KR101209111B1 - Glycopegylated factor ix - Google Patents

Abstract

The present invention provides a conjugate between Factor IX and a PEG moiety. This conjugate is inserted between the peptide and the modifying group and is linked through an intact glycosyl linking group covalently attached to them. This conjugate is formed from glycosylated peptide by the action of glycosyltransferases. Glycosyltransferases link modified sugar sites on glycosyl residues on peptides. The present invention also provides a method of making the conjugate, a method of treating various disease states using the conjugate, and a pharmaceutical formulation comprising the conjugate.

Glycopezylation, Factor IX, Glycosyltransferases, Conjugates, Blood Coagulation, Hemophilia

Description

Glycopezylation Factor Nine {GLYCOPEGYLATED FACTOR IX}

(Cross-reference to related applications)

This application is U.S. Provisional Patent Application No. 60 / 527,089, filed Dec. 3, 2003, U.S. Provisional Patent Application No. 60 / 539,387, filed Jan. 26, 2004; United States Provisional Patent Application 60 / 592,744, filed Jan. 29, 2004; United States Provisional Patent Application 60 / 614,518, filed September 29, 2004; And US Provisional Patent Application No. 60 / 623,387, filed October 29, 2004, each of which is incorporated herein by reference for all purposes.

The present invention relates to a conjugate between Factor IX and PEG moieties. This conjugate is inserted between the peptide and the modifying group and is linked through an intact glycosyl linking group covalently attached to them. This conjugate is formed from glycosylated peptide by the action of glycosyltransferases. Glycosyltransferases link modified sugar sites on glycosyl residues on peptites. The invention also relates to methods of making the conjugates, methods of treating various disease states using the conjugates, and pharmaceutical formulations comprising the conjugates.

Vitamin K-dependent proteins (eg Factor IX) contain 9 to 13 gamma-carboxyglutamic acid residues (Gla) at 45 residues of their amino termini. Gla residues are produced by enzymes in the liver that carboxylate the side chains of glutamic acid residues in the protein precursor with vitamin K. Vitamin K-dependent proteins are involved in many biological processes, the best described of which is blood coagulation (reviewed in Neelsestuen, Vitam. Horm. 58 : 355-389 (2000)). Dependent proteins include protein Z, protein S, prothrombin (factor II), factor X, factor IX, protein C, factor VII, Gas6, and matrix GLA proteins Factor VII, IX, X, and II are procoagulations. Protein C, protein S and protein Z act as anticoagulants, while Gas6 is a growth arrest hormone encoded by growth stop-specific gene 6 (gas6) and is associated with protein S (see : Manfioletti et al. Mol. Cell. Biol. 13 : 4976-4985 (1993)) Matrix GLA proteins are typically present in bone and are important for preventing soft tissue calcification in circulation (Luo et al. Nature 386 : 78-81 (1997).

Control of blood coagulation is a process that manifests a number of major health problems, including both the failure of blood clots and clots to form and unwanted blood clots. Agents to prevent unwanted blood clots are used in a number of situations, and a variety of drugs are available. Unfortunately, most current therapies have undesirable side effects. Orally administered anticoagulants, such as warfarin, inhibit the action of vitamin K in the liver to prevent complete carboxylation of glutamic acid residues in vitamin K-dependent proteins, lower the concentration of active proteins in the circulation, and form blood clots It works by lowering your ability to do so. Warfarin therapy is complicated by the competitive nature of the drug with its target. Changes in dietary vitamin K can lead to overdose or low doses of warfarin. Variation in coagulation activity is an undesirable consequence of this therapy.

Infused materials such as heparin, including low molecular weight heparin, are also commonly used anticoagulants. In addition, these compounds are prone to overdose and must be carefully monitored.

Newer categories of anticoagulants include active-site modified vitamin K-dependent coagulation factors such as Factors VIIa and IXa. Active sites are blocked by serine protease inhibitors, such as chloromethylketone derivatives of amino acids or short peptides. Active site-modified proteins retain the ability to form complexes with their respective cofactors, but are inactive, resulting in no enzymatic activity and preventing complex formation of each active enzyme and cofactor. After all, these proteins seem to provide the benefits of anticoagulant therapy without the side effects of other anticoagulants. Active site modified factor Xa is another possible anticoagulant of this group. His cofactor protein is the factor Va. Active site modified activating protein C (APC) can also form effective inhibitors of coagulation (Sorensen et al. J. Biol . Chem . 272 : 11863-11868 (1997)). Active site modified APC binds to factor Va to prevent factor Xa from binding.

The main limitation on the use of vitamin K-dependent coagulation factors is cost. Biosynthesis of vitamin K-dependent proteins depends on the intact glutamic acid carboxylation system present in a few animal cell types. Overproduction of these proteins is limited by this enzyme system. In addition, the effective dose of these proteins is large. A typical dosage is 1000 μg of peptide per kg body weight (Harker et al. 1977, supra).

Another phenomenon that inhibits the use of therapeutic peptides is the relatively short in vivo half-life exhibited by these peptides in a well-known aspect of protein glycosylation. Overall, the problem of short in vivo half-life means that therapeutic glycopeptides should be frequently administered at high doses and ultimately needed when more efficient methods of preparing longer-lasting and more effective glycoprotein therapeutics are available. This translates into greater health care costs than can be achieved.

For example, factor VIIa illustrates this problem. Factors VII and VIIa have a circulating half-life of about 2-4 hours in humans. That is, within 2-4 hours the concentration of peptide in serum is halved. When factor VIIa is used as a procoagulant to treat certain types of hemophilia, the standard protocol is to inject VIIa at high doses (45-90 .mu.g / kg body weight) every 2 hours ( See Hedner et al., Transfus.Med. Rev. 7: 78-83 (1993). Thus, using these proteins as coagulants or anticoagulants (for factor VIIa) requires high doses of the protein at frequent intervals.

One solution to the problem of providing cost effective glycopeptide therapeutics is to provide peptides with longer in vivo half-lives. For example, glycopeptide therapeutics with improved pharmacodynamic properties have been produced by attaching synthetic polymers to the peptide backbone. An example of a polymer conjugated to a peptide is poly (ethylene glycol) (“PEG”). The use of PEG to derivatize peptide therapeutics has been shown to reduce the immunogenicity of peptides. For example, US Pat. No. 4,179,337 (Davis et al.) Describes non-immunogenic polypeptides such as enzymes and peptides coupled to polyethylene glycol (PEG) or polypropylene glycol. In addition to reduced immunogenicity, the clearance time in the circulation is extended due to the increased size of the PEG-conjugate of the polypeptide in question.

The main mode of attachment of PEG and its derivatives to peptides is non-specific binding via peptide amino acid residues (see, eg, US Pat. No. 4,088,538, US Pat. No. 4,496,689, US Pat. No. 4,414,147, US Pat. No. 4,055,635). No., and PCT WO 87/00056). Another mode of attaching PEG to a peptide is through non-specific oxidation of glycosyl residues on glycopeptides (see eg WO 94/05332).

In these non-specific methods, poly (ethyleneglycol) is added to the reactive moiety on the peptide backbone in a random and non-specific manner. Of course, the random addition of PEG molecules has its drawbacks, including the lack of uniformity of the final product and the possibility of reducing the biological or enzymatic activity of the peptide. Thus, for the production of therapeutic peptides, the derivatization strategies that are specifically labeled and easily specified and which inevitably lead to the formation of uniform products are excellent. This method has been developed.

Specifically labeled homogeneous peptide therapeutics can be produced in vitro by the action of an enzyme. Unlike typical non-specific methods of attaching synthetic polymers or other labels to peptides, enzyme-based synthesis has the advantages of regioselectivity and stereoselectivity. Two major classes of enzymes for use in the synthesis of labeled peptides are glycosyltransferases (eg, sialyltransferases, oligosaccharyltransferases, N-acetylglucosamyltransferases), and glycosidases. These enzymes can be used for specific attachment of sugars which can later be modified to include therapeutic sites. Alternatively, glycosyltransferases and modified glycosidase can also be used to directly transfer modified sugars to the peptide backbone (see, eg, US Pat. No. 6,399,336 and US Patent Application Publication Nos. 20030040037, 20040132640, 20040137557, 20040126838 and 20040142856, each of which are incorporated herein by reference). Methods of binding both chemical and enzymatic syntheses are also known (see, eg, Yamamoto et al. Carbohydr. Res. 305: 415-422 (1998) and US Patent Application Publication No. 20040137557, incorporated herein by reference). number).

Factor IX is a very useful therapeutic peptide. Although therapeutically useful forms of Factor IX are used today, these peptides can be improved by modifications that enhance the pharmacodynamics of the resulting isolated glycoprotein product. Thus, there remains a need in the art for longer term sustained Factor IX peptides with improved efficacy and better pharmacodynamics. In addition, to be effective for the maximum number of individuals, it must be able to produce, on an industrial scale, factor IX peptides with improved therapeutic pharmacodynamics with predictable and essentially homogeneous structures that can be easily reproduced over and over again. do.

Fortunately, Factor IX peptides with improved pharmacodynamics and methods of making them are currently found. In addition to the Factor IX peptides with improved pharmacodynamics, the present invention also provides industrially practical and cost effective methods for the production of these Factor IX peptides. Factor IX peptides of the invention include modifying groups such as PEG moieties, therapeutic moieties, biomolecules and the like. Accordingly, the present invention satisfies the need for Factor IX peptides with improved therapeutic efficacy and improved pharmacodynamics for the treatment of conditions and diseases in which Factor IX provides an effective treatment.

In accordance with the present invention, it has been found that controlled modification of Factor IX with one or more poly (ethylene glycol) moieties provides novel Factor IX derivatives with improved pharmacodynamic properties. In addition, a reliable and cost-effective method of preparing the modified Factor IX peptides of the present invention has been discovered and developed.

In one aspect, the invention provides a Factor IX peptide comprising a moiety of the formula:

In the above formula, D is -OH or R ¹ -L-HN-. Symbol G represents R ¹ -L- or —C (O) (C ₁ -C ₆ ) alkyl. R ¹ is a site comprising a straight or branched poly (ethylene glycol) residue; L is a linker which is a member selected from bonded, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl. Generally, if D is OH, G is R ¹ -L-, and if G is -C (O) (C ₁ -C ₆ ) alkyl, D is R ¹ -L-NH-. As will be appreciated by those skilled in the art, COOH in the sialic acid homologs described herein also represents COO ^- or a salt thereof.

In another aspect, the present invention provides a method for preparing PEG-ylation factor IX comprising the above site. The method of the present invention comprises (a) contacting a substrate factor IX peptide with an enzyme that transfers the PEG-sialic acid donor, and PEG-sialic acid, onto an amino acid or glycosyl residue of the Factor IX peptide under conditions appropriate for the transfer. It comprises the step of. Examples of PEG-sialic acid donor sites have the formula:

In one embodiment, the host is a mammalian cell. In other embodiments, the host cell is an insect cell, plant cell, bacterium or fungus.

In another aspect, the present invention provides a method for treating a condition in a subject in need of treatment of a condition characterized by impaired coagulation. The method comprises administering to the subject an amount of a Factor IX peptide conjugate of the invention effective to ameliorate the condition in the subject. An example of a disease that can be treated by this method is hemophilia.

In another aspect, the invention provides a pharmaceutical formulation comprising a Factor IX peptide of the invention and a pharmaceutically acceptable carrier.

Still other objects and advantages of the invention will be apparent to those skilled in the art from the following detailed description.

1 is Asn 157, Asn 167; At Ser 53, Ser 61, Thr 159, Thr 169, and Thr 172 are structures of Factor IX that indicate the presence and location of potential glycosylation sites.

FIG. 2 is a diagram showing an exemplary embodiment of the invention wherein carbohydrate residues on Factor IX peptides have been remodeled and glycopezylated: (A) Sialic acid sites are removed by sialidase and resulting galactose residues Glycofezilated by the sialic acid derivative of FIG. 5; (B) the mannose residue is glycopeptylated by sialic acid PEG; (C) the sialic acid moiety of N-glycan is glycopeptylated by sialic acid PEG; (D) the sialic acid moiety of O-glycans is glycopeptylated by sialic acid PEG; (E) SDS PAGE gel of Factor IX from 2 (A); (F) SDS PAGE gel of Factor IX from the reaction producing 2 (C) and 2 (D).

FIG. 3 is a plot comparing the in vivo residence life of aglycosylation factor IX and enzymatically glycopeptylated factor IX.

4 is a table comparing the activity of the species shown in FIG.

5 is the amino acid sequence of factor IX.

FIG. 6 graphically depicts the pharmacodynamic properties of various glycofezylation factor IX molecules as compared to non-pezylation factor IX.

7 is a table of representative modified sugar classes for use in the present invention.

8 is a table of representative modified sugar classes for use in the present invention.

FIG. 9 is a table of sialyl transferases used to transfer modified sialic acid sites and unmodified sialic acid sites, as described herein, onto an acceptor.

