 (R﹠amp; D Systems, Minneapolis, MN) be used for FSH, FSH-SA-PEG (1kDa) and FSH-SA-PEG (10kDa) incubation after the quantitative level of estradiol.

This program is as follows: with 100 μ l estradiol standards (by test kit provide and according to the preparation of the specification sheets of test kit) or sample be drawn on the 17B-estradiol Elisa flat board; With 50 μ l 17B-estradiol conjugates (by test kit provide and according to the preparation of the specification sheets of test kit) add in each hole; With 50 μ l 17B-estradiol antibody-solutions (by test kit provide and according to the preparation of the specification sheets of test kit) add in each hole; Make dull and stereotyped in room temperature 200rpm incubation 2 hours; With liquid sucking-off from each hole; The hole is washed four times with washing soln; All liquid is removed from the hole; With 200 μ l pNPP substrates (by test kit provide and according to the preparation of the specification sheets of test kit) add to institute porose in and incubation 45 minutes; Add 50 μ l stop baths (by test kit provide and according to the preparation of the specification sheets of test kit) and read flat board (Figure 170) at 405nm.Although FSH-PEG (10kDa) shows the medium stimulation to sustenticular cell when 1 μ l/ml, FSH-PEG (1kDa) is higher by 50% than the FSH of PEGization not to the stimulation of sustenticular cell.

27. the Steelman-Pohley biological assay of Glycopegylated FSH activity in vivo

In the present embodiment, Steelman-Pohley biological assay (Steelman and Pohley, 1953, Endocrinology 53:604-615) is used for determine the activity in vivo of Glycopegylated FSH.The Steelman-Pohley biological assay utilizes the variation of rat ovary weight to measure the activity in vivo of FSH, this FSH and the common injection of human chorionic gonadotrophin.

The Steelman-Pohley biological assay is carried out according to the rules that people such as Christin-Maitre (2000, Methods 21:51-57) describe.(Charles River Laboratories, Wilmington MA) placed 5 in testing installation before beginning the mensuration program at least with the female Sprague-Dawley rat of 70 21-22 ages in days.In the whole procedure process, the weather of animal housing is controlled to be 18～26 ℃, 30～70% relative humidity and 12 hours artificial lighting/12 hour dark.All animals all use Certified Rodent Chow (Harlan Teklad, Madison WI) or equivalent and water to feed, and all can arbitrarily obtain.At CalvertPreclinical Services, (Olyphant PA) carries out the animal program to Inc..

Recombinant FSH is expressed in the Chinese hamster ovary celI, carries out purifying and carries out Glycopegylated with PEG (1kDa) by standard technique.Rat is divided into 7 experimental group, every group of 10 animals.On-1 and 0, the animal of all groups all used the 20I.U. human chorionic gonadotrophin (HCG) among 0.5ml 0.9% NaCl to carry out subcutaneous injection.On 1st, 2 and 3, control animal is used in the 0.5ml dosage that contains 20I.U.HCG among 0.9% NaCl and carries out subcutaneous injection, and in other groups, HCG dosage increases along with the rFSH of 0.14 μ g, 0.4 μ g or 1.2 μ g in each administration or rFSH-SA-PEG (1kDa).On 4th, pass through CO ₂Suck painless execution animal.Obtain ovary, repair and weigh.Determine the average ovary weight of each group.

The average ovary weight of experimental group when Figure 171 represents 4.The group of accepting the group (contrast) of HCG separately or accepting low dosage (0.14 μ g) rFSH or rFSH-SA-PEG (1kDa) has the ovary weight that approximately equates.The group of accepting medium (0.4 μ g) or high (1.2 μ g) dosage rFSH or rFSH-SA-PEG (1kDa) has the ovary weight that approximately doubles control group.When median dose (0.4 μ g), Glycopegylated rFSH has about identical activity in vivo (being determined by ovary weight) with the rFSH of non-PEGization.When high dosage (1.2 μ g), Glycopegylated rFSH has the activity in vivo slightly higher than the rFSH of non-PEGization.

G-CSF

28. The G-CSF's that produces in the Chinese hamster ovary celI is Glycopegylated

The preparation of asialylated-granulocyte colony-stimulating factor (G-CSF).The G-CSF that produces in the Chinese hamster ovary celI is dissolved in 50mM Tris-HCl pH7.4,0.15M NaCl, 5mM CaCl with 2.5mg/mL ₂In and in Centricon Plus 20 centrifugal filters, be concentrated into 500 μ L.Make this solution and 300mU/ml neuraminidase II (vibrio cholerae) in 32 ℃ of incubations 16 hours.In order to monitor reaction, the little sample aliquot of reactant is diluted with suitable damping fluid and operation IEF gel analysis.Then reaction mixture is added into N-(p-aminophenyl) oxaminic acid-agarose conjugate (800 μ L/mL reaction volume) of washing in advance, and the pearl of washing was rotated 24 hours gently in 4 ℃.In 10,000rpm is centrifugal and collect supernatant liquor with mixture.Pearl with Tris-EDTA damping fluid washing 3 times, once and with the 0.2mLTris-EDTA damping fluid is washed once with the washing of 0.4mL Tris-EDTA damping fluid, and collects all supernatant liquors.With supernatant liquor in 4 ℃ to 50mMTris-HCl pH7.4,1M NaCl, 0.05% NaN ₃Dialyse, and then to 50mM Tris-HClpH7.4,1M NaCl, 0.05% NaN ₃Dialyse twice.The solution of dialysis concentrates with CentriconPlus 20 centrifugal filters then and is stored in-20 ℃.The condition of IEF gel is to move according to program that is provided by Invitrogen and reagent.Natural and asialylated G-CSF sample is dialysed to water and analyze by MALDI-TOF MS.

The preparation of G-CSF-(α 2,3)-sialic acid-PEG.Asialylated G-CSF is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The ST3Gal1 that makes this solution and 1mM cmp sialic acid-PEG and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-PEG, added the CMP-SA-PEG-fluorescent ligand to the little sample aliquot of reactant; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).The fluorescent mark that is integrated in the peptide is to carry out quantitative with built-in fluorescent probe.After 2 days, reaction mixture is prepared the type post with the Toso Haas G3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make G-CSF sample natural and PEGization dialyse to water and analyze by MALDI-TOF MS.

The preparation of G-CSF-(α 2,8)-sialic acid-PEG.Contain α 2 with what produce in the Chinese hamster ovary celI, the G-CSF that the sialylated O-of 3-connects glycan is dissolved in 50mMTris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The CST-II that makes this solution and 1mM cmp sialic acid-PEG and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-PEG, added the CMP-SA-PEG-fluorescent ligand to the little sample aliquot of reactant; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).The fluorescent mark that is integrated in the peptide is to carry out quantitative with built-in fluorescent probe.After 2 days, reaction mixture is prepared the type post with the Toso Haas G3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make G-CSF sample natural and PEGization dialyse to water and analyze by MALDI-TOF MS.

The preparation of G-CSF-(α 2,6)-sialic acid-PEG.The G-CSF that only contains the GalNAc of O-connection is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.Make the ST6GalNAcI of this solution and 1mM cmp sialic acid-PEG and 0.1U/mL or II in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-PEG, added the CMP-SA-PEG-fluorescent ligand to the little sample aliquot of reactant; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).The fluorescent mark that is integrated in the peptide is to carry out quantitative with built-in fluorescent probe.After 2 days, reaction mixture is prepared the type post with the Toso HaasG3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make G-CSF sample natural and PEGization dialyse to water and analyze by MALDI-TOF MS.

The G-CSF that will produce in Chinese hamster ovary celI handles with genus arthrobacter (Arthrobacter) sialidase, and on Superdex 75, carry out purifying by size exclusion, handle with ST3Gal1 or ST3Gal2, handle with CMP-SA-PEG 20kDa then.The molecule of gained carries out purifying by ion-exchange and gel-filtration as a result, and is completely by the analytical proof PEGization of SDS PAGE.This is to prove Glycopegylated for the first time of the glycan that connects of O-.

Glucocerebrosidase

29. Glucocerebrosidase-the Man-6-P that produces in the Chinese hamster ovary celI

Present embodiment has proposed to make the peptide such as the glucocerebrosidase that produce in Man-6-P and the Chinese hamster ovary celI to carry out the program that sugar is puted together.

The preparation of asialylated glucocerebrosidase.The glucocerebrosidase that produces in the Chinese hamster ovary celI is dissolved among 50mM Tris-HCl pH7.4, the 0.15M NaCl with 2.5mg/mL, and makes itself and 300mU/mL sialidase-agarose conjugate in 32 ℃ of incubations 16 hours.In order to monitor reaction, with the little sample aliquot of reactant with suitable damping fluid dilution and according to program run IEF gel and the SDS-PAGE of Invitrogen.In 10,000rpm is centrifugal and collect supernatant liquor with mixture.Pearl with Tris-EDTA damping fluid washing 3 times, once and with 0.2mL Tris-EDTA damping fluid is washed once with the washing of 0.4mLTris-EDTA damping fluid.Collect all supernatant liquors.With supernatant liquor in 4 ℃ to 50mM Tris-HCl pH7.4,1M NaCl, 0.05% NaN ₃Dialyse, and then to 50mM Tris-HCl pH7.4,1M NaCl, 0.05%NaN ₃Dialyse twice.The solution of dialysis concentrates with Centricon Plus 20 centrifugal filters then.Reaction product is to analyze with SDS-PAGE and IEF according to program and reagent that Invitrogen provides.Sample is dialysed to water and analyze by MALDI-TOF MS.

The preparation of glucocerebrosidase-SA-linker-Man-6-P (program 1).Above-mentioned asialylated glucocerebrosidase is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The ST3Gal3 that makes this solution and 1mM cmp sialic acid-linker-Man-6-phosphoric acid and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-linker-Man-6-phosphoric acid, added the CMP-SA-PEG-fluorescent ligand to the little sample aliquot of reactant; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas TSK-gel-3000 analysis mode post of using PBS damping fluid (pH7.1).The fluorescent mark that is integrated in the peptide is to carry out quantitative with built-in fluorescent probe.After reaction finishes, reaction mixture is carried out purifying and absorbs the collection fraction based on UV with the Toso Haas TSK-gel-3000 preparation type post of using PBS damping fluid (pH7.1).Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

The preparation of glucocerebrosidase-SA-linker-Man-6-P (program 2).With produce among the CHO but not exclusively sialylated glucocerebrosidase be dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The ST3Gal3 that makes this solution and 1mM cmp sialic acid-linker-Man-6-phosphoric acid and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-linker-Man-6-phosphoric acid, added the CMP-SA-PEG-fluorescent ligand to the little sample aliquot of reactant; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas TSK-gel-3000 analysis mode post of using PBS damping fluid (pH7.1).The fluorescent mark that is integrated in the peptide is to carry out quantitative with built-in fluorescent probe.After reaction finishes, reaction mixture is carried out purifying and absorbs the collection fraction based on UV with the Toso HaasTSK-gel-3000 preparation type post of using PBS damping fluid (pH7.1).Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

30. glucocerebrosidase-transferrin

Present embodiment has proposed protein is carried out the program that sugar is puted together, and particularly transferrin and glucocerebrosidase is carried out sugar and puts together.On glucocerebrosidase, generated the GlcNAc-ASN structure, and the GlcNAc-ASN structure of transferrin-SA-linker-Gal-UDP and GDNF has been puted together with galactosyltransferase.

