WO2006094826A2 - Procede permettant de coupler des glycoconjugues actives sur le plan enzymatique a un compose de modification - Google Patents

Procede permettant de coupler des glycoconjugues actives sur le plan enzymatique a un compose de modification Download PDF

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Publication number
WO2006094826A2
WO2006094826A2 PCT/EP2006/002236 EP2006002236W WO2006094826A2 WO 2006094826 A2 WO2006094826 A2 WO 2006094826A2 EP 2006002236 W EP2006002236 W EP 2006002236W WO 2006094826 A2 WO2006094826 A2 WO 2006094826A2
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group
glycoconjugate
linker
polymer
hydroxyalkyl starch
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PCT/EP2006/002236
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WO2006094826A3 (fr
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Jürgen Hemberger
Dirk Merkel
Andreas Mitsch
Michele Orlando
Jeanne Delbos-Krampe
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Fresenius Kabi Deutschland Gmbh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof

Definitions

  • the present invention relates to a method for coupling of an enzymatically activated glycoconjugate to a modifying compound as well as a glycoconjugate coupled to a modifying compound obtainable by said method. Furthermore, the present invention is directed to the use of an oxidase enzyme in said method and also to pharmaceutical compositions comprising a coupled glycoconjugate obtained by said method.
  • the conjugation of proteins typically involves reactive groups derived from the side chains of sterically available amino acids. Some of the most common amino acid targets for this purpose are lysine, cysteine and arginine. Because a protein usually comprises more than one of the target amino acids, the probability is high that conjugation will take at different position(s) on the protein surface. This can lead to considerable heterogeneity of the coupling products.
  • At least one of the reaction partners i.e. protein or water-soluble polymer
  • requires prior activation i.e. protein or water-soluble polymer.
  • activation methods are not suited to distinguish among several different amino acids of the same species (for example lysines) on the protein's surface.
  • lysines amino acids of the same species
  • a complex mixture of positional isomers of the conjugation product is generally obtained.
  • the separation of these isomers is a very complex and work-intensive task. In some instances the necessary isolation cannot be accomplished with standard chromatography techniques.
  • glycoconjugates particularly proteins with carbohydrate chains on their surface, i.e. glycoproteins
  • a different approach for coupling polymers is available. Because most glycoproteins only have a limited number of glycosylation sites, in some cases only one site, coupling via carbohydrate chains may provide a higher level of coupling specificity than coupling to the more abundant amino acids.
  • a further advantage of this strategy is that coupling takes place at some distance from the protein because the carbohydrate chain of the glycoprotein acts as a natural spacer. As a consequence, the coupling to carbohydrate moieties typically results in no or minor modifications for the tertiary protein structure and yields a more homogeneous coupling product than amino acid targeted coupled polymers.
  • the carbohydrate residues generally require a previous activation step.
  • the activation step is typically an oxidation of vicinal diols to dialdehydes with periodate and interferes with a number of amino acids residues such as, for instance, methionine.
  • This unselective oxidation method will create more than one activation site and, therefore, more than one coupling site. Consequently, a heterogeneous coupling product is often encountered.
  • these strong oxidizing conditions may partially modify the protein's chemistry (e.g., by oxidation of Met). Therefore, chemical oxidation can lead to loss of activity.
  • the enzyme oxygen 6-oxidoreductase (E.G. 1.1.3.9) isolated from Dactylium dendroides, also known as galactose oxidase (GAO) is a copper enzyme catalyzing the following reaction:
  • GAO has been used in a number of diagnostic and biosensor applications (e.g., Vega FA et al., Anal. Chim. Acta, 373, p.57-62 (1998); Tkac J. et al., Biotechnol. Tech., 13, p. 931-936 (1999); Szabo EE et al., Biosens. Bioelectron., H, p. 1051- 1058 (1996)). It was also used to introduce a label into polysaccharides on cell surfaces (Calderhead DM et al., J. Biol. Chem., 263, p.
  • GAO has also been used to modify galactose-containing polysaccharides in order to reduce molecular weight and/or viscosity (e.g., US2002/0076769A1 or WO01/62938).
  • the object of the present invention is to provide an improved method for coupling glycoproteins to modifying compounds such as polymers, preferably water soluble polymers such as PEG or starch derivatives. It is another object of the invention to achieve an improved selectivity for carbohydrate-based coupling of glycoconjugates and a further object to provide a mild and selective activation method so that the conjugation reaction does not interfere with the glycoprotein's original protein structure and the biological activity of the original glycoprotein is maintained in the coupling product.
  • the object of the invention is solved by a method for coupling an enzymatically activated glycoconjugate to a modifying compound, comprising the steps of: a) activating by enzymatic oxidation of at least one primary and/or secondary hydroxyl group of at least one oligo- or polysaccharide moiety in a glycoconjugate to an aldehyde or ketone group, and
  • the activation by enzymatic oxidation of the primary and/or secondary hydroxyl groups of the oligo- or polysaccharide moiety of a glycoprotein in step a) according to the invention is accompanied by mild reaction conditions and high selectivity.
  • the non-saccharide part of the glycoconjugate, its structure and its function typically remains unchanged due to the mild reaction conditions and the selectivity of the respective enzyme.
  • glycoconjugate is defined as any substance having an oligo- or polysaccharide moiety with primary and/or secondary hydroxyl groups.
  • Said substance can be a protein, an oligo- or polypeptide or a lipid.
  • said glycoconjugate is a glycoprotein, more preferably a biologically active glycoprotein, most preferred a therapeutically active glycoprotein. It is also preferred that the glycoprotein is a human or human-derived glycoprotein.