Abbreviation

PEG, poly (ethylene glycol); PPG, poly (propylene glycol); Ara, arabinosyl; Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl; GalNAc, N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc, N-acetylglucosaminyl; Man, mannosyl; ManAc, mannosaminyl acetate; Xyl, xyloxyl; And NeuAc, sialyl (N-acetylneuraminil); M6P, mannose-6-phosphate; Sia, sialic acid, N-acetylneuraminil, and derivatives and homologues thereof.

Justice

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used in the present invention and the laboratory methods of cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization are well known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Techniques and methods are generally described in the various fields of the art and throughout this specification [General Reference: Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is hereby incorporated by reference. The nomenclature used herein and the laboratory methods of analytical chemistry and organic synthesis described below are well known and commonly used in the art. Standard techniques or variants thereof are used for chemical synthesis and chemical analysis.

All oligosaccharides described herein have names or abbreviations for non-reducing saccharides (ie Gal) followed by glycoside bonds (α or β), ring bonds (1 or 2), reducible saccha contained in the bonds. The ring position of the ride, followed by the name or abbreviation of the reducing saccharide (ie, GlcNAc). Each saccharide is preferably pyranose. For a review of standard glycobiology nomenclature, see Essentials of Glycobiology Varki et al. eds. CSHL Press (1999).

Oligosaccharides are believed to have reducing and non-reducing ends, whether or not the saccharide at the reducing end is actually a reducing sugar. According to the nomenclature adopted, oligosaccharides are here expressed as having non-reducing ends on the left and reducing ends on the right.

The term "sialic acid" means all members of the family of 9-carbon carboxylated sugars. The most common members of the sialic acid family are N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononeupyranoz-1- On acid (often abbreviated as Neu5Ac, NeuAc, or NANA). The second member of this family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), where the N-acetyl group of NeuAc is hydroxylated. The third sialic acid family member is 2-keto-3-deoxy-nonurosonic acid (KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265: 21811-21819 (1990). Furthermore, 9-O-C1-C6 acyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-de, such as 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac Also included are 9-substituted sialic acids such as oxy-Neu5Ac. A review of the sialic acid family is described, for example, in Varki, Glycobiology 2: 25-40 (1992); Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New York (1992)). The synthesis and use of sialic acid compounds in the sialylation method is described in international application WO 92/16640, published October 1, 1992.

"Peptide" means a polymer in which the monomer is an amino acid, linked via an amide bond, and in other words called a polypeptide. In addition, non-natural amino acids such as β-alanine, phenylglycine and homoarginine are also included. Non-genetically encoded amino acids can also be used in the present invention. In addition, amino acids modified to include reactive groups, glycosylation sites, polymer therapeutic sites, biomolecules and the like can also be used in the present invention. All amino acids used in the present invention may be d- or l-isomers. 1-isomers are generally preferred. In addition, other peptidomimetics are also useful in the present invention. As used herein, "peptide" refers to both glycosylated and aglycosylated peptides. Also included are peptides that are incompletely glycosylated by a system that expresses the peptide. General reviews are described in Patola, A. F., in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).

The term “peptide conjugate” means a species of the invention wherein the peptide is conjugated with a modified sugar as described herein.

The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid analogs that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are not only those encoded by the genetic code, but also later modified amino acids such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid homologs are compounds having the same basic chemical structure as the naturally occurring amino acids, i.e., α carbon, carboxyl group, amino group and R group bound to hydrogen, for example homoserine, norleucine, methionine sulfoxide, methionine Mean methyl sulfonium. Such homologues have a modified R group (eg noroicine) or a modified peptide backbone, but retain the same basic chemical structure as the naturally occurring amino acids. An amino acid analogue refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid, but which acts in a similar manner to naturally occurring amino acids. As used herein, "amino acid" means both the D- and L-isomers of an amino acid, and mixtures of these two isomers, whether they are as linkers or as components of peptide sequences.

As used herein, the term "modified sugar" refers to naturally- and non-naturally-present carbohydrates that are enzymatically added onto glycosyl residues of amino acids, or peptides in the methods of the invention. it means. Modified sugars include sugar nucleotides (mono-, di- and tri-phosphates), activated sugars (e.g., glycosyl halides, glycosyl mesylate) and sugars that are neither activated nor nucleotides, provided they are But not limited to a plurality of enzyme substrates. A "modified sugar" is covalently functionalized by a "modifying group". Useful modifying groups include, but are not limited to, PEG moieties, therapeutic sites, diagnostic sites, biomolecules, and the like. The modifying group is preferably a naturally occurring or unmodified carbohydrate. The site of functionalization by the modifying group is chosen so that it does not interfere with the enzymatic addition of the "modified sugar" to the peptide.

The term "water soluble" means a site having some detectable degree of solubility in water. Methods for detecting and / or quantifying water solubility are well known in the art. Examples of water soluble polymers include peptides, saccharides, poly (ethers), poly (amines), poly (carboxylic acids) and the like. The peptide may have a mixed sequence composed of a single amino acid, for example poly (lysine). Poly (ethylene imine) is an example of a polyamine, and poly (acrylic) acid is a representative poly (carboxylic acid).

The polymer backbone of the water soluble polymer may be poly (ethylene glycol) (ie PEG). However, other related polymers are also suitable for use in the practice of the present invention, and it should be understood that the use of the term PEG or poly (ethylene glycol) is intended to be included in this respect and is not intended to be exclusive. . The term PEG is alkoxy PEG, bifunctional PEG, PEG with multiple arms, bifurcated PEG, branched PEG, pendant PEG (ie PEG with one or more functional groups suspended from the polymer backbone or related polymers). ), Or all forms of poly (ethylene glycol), including PEG with degradable bonds.

The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. In general, a branched polymer has a branch core site in the center, and a plurality of linear polymer chains connected to the central branch core. PEG is commonly used in branched form, which can be prepared by adding ethylene oxide to various polyols such as glycerol, pentaerythritol and sorbitol. The central branch core can also be derived from several amino acids, such as lysine. Branched poly (ethylene glycol) can be represented by the general formula R (-PEG-OH) _m , where R represents a core site, such as glycerol or pentaerythritol, and m represents the number of arms. PEG molecules with multiple arms, such as those described in US Pat. No. 5,932,462, incorporated herein by reference in its entirety, may also be used as polymer backbone.

Many other polymers are also suitable for the present invention. Particularly useful in the present invention are polymer backbones having 2 to about 300 termini, which are non-peptidyl and water soluble. Examples of suitable polymers include poly (propylene glycol) (“PPG”), other poly (alkylene glycols) such as copolymers of ethylene glycol and propylene glycol, poly (oxyethylated polyols), poly (olefinic alcohols), poly (Vinylpyrrolidone), poly (hydroxypropylmethacrylamide), poly (α-hydroxy acid), poly (vinyl alcohol), polyphosphazene, polyoxazoline, poly (N-acryloylmorpholine) Such as, but not limited to, those described in US Pat. No. 5,629,384, incorporated herein by reference, and copolymers, terpolymers, and mixtures thereof. The molecular weight of each chain of the polymer backbone can vary, but this generally ranges from about 100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da.

As used herein in connection with administering a peptide drug to a patient, "subsurface area" or "AUC" is defined as the total area below the curve representing the concentration of the drug in the large circulation in the patient as a function of time from zero to infinity. do.

The term "half life" or "t½" as used herein in connection with administering a peptide drug to a patient is defined as the time required to halve the plasma concentration of the drug in the patient. Here, there may be one or more half-lives associated with the peptide drug according to a number of vacuum cleaners, redistributions and other mechanisms well known in the art. Typically, alpha and beta half-lives are defined such that alpha phase is associated with redistribution and beta phase is associated with clearance. However, for protein drugs that are mostly limited to blood flow, there may be at least two clearance half-lives. For some glycosylated peptides, rapid beta phase clearance may be mediated through macrophages or receptors on endothelial cells that recognize terminal galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose or fucose. Can be. Slower beta phase clearance may occur by renal glomerular filtration in the case of molecules having specific or non-specific uptake and metabolism within an effective radius of <2 nm (about 68 kD) and / or tissue. Glycopezilation can cap terminal sugars (eg, galactose or N-acetylgalactosamine) and thus block alpha phase clearance through receptors that recognize these sugars. It also provides a larger effective radius, which can extend the late beta phase by reducing the volume of distribution and tissue uptake. Thus, the precise effect of glycopelation on alpha phase and beta phase half-life may vary depending on the size, state, and other parameters of glycosylation, as is well known in the art. Further description of "half-life" can be found in Pharmaceutical Biotechnology (1997, DFA Crommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp 101-120).

As used herein, the term “glycoconjugation” refers to enzymatically mediated conjugation of a modified sugar type to an amino acid or glycosyl residue of a peptide, eg, a Factor IX peptide substrate. The subgenus of “glycoconjugation” is that the modified group of the modified sugar is poly (ethylene glycol), and alkyl derivatives thereof (eg m-PEG) or reactive derivatives thereof (eg H ₂ N-PEG, HOOC-PEG), "glyco-pezylation".

The terms “large scale” and “industrial-scale” are used interchangeably and at least about 250 mg, preferably about 500 mg, more preferably at least about gram of glycoconjugate at the completion of a single reaction cycle. Means a reaction fraud to produce.

As used herein, the term “glycosyl linking group” refers to a glycosyl residue to which a modifying group (eg, PEG moiety, therapeutic moiety, biomolecule) is covalently attached, and glycosyl linking The group connects the deformable group to the rest of the conjugate. In the methods of the invention, a "glycosyl linking group" is covalently attached to a glycosylated or aglycosylated peptide to link the agent to amino acids and / or glycosyl residues on the peptide. A "glycosyl linking group" is generally derived from a "modified sugar" by enzymatic attachment of a "modified sugar" to amino acids and / or glycosyl residues of the peptide. The glycosyl linking group may be a saccharide-derived structure that degrades during the formation of a modified group-modified sugar cassette (e.g., oxidation → Schiff base formation → reduction), or the glycosyl linking group is It may be intact. An "intact glycosyl linking group" refers to a saccharide monomer that links the modifying group to the rest of the conjugate, for example, from an unoxidized glycosyl site that is degraded by, for example, sodium metaperiodate. Refers to a group of connections derived. "Intact glycosyl linking groups" of the invention can be derived from naturally occurring oligosaccharides by adding glycosyl unit (s) or by removing one or more glycosyl units from the parent saccharide structure.

As used herein, the term "targeting moiety" refers to a species that selectively internationalizes to a specific tissue or part of the body. Localization is mediated by specific recognition of molecular determinants, molecular size of targeting agents or conjugates, ionic interactions, hydrophobic interactions, and the like. Other mechanisms for targeting medicaments to specific tissues or parts are known to those of skill in the art. Examples of targeting sites include antibodies, antibody fragments, transferrins, HS-glycoproteins, aggregate factors, serum proteins, β-glycoproteins, G-CSF, GM-CSF, M-CSF, EPO, serum proteins (eg, factors VII, VIIa, VIII, IX, and X) and the like.

As used herein, "therapeutic site" means all agents useful for treatment, including but not limited to antibodies, anti-inflammatory agents, anti-tumor agents, cytotoxins and radiopharmaceuticals. “Therapeutic site” includes prodrugs of bioactive agents, constructs in which one or more therapeutic sites are bound to a carrier, eg, multivalent agents. Therapeutic sites also include proteins and constructs containing the proteins. Examples of proteins include granulocyte colony stimulator (GCSF), granulocyte macrophage colony stimulator (GMCSF), interferon (eg interferon-α, -β, -γ), interleukin (eg interleukin II), serum Proteins (eg, factors VII, VIIa, VIII, IX, and X), human chorionic gonadotropin (HCG), follicle stimulating hormone (FSH) and luteinizing hormone (LH) and antibody fusion proteins (eg Tumor necrosis factor receptor ((TNFR) / Fc region fusion protein)), but is not limited to these.

As used herein, a "pharmaceutically acceptable carrier" includes any substance that, when combined with a conjugate, maintains the conjugate's activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil / water emulsions, and various types of wetting agents. Other carriers may also include sterile solutions, tablets including coated tablets, and capsules. Generally, such carriers are starches, milk, sugars, certain types of clays, gelatin, stearic acid or its salts, magnesium or calcium stearate, talc, vegetable fats or oils, rubber, glycols or other known Excipients such as excipients. Such carriers may also include flavoring and coloring additives, or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods.

As used herein, "administration" refers to oral administration to a subject, administration as a suppository, local contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, or sustained release device, eg For example, it means the implantation of a mini-osmotic pump. Administration can be by any route including parenteral and transmucosal (eg, orally, intranasally, vaginally, rectally or transdermally). Parenteral administration includes, for example, intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Moreover, where the injection is for treating a tumor, eg, inducing apoptosis, administration may be directly to the tumor and / or into the tissue surrounding the tumor. Other modes of delivery include, but are not limited to, the use of liposome preparations, intravenous infusions, transdermal patches, and the like.