GlcNAc-glucocerebrosidase (Cerezyme ^TM ) preparation.With the Cerezyme that produces in the Chinese hamster ovary celI ^TM(glucocerebrosidase) is dissolved among 50mM Tris-HClpH7.4, the 0.15M NaCl with 2.5mg/mL, and makes itself and 300mU/mL Endo-H-agarose conjugate in 32 ℃ of incubations 16 hours.In order to monitor reaction, with the little sample aliquot of reactant with suitable damping fluid dilution and according to program run IEF gel and the SDS-PAGE of Invitrogen.In 10,000rpm is centrifugal and collect supernatant liquor with mixture.Pearl with Tris-EDTA damping fluid washing 3 times, once and with 0.2mL Tris-EDTA damping fluid is washed once with the washing of 0.4mLTris-EDTA damping fluid, and collects all supernatant liquors.With supernatant liquor in 4 ℃ to 50mM Tris-HCl pH7.4,1M NaCl, 0.05% NaN ₃Dialyse, and then to 50mM Tris-HCl pH7.4,1M NaCl, 0.05%NaN ₃Dialyse twice.The solution of dialysis concentrates with Centricon Plus 20 centrifugal filters then.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Sample is dialysed to water and analyze by MALDI-TOF MS.

The preparation of transferrin-SA-linker-Gal-glucocerebrosidase.Above-mentioned transferrin-SA-linker-Gal-UDP is dissolved in 50mM Tris-HCl, 0.15M NaCl, 5mM MnCl with 2.5mg/mL ₂, 0.05% NaN ₃, among the pH7.2.Make this solution and 2.5mg/mL GlcNAc-glucocerebrosidase and 0.1U/mL galactosyltransferase in 32 ℃ of incubations 2 days.In order to monitor the integration of glucocerebrosidase, peptide is separated by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1), and product is surveyed by the UV absorption.Then reaction mixture being prepared the type post with the Toso Haas G3000SW that uses PBS damping fluid (pH7.1) carries out purifying and absorbs the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

GM-CSF

31. The generation and the PEGization of GlcNAc-ASN structure: saccharomyces (Saccharomyces) The middle GM-CSF that produces

Present embodiment has proposed the preparation to the tissue-type activator of the GlcNAc-Asn structure with PEGization.

The leukine expection of expressing in the yeast contains the glycan that is connected with 2 O-that 2 N-connect.The glycan that N-connects should be a ramose seminose type.To this recombinant glycoprotein with the endoglycosidase individual curing that is selected from endoglycosidase H, endoglycosidase-F1, endoglycosidase-F2, endoglycosidase-F3, endoglycosidase-M or with mannosidase I, II and III combined treatment on the l-asparagine on peptide/protein main chain (Asn) residue, to generate the GlcNAc tubercle.

Then the GlcNAc-Asn structure on peptide/protein main chain can be respectively with UDP-semi-lactosi or UDP-semi-lactosi-6-PEG and galactosyltransferase such as GalT1 carries out semi-lactosi or semi-lactosi-PEG modifies.In one case, semi-lactosi-PEG is a terminal residue.Under second kind of situation, semi-lactosi can be further modified with SA-PEG with CMP-SA-PEG donor and sialyltransferase such as ST3GalIII.In another embodiment, GlcNAc-Asn structure on peptide/protein main chain can be carried out galactosylation and sialylated as mentioned above, further use CMP-SA-PEG and α 2 then, the 8-sialyltransferase carries out sialylated, as the enzyme by the cst-II genes encoding of campylobacter jejuni jejunum subspecies.

Herceptin ^TM

32. Mithramycin withHerceptin ^TM Sugar put together

Present embodiment has proposed to make the Fc zone glycan of the antibody molecule that produces in small molecules such as mithramycin and the mammalian cell to carry out the program that sugar is puted together.At this, used antibody Herceptin ^TM, but it should be appreciated by those skilled in the art that this method can be applicable to many other antibody.

Herceptin ^TM The preparation of-Gal-linker-mithramycin.With Herceptin ^TMBe dissolved in 50mM Tris-HCl, 0.15M NaCl, 5nM MnCl with 2.5mg/mL ₂, 0.05% NaN ₃, among the pH7.2.Make this solution and 1mM UDP-semi-lactosi-linker-mithramycin and 0.1U/mL galactosyltransferase in 32 ℃ of incubations 2 days in the glycan in Fc zone, to introduce mithramycin.In order to monitor the integration of semi-lactosi, added to the little sample aliquot of reactant ¹⁴C-semi-lactosi-UDP part; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).The radio-labeling that is integrated in the peptide is to carry out quantitative with built-in radioactivity seeker.

When reaction finishes, reaction mixture is carried out purifying and absorbs the collection fraction based on UV with the TosoHaas TSK-gel-3000 preparation type post of using PBS damping fluid (pH7.1).The fraction that will contain product merges, concentrated, exchange buffering liquid, freeze-drying then.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

Interferon alpha and interferon beta

33. Expressed protein is Glycopegylated in Mammals or the insect system: EPO, Interferon, rabbit α and interferon beta

Present embodiment has proposed being expressed in the preparation of the PEGization peptide in Mammals and the insect system.

Preparation from the acceptor of mammalian expression system.Carry out Glycopegylated peptide with cmp sialic acid PEG and need have the glycan that ends up with semi-lactosi.Great majority will have from the peptides of mammalian expression system at first needs the terminal sialic acid removed.

Sialidase digestion.Carry out asialylated with sialidase peptide.General program comprises: make the 1mg/mL peptide solution add 5mM CaCl ₂The Tris-buffered salt of pH7.2 in 0.2U/mL from the fixed sialidase (Calbiochem) of vibrio cholerae in 32 ℃ of incubations 24 hours.Microorganism growth can or comprise 0.02% sodiumazide by sterile filtration to be stopped.Remove this resin by centrifugal or filtration then, and wash the peptide of catching with recovery.At this moment, EDTA can be added in the solution to suppress any sialidase of seepage from resin.

Preparation from insect expression system.EPO, interferon alpha and interferon beta also can be expressed in the dried nonmammalian system, in yeast, plant or insect cell.Carry out Glycopegylated peptide with cmp sialic acid PEG and need have the glycan that ends up with semi-lactosi.It is three seminose cores for example that great majority are expressed in glycan on the peptide in the insect cell.These glycan at first were configured to the glycan with the semi-lactosi ending before becoming the acceptor of sialyltransferase.

Make up the acceptor glycan from three seminose cores.Make peptide (1mg/mL) contain 5mM MnCl ₂, 5mM UDP-GlcNAc, 0.05U/mL GLCNACT I, 0.05U/mL GLCNACTII the Tris-buffering salt of pH7.2 in finish in 32 ℃ of incubations 24 hours or up to reaction is basic.Microorganism growth can or comprise 0.02% sodiumazide by sterile filtration to be stopped.After exchange buffering liquid is with removal UDP and other small molecules, with UDP-semi-lactosi and MnCl ₂Be added into 5mM separately, galactosyltransferase is added into 0.05U/mL, and in 32 ℃ of incubations 24 hours or up to the basic end of reaction.Microorganism growth can or comprise 0.02% sodiumazide by sterile filtration to be stopped.This peptide can be used for carrying out Glycopegylated then.

Make up O-and connect glycan.Similarly strategy can be applicable to interferon alpha and produces the O-glycan Gal-GalNAc that wants with enzymatic.If desired, available suitable peptide GalNAc transferring enzyme (as GalNAc Tl, GalNAc T2, T3, T4 etc.) and UDP-GalNAc add the GalNAc that is connected on Serine or the Threonine on the peptide to.Similarly, if desired, available galactosyltransferase and UDP-semi-lactosi add semi-lactosi.

Carry out Glycopegylated with sialyltransferase.(for the N-glycan on EPO and the interferon beta is ST3Gal3 or ST3Gal4 to make the glycopeptide with terminal galactose (1mg/mL) and CMP-SA-PEG (0.75mM) in Tris buffered saline+0.02% sodiumazide and 0.4U/mL sialyltransferase; For the O-glycan on the interferon alpha is ST3Gal4 or ST3Gal1) in 32 ℃ of incubations 24 hours.Other available transferring enzymes comprise

α

2,6 sialyltransferases from Mermaid luminous bacillus (Photobacteriumdamsella).The acceptor peptide concentration is most preferably the solubility limit of 0.1mg/mL to peptide.The concentration of CMP-SA-PEG should be enough to surpass the available site, but should be too not high and cause peptide solubility problem (PEG causes), and can be 50 μ M～5mM, and temperature can be 2 ℃～40 ℃.The time of finishing reaction needed will depend on temperature, enzyme to the relative quantity of receptor substrate, the concentration and the pH of donor substrate.

34. The interferon alpha that produces in the Chinese hamster ovary celI Glycopegylated

The preparation of asialylated-interferon alpha.The interferon alpha that will produce from Chinese hamster ovary celI is dissolved in 50mM Tris-HCl pH7.4,0.15M NaCl, 5mM CaCl with 2.5mg/mL ₂In and in Centricon Plus 20 centrifugal filters, be concentrated into 500 μ L.Make this solution and 300mU/mL neuraminidase II (vibrio cholerae) in 32 ℃ of incubations 16 hours.In order to monitor reaction, the little sample aliquot of reactant is diluted with suitable damping fluid and operation IEF gel analysis.Then reaction mixture is added into N-(p-aminophenyl) oxaminic acid-agarose conjugate (800 μ L/mL reaction volume) of washing in advance, and the pearl of washing was rotated 24 hours gently in 4 ℃.In 10,000rpm is centrifugal and collect supernatant liquor with mixture.Pearl with Tris-EDTA damping fluid washing 3 times, once and with 0.2mL Tris-EDTA damping fluid is washed once with the washing of 0.4mL Tris-EDTA damping fluid, and collects all supernatant liquors.With supernatant liquor in 4 ℃ to 50mM Tris-HCl pH7.4,1M NaCl, 0.05% NaN ₃Dialyse, and then to 50mM Tris-HCl pH7.4,1M NaCl, 0.05% NaN ₃Dialyse twice.The solution of dialysis concentrates with Centricon Plus 20 centrifugal filters then and is stored in-20 ℃.The condition of IEF gel is to move according to program that is provided by Invitrogen and reagent.Natural and asialylated G-CSF sample is dialysed to water and analyze by MALDI-TOF MS.