  • the oligo- or polysaccharide moiety of the glycoconjugate comprises at least one saccharide selected from the group comprising of N-acetyl glucosamine, N-acetyl galactosamine, mannose, galactose, sialic acids and fucose.
  • At least one primary hydroxyl group of at least one oligo- or polysaccharide moiety in a glycoconjugate is enzymatically oxidized to an aldehyde group.
  • the enzymatic oxidation (activation) of the glycoconjugate is performed by a saccharide-specific oxidase.
  • Saccharide-specific oxidases require mild, often physiological conditions and demonstrate high selectivity for the saccharide.
  • saccharide-specific oxidases the saccharide specificity as well as the saccharide- specific content of the oligo- or polysaccharide of the glycoconjugate will determine the number of aldehyde groups in the activated glycoconjugate.
  • the saccharide-specific oxidase is a galactose-specific oxidase.
  • galactose-specific oxidases 6-oxidoreductases are particularly preferred.
  • a galactose oxidase (GAO) was originally isolated from Dactylium dendroides by Avigad et al. (J. Biol. Chem, 237, p. 2736-2743 (1962)).
  • the 6-oxidoreductase for use in the invention is derived from Dactylum dendroides.
  • GAO is also available as a recombinant protein from E. coli (Sun et al., Protein Eng., 14. P- 699-704 (2001)), Pichia pastoris (Whittaker et al., Protein Expr. Purif., 20, p. 105-111 (2000)), mammalian cells (Maffia et al., WO01/62938) and others such as Fusarium species.
  • the recombinant GAO enzymes are preferred for use in the method according to the invention.
  • the 6-oxidoreductase employed is a recombinant protein, most preferably one produced by E. coli, Pichia pastoris or Fusarium species.
  • Alternative preferred GAOs may be produced by other yeasts, such as Saccharomyces cerevisiae or Hansenula polymorpha, by mammalian or by insect cells.
  • the galactose- specific oxidase is galactose oxidase EC 1.1.3.9 or another oxygen 6- oxidoreductase.
  • the enzyme in an immobilized form.
  • a insoluble matrix such as a bead, a sheet or a membrane.
  • the insoluble matrix material may be glass, a polymer or any other inert insoluble matrix material available to the skilled person.
  • the enzyme should be firmly attached to the insoluble matrix, e.g. by covalent bonds. Methods for attaching protein to commercially available matrices are known to the skilled person.
  • GAO has quite a broad specificity for primary hydroxyl groups, but oxidation in the presence of C6-OH groups in galactose is much preferred over other primary OH- groups.
  • the wild type enzyme is a glycoprotein of about 68 kD consisting of 639 amino acids with a carbohydrate content of about 1.7% by weight. It has a single polypeptide chain with a type 2 copper centre as prosthetic group.
  • the catalysed reaction is the following:
  • a tyrosin is located in the active site of GAO, which is involved in the redox mechanism as tyrosyl radical / tyrosyl anion.
  • tyrosyl radical / tyrosyl anion In combination with a copper ion a two- electron transfer is accomplished:
  • the electrons released during the oxidation reaction are transferred to oxygen.
  • the Cu(ll)/Tyr " system is regenerated to the Cu(l)/Tyr system by the oxidation of the primary OH-group at the C6 of the galactose to the aldehyde.
  • D-galactose D-gulose and D-talose may also act as substrates, whereas D-glucose or D-mannose are not recognized by this enzyme. This is also true for derivatives of D-galactose with substituents in the C4 position, for which a loss of activity results. Substituents in the C2 position are much better tolerated, although with a reduced reaction rate. Some galactosides are more rapidly oxidized than galactose itself, especially oligo- and polysaccharides of the guaran type.
  • the intraglycosidic bond between a terminal galactose and a subterminal glycosyl unit may be ⁇ (1->6), ⁇ (1->6) or ⁇ (1->4).
  • ⁇ (1->4) linkages have a much lower affinity for the enzyme.
  • Galactose oxidase can be completely inhibited by hydroxylamine, hydrazine and cyanide.
  • the enzyme is also strongly inhibited by its product hydrogen peroxide above a concentration of 2 mM. At 10 mM the inhibition is complete.
  • GAO is quite stable at room temperature but rapidly looses activity at higher temperatures or pH values above 8.0.
  • the method of the invention further comprises the step of degrading any produced hydrogen peroxide.
  • the hydrogen peroxide is degraded by catalase and/or peroxidase (POD).
  • POD peroxidase
  • step a) of the method of the present invention degrades the hydrogen peroxide formed by the GAO reaction into oxygen and water.
  • a considerable portion of the GAO molecules will remain in a semi-active state after the catalysis reaction, a phenomenon which is not well understood despite the availability of 3D structures of the enzyme. It has been suggested, that the use of "one-electron-oxidizers” would be able to convert the enzyme into an active state again.
  • oxidizing agents are Fe- cyanide, [Co(Phen) 3 ] 3" and others.
  • the enzyme peroxidase POD
  • peroxidase from horse radish
  • the method of the invention is one wherein the hydrogen peroxide is degraded by catalase and peroxidase (POD) so that the hydrogen peroxide is degraded by catalase and at the same time GOA is reactivated by POD.
  • POD peroxidase
  • step a) involves at least the three enzymes oxygen 6-oxidoreductase, preferably GAO, catalase and peroxidase (POD).
  • oxygen 6-oxidoreductase preferably GAO
  • catalase preferably catalase
  • the reaction conditions of the above complex system can be optimized by employing raffinose as a substrate.