The term "improvement" or "improvement" refers to any indication of success in treating a pathology or condition, including objective or subjective parameters such as alleviation, loss or reduction of symptoms, or the physical or mental well-being of a patient. It means improvement. Improvement of symptoms may be based on objective or subjective parameters, including results of physical examinations and / or psychiatric evaluations.

The term "therapy" may be predisposed to a disease, but prevents the appearance of the disease or condition (prophylactic treatment) in animals that do not yet experience or exhibit symptoms of the disease (prophylactic treatment), inhibits the disease (slows or stops its occurrence). Means "treating" or "treatment" of the disease or condition, including providing relief of symptoms or side effects of the disease (relaxing treatment), and alleviating the disease (causing regression of the disease). do.

The term "effective amount" or "effective amount for" or "therapeutically effective amount" or grammatically equivalent, means any amount sufficient to achieve treatment for a disease when administered to an animal to treat the disease.

The term "isolated" means a substance that is substantially or essentially free of components used to produce the substance. In the case of the peptide conjugates of the present invention, the term "isolated" means a substance that is substantially or essentially free of components that accompany the substances in the mixture typically used to prepare the peptide conjugate. "Isolated" and "pure" are used interchangeably. In general, the isolated peptide conjugates of the present invention have a level of purity that is preferably expressed in a range. The lower limit of purity for the peptide conjugate is about 60%, about 70% or about 80% and the upper limit of purity is about 70%, about 80%, about 90% or about 90% or more.

If the peptide conjugates are at least about 90% pure, their purity is also preferably expressed in ranges. The lower limit of the range of purity is about 90%, about 92%, about 94%, about 96% or about 98%. The upper limit of the range of purity is about 92%, about 94%, about 96%, about 98% or about 100% purity.

Purity is determined by any method of analysis known in the art (eg, band strength on silver stained gels, polyacrylamide gel electrophoresis, HPLC, or similar means).

As used herein, "essentially each member of the population" describes the characteristics of the population of peptide conjugates of the invention in which selected percentages of the modified sugars added to the peptide are added to multiple identical receptor sites on the peptide. “Essentially each member of the population” refers to “uniformity” of the site on the peptide conjugated to the modified sugar, which is at least about 80%, preferably at least about 90%, more preferably at least about 95 By% uniform the conjugate of the invention is meant.

"Homogeneity" means structural consistency across a population of receptor sites at which modified sugars are conjugated. Thus, in the peptide conjugates of the invention where each modified sugar site is conjugated to a receptor site having the same structure as the receptor site to which all other modified sugars are conjugated, the peptide conjugate is said to be about 100% homogeneous. do. Uniformity is generally expressed in ranges. The lower limit of the uniformity range for the peptide conjugate is about 60%, about 70% or about 80% and the upper limit of purity ranges from about 70%, about 80%, about 90% or about 90%.

If the peptide conjugates are about 90% or more homogeneous, their uniformity is also preferably expressed in a range. The lower limit of the range of uniformity is about 90%, about 92%, about 94%, about 96% or about 98%. The upper limit of the range of purity is about 92%, about 94%, about 96%, about 98% or about 100% uniformity. Uniformity of the peptide conjugate is generally one or more methods known to those skilled in the art, for example, liquid chromatography-mass spectrometry (LC-MS), matrix of flight spectrometry. Assisted laser desorption mass time (MALDITOF), capillary electrophoresis and the like. The above considerations are equally appropriate for other O-glycosylation and N-glycosylation sites.

"Substantially uniform glycoform" or "substantially uniform glycosylation pattern" when referring to a glycopeptide species is glycosylated by an important glycosyltransferase (eg, fucosyltransferase). It means the percentage of the receptor site. For example, in the case of α1,2 fucosyltransferase, substantially all (as defined below) Galβ1,4-GlcNAc-R and sialylated homologues thereof are substantially fucosylated in the peptide conjugates of the present invention. There is a uniform fucosylation pattern. It will be appreciated by those skilled in the art that the starting material may contain glycosylated receptor sites (eg, fucosylated Galβ1,4-GlcNAc-R sites). Thus, the calculated percent glycosylation will include glycosylated receptor sites and glycosylated receptor sites already in the starting material by the method of the present invention.

The term "substantially" in the above definition of "substantially uniform" generally refers to at least about 40%, at least about 70%, at least about 80%, or more preferably at least about, about a receptor site for a particular glycosyltransferase. 90%, even more preferably at least about 95% is glycosylated. For example, if the Factor IX peptide conjugate comprises a Ser linked glycosyl residue, then at least about 70%, 80%, 90%, 95%, 97%, 99%, 99.2%, 99.4%, 99.6 of the peptides in the population %, Or more preferably 99.8% will have the same glycosyl residue covalently linked to the same Ser residue.

Where substituent groups are embodied by their usual formula written from left to right, they also include chemically identical substituents which may be represented by writing from right to left, for example, -CH ₂ O- OCH ₂ -is also intended to explain.

The term "alkyl" by itself or as part of another substituent, unless stated otherwise, refers to the number of designated carbon atoms which may be fully saturated, mono- or polyunsaturated and may include divalent and polyvalent radicals. Having (ie, C ₁ -C ₁₀ means 1 to 10 carbons) means straight or branched chain or cyclic hydrocarbon radicals or combinations thereof. Examples of saturated hydrocarbon radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, secondary-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, for example Examples include, but are not limited to, groups such as homologues and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl) , Ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers are included, but are not limited to these. The term "alkyl", unless otherwise indicated, is also meant to include derivatives of alkyl as defined in more detail below, such as "heteroalkyl". Alkyl groups that are limited to hydrocarbon groups are referred to as "homoalkyl".

The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkane, exemplified by, but not limited to, —CH ₂ CH ₂ CH ₂ CH ₂ —, Further include the groups described below as "heteroalkylene". In general, alkyl (or alkylene) groups can have from 1 to 24 carbon atoms, with those groups having 10 or less carbon atoms being preferred in the present invention. "Lower alkyl" or "lower alkylene" is generally a shorter chain alkyl or alkylene group having 8 or less carbon atoms.

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their usual concepts and refer to alkyl groups attached to the remainder of the molecule, respectively, via an oxygen atom, an amino group, or a sulfur atom. do.

The term “heteroalkyl” by itself or in combination with another term, unless otherwise stated, is a stable straight chain composed of a specified number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S Or branched chain, or cyclic hydrocarbon radical, or a combination thereof, wherein nitrogen and sulfur atoms can be optionally oxidized, and nitrogen heteroatoms can be optionally quaternized. The heteroatom (s) O, N and S and Si may be placed anywhere within the heteroalkyl group or at the position at which the alkyl group is attached to the rest of the molecule. Examples thereof include -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N (CH ₃ ) -CH ₃ , -CH ₂ -S-CH ₂ -CH ₃ , -CH ₂ -CH ₂ , -S (O) -CH ₃ , -CH ₂ -CH ₂ -S (O) ₂ -CH ₃ , -CH = CH-O-CH ₃ , -Si ( CH 3) ₃ , —CH ₂ —CH═N—OCH ₃ , and —CH═CH—N (CH ₃ ) —CH ₃ , including but not limited to. For example, up to _two heteroatoms, such as —CH ₂ —NH—OCH ₃ and —CH ₂ —O—Si (CH 3) ₃ , may be present in series. Similarly, the term “heteroalkylene”, as such or as part of another substituent, refers to —CH ₂ —CH ₂ —S—CH ₂ —CH ₂ — and —CH ₂ —S—CH ₂ —CH ₂ —NH— By exemplified by CH _2- , but not limited to, divalent radicals derived from heteroalkyl. In the case of heteroalkylene groups, heteroatoms may also occupy either or both of the chain termini (eg, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino and the like). In addition, in the case of alkylene and heteroalkylene linking groups, the orientation of the linking groups is not implied by the direction in which the formula of the linking group is written. For example, the formula -C (O) ₂ R'- represents both -C (O) ₂ R 'and -R'C (O) _2- .

The terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms refer to cyclic variants of "alkyl" and "cycloalkyl", respectively, unless stated otherwise. In addition, in the case of heterocycloalkyl, the heteroatom may occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyls include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morph Polyyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. However, they are not limited thereto.

The term "halo" or "halogen" by themselves or as part of another substituent means a fluorine, chlorine, bromine or iodine atom unless stated otherwise. In addition, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C4) alkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like, but these It is not limited to.

Unless stated otherwise, the term "aryl" refers to a polyunsaturated aromatic substituent which may be a single ring or multiple rings (preferably one to three rings) fused together or covalently linked. The term "heteroaryl" means an aryl group (or ring) containing 1 to 4 heteroatoms selected from N, O and S, wherein nitrogen and sulfur atoms are optionally oxidized, and nitrogen atom (s) are optionally 4 It is expedited. Heteroaryl groups may be attached to the rest of the molecule via heteroatoms. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- Imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5 Isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4- Pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolinyl, 5-isoquinolinyl, 2- Quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazolyl, benzo [b] furanyl, benzo [b] thienyl, 2,3-dihydrobenzo [1,4] dioxine6-yl, Benzo [1,3] dioxol-5-yl and 6-quinolyl. Substituents for each of the aryl and heteroaryl ring systems set forth above are selected from the group of acceptable substituents described below.

In summary, the term “aryl” (eg, aryloxy, arylthioxy, arylalkyl) when used in combination with other terms includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" means that the aryl group is a carbon atom (eg methylene group), for example an oxygen atom (eg phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naph) Radicals (eg, benzyl, phenethyl, pyridylmethyl, etc.) attached to an alkyl group including an alkyl group substituted by butyloxy) propyl, etc.).

Each of the above terms (eg, "alkyl", "heteroalkyl", "aryl" and "heteroaryl") is meant to include both substituted and unsubstituted forms of the radicals indicated. Preferred substituents for each radical type are given below.

Substituents for alkyl and heteroalkyl radicals (often including groups called alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) Generally referred to as "alkyl group substituents", they are -OR ', = O, = NR', = N of a number ranging from 0 to (2m '+ 1), where m' is the total number of carbon atoms of these radicals -OR ', -NR'R ", -SR', -halogen, -SiR'R" R '", -OC (O) R', -C (O) R ', -CO ₂ R', -CONR 'R', -OC (O) NR'R ", -NR" C (O) R ', -NR'C (O) NR "R'", -NR "C (O) ₂ R ', -NR -C (NR'R "R '") = NR "", -NR-C (NR'R ") = NR'", -S (O) R ', -S (O) ₂ R', -S (O) ₂ NR'R ", -NRSO ₂ R ', -CN and -NO ₂ may be one or more of various groups selected from, but not limited to. Each of R ′, R ″, R ′ ″ and R ″ ″ is preferably independently hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, eg, aryl substituted with 1-3 halogens , Substituted or unsubstituted alkyl, alkoxy or thioalkoxy group, or arylalkyl group. Where the compounds of the present invention comprise more than one R group, for example, each R group is each of these groups where at least one of the groups R ', R ", R'" and R "" is present Are independently selected. When R 'and R "are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 5-, 6- or 7-membered ring. For example, -NR'R" is 1-blooded. It includes, but is not limited to, lollidinyl and 4-morpholinyl. From the above review of substituents, a person skilled in the art will appreciate that the term "alkyl" refers to haloalkyl (eg -CF ₃ and -CH ₂ CF ₃ ) and acyl (eg -C (O) It will be understood that it is meant to include groups containing carbon atoms bonded to groups other than hydrogen atoms, such as CH ₃ , -C (O) CF ₃ , -C (O) CH ₂ OCH _3, etc.).

Similar to the substituents described for alkyl radicals, substituents for aryl and heteroaryl groups are generally referred to as "aryl group substituents". Substituents may be, for example, halogens in the range from 0 to the total number of open valences on the aromatic ring system, -OR ', = O, = NR', = N-OR ', -NR'R " , -SR ', -halogen, -SiR'R "R'", -OC (O) R ', -C (O) R', -CO ₂ R ', -CONR'R ", -OC (O) NR'R ", -NR" C (O) R ', -NR'-C (O) NR "R'", -NR "C (O) ₂ R ', -NR-C (NR'R" R '") = NR"", -NR-C (NR'R") = NR'", -S (O) R ', -S (O) ₂ R', -S (O) ₂ NR'R" From -NRSO ₂ R ', -CN and NO ₂ , -R' -N ₃ , -CH (Ph) ₂ , fluoro (C ₁ -C ₄ ) alkoxy, and fluoro (C ₁ -C ₄ ) alkyl Wherein R ', R ", R'" and R "" are preferably hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted Independently selected from heteroaryl. Where the compounds of the present invention comprise more than one R group, for example, each R group is each of these groups where at least one of the groups R ', R ", R'" and R "" is present Are independently selected. In the following schemes, the symbol X represents "R" as described above.