The preparation of interferon alpha-(α 2,3)-sialic acid-PEG.Asialylated interferon alpha is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The ST3Gal1 that makes this solution and 1mM cmp sialic acid-PEG and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-PEG, added the CMP-SA-PEG-fluorescent ligand to the little sample aliquot of reactant; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).The fluorescent mark that is integrated in the peptide is to carry out quantitative with built-in fluorescent probe.After 2 days, reaction mixture is prepared the type post with the Toso Haas G3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make natural and asialylated interferon alpha sample dialyse to water and analyze by MALDI-TOF MS.

The preparation of interferon alpha-(α 2,8)-sialic acid-PEG.Contain α 2 with what produce in the Chinese hamster ovary celI, the interferon alpha that the sialylated O-of 3-connects glycan is dissolved in 50mMTris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The CST-II that makes this solution and 1mM cmp sialic acid-PEG and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-PEG, added the CMP-SA-PEG-fluorescent ligand to the little sample aliquot of reactant; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).The fluorescent mark that is integrated in the peptide is to carry out quantitative with built-in fluorescent probe.After 2 days, reaction mixture is prepared the type post with the Toso HaasG3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make interferon alpha sample natural and PEGization dialyse to water and analyze by MALDI-TOF MS.

The preparation of interferon alpha-(α 2,6)-sialic acid-PEG.The interferon alpha that only contains the GalNAc of O-connection is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.Make the ST6GalNAcI of this solution and 1mM cmp sialic acid-PEG and 0.1U/mL or II in 32 ℃ of incubations 2 days.In order to monitor the integration of sialic acid-PEG, added the CMP-SA-PEG-fluorescent ligand to the little sample aliquot of reactant; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).The fluorescent mark that is integrated in the peptide is to carry out quantitative with built-in fluorescent probe.After 2 days, reaction mixture is prepared the type post with the Toso Haas G3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make interferon alpha sample natural and PEGization dialyse to water and analyze by MALDI-TOFMS.

35. With Glycopegylated to interferon-beta-1a of PEG (10kDa) and PEG (20kDa)

Present embodiment has been illustrated the program of interferon-beta being carried out PEGization with PEG (10kDa) or PEG (20kDa).

In brief, interferon-beta-1a (INF-β) is available from Biogen (Avonex ^TM).INF-β at first carries out purifying by Superdex-75.INF-β carries out asialylated with the vibrio cholerae sialidase then.INF-β carry out PEGization with SA-PEG (10kDa) and SA-PEG (20kDa) then, and carries out purifying with Superdex-200.

The Superdex-75 chromatography purification. INF-β (150 μ g) is applied on the Superdex-75 post (Amersham Biosciences, Arlington Heights IL), and carry out wash-out with the PBS with 0.5M NaCl, 0.02 Tween-20,20mM Histidine and 10% glycerine.The monitoring elutriant is in the absorption (Figure 172 A and 172B) of 280nm, and the collection

fraction.Compile peak

4 and 5, Amicon Ultra 15 rotary filters (Millipore, Billerica MA) go up to concentrate, and with buffer-exchanged for having 0.5M CaCl ₂, 0.02% Tween-20,20mM Histidine and 10% glycerine TBS.

The sialidase reaction. have 5mM CaCl then ₂, 0.02% Tween-20,20mM Histidine and 10% glycerine TBS in agarose on the vibrio cholerae sialidase (70mU/ml,

EMD Biosciences, Inc., San Diego CA) carries out asialylated to INF-β.Being reflected at 32 ℃ carried out 18 hours.With 0.22 μ m Spin-X ^TM(Norcross GA) obtains INF-β from agarose to filter for CorningTechnology, Inc..Figure 173 A has described from the maldi analysis of the glycan of natural INF-β release.Natural INF-β has many sugar forms that contain terminal sialic acid part.Figure 173 B has described from the maldi analysis of the glycan that takes off sialic INF-β release.Take off sialic INF-β and mainly have a kind of two feeler sugar forms that contain terminal sialic acid part.

Sialylated lectin Dot blot is analyzed. the INF-β sample point trace of autospasy sialidase reaction in the future is on soluble cotton, use Tris buffered saline (TBS:0.05MTris then, 0.15M NaCl pH7.5) seals with DIG test kit (available from the glycan difference test kit of Roche#1 210 238) sealing damping fluid.With some traces with digoxigenin (DIG) (Roche Applied Science, Indianapolis, IL) the bosom sophora japonica lectin (MAA) of mark incubation together, to survey the α 2 of INF-β, 3-is sialylated.These traces are washed with TBS, wash then with anti-digitonin incubation, and then with TBS, and develop with the NBT/X-phosphate solution with alkali phosphatase enzyme mark, wherein NBT is the blue tetrazolium chloride of 4-nitro, and X-phosphoric acid salt is 5-bromo-4-chloro-3 indoles phosphoric acid salt.The result of the MAA trace of asialylated reaction back INF-β has been described in the left side of Figure 174.INF-β is that part is asialylated, and this is by comparing with natural INF-β in the asialylated sample, and weakening in trace develops is shown.

With other traces with (CA) the Erthrina cristagalli lectin (ECL) of mark incubation together is to survey the galactose residue that exposes on INF-β for Vector Laboratories, Burlingame with vitamin H.Behind 2.5 μ g/ml ECL incubations, in TBS, wash trace, and make it with streptavidin incubation with alkali phosphatase enzyme mark.And then the washing trace, and develop.The ECL trace after developing has been described on the right side of Figure 174.The intensity of comparing the marking increase of asialylated INF-β with natural INF-β shows the galactose moiety of more exposures, thereby shows asialylated fully.

With the PEGization of SA-PEG (10kDa) to asialylated INF-β. in suitable damping fluid, use ST3Gal3 (50mU/ml) and CMP-SA-PEG (10kDa) (250 μ M) to asialylated INF-β (0.05mg/ml) in 32 ℃ of PEGization 50 hours, described damping fluid is TBS+5mM CaCl ₂, 0.02% Tween 20,20mM Histidine, 10% glycerine.The SDS-PAGE that Figure 175 has described reaction product analyzes, and has shown the INF-β of the PEGization of about 98kDa.

With the PEGization of SA-PEG (20kDa) to asialylated INF-β. in suitable damping fluid, use ST3Gal3 (l70mU/ml) and CMP-SA-PEG (20kDa) to asialylated INF-β (0.5mg/ml) in 32 ℃ of PEGization 50 hours, described damping fluid is TBS+5mM CaCl ₂, 0.02% Tween 20,20mM Histidine, 10% glycerine.The SDS-PAGE that Figure 176 has described the PEGization reaction product analyzes.The INF-β of PEGization has many more high-molecular weight bands of not seeing in the INF-of unmodified β, this shows that it has carried out PEGization more fully.

Superdex-200 purifying with the INF-β of PEG (10kDa) PEGization.PEG change reaction product (Amersham Biosciences on the Superdex-200 post, ArlingtonHeights, IL) separate, this pillar is used the PBS with 0.5M NaCl, 0.02 Tween-20,20mM Histidine and 10% glycerine, and flow velocity is 1ml/ minute and 30cm/ hour.The monitoring elutriant is in the absorption (Figure 177) of 280nm, and the collection

fraction.Compile peak

3 and 4, and on AmiconUltra 15 rotary filters, concentrate.

Biological assay with the INF-β of PEG (10kDa) PEGization.

This test is a lung cancer cell line A549 inhibition of proliferation.A549 clone is at 37 ℃ of 5%CO ₂Grow in the lung cancer attached cell among the RPMI+10%FBS.They can obtain from ATCC#CCL-185.With 10ml PBS washed cell, and remove PBS.Add 5ml trypsinase, in room temperature incubation 5 minutes, perhaps in 37 ℃ of incubations 2 minutes.When cell during, be resuspended in the 25ml substratum and pair cell is counted from wall.With the concentration dilution cell of 10000 cells/ml, and with 200 μ l/ holes interpolations (96 hole flat board).At 37 ℃ of 5% CO ₂Incubation 4 hours.Prepared at concentrations 1mlINF-β with 0.1 μ g/ml.Under stink cupboard, filter with 0.2 μ m filter.100 μ l (8 repetition=1 roads) are added in every hole.Incubation 3 days (cell is joined).Remove 200 μ l substratum (the only surplus 100 μ l in every hole).Add 25 μ l MTT (Sigma) (5mg/ml has carried out 0.22 μ m and filtered).At 37 ℃ and 5% CO ₂Incubation 4 hours.Sucking-off substratum and add the mixture of 100 μ l Virahols (100ml) and 6N HCl gently.Suction is so that Viola crystallina homogenate up and down.Read 570

The OD of nm (remove 630 or the background of 690nm).

Figure 178 has described the biological assay from the peak of the INF-β that contains useful PEG (10kDa) PEGization of Superdex-200 post wash-out.

Superdex-200 purifying with the INF-β of PEG (20kDa) PEGization.PEG (20kDa) PEGization reaction product (Amersham Biosciences on the Superdex-200 post, Arlington Heights, IL) separate, this pillar is used the PBS with 0.5NaCl, 0.02Tween-20,20mM Histidine and 10% glycerine, and flow velocity is 1ml/ minute.The monitoring elutriant is in the absorption (Figure 179) of 280nm, and the collection fraction.Peak 3 contains most of INF-β with PEG (20kDa) PEGization.

Intracellular toxin test with the INF-β of PEG (20kDa) PEGization.

Carried out limulus test, Bio Whittaker#50-647U

The table 24. intracellular toxin test-results of the INF-β of PEG (20kDa) PEGization.

Remicade ^TM

36.Remicade ^TM Antibody Glycopegylated

Present embodiment has proposed by introducing the PEG molecule and recombinant antibody molecule is carried out Glycopegylated program to the glycan in Fc zone.At this, Remicade ^TM, promptly TNF-R:IgG Fc region fusion protein matter is exemplary peptide.