  • a yield of 80% aldehyde groups was achieved at room temperature with a reaction time of 45 hrs in a reaction mix with 50 mM potassium phosphate buffer, pH 6, 0.1 mM CuCI 2 , 10 mM raffinose and 0.1 U GAO, 2 U catalase and 0.1 U peroxidase per 1 ⁇ mol substrate, respectively. It is important to provide sufficient oxygen by bubbling O 2 through the reaction mix during the incubation.
  • step a) of the method of the invention is performed at a temperature in the range of 0 to 50 0 C for a time period of 30 min to 120 hours in the presence of sufficient oxygen in the reaction mixture.
  • step a) is performed at a temperature in the range of 15 to 40 0 C for a time period of 2 to 30 hours.
  • the yield calculated as mol aldehyde per mol protein was below 10 %, confirming that galactose residues subterminal to sialic acids are poor substrates for the GAO. Because fetuin contains up to three sialic acid residues per glycosylation site, it should be desialylated to become an improved substrate for the galactose oxidase. Desialylation with sialidase according to standard methods is described in the literature (e.g., Schauer: Sialic Acids: Chemistry, Metabolism and Function: Cell Biology Monographs (1983).
  • Asialofetuin isolated after sialidase treatment and oxidized with GAO / catalase / POD under optimized conditions will yield about 50 % oxidation of the available galactose units.
  • non-sialylated or desialylated glycoproteins are employed for the method of the present invention.
  • the oxidizing enzymes can be employed in an immobilized form. This is highly advantageous in that it enables one to reuse the enzymes for several biotransformations due to the higher stability of the enzymes in the immobilized form.
  • Each enzyme can be immobilized separately, so that different ratios of the individual enzymes can be used if desired by mixing the appropriate amounts of immobilized enzymes.
  • As a suitable immobilization technique many of the methods known in the art are suitable (e.g.: Chibata, I: Immobilized Enzymes (Kodansha Scientific Books, 1979). N-hydroxy succinimide activation or azlacton coupling to agarose, glass or polymer beads are preferred techniques of immobilization of enzymes for oxidation. The immobilized enzymes can then easily be removed from reaction mixture by filtration.
  • the oxidizing enzyme is immobilized on a solid insoluble matrix in order to simplify the purification of the substrate, e.g by filtration.
  • Preferred ratios of the three preferred enzymes 6- oxidoreductase, preferably GAO, catalase and peroxidase are in the range of 1-10 : 10-50 : 1-10 for 6-oxidoreductase : catalase : POD, more preferred in the range of 1- 2 : 5-20 : 2-4. Further preferred ratios are in the range of 1 : 0.1 to 100 for GAO : catalase and 1 : 0.5 to 50 for GAO : peroxidase, preferably horseradish peroxidase.
  • modifying compound means any compound that will modify the biological properties of the original glycoconjugate when coupled according to the invention.
  • said modification may influence the glycoconjugate 's own biological activity, its stability in solution and/or pharmacokinetic behaviour in vivo, such as, e.g. bioavailability, half-life and rapid degradation due to immune reactivity.
  • the modifying compound does not merely modify physical properties of the glycoconjugate, e.g. change the molecular weight or provide a label for detection, but actually modifies the biological activity of the glycoconjugate in at least one way.
  • the nucleophilic group of the modifying compound is selected from amine group, a hydroxyl group, a thiol group, hydrazide and a guanidino group.
  • X covalently attached to the modifying compound, wherein X is a bifunctional linker comprising a nucleophilic group or a combination of such bifunctional linkers.
  • X is a bifunctional linker comprising a nucleophilic group or a combination of such bifunctional linkers.
  • Suitable bifunctional linkers are well known in the art and can be found, for example, in the catalog of the Pierce company, Rockford, IL, USA (Pierce 2005-2006 Applications Handbook & Catalog at www.piercenet.com).
  • the linker is a sulfhydryl-reactive linker, preferably N-[ ⁇ - maleimidopropionic acid]hydrazide » TFA (MPH), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), N-[e-maleimidocaproic acid] hydrazide (EMCH), 4-(N-maleimido- methyl)cyclohexan-1-carboxylhydrazid HCI (M 2 C 2 H), 3-(2-pyridyldithio) propionyl- hydrazid (PDPH) or N-[k-maleimidoundecanoic acid]-hydrazide (KMUH).
  • MPH N-[ ⁇ - maleimidopropionic acid]hydrazide » TFA (MPH), 4-(4-N-maleimidophenyl)butyric acid hydrazide
  • EMCH N-[e-maleimidocaproic acid
  • the linker connects an SH- and an NH-group.
  • Linkers connecting an SH- and an NH-group are e.g. linkers selected from AMAS (N- ⁇ (maleimidoacetoxy)succinimidester), BMPS (N- ⁇ (maleimidopropyloxy)succinimid- ester), GMBS (N- ⁇ (maleimidobuty ⁇ y ⁇ oxy) succinimidester), EMCS (N- ⁇ (maleimido- caproyloxy)succinimidester), MBS (m-(maleimidobenzoyl)-N-hydroxysuccinimid- ester), SMCC (succinimidyl- ⁇ N-maleimidomethyl) cyclohexan-1-carboxylat), SMPB (succinimidyl-4-(p-maleimidophenyl) butyrat), SPDP (succinimidyl-S- ⁇ -pyridyld
  • linkers are used which connect two SH- groups.
  • linkers having that ability are selected from BMB (1.4-bis- maleimidobutan), BMDB (1.4-bis-maleimido-2.3-dihydroxybutan), BMH (bis-maleimi- dohexan), BMOE (bis-maleimidoethan), DTME (dithio-bis-maleimidoethan), HBVS (1.6-hexan-bis-vinylsulfon), BM(PEO) 3 (1.8-bis-maleimidotriethylenglycol) and BM (PEO) 4 (1.11-bis-maleimidotetraethylenglycol).