Two substituents on adjacent atoms of an aryl or heteroaryl ring may be optionally substituted by substituents of the formula -TC (O)-(CRR ') qU-, wherein T and U are independently -NR-, -O- , -CRR'- or a single bond, q is an integer of 0-3. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may be optionally substituted by substituents of the formula -A- (CH ₂ ) rB-, wherein A and B are independently -CRR'-, -O -, -NR-, -S-, -S (O)-, -S (O) _2- , -S (O) ₂ NR'- or a single bond, r is an integer of 1 to 4. One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may be optionally substituted by substituents of the formula-(CRR ') sX- (CR "R'") d-, wherein s and d are independently Is an integer from 0 to 3, and X is -O-, -NR'-, -S-, -S (O)-, -S (O) _2- , or -S (O) ₂ NR'-. Substituents R, R ', R "and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (C1-C6) alkyl.

As used herein, the term “heteroatom” means to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

As mentioned above, factor IX is important in the blood coagulation stage. The structure and sequence of factor IX are shown in FIG. 1. Deficiency of factor IX in the body specifies the type of hemophilia (type B). Treatment of this disease is typically limited to intravenous transfusion of human plasma protein concentrate of Factor IX. However, in addition to the practical disadvantages of time and cost, transfusions of blood concentrates include the risk of transmitting viral hepatitis, acquired immunodeficiency syndrome, or thromboembolic disease to recipients.

Although Factor IX itself has proven to be an important and useful compound for therapeutic applications, the proposed method of producing Factor IX from recombinant cells (US Pat. No. 4,770,999) is rather shorter biological half-life, and potentially immunogenic, It provides a product with an incorrect glycosylation pattern that can cause loss of function, increased need for both larger doses and more frequent dosing to achieve the same effect.

In order to improve the effectiveness of recombinant factor IX used for therapeutic purposes, the present invention provides glycosylated and aglycosylated factor IX peptides and polymers such as PEG (m-PEG), PPG (m-PPG) and the like. Provide a conjugate. This conjugate may be further or instead modified by additional conjugation with various species, such as therapeutic sites, diagnostic sites, targeting sites, and the like.

Conjugates of the invention are formed by enzymatic attachment of modified sugars to glycosylated or aglycosylated peptides. Glycosylation sites and glycosyl residues provide a location for conjugating the modifying group to the peptide, eg, by glycoconjugation. Examples of deformable groups are water soluble polymers such as poly (ethylene glycol), for example methoxy-poly (ethylene glycol). Modification of the Factor IX peptide may improve the stability and retention time of recombinant Factor IX in the patient's circulation and / or reduce the antigenicity of recombinant Factor IX.

The method of the present invention makes it possible to assemble a glycopeptide with a peptide having a substantially uniform derivatization pattern. The enzymes used in the present invention are generally selective for specific amino acid residues, combinations of amino acid residues, or for specific glycosyl residues of the peptide. This method is also practical for large scale production of modified peptides and glycopeptides. Thus, the method of the present invention provides a practical means for the large scale preparation of glycopeptides having a preselected uniform derivatization pattern.

The present invention also provides conjugates of glycosylated and aglycosylated peptides with increased therapeutic half-life due to, for example, reduced clearance or reduced uptake by the immune system or retinal endothelial system (RES). Moreover, the methods of the present invention provide a means for reducing or eliminating a host immune response to a peptide by masking antigenic determinants on the peptide. Selective attachment of targeting agents may also be used to target peptides to specific tissue or cell surface receptors that are specific for a particular targeting agent.

Conjugate

In a first aspect, the present invention provides a conjugate between a selected modifying group and a Factor IX peptide.

The linkage between the peptide and the modifying group includes a glycosyl linkage group inserted between the peptide and the selected site. As discussed herein, the selected site is essentially any species capable of producing a "modified sugar" that is recognized by a suitable transferase enzyme that is attached to the saccharide unit to add a modified sugar on the peptide. . The saccharide component of the modified sugar when inserted between the peptide and the selected site is a "glycosyl linking group", eg, an "intact glycosyl linking group". Glycosyl linking groups are formed from all mono- or oligo-saccharides that are substrates for enzymes that, after modification by a modifying group, add a modified sugar to an amino acid or glycosyl residue of the peptide.

The glycosyl linking group may be or include a saccharide site that is degradatively modified before or during addition of the modifying group. For example, glycosyl linking groups are derived from saccharide residues produced by oxidative degradation of the corresponding aldehyde, for example, by the action of metaperiodate, followed by the appropriate amine to the Schiff base. Can be converted, which is then reduced to the corresponding amine.

Examples of conjugates of the invention correspond to structures of the formula:

In which the symbols a, b, c, d and s represent a nonzero positive integer; t is zero or a positive integer. “Pharmaceuticals” are therapeutic agents, bioactive agents, detectable labels, water soluble sites (eg, PEG, m-PEG, PPG, and m-PPG) and the like. A "pharmaceutical" can be a peptide, eg, an enzyme, an antibody, an antigen, or the like. The linker may be any of the following broad arrays of link groups. Alternatively, the linker may be a single bond or a "zero order linker".

In an exemplary embodiment, the selected modifying group is a water soluble polymer, eg, m-PEG. The water soluble polymer is covalently attached to the peptide via a glycosyl linking group. Glycosyl linking groups are covalently attached to amino acid residues or glycosyl residues of the peptide. The invention also provides conjugates in which the amino acid residues and glycosyl residues are modified by glycosyl linking groups.

Examples of water soluble polymers are poly (ethylene glycol) such as methoxy-poly (ethylene glycol). The poly (ethylene glycol) used in the present invention is not limited to any particular form or molecular weight range. In the case of unbranched poly (ethylene glycol) molecules the molecular weight is preferably between 500 and 100,000. Molecular weights of 2,000-60,000 daltons are preferably used, more preferably about 5,000 to about 30,000 daltons.

In another embodiment, the poly (ethylene glycol) is a branched PEG with one or more PEG moieties attached. Examples of branched PEGs are described in US Pat. No. 5,932,462; US Patent No. 5,342,940; U.S. Patent 5,643,575; US Patent No. 5,919,455; US Patent No. 6,113,906; US Patent No. 5,183,660; WO 02/09766; Kodera Y., Bioconjugate Chemistry 5 : 283-288 (1994); And Yamasaki et al., Agric. Biol. Chem., 52 : 2125-2127, 1998. Additional useful branched polymer species are described herein.

In a preferred embodiment, the molecular weight of each poly (ethylene glycol) of the branched PEG is about 2,000, 5,000, 10,000, 15,000, 20,000, 40,000, 50,000 and 60,000 daltons or more.

In addition to providing conjugates formed through enzymatically added glycosyl linking groups, the present invention provides conjugates with very homogeneous substitution patterns thereof. Using the methods of the invention, it is possible to form peptide conjugates wherein essentially all of the modified sugar sites are attached to multiple copies of structurally identical amino acids or glycosyl residues across the population of conjugates of the invention. Accordingly, in a second aspect the present invention provides a peptide conjugate having a population of water soluble polymer moieties covalently bound to the peptide via intact glycosyl linking groups. In a preferred conjugate of the invention, essentially each member of the population is linked to a glycosyl residue of the peptide via a glycosyl linking group, wherein each glycosyl residue of the peptide to which the glycosyl linking group is attached has the same structure .

Also provided are peptide conjugates having a population of water soluble polymer moieties covalently attached via a glycosyl linking group. In a preferred embodiment, essentially all members of the population of water soluble polymer moieties are linked to the amino acid residues of the peptide via a glycosyl linking group, wherein each amino acid residue having a glycosyl linking group attached thereto has the same structure.

The present invention also provides a conjugate similar to that described above wherein the peptide is conjugated to a therapeutic site, a diagnostic site, a targeting site, a toxin site or the like via an intact glycosyl linking group. Each site mentioned above may be a small molecule, a natural polymer (eg polypeptide) or a synthetic polymer. Peptides of the invention comprise at least one N- or O-linked glycosylation site glycosylated by a glycosyl residue comprising a PEG moiety. PEG is covalently attached to Factor IX peptides via intact glycosyl linking groups. Glycosyl linking groups are covalently attached to amino acid residues or glycosyl residues of the Factor IX peptide. Instead, glycosyl linking groups are attached to one or more glycosyl units of the glycopeptide. The invention also provides conjugates wherein the glycosyl linking group is attached to both amino acid residues and glycosyl residues.

In an exemplary embodiment, the Factor IX peptide comprises a moiety having the formula:

In the above formula, D is a member selected from -OH and R ¹ -L-HN-; G is a member selected from R ¹ -L- and -C (O) (C ₁ -C ₆ ) alkyl; R ¹ is a moiety comprising a member of a selected moiety comprising a straight or branched poly (ethylene glycol) moiety; L is a linker that is a member selected from a bond, a substituted or unsubstituted alkyl and a substituted or unsubstituted heteroalkyl, so when D is OH, G is R ¹ -L- and G is -C (O) ( In the case of C ₁ -C ₆ ) alkyl, D is R ¹ -L-NH-.

In one embodiment, R ¹ -L has the structure of:

Wherein a is an integer from 0 to 20.

In an exemplary embodiment, R ¹ has the structure of a member selected from the following formulas:

Wherein e and f are integers independently selected from 1 to 2500; q is an integer of 1-20. In other embodiments, R ¹ has a structure that is a member selected from the formula:

Wherein e, f and f 'are independently integers selected from 1 to 2500; q and q 'are independently integers selected from 1-20.

In another embodiment, the present invention provides a Factor IX peptide conjugate having a structure wherein R ¹ is a member selected from the following formula:

Wherein e, f and f 'are independently integers selected from 1 to 2500; q, q 'and q "are independently integers selected from 1-20.

In another embodiment, R ¹ has a structure that is a member selected from the formula:

Where e and f are independently integers selected from 1 to 2500.

In another exemplary embodiment, the invention provides a peptide comprising a moiety having the formula:

Gal can be attached to an amino acid, or to a glycosyl residue attached directly or indirectly (eg, via a glycosyl residue) to an amino acid.

In another embodiment, the moiety has the structure of:

Gal can be attached to an amino acid, or to a glycosyl residue attached directly or indirectly (eg, via a glycosyl residue) to an amino acid.

In an exemplary embodiment, this structure is associated with glycofezilation of the O-glycosylation site on Factor IX (FIG. 2B).

In yet another exemplary embodiment, the peptide comprises a site according to the formula:

Where AA is the amino acid residue of the peptide and in each of the structures D and G are as described in the present invention.

Examples of amino acid residues of the peptides that can be conjugated to one or more of these species are serine and threonine, for example SEQ. ID. Serine 53 or 61 or threonine 159, 162 or 172 of NO: 1.

In another exemplary embodiment, the present invention provides a Factor IX conjugate comprising a glycosyl residue having the structure of:

Wherein a, b, c, d, i, r, s, t and u are integers independently selected from 0 and 1. The index q is one. The indices e, f, g and h are independently selected from integers from 0 to 6. The indices j, k, l and m are independently selected from integers of 0 and 100. The indices v, w, x and y are independently selected from 0 and 1 and at least one of v, w, x and y is 1. Symbol AA represents the amino acid residue of the Factor IX peptide.

The symbol Sia- (R) represents a group having the structure of:

Wherein D is selected from -OH and R ¹ -L-HN-. Symbol G represents R ¹ -L- or —C (O) (C ₁ -C ₆ ) alkyl. R ¹ represents a site comprising straight or branched poly (ethylene glycol) residues. L is a linker which is a member selected from bonded, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl. Generally, G is R ¹ -L- when D is OH, and D is R ¹ -L-NH- when G is -C (O) (C ₁ -C ₆ ) alkyl.

In another exemplary embodiment, the PEG-modified sialic acid moiety in the conjugate of the invention has the structure:

The index "s" here represents an integer of 0 to 20, and n is an integer of 1 to 2500. In selected embodiments, s is 1 and PEG is about 20 kD.

In yet further exemplary embodiments, the PEG-modified sialic acid has the structure of:

Wherein L is a substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl linker moiety that contends the sialic acid moiety with the PEG moiety.

In an exemplary embodiment wherein the glycosyl moiety has the structure set forth above, it is conjugated to one or both of Asn 157 and 167.

Factor IX was cloned and sequenced. Essentially any Factor IX peptide having any sequence is used as the Factor IX peptide component of the conjugate of the present invention. In an exemplary embodiment, the peptide has the sequence represented by SEQ ID NO: 1 in the present invention:

The invention is not limited in any way to the sequences described herein. Factor IX variants are described, for example, in US Pat. Nos. 4,770,999, 5,521,070, where tyrosine is replaced by alanine in the first position, US Pat. No. 6,037,452, where Factor XI is linked to an alkylene oxide group. ), And US Pat. No. 6,046,380, wherein DNA encoding factor IX is modified at at least one splice site. As demonstrated herein, variants of Factor IX are well known in the art and include the known variants or those that will be developed or discovered in the future.