Remicade ^TM The preparation of-Gal-PEG (10kDa).With Remicade ^TMBe dissolved in 50mM Tris-HCl, 0.15M NaCl, 5mM MnCl with 2.5mg/mL ₂, 0.05% NaN ₃, among the pH7.2.Make this solution and 1mM UDP-semi-lactosi-PEG (10kDa) and 0.1U/mL galactosyltransferase in 32 ℃ of incubations 2 days in the glycan in Fc zone, to introduce PEG.In order to monitor the integration of semi-lactosi, added to the little sample aliquot of reactant ¹⁴C-semi-lactosi-UDP part; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).The radio-labeling that is integrated in the peptide is to carry out quantitative with built-in radioactivity seeker.

When reaction finishes, reaction mixture is carried out purifying and absorbs the collection fraction based on UV with the TosoHaas TSK-gel-3000 preparation type post of using PBS damping fluid (pH7.l).The fraction that will contain product merges, concentrated, exchange buffering liquid, freeze-drying then.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

Rituxan ^TM

37. Geldanamycin and Rituxan ^TM Sugar put together

Present embodiment has proposed to make antibody such as the Rituxan that produces in small molecules such as geldanamycin and the Chinese hamster ovary celI ^TMFc zone glycan carry out the program that sugar is puted together.At this, used antibody Rituxan ^TM, but it should be appreciated by those skilled in the art that this method can be applicable to many other antibody.

Rituxan ^TM The preparation of-Gal-linker-geldanamycin.With Rituxan ^TMBe dissolved in 50mM Tris-HCl, 0.15M NaCl, 5mM MnCl with 2.5mg/mL ₂, 0.05% NaN ₃, among the pH7.2.Make this solution and 1mM UDP-semi-lactosi-linker-geldanamycin and 0.1U/mL galactosyltransferase in 32 ℃ of incubations 2 days in the glycan in Fc zone, to introduce geldanamycin.In order to monitor the integration of semi-lactosi, added to the little sample aliquot of reactant ¹⁴C-semi-lactosi-UDP part; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso HaasG3000SW analysis mode post of using PBS damping fluid (pH7.1).The radio-labeling that is integrated in the peptide is to carry out quantitative with built-in radioactivity seeker.

Rnase

38. High mannose N-glycan is reconstructed into N-glycan heterozygosis and complexity: ox pancreas RNase

Present embodiment has proposed the preparation to the ox pancreas RNase with N-glycan heterozygosis or complicated.The high mannose N-of RNase connected glycan carries out enzymatic digestion and the N-that handles with the generation heterozygosis is connected glycan.In addition, can carry out enzymatic digestion to the high mannose N-connection glycan of RNase and be connected glycan with handling to generate complicated N-.

The high mannose structures of N-connection oligosaccharides can be modified to heterozygosis or complicated form with the combination of alpha-Mannosidase and glycosyltransferase in the glycopeptide.Present embodiment has been summed up with the result of simple N-glycan as this effort of model substrates.

The glycopeptide that the ribonuclease B of purifying (RNaseB) (Sigma) is made up of 124 amino-acid residues from Pancreas Bovis seu Bubali.It has the single potential N-glycosylation site of being modified by high mannose structures.Because its simplicity and lower molecular weight (13.7kDa～15.5kDa), so the good candidate of the possibility of ribonuclease B is a proof connects oligosaccharides to heterozygosis or complicated N-from high mannose structures N-glycan reconstruct.The MALDI-TOF of RNaseB spectrum (Figure 180 A) with show by the HPLC figure (Figure 180 B) of N-glycanase from the oligosaccharides of RNaseB cutting: except that the small portion of the peptide of unmodified, most of N-glycosylation sites of peptide all use the high mannose oligosaccharides of being made up of 5～9 mannose residues to modify.

High mannose N-glycan is to the transformation of the N-of heterozygosis glycan.High mannose N-glycan is with α 1,2-mannosidase, GlcNAcT-I (β-1, the 2-N-acetylgucosamine transferase), (β 1 for GalT-I, the 4-galactosyltransferase) and α 2, the 3-sialyltransferase/or α 2, the combination of 6-sialyltransferase changes the N-glycan of heterozygosis into, shown in Figure 181.

As an example, the high mannose structures among the RNaseB is successfully changed into the structure of heterozygosis.

Man ₅GlcNAc ₂-R is the single α 1 that uses from the Trichodermareesei clone, 2-mannoside enzyme catalysis Man _5-9GlcNAc ₂(Figure 182) that-R obtains.Make the α 1 of RNaseB (1g, about 67 μ mol) and 15mU reorganization Trichodermareesei, the 2-mannosidase the MES damping fluid (50mM, pH6.5) in the 10mL cumulative volume in 30 ℃ of incubations 45 hours.Man _6-9GlcNAc ₂-protein structure successfully changes Man into efficiently with the reorganization mannosidase ₅GlcNAc ₂-protein.

Selectively, Man5GlcNAc ₂-R is the single α 1 that uses from the Aspergillussaitoi purifying, 2-mannoside enzyme catalysis Man _5-9GlcNAc ₂(Figure 183) that-R obtains.Make the α 1 of the commercial A.saitoi of RNaseB (40 μ g, about 2.7nmol) and 25 μ U, 2-mannosidase (Glyko or CalBioChem) the NaOAC damping fluid (100mM, pH5.0) in 20 μ L cumulative volumes in 37 ℃ of incubations 42.5 hours.Man _6-9GlcNAc ₂-protein structure successfully changes Man into the commercial mannosidase of buying ₅GlcNAc ₂-protein.Yet, proteinic new peak in spectrum, occurred, thereby shown the pollution of endoglycosidase H possible in the preparation corresponding to GlcNAc-.Although need several Mammals alpha-Mannosidases to realize this step, fungi α 1,2-mannosidase are for all α 1 of removal, and the mannose residue of 2-connection is very effective.

GlcNAcT-I adds the GlcNAc residue to Man then ₅GlcNAc ₂Among-the R (Figure 184).α 1 at Trichodermareesei, make after the enzyme reaction of 2-mannoside and contain RNaseB (600 μ g, the reorganization GlcNAcT-I (34mU) of reaction mixture about 40nmol) and non-purifying is at MES damping fluid (50mM, pH6.5) in the cumulative volume of 400 μ L in 37 ℃ of incubations 42 hours, this MES damping fluid contains MnCl ₂(20mM) and UDP-GlcNAc (5mM).The GlcNAc residue is to add Man quantitatively to by reorganization GlcNAcT-I ₅GlcNAc ₂In-the protein.

Add Gal residue (Figure 185) with GalT1 then.After the GnT-I reaction, make and contain RNaseB (120 μ g, about 8nmol) reaction mixture and 3.3mU reorganization GalT-1 are at Tris-HCl damping fluid (100mM, pH7.3) in the cumulative volume of 100 μ L in 37 ℃ of incubations 20 hours, this Tris-HCl damping fluid contains UDP-Gal (7.5mM) and MnCl ₂(20mM).The Gal residue is to add about 98% GlcNAc-Man to by reorganization GalT1 ₅GlcNAc ₂In-the protein.

Next procedure is with α 2,3-sialyltransferase or α 2, and the 6-sialyltransferase adds sialic acid (Figure 186).As an example, use ST3GalIII, promptly a kind of α 2,3-sialyltransferase.After the GalT-1 reaction, make and contain RNaseB (13 μ g, about 0.87nmol) reaction mixture and 8.9mU reorganization ST3GalIII are at Tris-HCl damping fluid (100mM, pH7.3) in the cumulative volume of 20 μ L in 37 ℃ of incubations 16 hours, this Tris-HCl damping fluid contains cmp sialic acid (5mM) and MnCl ₂(20mM).Sialic acid residues is to add about 90% Gal-GlcNAc-Man to as donor with CMP-SA by reorganization ST3GalIII ₅GlcNAc ₂In-the protein.This output can further be improved by the conditioned reaction condition.

For simplicity, after above-mentioned each reaction, do not need purifying or dialysis.What is interesting is that more GnT-I and ST3GalIII can be combined in one (one pot) reaction.Can obtain similar output with isolating reacting phase ratio.After the GlcNAcT-I reaction, make and contain RNaseB (60 μ g, about 4nmol) reaction mixture and 1.7mU reorganization GalT 1,9.8mU reorganization ST3Gal III are at Tris-HCl damping fluid (100mM, pH7.3) in the cumulative volume of 60 μ L in 37 ℃ of incubations 20 hours, this Tris-HCl damping fluid contains UDP-Gal (7.5mM), cmp sialic acid (5mM) and MnCl ₂(20mM).

Shown in Figure 187, SA-PEG (10kDa) is successfully added among the RNaseB.After GalT-1 reaction, make contain RNaseB (6.7 μ g, about 0.45nmol) reaction mixture in room temperature to H ₂O dialysis 1 hour, and with 55mU reorganization ST3GalIII the Tris-HCl damping fluid (50mM, pH7.3) in the cumulative volume of 20 μ L in 37 ℃ of incubations 15.5 hours, this Tris-HCl damping fluid contains CMP-SA-PEG (10kDa) (0.25mM) and MnCl ₂(20mM).The sialic acid residues that PEG-modifies is successfully to add Gal-GlcNAc-Man to by reorganization ST3GalIII ₅GlcNAc ₂In-the peptide.This output can further be improved by the conditioned reaction condition.

High mannose N-glycan is to the transformation of complicated N-glycan.In order to realize this transformation, obtained GlcNAc β 1,2Man ₃GlcNAc ₂-peptide intermediate.Shown in Figure 188, have at least 4 feasible routes to realize from Man ₅GlcNAc ₂-peptide is to the reaction of this intermediate:

Route I: by the Man of

fungi α

1,2 mannosidase generation ₅GlcNAc ₂-peptide is the substrate that adds the GlcNAc transferase I (GlcNAcT-I, enzyme 2) of a GlcNAc.GlcNAcMan ₅GlcNAc ₂The terminal α 1 of-peptide, 3-and α 1, the mannose residue that 6-connects can be removed by golgi body α mannosidase II (ManII, enzyme 5).This route is the part that the processing N-that carries out in the higher organism connects the natural approach of oligosaccharides.

Route II: at first use α mannosidase (enzyme 6) to remove 2 mannose residues, use GlcNAcT-I (enzyme 2) to add GlcNAc then.Remove its natural receptor Man5GlcNAc ₂Outside-the R, GlcNAcT-I also can discern Man ₃GlcNAc ₂-R adds on the seminose core texture to form GlcNAcMan as its substrate and with a GlcNAc ₃GlcNAc ₂-peptide.