  • linkers may be used, which connect an NH- and an NH-group.
  • linkers are selected from BSOCOES (bis-(2-(succinimidyloxycarbonyloxy) ethyl) sulfon, BS 3 (bis-(sulfosuccinimidyl) suberat), DFDNB (1.5-difluor-2.4-dinitrobenzol), DMA (dimethyladipimidat HCI)), DSG (disuccinimidylglutarat), DSS (disuccinimidylsuberat) and EGS (ethylenglycol-bis- (succinimidylsuccinat).
  • BSOCOES bis-(2-(succinimidyloxycarbonyloxy) ethyl) sulfon
  • BS 3 bis-(sulfosuccinimidyl) suberat
  • DFDNB 1.5-difluor-2.4-dinitrobenzol
  • the linker transforms an amino group into a sulfhydryl group and is preferably 2-iminothiolane-HCI (Traut ' s reagent).
  • the modifying compound is bound to the linker by a carbonyl group or a carboxyl group.
  • a group of the formula -Y-NH 2 is covalently attached to the modifying compound, wherein Y is a bifunctional linker selected from the bifunctional linkers recited in any of claims 16 to 25,
  • a group of the formula -Y-NH-NH 2 is covalently attached to the modifying compound, wherein Y is a bifunctional linker selected from the bifunctional linkers recited in any of claims 16 to 25, and in still another embodiment a group of the formula -Y-CO-NH-NH 2 is covalently attached to the modifying compound, wherein Y is a bifunctional linker selected from the bifunctional linkers recited in any of claims 16 to 25.
  • the modifying compound is a polymer, more preferably a biocompatible polymer, most preferably a water-soluble biocompatible polymer.
  • the modifying compound preferably carries a nucleophilic group.
  • the polymer is selected from the group consisting of starch, gelatine, dextran and albumin.
  • the polymer is hydroxyalkyl starch (HAS), an amino-functionalized hydroxyalkyl starch or a sulfhydryl functionalized hydroxyalkyl starch being most preferred.
  • HAS hydroxyalkyl starch
  • amino-functionalized hydroxyalkyl starch or a sulfhydryl functionalized hydroxyalkyl starch being most preferred.
  • a very preferred polymer for practicing this invention is a semi-synthetic polysaccharide hydroxyalkyl starch.
  • Particularly preferred among hydroxyalkyl starches is hydroxyethyl starch.
  • the hydroxyalkyl starch Due to the natural raw starting material, amylopectin, and the production process during which a certain extent of cleavage of the polymer chains is required, the hydroxyalkyl starch is produced as a molecular homogeneous substance with defined molecular weight but comprises a mixture of molecules of different sizes which may also be differently substituted by hydroxyalkyl groups. The characterization of such mixtures requires statistical measures. For the weight average molecular weight the mean molecular weight (Mw) is called upon. This value is generally determined by means of a light-scattering detector that provides the average molecular weight of all components of a polydisperse mixture.
  • Mw mean molecular weight
  • the average value (Mn) indicates the number average molecular weight currently used for calculating the real number of moles in a specimen. This value is measured by its effect on osmotic pressure by means of a membrane osmometer.
  • substitution degree (MS, molar substitution) is defined as the average number of hydroxyethyl groups per anhydroglucose unit. It is determined from the total number of hydroxyethyl groups in a specimen by ether cleavage and subsequent quantitative determination of ethyl iodide and ethylene.
  • the substitution degree DS (degree of substitution) is defined as the proportion of the substituted anhydroglucose units of all anhydroglucose units. It can be determined from the measured amount of the unsubstituted glucose after hydrolysis of a specimen. It follows from these definitions that the MS is normally greater than the DS. In the case where only a monosubstitution is present, each substituted anhydroglucose unit carries only one hydroxyethyl group and MS equals DS.
  • ⁇ -amylase is the major mechanism of in vivo breakdown of hydroxyalkyl starches. This enzyme preferably cleaves unsubstituted anhydroglucose units, whereas substituted anhydroglucose units are split much slower. Additionally, hydrolysis by ⁇ -amylase is more retarded by substitution on C2-OH than on C6-OH, because the C2 position is much closer to the actual cleavage site at C1.
  • HAS-pharmacokinetic behaviour is determined by the above physicochemical properties (MW, MS, DS and C2/C6 ratio).
  • the main mechanisms governing HAS-pharmacokinetic behaviour are the renal clearance (related to the polymer size) and the degradation rate by ⁇ -amylase (in relation with the extent of substitution).
  • the polymer for practicing the method of the invention is hydroxyalkyl starch (HAS) with a molecular weight in the range of 1 kD to 500 kD.
  • HAS hydroxyalkyl starch
  • the hydroxyalkyl starch has a molecular weight in the range of 5 kD to 200 kD.
  • the molar substitution of hydroxyalkyl starch for use according to the invention is in the range of 0.1 to 0.9, more preferably in the range of 0.2 to 0.6.
  • the C2/C6 ratio of hydroxyalkyl starches for use in the present invention is in the range of 1 to 10.
  • HES hydroxyalkyl starch
  • HES hydroxyethyl starch
  • HES has been used for 15 years in volume substitution therapy as so called plasma expander.
  • the broad clinical experience with this polymer has shown an extra- ordinary high biocompatibility with respect to toxicity, antigenicity, immunogenicity and rate of clinical side effects.