Methods for measuring the activity of mutants or modified Factor IX are described, for example, in Biggs (1972, Human Blood Coagulation Haemostasis and Thrombosis (Ed. 1), Oxford, Blackwell, Scientific, pg. 614). It can be performed using methods known in the art, such as one stage activated partial thromboplastin time assay as described. Briefly, in order to test the biological activity of Factor IX molecules developed according to the method of the present invention, the test was performed using an equivalent volume of activated partial thromboplastin reagent, sterile blood donation techniques that are well known in the art. Factor IX deficient plasma isolated from a patient with B, and plasma collected normally as a standard, or sample. In this test, one unit of activity is defined as the amount present in one milliliter of normally collected plasma. In addition, tests for biological activity based on the ability of Factor IX to reduce the clotting time of plasma from Factor IX-deficient patients to normal have been described, for example, in Proctor and Rapaport ( Amer . J. Clin. Path . 36 : 212 (1961).

Peptides of the invention comprise at least one N-linked or O-linked glycosylation site, at least one of which is conjugated to a glycosyl residue comprising a PEG site. PEG is covalently attached to the peptide via an intact glycosyl linking group. Glycosyl linking groups are covalently attached to amino acid residues or glycosyl residues of the peptide. Instead, glycosyl linking groups are attached to one or more glycosyl units of the glycopeptide. The invention also provides conjugates wherein the glycosyl linking group is attached to both amino acid residues and glycosyl residues.

The PEG moiety is attached to the intact glycosyl linker either directly or through a non-glycosyl linker, eg, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl.

A modified party

The present invention uses modified sugars and modified sugar nucleotides to form conjugates of modified sugars. In the modified sugar compounds of the present invention, the sugar moiety is preferably a saccharide, deoxy-saccharide, amino-saccharide, or N-acyl saccharide. The terms "saccharide" and its equivalents "saccharyl", "sugar" and "glycosyl" refer to monomers, dimers, oligomers and polymers. Sugar sites are also functionalized by deformable groups. The modifying group is generally conjugated to the sugar moiety via conjugation with an amine, sulfhydryl or hydroxyl, eg, primary hydroxyl, moiety on the sugar. In an exemplary embodiment, the modifying group is attached via an amine moiety on the sugar, eg, through an amide, urethane or urea formed through the reaction of an amine with a reactive derivative of the modifying group.

Any sugar can be used as the sugar core of the conjugate of the present invention. Examples of sugar cores useful for forming the compositions of the present invention include, but are not limited to, glucose, galactose, mannose, fucose and sialic acid. Other useful sugars include amino sugars such as glucosamine, galactosamine, mannosamine, 5-amine homologues of sialic acid, and the like. The sugar core may be a structure that exists in nature or may be modified to provide a site for conjugating the modifying group. For example, in one embodiment the invention provides sialic acid derivatives in which the 9-hydroxy moiety is replaced by an amine. Amines are readily derivatized by activated homologues of the selected modifying groups.

In an exemplary embodiment, the present invention utilizes modified sugar amines having the structure:

Wherein G is a glycosyl moiety, L is a bond or a linker, and R ¹ is a modifying group. An example of a bond is one formed between NH ₂ on the glycosyl moiety and a group of complementary reactivity on the modifying group. Thus, examples of binding include, but are not limited to, NHR ¹ , OR ¹ , SR ^1, and the like. For example, when R ¹ comprises a carboxylic acid moiety, this moiety can be activated and coupled with the NH ₂ moiety on the glycosyl moiety to provide a bond having the structure of formula NHC (O) R ¹ . Similarly, OH and SH groups can be converted to the corresponding ether or thioether derivatives, respectively.

Examples of linkers include alkyl and heteroalkyl moieties. Linkers include linking groups such as acyl-basic linking groups such as -C (O) NH-, -OC (O) NH- and the like. A linking group is a bond formed between component species of the invention, for example, between a glycosyl moiety and a linker (L), or between a linker and a modifying group (R ¹ ). Other linking groups are ethers, thioethers and amines. For example, in one embodiment the linker is an amino acid residue, such as a glycine residue. The carboxylic acid moiety of glycine is converted to the corresponding amide by the action of an amine on the glycosyl moiety, and the amine of the glycine is converted to the corresponding amide or urethane by reaction with the activated carboxylic acid or carbonate of the modifying group.

Another example of a linker is a PEG moiety, or a PEG moiety functionalized by an amino acid residue. PEG is linked to a glycosyl group through amino acid residues at one PEG terminus and to R ¹ through another PEG terminus. Instead, amino acid residues are linked to R ¹ and PEG ends not linked to amino acids are linked to glycosyl groups.

Examples of species for NH-LR ¹ have the formula:

-NH {C (O) (CH ₂ ) _a NH} _s {C (O) (CH ₂ ) _b (OCH ₂ CH ₂ ) _c O (CH ₂ ) _d NH} _t R ¹

Here, the indices s and t are independently 0 or 1. The indices a, b and d are independently integers from 0 to 20, and c is an integer from 1 to 2500. Other similar linkers are based on species in which the -NH moiety is replaced by another group, for example -S, -O or -CH ₂ .

More particularly, the present invention utilizes compounds in which NH-LR ¹ is as follows: NHC (O) (CH ₂ ) _a NHC (O) (CH ₂ ) _b (OCH ₂ CH ₂ ) _c O (CH ₂ ) _d NHR ¹ , NHC (O) (CH ₂ ) _b (OCH ₂ CH ₂ ) _c O (CH ₂ ) _d NH-R ¹ , NHC (O) O (CH ₂ ) _b (OCH ₂ CH ₂ ) _c O (CH ₂ ) _d NHR ¹ , NH (CH ₂ ) _a NHC (O) (CH ₂ ) _b (OCH ₂ CH ₂ ) _c O (CH ₂ ) _d NH-R ¹ , NHC (O) (CH ₂ ) _a NHR ¹ , NH (CH ₂ ) _a NHR ¹ , and NHR ¹ . In these formulae, the indices a, b and d are independently selected from integers from 0 to 20, preferably from 1 to 5. Index c is an integer from 1 to 2500.

In the following description, the invention is described in terms of the use of selected derivatives of sialic acid. Those skilled in the art will appreciate that the focus of the description is to clarify the description, and the described structures and compositions generally refer to saccharide groups, modified saccharide groups, activated modified saccharide groups and modified saccharides. It will be appreciated that it is generally applicable across the kind of conjugates in a group.

In an illustrative embodiment G is sialic acid and the compound selected for use in the present invention has the formula:

As will be appreciated by those skilled in the art, sialic acid sites in the exemplary compounds include, but are not limited to, glucosamine, galactosamine, mannosamine, N-acetyl derivatives thereof, and the like. May be replaced by any other amino-saccharide.

In another illustrative embodiment, the primary hydroxyl site of the sugar is functionalized by a modifying group. For example, the 9-hydroxyl of sialic acid can be converted to the corresponding amine and functionalized to provide the compounds according to the invention. Formulas according to this embodiment include:

In further exemplary embodiments, the present invention utilizes modified sugars in which the 6-hydroxyl position is converted to the corresponding amino moiety having a linker-modified group cassette as set forth above. Examples of saccharyl groups that can be used as cores of these modified sugars include Gal, GalNAc, Glc, GlcNAc, Fuc, Xyl, Man and the like. Representative modified sugars according to the invention have the formula:

Wherein R ³ -R ⁵ and R ⁷ are members independently selected from H, OH, C (O) CH ₃ , NH, and NH C (O) CH ₃ . R ⁶ is OR ¹ , NHR ¹ or NH-LR ¹ as described above.

Selected conjugates useful in the present invention are based on a kind having stereochemistry of mannose, galactose or glucose, or mannose, galactose or glucose. The general formula of these conjugates is as follows:

In another exemplary embodiment, the present invention utilizes a compound as set forth above, activated with the corresponding nucleotide sugar. Examples of sugar nucleotides used in the present invention in their modified forms include nucleotide mono-, di- or triphosphates or their analogs. In a preferred embodiment, the modified sugar nucleotide is selected from UDP-glycoside, CMP-glycoside or GDP-glycoside. Even more preferably, the sugar nucleotide portion of the modified sugar nucleotide is UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid or CMP -NeuAc. In an exemplary embodiment, the nucleotide phosphate is attached to C-1.

Thus, in an exemplary embodiment wherein the glycosyl moiety is sialic acid, the present invention utilizes a compound having the formula:

Where LR ¹ is as set forth above and L ¹ -R ¹ represents a linker bound to a deformable group. As in the case of L, examples of linker species according to L ¹ include linkage, alkyl or heteroalkyl moieties. Examples of modified sugar nucleotide compounds according to these embodiments are shown in FIGS. 7 and 8.

In another exemplary embodiment, the present invention provides a conjugate formed between the modified sugar of the invention and the substrate factor IX peptide. In such embodiments, the sugar moiety of the modified sugar is a glycosyl linking group inserted between the substrate and the modifying group. Examples of glycosyl linking groups include intact glycosyl linkages in which the glycosyl moiety or sites forming the linking group are not degraded by chemical (eg sodium periodate) or enzymatic processes (eg oxidase). Group. Selected conjugates of the invention include modifying groups attached to amine sites such as aminosaccharides such as mannoseamine, glucosamine, galactosamine, sialic acid, and the like. Examples of modifying group-intact glycosyl linking group cassettes according to this motif are based on sialic acid structures such as those having the formula:

R ¹ and L ¹ in the above formula are as described above.

In yet further exemplary embodiments, the conjugate is formed between the substrate factor IX and the saccharyl moiety to which the modifying group is attached via a linker at the 6-carbon position of the saccharyl moiety. Thus, exemplary conjugates according to this embodiment have the formula:

The radicals here are as described above. The skilled practitioner will recognize that the modified saccharyl moiety described above may also be conjugated to the substrate via oxygen or nitrogen atoms at 2, 3, 4, or 5 carbon atoms.

Exemplary compounds useful in this embodiment include compounds having the formula:

Here, the R groups and indices are as described above.

The invention also provides the use of sugar nucleotides modified with LR ¹ at the 6-carbon position. Examples of the kind according to this embodiment include:

Where R group and L represent the sites as described above. The index "y" is zero, one or two.

Additional exemplary nucleotide sugars useful in the present invention are based on a species having stereochemistry of GDP mannose. Examples of the kind according to this embodiment have the formula:

In yet another exemplary embodiment, the present invention provides a conjugate wherein the modified sugar is based on the stereochemistry of UDP galactose. Examples of nucleotide sugars useful in the present invention have the structure:

In another exemplary embodiment, the nucleotide sugar is based on the stereochemistry of glucose. Examples of the kind according to this embodiment have the formula:

The modifying group R ¹ may be any of a number of types, including but not limited to water soluble polymers, water-insoluble polymers, therapeutic agents, diagnostic agents and the like. The nature of the deformable groups illustrated is described in more detail below.

Deformable group

receptivity Polymer

Many water soluble polymers are known to those skilled in the art and are useful in practicing the present invention. The term water soluble polymers includes saccharides (eg, dextran, amylose, hyaluronic acid, poly (sialic acid), heparan, heparin, etc.); Poly (amino acids) such as poly (aspartic acid) and poly (glutamic acid); Nucleic acids; Synthetic polymers (eg, poly (acrylic acid), poly (ether), eg, poly (ethylene glycol); peptides, proteins, etc. The present invention may be carried out using any water soluble polymer). The only constraint is that the polymer must contain a point to which the rest of the conjugate can be attached.

Methods for activating polymers are also described in WO 94/17039, US Pat. No. 5,324,844, WO 94/18247, WO 94/04193, US Pat. No. 5,219,564, US Pat. No. 5,122,614, WO 90/13540, US Pat. No. 5,281,698 No. and also WO 93/15189, and for conjugation between activated polymers and peptides, see, for example, coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrier molecules (US Pat. 4,412,989), ribonucleases and superoxide dismutases (Veronese at al., App. Biochem. Biotech. 11: 141-45 (1985)).

Preferred water soluble polymers are those whose molecular weight is approximately equal in molecular weight to a substantial proportion of the polymer molecules in the polymer sample, and such polymers are “homodisperse”.

The present invention is further described with reference to poly (ethylene glycol) conjugates. Several reviews and papers on the functionalization and conjugation of PEG are available. See, eg, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); And Bhadra, et al., Pharmazie, 57: 5-29 (2002). Pathways for preparing reactive PEG molecules and using the reactive molecules to form conjugates are known in the art. For example, US Pat. No. 5,672,662 discloses active esters of polymeric acids selected from linear or branched poly (alkylene oxides), poly (oxyethylated polyols), poly (olefinic alcohols) and poly (acrylomorpholines). Water-soluble and separable conjugates of are described.