Route III: α 1, the seminose that 6-connects is by α 1, and the 6-mannosidase is removed, and adds GlcNAc by GlcNAcT-I subsequently and reaches by α 1, and the 3-mannosidase is removed terminal α 1, the seminose that 3-connects.From the experimental data that obtains, GlcNAcT-I can discern this Man ₄GlcNAc ₂-peptide is as acceptor and add a GlcNAc residue to form GlcNAcMan ₄GlcNAc ₂-peptide.

Route IV: III is similar to route, and α 1, and the seminose that 3-connects is by α 1, and the 3-mannosidase is removed, and is the GlcNAcT-I reaction subsequently.Terminal then α 1, the seminose that 6-connects can be by α 1, and the 6-mannosidase is removed.

(be responsible on the seminose core, adding and α 1 at GlcNAcT-I, the GlcNAc that 3-

seminose β

1,2 connects) and GlcNAcT-II (be responsible on the seminose core, adding second and α 1,6-

seminose β

1,2 GlcNAc that connect) after the effect, GlcNAc ₂Man ₃GlcNAc ₂-peptide can be processed to form the complicated N-glycan of two feelers by GalT1 and sialyltransferase.Other GlcNAc transferring enzymes such as GlcNAcT-IV, GlcNAcT-V and/or GlcNAcT-VI (Figure 188 and Figure 189) also can be to GlcNAc ₂Man ₃GlcNAc ₂-peptide carries out glycosylation.The extra glycosylation of being undertaken by GalT1 and sialyltransferase will form the complicated N-glycan of many feelers.The insertion of the GlcNAc of enzyme GlcNAcT-III catalysis five equilibrium, thus the effect of ManII and the effect of transferring enzyme GlcNAcT-II, GlcNAcT-IV and GlcNAcT-V subsequently stoped.

Tissue plasminogen activator (TPA)

39. TPA is carried out fucosylation to generate sialylated Lewis X

Present embodiment has proposed to have the preparation of the antigenic tissue plasminogen activator of sialylated Lewis X (TPA) that N-connects.

Sialylated. the TPA that expresses in mammalian cell usually contains the glycan of great majority with sialic acid ending, but in order to ensure sialylated completely, at first carry out external sialylated be favourable.Make suitable damping fluid (most preferably in pH5.5～9, Tris buffered salt for example, pH7.2) TPA in and CMP sialic acid and sialyltransferase incubation time enough are to change the sialic glycan of any shortage into sialylated kind.General condition is 1mg/mL TPA, 3mM CMP sialic acid, 0.02U/mL ST3Gal3, and 32 ℃ were carried out 24 hours.Microorganism growth can or comprise 0.02% sodiumazide by sterile filtration and suppress.TPA concentration is most preferably the solubility limit of 0.1mg/mL to peptide.The concentration of CMP-SA should be enough to surpass the available site, and can be at 50 μ M～50mM, and temperature is 2 ℃～40 ℃.Finish reaction and the needed time will depend on temperature, enzyme the relative quantity of receptor substrate, the concentration and the pH of donor substrate.Can add sialic other sialyltransferases with 2,3 keys and comprise ST3Gal4; But also using microbe transferring enzyme.

Fucosylation.The general condition of fucosylation is 1mg/mL TPA, 3mM GDP-Fucose, 0.02U/mL FTVI, 5mM MnCl ₂, in Tris buffered salt, carried out 24 hours in 32 ℃.Microorganism growth can or comprise 0.02% sodiumazide by sterile filtration and suppress.TPA concentration is most preferably the solubility limit of 0.1mg/mL to peptide.The concentration of GDP-Fucose should be enough to surpass the available site, and can be at 50 μ M～50mM, and temperature is 2 ℃～40 ℃.Finish reaction and the needed time will depend on temperature, enzyme the relative quantity of receptor substrate, the concentration and the pH of donor substrate.Other fucosyltransferases that can prepare sialylated Lewis X comprise FTVII, FTV, FTIII, but also using microbe transferring enzyme.

40. Pruning high mannose is three seminose core textures: the tissue that produces among the CHOType Plasmin Former activator.

Present embodiment has proposed to prepare the tissue plasminogen activator with three seminose cores by pruning high mannose glycans.

The oligosaccharides of the high mannose N-connection of low amount is produced and is contained in the preparation of tissue plasminogen activator (TPA) at present in Chinese hamster ovary (CHO) cell.This seminose can be pruned with various specific mannosidases.First step is to generate Man5GlcNAc2 (Fuc0-1) from Man9GlcNAc2 (Fuc0-1).This available mannosidase I carries out.Then GlcNAcT1 (GlcNAc transferase I) is used to prepare GlcNAc1Man5GlcNAc2 (Fuc0-1) or mannosidase III is used to prepare Man3GlcNAc2 (Fuc0-1).The available GlcNAcT1 of GlcNAc1Man3GlcNAc2 (Fuc0-1) is from Man3GlcNAc2 (Fuc0-1) preparation, and perhaps the available mannosidase II of GlcNAc1Man3GlcNAc2 (Fuc0-1) prepares from GlcNAc1Man5GlcNAc2 (Fuc0-1).Use GlcNAc transferase I I (GlcNAcTII) to change GlcNAc1Man3GlcNAc2 (Fuc0-1) into GlcNAc2Man3GlcNAc2 (Fuc0-1) then.Two available then GalTI of terminal GlcNAc residue carry out galactosylation, and carry out sialylated with ST3Gal III with SA-PEG then.

On the contrary, TPA can produce in yeast or fungal systems.To need similar process for fungi deutero-material.

41. The generation and the PEGization of GlcNAc-ASN structure: the TPA that produces in the yeast

Present embodiment has proposed the TPA that expresses in peptide such as the yeast is gone up the preparation of the GlcNAc-Asn structure of PEGization.

Yeast expression can access the TPA of the seminose type structure that contains single N-connection.This recombinant glycoprotein is at first handled to generate the GlcNAc structure on the l-asparagine on the peptide (Asn) residue with endoglycosidase H.

GlcNAc-Asn structure on peptide/protein main chain is respectively with UDP-semi-lactosi or UDP-semi-lactosi-6-PEG and galactosyltransferase such as GalT1 carries out semi-lactosi or semi-lactosi-PEG modifies then.In one case, semi-lactosi-PEG is a terminal residue.Under second kind of situation, semi-lactosi can be further modified with SA-PEG with CMP-SA-PEG donor and sialyltransferase such as ST3Gal III.In another embodiment, GlcNAc-Asn structure on peptide/protein main chain can be carried out galactosylation and sialylated as mentioned above, further use CMP-SA-PEG and α 2 then, the 8-sialyltransferase carries out sialylated, as the enzyme by the cst-II genes encoding of campylobacter jejuni jejunum subspecies.

Transferrin

42. Transferrin Glycopegylated

Present embodiment proposed to the preparation of asialylated transferrin and with the PEG-CMP-sialic acid carry out sialylated.

The preparation of asialylated transferrin.The complete transferrin (10mg) in people source is dissolved in 50mM NaOAc, the 5mM CaCl of 500 μ L ₂, among the pH5.5.In this solution, add 500mU neuraminidase II (vibrio cholerae), and reaction mixture was shaken 20.5 hours gently in 37 ℃.Reaction mixture is joined in N-(p-aminophenyl) oxaminic acid-agarose conjugate (600 μ L) of washing in advance, and the pearl of washing was rotated 24 hours gently in 4 ℃.In 10,000rpm is centrifugal and collect supernatant liquor with mixture.By adding the 30mM EDTA of 100 μ l to the pearl of washing, thereby reaction mixture is adjusted to 5mM EDTA, this pearl rotated 20 hours gently at 4 ℃ then.With suspension 10, centrifugal 2 minutes of 000rpm, and collect supernatant liquor.With 50mM NaOAc, the 5mM CaCl of pearl with 0.35mL ₂, 5mM EDTA, pH5.5 washing is also collected all supernatant liquors 5 times.Enzyme solution is dialysed in 15mM Tris-HCl, 1MNaCl, among the pH7.4 for twice in 4 ℃.Obtain 0.3mL transferrin solution (3.3mL altogether) and to twice of water dialysis.Residuum is dialysed twice to phosphate buffered saline (PBS) in 4 ℃ once more.The solution of dialysis is stored in-20 ℃.Protein example is by the IEF electrophoretic analysis.Sample (9 μ L, 25 μ g) is mixed with the dilution of 16 μ L Tris damping fluids and with 25 μ L sample pipetting volume damping fluids, and be applied to Isoelectric Focusing Gels (pH3-7).Gel is with standard program operation and fixed.Gel dyes with Colloidal Blue Stain.

Sialic acid-the PEGization of asialylated transferrin.Asialylated transferrin (250 μ g) and cmp sialic acid or CMP-SA-PEG (1kDa or 10kDa) (0.05 μ mol) are dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN of 69 μ L in the 1.5mL plastic test tube ₃, among the pH7.2.With test tube vortex and add 100mU ST3Gal3 (90 μ L) (250 μ L cumulative volume) simply.Test tube is carried out vortex once more, and mixed 24 hours in 32 ℃ gently.This reaction is by in-80 ℃ of freezing stopping.Novex Tris-glycine 8-16%1mm gel is used for SDS-PAGE analyzes (Figure 190).With sample (25 μ L, 25 μ g) and 25 μ L sample pipetting volume damping fluids and mixed 85 ℃ of heating 6 minutes that are incorporated in of 0.4 μ L beta-mercaptoethanol.Gel is also dyeing with Colloidal Blue Stain with the standard conditions operation.The IEF gel also carries out (Figure 191) as mentioned above.Sample is also dialysed to water and is analyzed by MALDI-TOF.

The result.Also carried out MALDI.Natural transferrin (78729); Asialylated transferrin (78197); Sialylated once more transferrin (79626/80703); Has SA-PEG1k (79037 (1); 80961 (2); 82535 (3); 84778 (4)); Has SA-PEG5k (90003 (2); 96117 (3); 96117 (4)); Has SA-PEG10k (100336 (2); 111421 (3); 122510 (4)).

43. Transferrin-GDNF

Present embodiment has proposed protein is carried out the program that sugar is puted together, and particularly transferrin and GDNF is carried out sugar and puts together.Transferrin-SA-linker-Gal-UDP prepares from transferrin.Galactose residue is removed from the GDNF glycan, and transferrin-SA-linker-Gal-UDP and GDNF glycan are puted together with galactosyltransferase.