  • PEG hydroxyalkyl starch is a biodegradable polymer with a very well studied pharmacokinetic profile, which lead to predictable degradation rates according to the structural parameters MW, MS, DS and C2/C6 ratio as discussed above.
  • HES is commercially produced by hydroxyethylation of starch using ethylene oxide or 2-chloroethanol as described, for example, by Sommermeyer et al. (EP0402724). HES has been commonly employed for the production of colloidal plasma volume expanders. The field of volume substitution (e.g. in case of hemorrhagic shock) or hemodilution (e.g. for arterial occlusive disease, Fontaine Il B, III) is today inconceivable without the use of colloidal plasma substitutes. HES is highly accepted in clinical applications because of its very low antigenicity, immunogenicity and toxicity and its well known predictable pharmacokinetic behaviour.
  • Biologically active glycoprotein-HES conjugates are very valuable as therapeutic protein drugs, because of their increased stability, longer life span in vivo and reduced antigenic or immunogenic potential.
  • Conjugates produced according to the methods of the invention may also be used as diagnostic tools, for example, when antibodies or antibody fragments are used as the glycoprotein part.
  • the starch derivative may be selectively oxidized at the reducing end aldehyde to the corresponding acid by methods already described by Hemberger & Orlando in WO03/074087 and WO03/074088. This acid may be readily converted into the corresponding lacton by dehydration. To react with the aldehyde function of the activated glycoprotein, the HES lacton has to be further functionalized.
  • HES lacton with hydrazine or diaminobutane in a water-free organic solvent, preferably DMSO or DMF.
  • a water-free organic solvent preferably DMSO or DMF.
  • the resulting HES hydrazide may then be coupled to the enzymatically activated glycoprotein to form a stable hydrazone bond.
  • monosulfhydryl-HAS may be used, which has advantages in purification in comparison to the hydrazine derivative.
  • HES-SH may be reacted with 4-maleimidopropionic acid hydrazide (MPH) or 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH) to yield hydrazide derivatives which are coupled via the above mentioned linkers to the SH group of the HES-SH.
  • MPH 4-maleimidopropionic acid hydrazide
  • MPBH 4-(4-N- maleimidophenyl)butyric acid hydrazide
  • a hydrazide-functionalized HAS molecule one may start from the selectively oxidized HAS as described in WO03/074087 or the corresponding lacton. After deprotection the reaction with N-BOC-hydrazin results in the derivative HAS- CO-NHNH 2 without a linker molecule.
  • a bifunctional linker can be introduced very easily by standard methods if desired.
  • these linkers have biocompatible, non-toxic, non-antigenic and non-immunogenic properties.
  • an SH-functionalized HAS derivative will provide for higher reactivity and higher purity. Its synthesis may be accomplished via cysteamine coupling as described in the example section below. The HAS-SH is then reacted with a suitable SH-reactive linker to yield a hydrazide of the general formula HAS-X- CO-NHNH 2 , wherein X is the linker.
  • the pre-treated conjugation partners may be dissolved together in a suitable buffer system, e.g. 0.1 M sodium acetate solution (pH ⁇ 5.5) and be maintained under moderate stirring conditions for 20 to 24 hrs at 20°-25°C.
  • a suitable buffer system e.g. 0.1 M sodium acetate solution (pH ⁇ 5.5)
  • the buffer conditions have to be adjusted according with the protein target ' s specific tolerance.
  • the reaction product can be isolated and analyzed, for example, by gel permeation chromatography as well as by SDS-PAGE and subsequent protein- and glyco-specific staining.
  • the glycoprotein for use in the method of the present invention is selected from the group consisting of growth factors, preferably epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), differentiation factors, preferably erythropoetin (EPO), granulocyte- colony stimulating factor (G-CSF), granulocyte/macrophage-colony stimulating factor (GM-CSF), chemokines and cytokines, preferably interferons, more preferably ⁇ - interferon, ⁇ -interferon, ⁇ -interferon and their interferone subtypes, interleukins, more preferably interleukines 1 to 12, asialofetuin, and monoclonal antibodies as well as fragments thereof.
  • growth factors preferably epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), differentiation factors, preferably erythropoetin (EPO), granulocyte- colony stimulating factor (G-CSF
  • step b) the enzymatically and selectively oxidized (activated) glycoconjugates are coupled to at least one modifying compound by reacting at least one nucleophilic moiety of the modifying compound with at least one of the aldehyde and/or ketone moieties of the activated glycoconjugate.
  • the method of the invention further comprises the step of reducing the coupling bond resulting from step b), if further stabilization of the bond between the glycoconjugate and the modifying compound, e.g. HAS, is desired.
  • the modifying compound e.g. HAS
  • the coupling between the aldehyde and/or ketone function of the enzymatically activated glycoconjugate and the nucleophilic modifying compound will form a Schiff ' s base structure which may be reduced to a stable amine by suitable reduction agents such as boron hydride or more preferred cyanoboron hydride.
  • the method of the invention allows for the highly selective coupling of enzymatically activated aldehydes and/or ketones on glycoconjugates to nucleophilic modifying compounds in very high to moderate yields and retention of the biological activity of the glycoprotein target.
  • the major advantage of the method of the invention is that unlike to the methods of the prior art a predictable, selective, stable covalent coupling with good recovery of the biological activity is achieved, which yields a homogenous coupling product with respect to a 1 :1 stochiometry and the specific coupling site.
  • the present invention also provides new glycoconjugates coupled to a modifying compound, obtainable by a method according to the invention.
  • the polymer of said glycoconjugate is hydroxyalkyl starch (HAS).
  • HAS hydroxyalkyl starch
  • the polymer is hydroxyalkyl starch (HAS) with a molecular weight in the range of 1 kD to 500 kD.