US Pat. No. 6,376,604 describes a process for preparing water-soluble 1-benzotriazolylcarbonate esters of water-soluble and non-peptidyl polymers by reacting the terminal hydroxyl of the polymer with di (1-benzotriazolyl) carbonate in an organic solvent. have. This active ester is used to form conjugates with biologically active agents such as proteins or peptides.

WO 99/45964 describes conjugates comprising an active water-soluble polymer comprising a polymer backbone having at least one end linked to the polymer backbone via a stable bond and a biologically active agent, wherein at least one end is A branching moiety having a proximal reactive group linked to a branching moiety, wherein the biologically active agent is linked to at least one proximal reactive group. Other branched poly (ethylene glycol) is described in WO 96/21469, and US Pat. No. 5,932,462 describes conjugates formed by branched PEG molecules comprising branched ends comprising reactive functional groups. The free reactive group can react with a biologically active species such as a protein or peptide, forming a conjugate between the poly (ethylene glycol) and the biologically active species. US Pat. No. 5,446,090 describes bifunctional PEG linkers and their use in forming conjugates with peptides at each of the PEG linker ends.

Conjugates comprising degradable PEG bonds are described in WO 99/34833; And WO 99/14259 as well as US Pat. No. 6,348,558. Such degradable bonds are applicable in the present invention.

The above-mentioned methods of polymer activation known in the art are for the formation of the branched polymers described herein and for the conjugation of these branched polymers to other kinds, for example, sugars, sugar nucleotides and the like. It is useful in connection with the present invention.

Examples of poly (ethylene glycol) molecules useful in the present invention include, but are not limited to, those having the formula:

Wherein R ⁸ is H, OH, NH ₂ , substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroalkyl For example, acetal, OHC-, H ₂ N- (CH ₂ ) _q −, HS- (CH ₂ ) _q , or — (CH ₂ ) _q C (Y) Z ¹ . Index "e" represents an integer from 1 to 2500. The indices b, d, and q independently represent integers from 0 to 20. Symbols Z and Z ¹ independently represent an OH, NH ₂ , leaving group such as imidazole, p-nitrophenyl, HOBT, tetrazole, halide, SR ⁹ , alcohol portion of activated ester; -(CH ₂ ) _p C (Y ¹ ) V or-(CH ₂ ) _p U (CH ₂ ) _s C (Y ¹ ) _v . The symbol Y represents H (2), = O, = S, = NR ¹⁰ . The symbols X, Y, Y ¹ , A ¹ , and U independently represent sites O, S, NR ¹¹ . Symbol V represents OH, NH ₂ , halogen, SR ¹² , alcohol component of activated ester, amine component of activated amide, sugar-nucleotides and protein. The indices p, q, s and v are members independently selected from integers from 0 to 20. Symbols R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substitution Substituted or unsubstituted heteroaryl.

In still other exemplary embodiments, the poly (ethylene glycol) molecule is selected from the following compounds:

Poly (ethylene glycol) useful for forming the conjugates of the invention are linear or branched. Branched poly (ethylene glycol) molecules suitable for use in the present invention include, but are not limited to, compounds represented by the formula:

Wherein R ⁸ and R ^{8 ′} are members independently selected from the group defined for R ⁸ above. A ¹ and A ² are members independently selected from the group defined for A ¹ above. The indices e, f, o, and q are as described above. Z and Y are as described above. X ¹ and X ^{1 ′} are S, SC (O) NH, HNC (O) S, SC (O) O, O, NH, NHC (O), (O) CNH and NHC (O) O, OC (O Is a member independently selected from NH.

In another exemplary embodiment, the branched PEG is based on a cysteine, serine or di-lysine core. Thus, further examples of branched PEGs include the following compounds:

In another embodiment, the branched PEG moiety is based on a tri-lysine peptide. Tri-lysine may be modo-, di-, tri- or tetra-pezylated. Examples of the kind according to this embodiment have the formula:

Wherein e, f and f 'are independently integers selected from 1 to 2500; q, q 'and q "are independently integers selected from 1-20.

In an exemplary embodiment of the invention, the PEG is m-PEG (5 kD, 10 kD, 15 kD, 20 kD or 30 kD). Examples of branched PEG species are serine- or cysteine- (m-PEG) ₂ , where m-PEG is 20 kD m-PEG.

As will be apparent to the skilled artisan, the branched polymers useful in the present invention include variants on the subject matter described above. For example, the di-lysine-PEG conjugate shown above may comprise three polymeric subunits, and the third is bound to the α-amine shown as unmodified in the structure. Similarly, the use of tri-lysine functionalized with three or four polymeric units is within the scope of the present invention.

Additional exemplary classes useful in the present invention include compounds of the formula:

 And carbonates and active esters of these kinds, for example

Other activation or leaving groups suitable for activating linear PEGs useful for preparing the compounds described herein include, but are not limited to, the following types of formulas:

PEG molecules activated by these and other classes and methods for producing activated PEGs are described in WO 04/083259.

One skilled in the art will appreciate that one or more m-PEG arms of a branched polymer have a PEG moiety having different ends, for example, OH, COOH, NH ₂ , C ₂ -C ₁₀ -alkyl and the like. It will be appreciated that it can be replaced. In addition, the structure is easily modified by inserting an alkyl linker (or removing carbon atoms) between the α-carbon atom of the “amino acid” and the functional group of the side chain. Thus, "homo" derivatives and higher homologues and lower homologues are useful "amino acid" cores for branched PEGs useful in the present invention.

The branched PEG species described in the present invention are readily prepared by methods such as those shown in the following schemes:

Wherein X ^a is O or S and r is an integer from 1 to 5. The indices e and f are independently integers selected from 1 to 2500.

Thus according to the above scheme, a natural or unnatural amino acid is contacted with an activated m-PEG derivative to form 1 by alkylating the side chain heteroatoms X ^a in the case of tosylate. Branched m-PEG 2 is assembled by subjecting the mono-functionalized m-PEG amino acid to N-acylation conditions with a reactive m-PEG derivative. As will be appreciated by the skilled practitioner, the tosylate leaving group can be replaced by any suitable leaving group, for example, halogen, mesylate, triflate and the like. Similarly, the reactive carbonates used to acylate amines may be replaced with active esters such as N-hydroxysuccinimide or the like, or the acid may be dicyclohexylcarbodiimide, carbonyldiimine in situ. It may be activated using a dehydrating agent such as dazol or the like.

In an exemplary embodiment, the deformable group is a PEG moiety, but any deformable group may be incorporated into the glycosyl moiety, for example, through appropriate binding, such as water soluble polymers, water-insoluble polymers, therapeutic moieties, and the like. Modified sugars are formed by enzymatic, chemical, or combinations thereof to produce modified sugars. In an exemplary embodiment, the sugar is substituted by the active amine at any position that allows for attachment of the modifying site, but the sugar is still allowed to act as a substrate for an enzyme capable of coupling the modified sugar to the peptide. In an exemplary embodiment, where the galactosamine is a modified sugar, the amine moiety is attached to the carbon atom at the 6-position.

receptivity Polymer  Variation

Water soluble polymer modified nucleotide sugar classes in which the sugar moiety is modified by the water soluble polymer are useful in the present invention. Exemplary modified sugar nucleotides have sugar groups modified through the amine moiety on the sugar. Modified sugar nucleotides, such as sacaryl-amine derivatives of sugar nucleotides, are also useful in the methods of the present invention. For example, saccharyl amine (without a modifying group) can be enzymatically conjugated to a peptide (or other kind), followed by conjugation of the free saccharyl amine moiety to the desired modifying group. Alternatively, the modified sugar nucleotides can serve as substrates for enzymes that transfer the modified sugars to substrates, such as sacaryl receptors such as peptides, glycopeptides, lipids, aglycones, glycolipides, and the like.

In one embodiment where the saccharide core is galactose or glucose, R ⁵ is NHC (O) Y.

In an exemplary embodiment, the modified sugar is based on the 6-amino-N-acetyl-glycosyl moiety. As shown below for N-acetylgalactosamine, the 6-amino-sugar moiety is readily prepared by standard methods.

In the above scheme, the index n represents an integer of 1 to 2500, preferably 10 to 1500, more preferably 10 to 1200. The symbol "A" refers to an activating group such as halo, a component of an activated ester (eg N-hydroxysuccinimide ester), a component of a carbonate (eg p-nitrophenyl carbonate), and the like. Indicates. Those skilled in the art will appreciate that other PEG-amide nucleotide sugars are readily prepared by this and similar methods.

In another exemplary embodiment, the amide moiety is replaced by a group such as urethane or urea.

In yet further embodiments, R ¹ is branched PEG, eg, one of the aforementioned types. Examples of compounds according to this embodiment include the following compounds:

Wherein X ⁴ is a bond or O.

In addition, as discussed above, the present invention provides nucleotide sugars modified by water-soluble polymers that are linear or branched. For example, compounds having the formula shown below are included within the scope of the present invention:

Where X ⁴ is O or a bond.

Similarly, the present invention provides nucleotide sugars of these modified sugar types wherein the carbon at the 6-position is modified:

Where X ⁴ is a bond or O.

Also provided are conjugates of peptides and glycopeptides, lipids and glycolipides comprising the compositions of the present invention. For example, the present invention provides a conjugate having the formula:

Water-insoluble Polymer

In another embodiment, the modified sugars similar to those discussed above include water-insoluble polymers rather than water-soluble polymers. The conjugates of the present invention may also comprise one or more water-insoluble polymers. This embodiment of the invention is illustrated by the use of a conjugate as a vehicle to deliver a therapeutic peptide in a controlled manner. Polymeric drug delivery systems are well known in the art. See, eg, Dunn et al., Eds. Polymeric Drugs And Drug Delivery Systems, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C. 1991]. Those skilled in the art will recognize that any drug delivery system known in the art can be applied to the conjugates of the present invention.

Representative water-insoluble polymers include polyphosphazine, poly (vinyl alcohol), polyamides, polycarbonates, polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers , Polyvinyl ester, polyvinyl halide, polyvinylpyrrolidone, polyglycolide, polysiloxane, polyurethane, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), Poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), Poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate) polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene oxa De), poly (ethylene terephthalate), poly (vinyl acetate), polyvinyl chloride, polystyrene, polyvinylpyrrolidone, fluorenyl Optronics and polyvinyl phenols and include, but these copolymers, are not limited to.

Synthetically modified natural polymers useful in the conjugates of the present invention include, but are not limited to, alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, and nitrocellulose. Particularly preferred members of the broad class of synthetically modified natural polymers include methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate , Cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, and polymers of acrylic acid and methacrylic acid esters and alginic acid.

These and other polymers discussed in the present invention include Sigma Chemical Co., St. Louis, Mo., Polysciences, Warrenton, PA., Aldrich, Milwaukee, WI. .), Fluka (Fluka, Ronkonkoma, NY), and Biorad (BioRad, Richmond, CA) can be easily obtained from monomers, or else from monomers obtained from these suppliers using standard techniques. It may be synthesized.

Representative biodegradable polymers useful in the conjugates of the present invention include polylactide, polyglycolide and copolymers thereof, poly (ethylene terephthalate), poly (butyric acid), poly (valeric acid), poly (lactide-co- Caprolactone), poly (lactide-co-glycolide), polyanhydrides, polyorthoesters, blends and copolymers thereof, but are not limited to these. Particularly useful are compositions that form gels, such as those containing collagen, pluronix and the like.

Polymers useful in the present invention include "hybrid" polymers comprising water-insoluble materials having bioresorbable molecules in at least a portion of their structure. An example of such a polymer is one that includes a water-insoluble copolymer having a bioresorbable moiety, a hydrophilic moiety and a plurality of crosslinkable functional groups per polymer chain.

For the purposes of the present invention, "water-insoluble material" includes materials that are substantially insoluble in water or in a water-containing environment. Thus, even though certain portions or fragments of the copolymer may be hydrophilic or water soluble, the polymer molecules as a whole do not dissolve to any significant measure in water.

For the purposes of the present invention, the term "bioresorbable molecule" includes moieties that can be metabolized, degraded, reabsorbed and / or removed by the body through normal secretion pathways. Such metabolites or degradation products are preferably substantially nontoxic to the body.

The bioresorbable moiety can be hydrophobic or hydrophilic so long as the copolymer composition is not water soluble as a whole. Thus, the bioresorbable moiety is selected based on the condition that the polymer as a whole maintains water insolubility. Thus, the relative properties, ie the type of functional groups contained by the bioresorbable moiety and the hydrophilic moiety, and their relative proportions, are selected to ensure that the useful bioresorbable composition maintains water insolubility.

Examples of resorbable polymers include, for example, synthetically produced resorbable block copolymers of poly (α-hydroxy-carboxylic acid) / poly (oxyalkylene) (Cohn et al., US Patent No. 4,826,945). These copolymers are non-crosslinked and are water soluble so that the body can excrete the degraded block copolymer composition. Younes et al., J Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J Biomed. Mater. Res. 22: 993-1009 (1988).