Take off the preparation of glycosyl galactose (agalacto)-GDNF.The GDNF that produces in the NSO cell (NSO rat bone marrow tumour cell) is dissolved among 50mM Tris-HCl pH7.4, the 0.15M NaCl with 2.5mg/mL, and makes itself and 300mU/mL beta-galactosidase enzymes-agarose conjugate in 32 ℃ of incubations 16 hours.In order to monitor reaction, with the little sample aliquot of reactant with suitable damping fluid dilution and according to the program run IEF gel analysis of Invitrogen.In 10,000rpm is centrifugal and collect supernatant liquor with mixture.With supernatant liquor in 4 ℃ to 50mM Tris-HCl pH7.4,1M NaCl, 0.05% NaN ₃Dialyse, and then to 50mM Tris-HCl pH7.4,1M NaCl, 0.05%NaN ₃Dialyse twice.The solution of dialysis concentrates with Centricon Plus 20 centrifugal filters then and is stored in-20 ℃.The condition of IEF gel is to move according to program and reagent that Invitrogen provides.Sample is dialysed to water and analyze by MALDI-TOF MS.

The preparation of transferrin-SA-linker-Gal-UDP.Asialylated transferrin is dissolved in 50mM Tris-HCl, 0.15M NaCl, 0.05% NaN with 2.5mg/mL ₃, among the pH7.2.The ST3Gal3 that makes this solution and cmp sialic acid-linker-Gal-UDP (mole add up to the nucleotide sugar of 1 mole of equivalence is added on the transferrin) and 0.1U/mL was in 32 ℃ of incubations 2 days.In order to monitor sialic integration, added to the little sample aliquot of reactant ¹⁴The C-SA-UDP part; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso HaasG3000SW analysis mode post of using PBS damping fluid (pH7.1).The radio-labeling that is integrated in the peptide is to carry out quantitative with built-in radioactivity seeker.

The ST3Gal3 (so that any unreacted transferrin glycan is added cap) that makes this solution and 5mM cmp sialic acid and 0.1U/mL was in 32 ℃ of incubations 2 days.Integration in peptide is to carry out quantitative with built-in UV detector.After 2 days, reaction mixture is prepared the type post with the Toso Haas G3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

The preparation of transferrin-SA-linker-Gal-GDNF.Transferrin-SA-linker-the Gal-UDP of above-mentioned preparation is dissolved in 50mM Tris-HCl, 0.15M NaCl, 5mM MnCl with 2.5mg/mL ₂, 0.05% NaN ₃, among the pH7.2.Make this solution and 2.5mg/mL take off glycosyl galactose-GDNF and 0.1U/mL galactosyltransferase in 32 ℃ of incubations 2 days.In order to monitor the integration of semi-lactosi, added to the little sample aliquot of reactant ¹4C-semi-lactosi-UDP part; The mark that is integrated in the peptide is isolating with free label by gel-filtration on the Toso Haas G3000SW analysis mode post of using PBS damping fluid (pH7.1).The radio-labeling that is integrated in the peptide is to carry out quantitative with built-in radioactivity seeker.

When reaction finishes, make this solution and 5mM UDP-Gal and 0.1U/mL galactosyltransferase (so that any unreacted transferrin glycan is added cap) in 32 ℃ of incubations 2 days, add the ST3Gal3 of 5mM CMP-SA and 0.1U/mL subsequently.Behind extra 2 days, reaction mixture is prepared the type post with the Toso Haas G3000SW that uses PBS damping fluid (pH7.1) carry out purifying and absorb the collection fraction based on UV.Reaction product is to analyze with SDS-PAGE and IEF according to the program and the reagent that are provided by Invitrogen.Make sample dialyse to water and analyze by MALDI-TOF MS.

Herein each that quote and all patents, patent application and publication disclosure all herein integral body be incorporated herein by reference.

Although the present invention discloses with some specific embodiment, it is evident that and to design other embodiments of the present invention and modification by those skilled in the art and do not deviate from true spirit of the present invention and scope.Additional claim should be interpreted as comprising all this embodiments and modification of equal value.

Sequence table

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DeFrees，Shawn

Zopf，David

Bayer，Robert

Hakes，David

Chen，Xi

Bowe，Caryne

＜120〉Glycopegylated method and the proteins/peptides produced by this method

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<212>PRT

＜213〉homo sapiens

<400>48

<210>49

<211>963

<212>DNA

＜213〉homo sapiens

<400>49

<210>50

<211>320

<212>PRT

＜213〉homo sapiens

<400>50

<210>51

<211>107

<212>PRT

＜213〉homo sapiens

<400>51

<210>52

<211>107

<212>PRT

<213>Mus?musculus

<400>52

<210>53

<211>119

<212>PRT

＜213〉homo sapiens

<400>53

<210>54

<211>119

<212>PRT

<213>Mus?musculus

<400>54

<210>55

<211>214

<212>PRT

＜213〉homo sapiens

<400>55

<210>56

<211>448

<212>PRT

＜213〉homo sapiens

<400>56

<210>57

<211>8540

<212>DNA

＜213〉homo sapiens

<400>57

<210>58

<211>9209

<212>DNA

<213>Mus?musculus

<400>58

<210>59

<211>384

<212>DNA

<213>Mus?musculus

<400>59

<210>60

<211>128

<212>PRT

<213>Mus?musculus

<400>60

<210>61

<211>420

<212>DNA

<213>Mus?musculus

<400>61

<210>62

<211>140

<212>PRT

<213>Mus?musculus

<400>62

<210>63

<211>1395

<212>DNA

＜213〉homo sapiens

<400>63

<210>64

<211>464

<212>PRT

＜213〉homo sapiens

<400>64

<210>65

<211>1962

<212>DNA

＜213〉homo sapiens

<400>65

<210>66

<211>653

<212>PRT

＜213〉homo sapiens

<400>66

<210>67

<211>1290

<212>DNA

＜213〉homo sapiens

<400>67

<210>68

<211>429

<212>PRT

＜213〉homo sapiens

<400>68

<210>69

<211>351

<212>DNA

＜213〉homo sapiens

<400>69

<210>70

<211>116

<212>PRT

＜213〉homo sapiens

<400>70

<210>71

<211>498

<212>DNA

＜213〉homo sapiens

<400>71

<210>72

<211>165

<212>PRT

＜213〉homo sapiens

<400>72

<210>73

<211>165

<212>PRT

＜213〉homo sapiens

<400>73

<210>74

<211>588

<212>DNA

＜213〉homo sapiens

<400>74

<210>75

<211>195

<212>PRT

＜213〉homo sapiens

<400>75

Claims

1. peptide conjugate that comprises polyoxyethylene glycol, it externally puts together described polyoxyethylene glycol and precursor peptide makes by acellular, wherein use sialytransferase to make described polyoxyethylene glycol be attached to described precursor peptide by complete sialic acid linking group, described precursor peptide has following formula:

Wherein

AA is the end or the internal amino acid residue of described peptide;

X ¹-X ²Be the sugar covalently bound, wherein with described AA

X ¹It is first glycosyl residue; And

X ²Be and X ¹Covalently bound second glycosyl residue, wherein X ¹And X ²Be selected from monose and oligosaccharides residue, wherein said peptide conjugate is by acellular in vitro method preparation, and this method comprises:

(a) from this peptide, remove X ²Or its sugared subunit, thereby the glycan of formation brachymemma; With

(b) sialic acid donor that makes the glycan of described brachymemma, at least a sialytransferase and at least a modification contacts under the condition of glycan that sialic acid with the modification of the sialic acid donor of described at least a modification partly is transferred to described brachymemma being suitable for, the sialic acid of wherein said modification partly comprises at least one and is the modification group of polyoxyethylene glycol, thereby forms the described peptide conjugate that comprises polyoxyethylene glycol.

2. the peptide conjugate of claim 1, wherein said polyoxyethylene glycol has monodispersed substantially molecular weight distribution.

3. the peptide conjugate of claim 1, wherein said polyoxyethylene glycol is selected from linear polyethylene glycol and branch polyoxyethylene glycol.

4. the peptide conjugate of claim 1, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

5. the peptide conjugate of claim 1, wherein said sialytransferase is selected from ST3Gal3, CST-I and its combination.

6. the peptide conjugate of claim 1, described method also comprises:

(c) described peptide conjugate, sialic acid donor and sialyltransferase are contacted under the condition that described sialyltransferase is transferred to sialic acid residues described peptide conjugate being suitable for, thereby sialic acid partly is transferred to described peptide conjugate.

7. the peptide conjugate of claim 1, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

8. pharmaceutical composition comprises the peptide conjugate of claim 1.

9. peptide conjugate that comprises polyoxyethylene glycol, it externally puts together described polyoxyethylene glycol and precursor peptide makes by acellular, wherein use sialytransferase to make described polyoxyethylene glycol be attached to described precursor peptide by complete glycosyl linking group, described precursor peptide has following formula:

Wherein

X ³, X ⁴, X ⁵, X ⁶, X ⁷And X ¹⁷Be independently selected from monose and oligosaccharides residue; With

A, b, c, d, e and x are independently selected from integer 0,1 and 2, and prerequisite is that at least one is 1 or 2 among a, b, c, d, e and the x;

Wherein said peptide conjugate is by acellular in vitro method preparation, and this method comprises:

(a) from this peptide, remove X ³, X ⁴, X ⁵, X ⁶, X ⁷, X ¹⁷Or in its sugared subunit at least one, thereby form the glycan of brachymemma; With

10. the peptide conjugate of claim 9, wherein said polyoxyethylene glycol has monodispersed substantially molecular weight distribution.

11. the peptide conjugate of claim 9, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

12. the peptide conjugate of claim 9, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

13. the peptide conjugate of claim 9, wherein said sialytransferase is ST3Gal3.

14. the peptide conjugate of claim 9, described method also comprises:

15. the peptide conjugate of claim 9, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

16. a pharmaceutical composition comprises the peptide conjugate of claim 9.

17. peptide conjugate that comprises polyoxyethylene glycol, it externally puts together described polyoxyethylene glycol and precursor peptide makes by acellular, wherein use sialytransferase to make described polyoxyethylene glycol be attached to described precursor peptide by complete glycosyl linking group, described precursor peptide comprises the glycan with following formula:

Wherein

R, s and t are independently selected from 0 and 1 integer;

(a) sialic acid donor that makes described peptide, at least a sialytransferase and at least a modification contacts under the condition that sialic acid with the modification of the sialic acid donor of described at least a modification partly is transferred to described glycan being suitable for, the sialic acid of wherein said modification partly comprises at least one and is the modification group of polyoxyethylene glycol, thereby forms the described peptide conjugate that comprises polyoxyethylene glycol.