  • HAS hydroxyalkyl starch
  • the polymer is hydroxyalkyl starch (HAS) with a molecular weight in the range of 5 kD to 200 kD.
  • HAS hydroxyalkyl starch
  • the polymer is hydroxyalkyl starch (HAS) with a molar substitution in the range of 0.1 to 0.9, more preferably in the range of 0.2 to 0.6. Furthermore, it is preferred that the C2/C6 ratio of HAS is in the range of 1 to 10.
  • HAS hydroxyalkyl starch
  • the new glycoconjugate according to the invention is coupled to a modifying compound by means of a bifunctional linker.
  • hydroxyalkyl starch is hydroxyethyl starch (HES).
  • the hydroxyethyl starch is conjugated to a glycoprotein by means of a hydrazone bond.
  • the glycoconjugate produced according to the invention comprises a glycoprotein selected from the group consisting of growth factors, preferably epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), differentiation factors, preferably erythropoetin (EPO), granulocyte-colony stimulating factor (G-CSF), granulocyte/macrophage- colony stimulating factor (GM-CSF), chemokines and cytokines, preferably interferons, more preferably ⁇ -interferon, ⁇ -interferon, ⁇ -interferon and their interferone subtypes, interleukins, more preferably interleukines 1 to 12, asialofetuin, and monoclonal antibodies as well as fragments thereof.
  • growth factors preferably epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), differentiation factors, preferably erythropoetin (EPO), granulocyte-colony stimulating factor (G
  • a third aspect of the invention is directed to the use of an oxidase enzyme in a method according to the invention.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a coupled glycoconjugate produced according to the invention and optionally a pharmaceutically acceptable excipient.
  • the glycoconjugate in the pharmaceutical composition is preferably selected from the group consisting of growth factors, preferably epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), differentiation factors, preferably erythropoetin (EPO), granulocyte-colony stimulating factor (G-CSF), granulocyte/macrophage-colony stimulating factor (GM-CSF), chemokines and cytokines, preferably interferons, more preferably ⁇ -interferon, ⁇ -interferon, ⁇ - interferon and their interferone subtypes, interleukins, more preferably interleukines 1 to 12, asialofetuin and monoclonal antibodies as well as fragments thereof.
  • growth factors preferably epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), differentiation factors, preferably erythropoetin (EPO), granulocyte-colony stimulating factor (G-CSF
  • the pharmaceutical composition of the invention comprises a glycoconjugate coupled to a hydroxyalkyl starch (HAS), preferably a hydroxyethyl starch (HES).
  • HAS hydroxyalkyl starch
  • HES hydroxyethyl starch
  • Fig. 1 illustrates the substrate specificity of GAO in the presence of catalase and POD.
  • the yield is calculated from the estimation of the newly formed aldehyde groups by the BCA methods (for details see example 7).
  • Fig. 2 illustrates the absence of sialic acids in the desialylated fetuin by isoelectric focussing (for details see example 12)
  • Fig. 3 illustrates the oxidation of asialofetuin by GAO in the presence of catalase and POD.
  • the yield is calculated from the estimation of the newly formed aldehyde groups by the BCA methods (for details see example 13)
  • Fig. 4 demonstrates the formation of new aldehyde groups by glycan detection (for details see example 13)
  • Fig. 5 shows the analysis of the coupling product of oxidized asialofetuin with HES- MPH in example 15 by SDS-PAGE. From the results of the SDS-PAGE a coupling yield of about 50% was estimated, i.e. about 50% of the oxidized asialofetuin remained unchanged after the reaction (see example 15 for details).
  • Fig. 6 shows the GPC analysis of the coupling reaction mixture from example 14.
  • the following examples are presented for illustrating preferred embodiments of the present invention only. They are not intended to be construed as limiting to the scope of the present invention.
  • HES 70 Hydroxyethyl starch with a molecular weight of 70 kD and a degree of substitution of 0.5 (HES 70 ) was used for this experiment.
  • OxHES 70 in the lacton form was treated with a 2-fold molar excess of N-BOC-hydrazine in water-free DMSO under argon atmosphere for 24 hrs at 50 °C.
  • the reaction product was precipitated by adding an ice-cold mixture of acetone/methanol (4:1) and washed until no N-BOC-hydrazine was detected on TLC.
  • the precipitate was dissolved in water, treated with a strong cation exchanger to remove traces of the starting material and lyophilized.
  • the lyophilisate was dissolved in water/MeOH (3:1), cooled on ice and treated with gaseous HCI under moderate stirring.
  • the reaction was monitored with ninhydrin on TLC plates and stopped upon completion of the reaction by adding 1 M potassium phosphate until a pH of about 7.
  • the reaction mixture was extensively dialyzed against water under argon atmosphere at 4 °C.
  • the amount of hydrazide in the product was estimated using the TNBS method (Inman JK, Biochem., 8, p. 4074- 4082(1969)).
  • TNBS 2,4,6-trinitrobenzosulfonic acid
  • Sulfhydryl-functionalized HES 70 from example 2 was treated with a 10-fold excess of the crosslinker 4-maleimidopropionic acid hydrazide trifluoracetate (MPH) in dry DMSO under argon atmosphere for 4.5 hrs at room temperature.
  • MPH 4-maleimidopropionic acid hydrazide trifluoracetate
  • the HES derivative was precipitated by adding cold acetone, centrifuged and redissolved in water.
  • the product was purified by a Hi-prep SEC 26/10 column (Amersham Biotech). The high molecular weight fraction was pooled and lyophilized.
  • the yield of the modification reaction was determined by quantifying the hydrazide group with TNBS at 500 nm. The yield was about 75%.