Currently preferred bioresorbable polymers include poly (ester), poly (hydroxy acid), poly (lactone), poly (amide), poly (ester-amide), poly (amino acid), poly (anhydride), poly ( Orthoesters), poly (carbonates), poly (phosphazines), poly (phosphoesters), poly (thioesters), polysaccharides and mixtures thereof. Even more preferably, the bioresorbable polymer includes a poly (hydroxy) acid component. Among the poly (hydroxy) acids, polylactic acid, polyglycolic acid, polycaproic acid, polybutyric acid, polyvaleric acid and copolymers and mixtures thereof are preferred.

In addition to forming fragments that are absorbed ("bioresorbed") in vivo, preferred polymer coatings useful in the methods of the present invention may also form excretable and / or metabolizable fragments.

Higher copolymers may also be used in the present invention. For example, US Pat. No. 4,438,253 (Casey et al.), Issued March 20, 1984, describes a tree resulting from the transesterification of poly (glycolic acid) and hydroxyl-terminated poly (alkylene glycols). Tri-block copolymers are described. Such compositions are described for use as resorbable monofilament sutures. The flexibility of such compositions is controlled by incorporating aromatic orthocarbonates such as tetra-p-tolyl orthocarbonate into the copolymer structure.

Other polymers based on lactic acid and / or glycolic acid may be used. For example, US Pat. No. 5,202,413 to Spinu, issued April 13, 1993, discloses ring-opening polymerization of lactide and / or glycolide on oligomeric diol or diamine residues, followed by diisocyanate, diacylchloride or Biodegradable multi-block copolymers with successively ordered blocks of polylactide and / or polyglycolide produced by chain extension by bifunctional compounds such as dichlorosilanes are described.

The bioresorbable portion of the coating useful in the present invention may be designed to be hydrolytically and / or enzymatically degradable. For the purposes of the present invention, "hydrolytically degradable" refers to the susceptibility of copolymers, especially bioresorbable moieties, to hydrolysis in water or water-containing environments. Similarly, "enzymatically degradable" as used herein refers to the susceptibility of copolymers, particularly bioresorbable moieties, to degradation by endogenous or exogenous enzymes.

When placed in the body, the hydrophilic portion can be treated with excretable and / or metabolizable fragments. Thus, hydrophilic moieties include, for example, polyethers, polyalkylene oxides, polyols, poly (vinyl pyrrolidone), poly (vinyl alcohol), poly (alkyl oxazolines), polysaccharides, carbohydrates, peptides, proteins And copolymers and mixtures thereof. In addition, the hydrophilic portion may also be a poly (alkylene) oxide, for example. Such poly (alkylene) oxides may include, for example, poly (ethylene) oxide, poly (propylene) oxide and mixtures and copolymers thereof.

Polymers that are components of hydrogels are also useful in the present invention. Hydrogels are polymeric materials that can absorb relatively large amounts of water. Examples of hydrogel forming compounds include polyacrylic acid, sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, gelatin, carrageenan and other polysaccharides, hydroxyethylenemethacrylic acid (HEMA), and their Derivatives and the like are included, but are not limited to these. Stable, biodegradable and bioresorbable hydrogels can be produced. Moreover, the hydrogel composition may include subunits that exhibit one or more of these properties.

Bio-compatible hydrogel compositions whose integrity can be controlled through crosslinking are known and are currently preferred for use in the methods of the present invention. For example, Hubbell et al., US Pat. Nos. 5,410,016 (patented April 25, 1995) and 5,529,914 (patented June 25, 1996), show two hydrolytically labile extensions. A water soluble system is described that is a crosslinked block copolymer having a water soluble central block segment sandwiched between extensions. Such copolymers are also end-capped with photopolymerizable acrylate functionalities. When crosslinked, these systems become hydrogels. The water soluble central block of such copolymers may comprise poly (ethylene glycol); On the other hand, the hydrolytically labile extension may be a poly (α-hydroxy acid) such as polyglycolic acid or polylactic acid (Sawhney et al., Macromolecules 26 : 581-587 (1993)).

In another preferred embodiment, the gel is a thermally reversible gel. Pluronix, collagen, gelatin, hyalurouronic acid, polysaccharides, polyurethane hydrogels, polyurethane-urea hydrogels and combinations thereof are presently preferred.

In another exemplary embodiment, the conjugates of the present invention comprise a component of a liposome. Liposomes can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811 to Eppstein et al. (June 11, 1985). Can be. For example, a liposome preparation may dissolve an appropriate lipid (s) (eg, stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent and then evaporate it. By leaving a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound or a pharmaceutically acceptable salt thereof is then introduced into the container. The vessel is then swirled by hand to separate the lipid material from the sides of the vessel and disperse the lipid aggregates to form a liposome suspension.

The microparticles described above and methods of making the microparticles are presented by way of example and they are not intended to define the scope of microparticles useful in the present invention. It will be apparent to those skilled in the art that an array of particulates produced by various methods is useful in the present invention.

The structural forms discussed above in connection with both straight and branched water soluble polymers are generally applicable to water-insoluble polymers as well. Thus, for example, cysteine, serine, dilysine and trilysine branched cores can be functionalized by two water-insoluble polymer sites. The method used to produce these species is generally very similar to that used to produce water soluble polymers.

The PEG substitution degree of the conjugate can be controlled by selecting stereochemistry, the number of available glycosylation sites, selection of enzymes selective for a particular site, and the like (FIG. 2F). Glycopezylation Factor IX type shows increased circulatory half-life compared to unlabeled Factor IX (FIG. 3, FIG. 6).

Way

In addition to the conjugates discussed above, the present invention provides methods for making these and other conjugates. The present invention also provides a method of preventing, treating or ameliorating a disease state by administering the conjugate of the present invention to a subject at risk of developing the disease or to a subject having a disease.

Accordingly, the present invention provides a method of forming a covalent conjugate between a selected site and a Factor IX peptide.

In an exemplary embodiment, the conjugate is formed between a water soluble polymer, therapeutic site, targeting site or biomolecule and glycosylated or non-glycosylated factor IX peptide. Polymers, therapeutic sites, or biomolecules are inserted between the peptide and two modifying groups (eg, water soluble polymers) and conjugated to the peptide via glycosyl linkage groups covalently linked to them. The method comprises contacting a peptide with a mixture containing a modified sugar and an enzyme that conjugates the modified sugar to a substrate (eg, peptide, aglycone, glycolipid), eg, glycosyltransferase. Include. This reaction is carried out under conditions appropriate to form a covalent bond between the modified sugar and the Factor IX peptide.

Receptor factor IX peptides are generally newly synthesized or recombinant in prokaryotic cells (e.g., bacterial cells such as E. coli ) or eukaryotic cells such as mammalian, yeast, insect, fungal or plant cells. It is expressed as. Peptides may be full-length proteins or fragments. The peptide can also be a wild type or mutated peptide. In an exemplary embodiment, the peptide comprises a mutation that adds or removes one or more N- or O-linked glycosylation sites in the peptide sequence.

In an exemplary embodiment, Factor IX is O-glycosylated and functionalized by a water soluble polymer in the following manner. Peptides are produced with available amino acid glycosylation sites or, if glycosylated, the glycosyl sites are removed to expose the amino acids. For example, serine or threonine is α-1 N-acetyl amino galactosylated (GalNAc) and NAc-galactosylated peptide is sialylated with a sialic acid-modifying group cassette using ST6GalNAcT1. Instead, the NAc-galactosylated peptide is galactosylated using Core-1-GalT-1 and the product is sialylated with a sialic acid-modifying group cassette using ST3GalT1. An example of a conjugate according to this method has the following linkage: Thr-α-1-GalNAc-β-1,3-Gal-α2,3-Sia *, where Sia * is a sialic acid-modified group cassette to be).

In the methods of the invention as described above, individual glycosylation steps can be performed separately using multiple enzymes and saccharyl donors, or can be combined in a "single pot" reaction. For example, in the three enzymatic reactions described above, GalNAc transferases, GalT and SiaT and their donors can be combined in a single vessel. Alternatively, the GalNAc reaction can be performed alone, and both GalT and SiaT and the appropriate saccharyl donor can be added in a single step. Another mode of conducting the reaction involves sequentially adding each enzyme and the appropriate donor and carrying out the reaction in a "single port" motif. Combinations of each of the above described methods may be useful for making the methods of the present invention.

In the conjugates of the invention, in particular glycofezilated N-linked glycans, the Sia-modified group cassette can be linked to Gal by α-2,6 or α-2,3 bonds.

The methods of the present invention also provide modifications of recombinantly produced incompletely glycosylated factor IX peptides. Using modified sugars in the methods of the present invention, the peptides may be further glycosylated simultaneously and derivatized with, for example, water soluble polymers, therapeutic agents and the like. The sugar moiety of the modified sugar may be a moiety that can be appropriately conjugated to a receptor in a fully glycosylated peptide, or another sugar moiety with desirable properties.

Examples of methods for modifying peptides useful in the present invention are described in WO04 / 099231, WO 03/031464, and the references cited therein.

In an exemplary method, the present invention provides a method of preparing PEG-ylated factor IX comprising a moiety of the formula:

Wherein D is -OH or R ¹ -L-HN-. Symbol G represents R ¹ -L- or —C (O) (C ₁ -C ₆ ) alkyl. R ¹ is a site comprising a straight or branched poly (ethylene glycol) residue. The symbol L represents a linker selected from a bond, a substituted or unsubstituted alkyl and a substituted or unsubstituted heteroalkyl. Generally, if D is OH, G is R ¹ -L-, and if G is -C (O) (C ₁ -C ₆ ) alkyl, D is R ¹ -L-NH-. The method of the present invention comprises the steps of (a) contacting the substrate factor IX peptide with a PEG-sialic acid donor and an enzyme capable of transferring the PEG-sialic acid site from the donor to the substrate factor IX peptide.

Exemplary PEG-sialic acid donors are nucleotide sugars, such as those having the formula: and the enzyme transfers PEG-sialic acid onto amino acids or glycosyl residues of the Factor IX peptide under conditions suitable for transfer:

In one embodiment, the substrate factor IX peptide is expressed in a host cell prior to the formation of the conjugates of the invention. Examples of host cells are mammalian cells. In another embodiment, the host cell is an insect cell, plant cell, bacterium or fungus.

The method presented in the present invention is applicable for each of the Factor IX conjugates described in the above section.

Factor IX peptides modified by the methods of the invention may be synthetic or wild type peptides, or they may be mutated peptides produced by methods known in the art, such as site-directed mutagenesis. Can be. Glycosylation of peptides is generally either N-linked or O-linked. An example of an N-linkage is the attachment of a modified sugar to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X can be any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate sites to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide results in a potential glycosylation site. O-linked glycosylation is one sugar for the hydroxy side chain of hydroxyamino acids, preferably serine or threonine (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose or xyl). Rose), but unique or non-natural amino acids such as 5-hydroxyproline or 5-hydroxylysine may be used.

The addition of glycosylation sites to the peptide or other structure is conveniently performed by modifying the amino acid sequence to contain one or more glycosylation sites. The addition may also be made by incorporating one or more species, preferably serine or threonine residues, representing a -OH group in the sequence of the peptide (for O-linked glycosylation sites). The addition can be by mutation or by complete chemical synthesis of the peptide. Peptide amino acid sequences are preferably changed by mutating the DNA encoding peptides at preselected bases so as to produce codons that are translated into particularly preferred amino acids through changes in the DNA level. DNA mutation (s) is preferably made using methods known in the art.

In an exemplary embodiment, the glycosylation site is added by shuffling polynucleotides. Polynucleotides encoding candidate peptides can be modulated by DNA shuffling protocols. DNA shuffling is a process of repetitive recombination and mutation performed by random fragmentation of a pool of related genes, followed by reassembly of fragments by polymerase chain reaction-like processes. See, eg, Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747-10751 (1994); Stemmer, Nature 370: 389-391 (1994); And US Pat. Nos. 5,605,793, 5,837,458, 5,830,721 and 5,811,238.

Examples of how to add or remove glycosylation sites and add or remove glycosyl structures or substructures are described in detail in WO 04/099231, WO03 / 031464 and related US and PCT applications.

The invention also utilizes means for adding (or removing) one or more selected glycosyl residues to the Factor IX peptide, wherein the modified sugar is conjugated to at least one of the selected glycosyl residues of the peptide. This technique is useful, for example, when it is desirable to conjugate the modified sugar to selected glycosyl residues that are not present on the Factor IX peptide or are not present in the desired amount. Thus, prior to coupling the modified sugar to the peptide, the selected glycosyl moiety is conjugated to the peptide by enzymatic or chemical coupling. In another embodiment, the glycosylation pattern of the glycopeptide is altered prior to conjugation of the modified sugar by removing the carbohydrate moiety from the glycopeptide, eg WO 98/31826. For example, sialic acid groups can be removed from factor IX to form asialo-factor IX prior to glycopezilation with PEG modified sialic acid (FIG. 2F).