18. the peptide conjugate of claim 17, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

19. the peptide conjugate of claim 17, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

20. the peptide conjugate of claim 17, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

21. the peptide conjugate of claim 17, wherein said sialytransferase is ST3Gal3.

22. the peptide conjugate of claim 17, described method also comprises:

(b) described peptide conjugate, sialic acid donor and sialyltransferase are contacted under the condition that described sialyltransferase is transferred to sialic acid residues described peptide conjugate being suitable for, thereby sialic acid partly is transferred to described peptide conjugate.

23. the peptide conjugate of claim 17, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

24. a pharmaceutical composition comprises the peptide conjugate of claim 17.

25. peptide conjugate that comprises polyoxyethylene glycol, it externally puts together described polyoxyethylene glycol and precursor peptide makes by acellular, wherein use sialytransferase to make described polyoxyethylene glycol be attached to described precursor peptide by complete glycosyl linking group, described precursor peptide has following formula:

Wherein

AA is the end or the internal amino acid residue of described peptide;

X ¹Be the glycosyl covalently bound, be selected from monose and oligosaccharides residue with described AA; With

U is selected from 0 and 1 integer,

(a) sialic acid donor that makes described peptide, at least a sialytransferase and at least a modification contacts under the condition that sialic acid with the modification of the sialic acid donor of described at least a modification partly is transferred to described peptide being suitable for, the sialic acid of wherein said modification partly comprises at least one and is the modification group of polyoxyethylene glycol, thereby forms the described peptide conjugate that comprises polyoxyethylene glycol.

26. the peptide conjugate of claim 25, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

27. the peptide conjugate of claim 25, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

28. the peptide conjugate of claim 25, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

29. the peptide conjugate of claim 25, wherein said sialytransferase are selected from ST3Gal1, ST6GalNAcI, CST-I and its combination.

30. the peptide conjugate of claim 25, described method also comprises:

31. the peptide conjugate of claim 25, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

32. a pharmaceutical composition comprises the peptide conjugate of claim 25.

33. the covalent conjugates of peptide and polyoxyethylene glycol, wherein said polyoxyethylene glycol by the first intact glycosyl linking group in first glycosyl of described peptide or amino-acid residue place covalent attachment to described peptide, the wherein said first intact glycosyl linking group by the sialytransferase covalent attachment to described glycosyl or amino-acid residue.

34. the covalent conjugates of claim 33, wherein said polyoxyethylene glycol and intact glycosyl linking group precursor are connected to described peptide by the work of enzyme in order to covalently bound unit, described enzyme becomes described complete glycosyl linking group with described precursor conversion, thereby forms described covalent conjugates.

35. the covalent conjugates of claim 33, also comprise: be connected to second glycosyl of described peptide or the modification group of amino-acid residue by the second intact glycosyl linking group, the wherein said second intact glycosyl linking group passes through the sialytransferase covalent attachment to described glycosyl or amino-acid residue.

36. the covalent conjugates of claim 35, wherein said first residue is identical on the structure with described second residue.

37. the covalent conjugates of claim 35, wherein said first residue has different structures with described second residue.

38. the covalent conjugates of claim 35, wherein said first residue and described second residue are glycosyl residues.

39. the covalent conjugates of claim 35, wherein said first residue and described second residue are amino-acid residues.

40. the covalent conjugates of claim 33 wherein before forming described covalent conjugates, is modified described peptide to introduce the acceptor portion of described intact glycosyl linking group.

41. the covalent conjugates of claim 33, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

42. the covalent conjugates of claim 33, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

43. the covalent conjugates of claim 33, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

44. the covalent conjugates of claim 33, wherein said sialytransferase are selected from ST3Gal3, ST3Gal1, CST-I, CST-II and its combination.

45. the covalent conjugates of claim 33 also comprises:

Described covalent conjugates, sialic acid donor and sialyltransferase are contacted under the condition that described sialyltransferase is transferred to sialic acid residues described covalent conjugates being suitable for, thereby sialic acid partly is transferred to described covalent conjugates.

46. the covalent conjugates of claim 33, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

47. pharmaceutical composition, the covalent conjugates that comprises medicine acceptable diluent and polyoxyethylene glycol and glycosylation or non-glycosylated peptide, wherein said polyoxyethylene glycol is conjugated to described peptide by the intact glycosyl linking group, described intact glycosyl linking group by the sialytransferase covalent attachment to described peptide, and between described peptide and described polyoxyethylene glycol and covalently bound with both.

48. the pharmaceutical composition of claim 47, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

49. the pharmaceutical composition of claim 47, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

50. the pharmaceutical composition of claim 47, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

51. the pharmaceutical composition of claim 47, wherein said sialytransferase are selected from ST3Gal3, ST3Gal1, CST-I, CST-II and its combination.

52. the pharmaceutical composition of claim 47 also comprises:

53. the pharmaceutical composition of claim 47, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

54. be used for forming between the sugar of peptide and modification the composition of covalent conjugates, described composition comprises: the mixture of the sugar of modification, sialytransferase and peptide receptor substrate, the sugar of wherein said modification is covalently attached to polyoxyethylene glycol.

55. the composition of claim 54, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

56. the composition of claim 54, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

57. the composition of claim 54, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

58. the composition of claim 54, wherein said sialytransferase are selected from ST3Gal3, ST3Gal1, CST-I, CST-II and its combination.

59. the composition of claim 54 also comprises:

60. the composition of claim 54, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

61. a formation has the acellular in vitro method of covalent conjugates of the precursor peptide of following formula:

Wherein

AA is the end or the internal amino acid residue of described peptide;

X ¹-X ²Be the sugar covalently bound, wherein with described AA

X ¹It is first glycosyl residue; And

(b) sialic acid donor that makes the glycan of described brachymemma, at least a sialytransferase and at least a modification contacts under the condition of glycan that the sialic acid of described at least a sialytransferase with the modification of the sialic acid donor of described at least a modification partly be transferred to described brachymemma being suitable for, the sialic acid of wherein said modification partly comprises polyoxyethylene glycol, thereby forms the covalent conjugates of described peptide.

62. the method for claim 61 also comprises:

(c) before, remove the group that in the posttranslational modification process, is added on the described sugar in step (b).

63. the method for claim 62, wherein said group are selected from phosphoric acid, sulfuric acid, carboxylic acid and its ester.

64. the method for claim 61, wherein said peptide has following formula:

Wherein Z is selected from O, S, NH and linking agent.

65. the method for claim 61, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

66. the method for claim 61, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

67. the method for claim 61, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

68. the method for claim 61, wherein said sialytransferase are selected from ST3Gal1, ST3Gal3, CST-I and its combination.

69. the method for claim 61, described method also comprises:

(c) described covalent conjugates, sialic acid donor and sialyltransferase are contacted under the condition that described sialyltransferase is transferred to sialic acid residues described covalent conjugates being suitable for, thereby sialic acid partly is transferred to described covalent conjugates.

70. the method for claim 61, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, proconvertin a, plasma thromboplastin component, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, blood coagulation factor VIII, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

71. the method for claim 61, wherein said peptide has following formula:

Wherein

X ⁹And X ¹⁰Be independently selected from monose and oligosaccharides residue; With

M, n and f are selected from 0 and 1 integer.

72. the method for claim 61, wherein said peptide has following formula:

Wherein

X ¹¹And X ¹²It is the independent glycosyl part of selecting; With

R and x are independently selected from 0 and 1 integer.

73. the method for claim 72, wherein X ¹¹And X ¹²Be (seminose) _q,

Wherein

Q is the integer that is selected from 1-20, and when q be 3 or when bigger, (seminose) q is selected from linear and branched structure.

74. the method for claim 72, wherein said sialytransferase comprises: (i) GNT-1; (ii) GalT1; (iii) ST3Gal3.

75. the method for claim 61, wherein said peptide has following formula:

Wherein

X ¹³, X ¹⁴And X ¹⁵It is the independent glycosyl residue of selecting; With

G, h, i, j, k and p are independently selected from integer 0 and 1, and prerequisite is that at least one is 1 among g, h, i, j, k and the p.

76. the method for claim 75, wherein

X ¹⁴And X ¹⁵Be independently selected from GlcNAc and Sia; I and k are independently selected from integer 0 and 1, and prerequisite is that at least one is 1 among i and the k, and when k was 1, g, h and j were 0.

77. the method for claim 61, wherein said peptide has following formula:

X wherein ¹⁶Be selected from

With

Wherein

S, u and i are independently selected from integer 0 and 1.

78. the method for claim 61, wherein said removing utilized Glycosylase.

79. an acellular in vitro method that forms the covalent conjugates of precursor peptide, described peptide has following formula:

Wherein

AA is the end or the internal amino acid residue of described peptide;

X ¹Be the glycosyl residue covalently bound, be selected from monose and oligosaccharides residue with described AA; With

U is selected from 0 and 1 integer, and this method comprises:

(a) sialic acid donor that makes described peptide, at least a sialytransferase and at least a modification contacts under the condition that the sialic acid of described at least a sialytransferase with the modification of the sialic acid donor of described at least a modification partly be transferred to described peptide being suitable for, the sialic acid of wherein said modification partly comprises polyoxyethylene glycol, thereby forms the covalent conjugates of described peptide.

80. the method for claim 79, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

81. the method for claim 79, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

82. the method for claim 79, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

83. the method for claim 79, wherein said sialytransferase are selected from ST3Gal1, ST3Gal3, CST-I, CST-II and its combination.

84. the method for claim 79, described method also comprises:

(b) described covalent conjugates, sialic acid donor and sialyltransferase are contacted under the condition that described sialyltransferase is transferred to sialic acid residues described covalent conjugates being suitable for, thereby sialic acid partly is transferred to described covalent conjugates.

85. the method for claim 79, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, proconvertin a, plasma thromboplastin component, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, blood coagulation factor VIII, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP 1, FGF-7, FGF-21 and NT-3.

86. an acellular in vitro method that forms the covalent conjugates of precursor peptide, described peptide has following formula:

Wherein

R, s and t are independently selected from 0 and 1 integer, and this method comprises:

87. the method for claim 86, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

88. the method for claim 86, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

89. the method for claim 86, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

90. the method for claim 86, wherein said sialytransferase are selected from GalT, ST3Gal3, CST-II and its combination.