  • HES70 from example 2 was treated with a 10-fold excess of the crosslinker 4-(4-N-maleimidophenyl)butyric acid hydrazid x HCI (MPBH) in dry DMSO under argon atmosphere for 4.5 hrs at room temperature.
  • the HES derivative was precipitated by adding cold acetone, centrifuged and redissolved in water.
  • the product was purified by a Hi-prep SEC 26/10 column (Amersham Biotech). The high molecular weight fraction was pooled and lyophilized.
  • the yield of the modification reaction was determined by quantification of the hydrazide group with TNBS at 500 nm. The yield was about 80%.
  • Raffinose a non-reducing trisaccharide
  • 50 mM potassium phosphate buffer, pH 6.0 with 0.1 mM CuCI 2 at a concentration of 10 mM.
  • 0.1 u galactose oxidase, 2 u catalase from bovine liver and 0.1 u peroxidase from horse radish were added per 1 ⁇ mol raffinose, respectively.
  • the reaction mix was stirred under oxygen pressure for 20 hrs.
  • the yield determined by the BCA assay as described in example 5 was 40% under optimized conditions.
  • the carbohydrate chains may be either attached to Ser/Thr as O- glycosides or to Asn as N-glycosides.
  • a typical structure of O-glycosides is the Gal- containing disaccharide D-Gal- ⁇ [1-3]-D-GalNAc, whereas D-Gal- ⁇ [1-4]-D-GlcNAc represents a typical N-glycosidic structure.
  • Both structures as the corresponding O- methyl glycosides were oxidized by the three-enzyme system described above to determine a possible preference of the enzyme for one of these structures.
  • the results (Fig. 1) clearly show a strong preference for the D-Gal- ⁇ [1-3]-D-GalNAc structure over D-Gal- ⁇ [1 -4]-D-GlcNAc as substrate.
  • Enzymatic activity of the galactose oxidase was determined according to Avigad et al. (J. Biol. Chem, 237, p. 2736-2743 (1962)). Briefly, 1.7 ml 0.1 M potassium phosphate buffer, pH 6.0, containing 0.5% o-tolidine were added to 1.5 ml 10% galactose in H 2 O. After adjustment of the temperature to 25 °C 0.1 ml peroxidase (60 u/ml) was added and the reaction was started with 0.1 ml GAO preparation. The change of absorbance with time was read at 425 nm and the activity was calculated from the linear portion of the curve. For the immobilized enzyme an aliquot of the coupling product was used in the assay.
  • catalase The activity of catalase was measured as described by Beers & Sizer (J. Biol. Chem, 195, p. 133ff (1952)). Briefly, to 1.9 ml water, 1.0 ml of 0.059 M hydrogen peroxide was added and equilibrated to 25 0 C. The reaction was started by the addition of 0.1 ml catalase in 50 mM potassium phosphate buffer, pH 7.0. The change of absorbance at 240 nm with time was read and the enzymatic activity was calculated from the linear portion of the curve.
  • the azlactone method was also applied to the immobilization of peroxidase (POD). 10 mg POD were used per 10 ml activated gel under the same conditions described in example 8. This amount of POD was quantitatively coupled to the matrix. The yield in terms of enzymatic activity was about 80%.
  • Oxidation of fetuin was performed at concentrations up to 1 mg/ml in 50 mM potassium phosphate buffer, pH 6.0, with variable ratios of the enzymes GAO, catalase and POD at 25 °C for up to 120 hrs under sterile conditions.
  • GAO potassium phosphate buffer
  • POD POD
  • sialic acid residues of fetuin were removed. This was achieved by treatment with an appropriate sialidase enzyme. 1 ml immobilized sialidase from vibrio cholerae (Galab, Geesthacht, Germany) with 1 U/ml gel was used to treat 10 mg fetuin for 20 hrs at 37 0 C in 50 mM sodium acetate buffer, pH 5.5, containing 1 mM CaCI 2 . The absence of sialic acids in the treated fetuin was demonstrated by isoelectric focussing (Fig. 2)
  • the optimized incubation mix for the oxidation of asialofetuin in the three-enzyme approach consisted of 50 mM potassium phosphate buffer, pH 6.0, 1 U/ml GAO, 20 U/ml catalase, 1.5 u/ml POD and 0.5 mg/ml asialofetuin in a total volume of 1 ml.
  • the reaction was run at 25 0 C for up to 120 hrs under sterile conditions in an oxygen atmosphere.
  • the degree of oxidation was determined by the BCA method described in example 5. Because the presence of proteins will also cause an increase in the absorbance at 560 nm, although much lower than the effect of the aldehyde oxidation, this was corrected by measuring the calibration curve in the presence of protein.
  • Fig. 4 demonstrates the presence of newly formed aldehyde groups in the asialofetuin by glycan detection.
  • the reaction product was separated by SDS- PAGE and blotted onto a nitrocellulose membrane.
  • the membrane was treated with digoxigenin-3O-succinyl- ⁇ -aminocaproic acid hydrazide to form a stable hydrazone between aldehydes of asialofetuin and the hydrazid.
  • the presence of the digoxigenin derivative was detected by an digoxigenin-specific antibody coupled to alkaline phosphatase with NBT/X-phosphate as a substrate.
  • Example 14 Coupling of oxidized asialofetuin with HES-hydrazide
  • Example 16 Coupling of oxidized asialofetuin with HES- MPBH
  • Example 17 Oxidation of erythropoetin by GAO
  • EPO was desialylated by the same procedure as described in example 12 for fetuin.
  • Example 18 Coupling of oxidized erythropoetin with HES-MPBH
  • 0.5 mg of the oxidized asialo-EPO from example 17 can be dissolved in 1 ml 0.1 M potassium phosphate buffer, pH 5.5. HES 50 -MPBH in a 5-fold molar excess was added and the reaction was allowed to proceed for 48 hrs at room temperature. SDS- PAGE analysis and gel permeation chromatography show that the majority the EPO can be found in the high molecular range, i.e. is conjugated to the HES polymer.
  • the biological activity of the HES-EPO can be measured in vitro using the cell line NFS-60 according to Hara K et al. (Experimental Hematology, 16, 256-61 (1988)). Briefly, growth proliferation of this cell line was measured in response to EPO by a colorimetric method. Alamar blue (Biosource, Camarillo, CA) was used to stain the cells and measured at 570 nm.
  • Example 19 Oxidation of granulocyte-colony stimulation factor by GAO
  • 1 mg G-CSF may be desialylated by the same procedure described in example 12 for fetuin.
  • the degree of oxidation reached in this case is much higher compared to example 17.
  • Example 20 Coupling of oxidized granulocyte-colony stimulating factor with HES-hydrazide
  • HES ⁇ o-hydrazide prepared according to example 1 may be used as coupling partner for the oxidized asialo-G-CSF.
  • the reaction conditions applied in this case are similar to example 18.
  • 0.5 mg of the oxidized glycoprotein were dissolved in 1 ml 0.1 M potassium phosphate buffer, pH 6.5.
  • a 10-fold excess of the HES 70 -hydrazide is added and the reaction is allowed to proceed over night at room temperature.
  • SDS- PAGE and GPC were again used to analyze the coupling product.
  • the coupling efficiency was slightly lower than observed in example 18 with EPO, but still in an acceptable range.
  • Example 21 Oxidation of interleukin-2 by GAO
  • Recombinant human IL-2 produced in CHO cells was used as additional target for the enzymatic oxidation. In this case no desialylation reaction was performed, because analytical data had shown that this preparation was incompletely sialylated.
  • 100 ⁇ g of the glycoprotein were treated with the three enzymes in a ratio of 0.5 U/10 U/2 U for GAO/catalase/POD in 50 mM potassium phosphate buffer, pH 5.5, at 25 °C for 40 hrs under sterile conditions and with sterile oxygen bubbling through the suspension. The reaction was stopped and the product was analyzed by the BCA method. The oxidation yield was found to be about 45%.
  • Example 22 Coupling of oxidized interleukin-2 with HES-hydrazide
  • the coupling of the oxidized IL-2 was done with HES 7 o-hydrazide prepared according to example 1.
  • the reaction mix from example 21 was directly incubated with a 20-fold excess of HES 7 o-hydrazide and the reaction was run for 24 hrs at room temperature.
  • the coupling product was analyzed by SDS-PAGE and GPC as already shown for example 13 and 14. About half of the IL-2 present was found in the coupling product.
  • the in vitro bioactivity of the HES-IL-2 was estimated using a cell proliferation assay with the cell line CTLL-2, which specifically responds to active IL-2 (Weston L et al. Immunology and Cell Biology, 76, 190-192 (1998)).
  • the proliferation response to IL-2 is detected by measuring the absorbance of Alamar blue as described in example 18.
  • About 28 % of the original CTLL-2 proliferating activity was detected in the HES-IL-2 coupling product.

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Abstract

L'invention concerne un procédé permettant de coupler des glycoconjugués activés sur le plan enzymatique à des composés de modification, ainsi qu'un glycoconjugué couplé à un composé de modification pouvant être obtenu au moyen de ce procédé. De plus, l'invention concerne l'utilisation d'une enzyme oxydase dans ledit procédé et des compositions pharmaceutiques renfermant un glycoconjugué couplé obtenu au moyen du procédé.
PCT/EP2006/002236 2005-03-11 2006-03-10 Procede permettant de coupler des glycoconjugues actives sur le plan enzymatique a un compose de modification WO2006094826A2 (fr)

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WO2000017226A1 (fr) * 1998-09-23 2000-03-30 The Regents Of The University Of California Peptides de synthese, reactifs de conjugaison et procedes associes
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WO2000017226A1 (fr) * 1998-09-23 2000-03-30 The Regents Of The University Of California Peptides de synthese, reactifs de conjugaison et procedes associes
WO2003074087A1 (fr) * 2002-03-06 2003-09-12 Biotechnologie - Gesellschaft Mittelhessen Mbh Couplage de proteines avec un polysaccharide modifie
EP1398322A1 (fr) * 2002-09-11 2004-03-17 Fresenius Kabi Deutschland GmbH Polypeptides HASylés, en particulier érythropoiétine HASylée
WO2005014035A2 (fr) * 2003-08-08 2005-02-17 Novo Nordisk Health Care Ag Utilisation de galactose oxydase pour la conjugaison chimique selective de molecules d'extraction a des proteines d'interet therapeutique
WO2006094810A2 (fr) * 2005-03-11 2006-09-14 Fresenius Kabi Deutschland Gmbh Production de glycoproteines bioactives a partir d'un materiau de depart inactif
WO2006108052A2 (fr) * 2005-04-06 2006-10-12 Genzyme Corporation Ciblage de glycoproteines therapeutiques

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CN112924665A (zh) * 2021-02-19 2021-06-08 山东莱博生物科技有限公司 一种抗体辣根过氧化物酶标记物及其制备与应用
CN112924665B (zh) * 2021-02-19 2023-10-03 山东莱博生物科技有限公司 一种抗体辣根过氧化物酶标记物及其制备与应用

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