91. the method for claim 86, described method also comprises:

92. the method for claim 86, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, proconvertin a, plasma thromboplastin component, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, blood coagulation factor VIII, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

93. an acellular in vitro method that forms the covalent conjugates of precursor peptide, described peptide has following formula:

Wherein

AA is the end or the internal amino acid residue of described peptide;

X ¹-X ²Be the sugar covalently bound, wherein with described AA

X ¹It is first glycosyl residue; And

(a) remove X ¹And X ², expose described AA; With

(b) sialic acid donor that makes described peptide, at least a sialytransferase and at least a modification contacts under the condition that the sialic acid of described at least a sialytransferase with the modification of the sialic acid donor of described at least a modification partly be transferred to described peptide being suitable for, the sialic acid part of wherein said modification comprises polyoxyethylene glycol at least, thereby forms the covalent conjugates of described peptide.

94. the method for claim 93, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight.

95. the method for claim 93, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

96. the method for claim 93, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

97. the method for claim 93, wherein said sialytransferase are selected from ST3Gal1, ST3Gal3, CST-I, CST-II and its combination.

98. the method for claim 93, described method also comprises:

99. the method for claim 93, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, proconvertin a, plasma thromboplastin component, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, blood coagulation factor VIII, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

100. an acellular in vitro method that forms the covalent conjugates of precursor peptide, described peptide has following formula:

Wherein

This method comprises:

(a) from this peptide, remove X ³, X ⁴, X ⁵, X ⁶, X ⁷Or X ¹⁷In at least one or its sugared subunit, thereby form the glycan of brachymemma; With

(b) sialic acid donor that makes the glycan of described brachymemma, at least a sialytransferase and at least a modification contacts under the condition of glycan that sialic acid with the modification of the sialic acid donor of described at least a modification partly is transferred to described brachymemma being suitable for, the sialic acid of wherein said modification partly comprises polyoxyethylene glycol, thereby forms the covalent conjugates of described peptide.

101. the method for claim 100, wherein the described removal of step (a) has generated the glycan that b, c, e and x are 0 brachymemma.

102. the method for claim 100, wherein X ³, X ⁵And X ⁷Be selected from (seminose) _z(seminose) _z-(X ⁸) _y

Wherein

X ⁸It is the glycosyl part that is selected from monose and oligosaccharides;

Y is selected from 0 and 1 integer; With

Z is the integer of 1-20, wherein

When z is 3 or when bigger, (seminose) z is selected from linearity and apparatus derivatorius.

103. the method for claim 100, wherein X ⁴Be selected from GlcNAc and wood sugar.

104. the method for claim 100, wherein X ³, X ⁵And X ⁷Be (seminose) _u,

Wherein

U is selected from integer 1-20, when u is 3 or when bigger, (seminose) _uBe selected from linearity and apparatus derivatorius.

105. the method for claim 100, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

106. the method for claim 100, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

107. the method for claim 100, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

108. the method for claim 100, wherein said sialytransferase is ST3Gal3.

109. the method for claim 100, described method also comprises:

110. the method for claim 100, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, proconvertin a, plasma thromboplastin component, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, blood coagulation factor VIII, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

111. acellular in vitro method that between peg molecule and glycosylation or non-glycosylated precursor peptide, forms covalent conjugates, wherein said peg molecule is conjugated to described peptide by the intact glycosyl linking group, described intact glycosyl linking group is between described peptide and described polyoxyethylene glycol and covalently bound with both, and this method comprises:

(a) making described peptide is that the mixture of the sialytransferase of described nucleotide sugar contacts under the condition that the sialic acid of described at least a sialytransferase with the modification of described nucleotide sugar partly be transferred to described peptide being suitable for comprising the nucleotide sugar that is covalently attached on the described polyoxyethylene glycol and its substrate, the sialic acid of wherein said modification partly comprises polyoxyethylene glycol, thereby forms the covalent conjugates of described peptide.

112. the method for claim 111, wherein said glycosyl linking group is covalently attached to glycosyl residue, and described glycosyl residue is covalently attached to described peptide.

113. the method for claim 111, wherein said glycosyl linking group is covalently attached to the amino-acid residue of described peptide.

114. the method for claim 111, wherein said sialytransferase is selected from: sialyltransferase, gala sialytransferase, Portugal's sialytransferase, GalNAc transferring enzyme, GlcNAc transferring enzyme, rock algae sialytransferase and sweet dew sialytransferase.

115. being reorganization, the method for claim 111, wherein said sialytransferase produce.

116. the method for claim 115, wherein said sialytransferase are the former ribozymes of reorganization.

117. the method for claim 115, wherein said sialytransferase are reorganization eucaryon enzymes.

118. the method for claim 111, wherein said nucleotide sugar is selected from: UDP-glucosides, CMP-glucosides and GDP-glucosides.

119. the method for claim 118, wherein said nucleotide sugar is selected from: UDP-semi-lactosi, UDP-GalN, UDP-glucose, UDP-glycosamine, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-seminose, GDP-Fucose, cmp sialic acid and CMP-NeuAc.

120. the method for claim 111, wherein said peptide is a therapeutical agent.

121. the method for claim 111 wherein before described contact, makes the peptide moiety deglycation of described saccharification.

122. the method for claim 111, wherein said intact glycosyl linking group is a sialic acid residues.

123. the method for claim 111, wherein said method is carried out under acellular environment.

124. the method for claim 111 is wherein separated described covalent conjugates.

125. the method for claim 124 is wherein separated described covalent conjugates by membrane filter method.

126. the method for claim 111, wherein said polyoxyethylene glycol has the polymerization degree of about 1-20000.

127. the method for claim 126, wherein said polyoxyethylene glycol has the polymerization degree of about 1-5000.

128. the method for claim 127, wherein said polyoxyethylene glycol has the polymerization degree of about 1-1000.

129. the method for claim 111, wherein said polyoxyethylene glycol have monodispersed substantially molecular weight distribution.

130. the method for claim 111, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

131. the method for claim 111, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

132. the method for claim 111, wherein said sialytransferase are selected from ST3Gal1, ST3Gal3, CST-II and its combination.

133. the method for claim 111, described method also comprises:

134. the method for claim 111, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, proconvertin a, plasma thromboplastin component, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, blood coagulation factor VIII, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

135. method that between glycosylation or non-glycosylated first precursor peptide and glycosylation or non-glycosylated second precursor peptide, is connected to form covalent conjugates with polyoxyethylene glycol, wherein said polyoxyethylene glycol is conjugated to described first peptide by the first intact glycosyl linking group, the described first intact glycosyl linking group is between described first peptide and described polyoxyethylene glycol and covalently bound with the two, and described polyoxyethylene glycol is conjugated to described second peptide by the second intact glycosyl linking group, the described second intact glycosyl linking group is between described second peptide and described polyoxyethylene glycol and covalently bound with the two, and described method comprises:

(a) described first peptide is contacted with the precursor of described polyoxyethylene glycol, the precursor of described polyoxyethylene glycol comprises the precursor of the described first intact glycosyl linking group and the precursor of the described second intact glycosyl linking group;

(b) mixture that makes (a) and its substrate are that the sialytransferase of the described precursor of the described first glycosyl linking group becomes under the condition of the described first intact glycosyl linking group to contact being enough to described precursor conversion with the described first intact glycosyl linking group, thereby form first conjugate between described polyethylene glycol precursor and described first peptide;

(c) be that the sialytransferase of the described precursor of described second intact glycosyl becomes under the condition of the described second glycosyl linking group to contact being enough to described precursor conversion with the described second intact glycosyl linking group with described first conjugate, described second peptide and its substrate, thereby between described polyoxyethylene glycol and described glycosylation or nonglycosylated first peptide and described glycosylation or nonglycosylated second peptide, form described conjugate.

136. the method for claim 135, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

137. the method for claim 135, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

138. the method for claim 135, wherein said sialytransferase are selected from ST3Gal1, ST3Gal3 and its combination.

139. the method for claim 135, described method also comprises:

(d) described covalent conjugates, sialic acid donor and sialyltransferase are contacted under the condition that described sialyltransferase is transferred to sialic acid residues described covalent conjugates being suitable for, thereby sialic acid partly is transferred to described covalent conjugates.

140. the method for claim 135, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, proconvertin a, plasma thromboplastin component, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, blood coagulation factor VIII, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.

141. method that between glycosylation or non-glycosylated first precursor peptide and glycosylation or non-glycosylated second precursor peptide, is connected to form covalent conjugates by polyoxyethylene glycol, wherein said polyoxyethylene glycol covalency is conjugated to described first peptide, and described polyoxyethylene glycol is conjugated to described second peptide by the intact glycosyl linking group, described intact glycosyl linking group is between described second peptide and described polyoxyethylene glycol and covalently bound with both, and this method comprises:

(a) make the activatory precursor of described first peptide and described polyoxyethylene glycol, described precursor comprise with described first peptide on residue have the reactive functional groups of complementary interaction and the precursor of described intact glycosyl linking group, under the condition that forms covalent linkage between described reactive functional groups and the described residue, contact being enough to, thereby form first conjugate; With

(b) making described first conjugate, described second peptide and its substrate is that the sialytransferase of the described precursor of described intact glycosyl linking group becomes under the condition of described intact glycosyl linking group to contact being enough to precursor conversion with described intact glycosyl linking group, thereby is connected to form described conjugate by polyoxyethylene glycol between described glycosylation or non-glycosylated first peptide and glycosylation or non-glycosylated second peptide.

142. the method for claim 141, wherein said polyoxyethylene glycol are selected from linear polyethylene glycol and branch polyoxyethylene glycol.

143. the method for claim 141, wherein said polyoxyethylene glycol is mono methoxy-polyoxyethylene glycol.

144. the method for claim 141, wherein said sialytransferase are selected from ST3Gal1, ST3Gal3, CST-II and its combination.

145. the method for claim 141 also comprises:

146. the method for claim 141, wherein said peptide is selected from: granulocyte colony-stimulating factor, interferon alpha, interferon beta, proconvertin a, plasma thromboplastin component, follicle stimulating hormone, erythropoietin, rHuGM-CSF, interferon-gamma, α-1-proteinase inhibitor, beta-glucosidase enzyme, the tissue type plasminogen activation factor, interleukin II, blood coagulation factor VIII, the mosaic type Tumor Necrosis Factor Receptors, urokinase, the anti-glycoprotein iib/iiia antibody of mosaic type, the mosaic type Anti-HER 2, the mosaic type anti respiratory syncitial virus antibodies, the mosaic type anti-CD20 antibodies, DNase, the mosaic type D2E7, the insulin human, hepatitis B surface antigen, human growth hormone, BMP-7, FGF-7, FGF-21 and NT-3.