Examples of attachment points for selected glycosyl residues include (a) a consensus site for N-linked glycosylation, and a site for O-linked glycosylation; (b) a terminal glycosyl moiety that is a receptor for glycosyltransferases; (c) arginine, asparagine and histidine; (d) free carboxyl groups; (e) free sulfhydryl groups such as those present in cysteine; (f) free hydroxyl groups such as those present in serine, threonine or hydrociproline; (g) aromatic moieties such as those present in phenylalanine, tyrosine or tryptophan; Or (h) amide groups of glutamine, but are not limited to these. Examples of methods useful in the present invention are described in WO 87/05330 (published Sep. 11, 1987) and in Aplin and Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981).

PEG modified sugars are conjugated to glycosylated or non-glycosylated peptides using appropriate enzymes that mediate conjugation. Preferably, the concentrations of the modified donor sugar (s), enzyme (s) and receptor peptide (s) are chosen such that glycosylation proceeds until the receptor is consumed. The considerations discussed below are described in relation to sialyltransferases but are generally applicable to other glycosyltransferase reactions.

Many methods of using glycosyltransferases to synthesize preferred oligosaccharide structures are known and generally applicable to the present invention. Examples of methods are described, for example, in WO 96/32491, Ito et al., Pure Appl. Chem. 65 : 753 (1993), US Pat. Nos. 5,352,670, 5,374,541, 5,545,553 and commonly owned US Pat. Nos. 6,399,336 and 6,440,703, which are incorporated herein by reference.

The present invention is carried out using a single glycosyltransferase or a combination of glycosyltransferases. For example, a combination of sialyltransferase and galactosyltransferase can be used. In these embodiments using one or more enzymes, the enzyme and the substrate are preferably combined into an initial reaction mixture, or once the first enzymatic reaction is completed or nearly complete, the enzymes and reagents for the second enzymatic reaction are added to the reaction medium. Add. By performing two enzymatic reactions sequentially in a single vessel, the overall yield is improved over the method of separating intermediates. Moreover, cleanup and disposal of excess solvent and by-products is reduced.

In a preferred embodiment, each of the first and second enzymes is glycosyltransferase. In another preferred embodiment one enzyme is endoglycosidase. In a further preferred embodiment two or more enzymes are used to cope with the modified glycoprotein of the invention. Enzymes are used to alter the saccharide structure on the peptide at any point before or after adding the modified sugar to the peptide.

In another embodiment, the method uses one or more exo- or endoglycosidases. Glycosidase is generally a mutant engineered to form rather than break glycosyl bonds. Mutant glycanases generally include substitution of amino acid residues for active site acidic amino acid residues. For example, when the endoglycanase is endo-H, the substituted active site residue may typically be Asp at position 130, Glu at position 132, or a combination thereof. Amino acids are generally replaced with serine, alanine, asparagine or glutamine.

Mutant enzymes typically promote the reaction by a synthetic step similar to the reverse reaction of the endoglycanase hydrolysis step. In these embodiments, the glycosyl donor molecule (eg, the desired oligo- or mono-saccharide structure) contains a leaving group and the reaction is performed by adding the donor molecule to the GlcNAc residue on the protein. For example, the leaving group may be a halogen such as fluoride. In another embodiment, the leaving group is an Asn, or Asn-peptide site. In yet another embodiment, the GlcNAc residue on the glycosyl donor molecule is modified. For example, the GlcNAc residue may comprise a 1,2 oxazoline moiety.

In a preferred embodiment, each enzyme used to produce the conjugates of the invention is present in catalytic amounts. The catalytic amount of a particular enzyme varies depending on the concentration of the substrate of the enzyme and reaction conditions such as temperature, time and pH values. Methods of determining the catalytic amount for a given enzyme under preselected substrate concentrations and reaction conditions are well known to those skilled in the art.

The temperature at which the method is performed may range from the temperature just before freezing to the temperature at which most sensitive enzymes denature. Preferred temperature ranges are from about 0 ° C to about 55 ° C, more preferably from about 20 ° C to about 37 ° C. In another exemplary embodiment, one or more components of the method are performed at a corresponding temperature using a heat resistant enzyme.

The reaction mixture forms the desired conjugate by holding it for a time sufficient to be glycosylated with respect to the receptor. Some of the conjugates can often be detected after several hours, and recoverable amounts are typically obtained within 24 hours or less. It will be appreciated by those skilled in the art that the rate of reaction depends on a number of variable factors optimized for the selected system (eg, enzyme concentration, donor concentration, receptor concentration, temperature, solvent volume).

The invention also provides for industrial-scale production of modified peptides. As used herein, the industrial scale generally produces at least 250 mg, preferably at least 500 mg, more preferably at least 1 gram of processed purified conjugate.

In the following description, the invention is illustrated by the conjugation of modified sialic acid sites to glycosylated peptides. Examples of modified sialic acid are labeled with PEG. The focus of the following description on PEG-modified sialic acid and glycosylated peptides is for clarity and is not meant to limit the invention to the conjugation of these two partners. It is understood by the skilled artisan that this description is generally applicable to the addition of modified glycosyl moieties other than sialic acid. Moreover, this description is equally applicable to modification of glycosyl units by agents other than PEG, including other PEG moieties, therapeutic moieties and biomolecules.

Enzymatic methods can be used to selectively introduce PEGylated or PPGylated carbohydrates on peptides or glycopeptides. This method utilizes modified sugars containing PEG, PPG, or masked reactive functional groups and is combined with appropriate glycosyltransferases or glycosinase. By selecting a glycosyltransferase capable of making the desired carbohydrate bond and using a modified sugar as the donor substrate, PEG or PPG can be added to the peptide backbone, onto existing sugar residues of the glycopeptide, or to the peptide. It can be doled directly onto sugar residues.

Receptors for sialyltransferase are present on the peptide to be modified by the methods of the invention as naturally occurring structures or recombinantly, enzymatically or chemically arranged. Suitable receptors include, for example, Galβ1,4GlcNAc, Galβ1,4GalNAc, Galβ1,3GalNAc, Lacto-N-tetraoz, Galβ1,3GlcNAc, Galβ1,3Ara, Galβ1,6GlcNAc, Galβ1,4Glc (lactose), and the art Galactosyl receptor groups such as other receptors known to those skilled in the art. See, eg, Paulson et al., J. Biol. Chem. 253 : 5617-5624 (1978).

In one embodiment, the receptor for sialyltransferase is on the glycopeptide to be modified upon in vivo synthesis of the glycopeptide. Such glycopeptides can be sialylated using the claimed method without modifying the glycosylation pattern of the glycopeptide in advance. Alternatively, the methods of the present invention can be used to sialylate a peptide that does not contain a suitable receptor; The peptide is first modified to include a receptor by methods known to those skilled in the art. In an exemplary embodiment, GalNAc residues are added by the action of GalNAc transferase.

In an exemplary embodiment, the galactosyl receptor is assembled by attaching a galactose moiety to a suitable receptor linked to the peptide, eg, GlcNAc. This method comprises a suitable amount of galactosyltransferase (eg galβ1,3 or galβ1,4), and a suitable galactosyl donor (eg UDP-galactose) for the peptide to be modified. Culturing with the reaction mixture. The reaction is allowed to proceed until substantially complete, or instead the reaction ends when a preselected amount of galactose residue is added. Other methods of assembling the selected saccharide receptor will be apparent to those skilled in the art.

In another embodiment, the glycopeptide-linked oligosaccharides are first or totally “trimmed” to add a receptor for sialyltransferase, or one or more suitable residues to obtain a suitable receptor. Expose areas where possible. Enzymes such as glycosyltransferases and endoglycosidases (see, eg, US Pat. No. 5,716,812) are useful for attachment and ordering reactions.

In the following description the method of the invention is illustrated by using a modified sugar to which a PEG moiety is attached. The focus of the explanation is to go for a clear explanation. It will be apparent to those skilled in the art that this modified description applies equally to these embodiments, including therapeutic sites, biomolecules, and the like.

In an exemplary embodiment of the invention where the carbohydrate moiety is "arranged" before adding the modified sugar, the higher mannose is reversed into a primary producing biantennary structure. Modified sugars containing a PEG moiety are conjugated to one or more sugar residues exposed by "trimming back". In one example, the PEG moiety is added via a GlcNAc site conjugated to the PEG moiety. The modified GlcNAc is attached to one or both terminal mannose residues of the biantennary structure. Alternatively, unmodified GlcNAc can be added to one or both ends of the branched species.

In another exemplary embodiment, the PEG moiety is attached to one or both of the terminal mannose residues of the biantennary structure via a modified sugar having a galactose residue conjugated to a GlcNAc residue added on the terminal mannose residue. Is added. Alternatively, unmodified Gal can be added to one or both terminal GlcNAc residues.

In yet further examples, the PEG moiety is added onto the Gal residue using a modified sialic acid.

In another exemplary embodiment, the higher mannose structure is reversed to the mannose from which the biantennary structure is branched. In one example, PEG moieties are added via GlcNAc modified by a polymer. Instead, unmodified GlcNAc is added to mannose followed by Gal with an attached PEG moiety. In yet further embodiments, the unmodified GlcNAc and Gal residues are added sequentially to mannose followed by the addition of a sialic acid site modified with a PEG site.

In further exemplary embodiments, the higher mannose is reversed to GlcNAc, to which the first mannose is attached. GlcNAc is conjugated to a Gal residue containing a PEG moiety. Instead, unmodified Gal is added to GlcNAc followed by sialic acid modified with water soluble sugars. In yet further embodiments, the terminal GlcNAc is conjugated with Gal, and the GlcNAc is then fucosylated with a modified fucose containing a PEG moiety.

Higher mannose may also be reversely identified as the first GlcNAc attached to the Asn of the peptide. As one example, GlcNAc of the GlcNAc- (Fuc) _a residue is modified by Gal containing a water soluble polymer. In yet another embodiment, GlcNAc is modified by Gal and then conjugated to Gal of sialic acid modified with PEG sites.

Other exemplary embodiments include those commonly owned US published patent application 20040132640; 20040063911; 20040137557; US Patent Application No. 10 / 369,979; 10 / 410,913; 10 / 360,770; 10 / 410,945 and PCT / US02 / 32263, each of which is incorporated herein by reference.

The above example provides a description of the power of the method described in the present invention. Using the methods described herein, carbohydrate moieties of substantially the desired structure can be "reversed" and formed. The modified sugar can be added at the end of the carbohydrate moiety as described above, or it can be an intermediate between the peptide core and the end of the carbohydrate.

In an exemplary embodiment, the mechanism sialic acid is removed from the Factor IX glycopeptide using sialidase to expose all or most of the basic galactosyl residues. Instead, the peptide or glycopeptide is labeled with a galactose residue, or an oligosaccharide residue ending with a galactose unit. After exposing or adding the galactose moiety, modified sialic acid is added using an appropriate sialyltransferase. This method is summarized in Scheme 1.

In yet further methods summarized in Scheme 2, the masked reactive functional groups are present on sialic acid. The masked reactive group is preferably unaffected by the conditions used to attach the modified sialic acid to the factor IX. After covalently attaching the modified sialic acid to the peptide, the mask is removed and the peptide is conjugated with an agent such as PEG. The agent is conjugated to the peptide in a specific manner by reaction of the agent with an exposed reactive group on the modified sugar moiety.

All modified sugars described herein can be used with their appropriate glycosyltransferases depending on the terminal sugars of the oligosaccharide side chains of the glycopeptides (Table 1). As described above, the terminal sugars of the glycopeptides required for the introduction of the phylated structure can be introduced naturally during expression, or they can be incorporated into the appropriate glycosidase (s), glycosyltransferase (s) or glycosidase ( (S) and glycosyltransferase (s) can be used to produce post-expression.

In a further exemplary embodiment, UDP-galactose-PEG transfers the modified galactose to the appropriate terminal N-acetylglucosamine structure by reacting with milk β1,4-galactosyltransferase. Because terminal GlcNAc residues on glycopeptides may be present in expression systems such as mammals, insects, plants or fungi, they may not only be generated during expression, but also glycopeptides may be sialidase and / or glycosidase and And / or by treatment with glycosyltransferase.

In another exemplary embodiment, PEGylated-GlcN is transferred to a terminal mannose residue on the glycopeptide using a GlcNAc transferase such as GNT1-5. In yet further exemplary embodiments, N- and / or O-linked glycan structures are enzymatically removed from glycopeptides to expose amino acid or terminal glycosyl residues, which are then conjugated with modified sugars. For example, endoglycanase is used to remove the N-linked structure of the glycopeptide to expose the terminal GlcNAc as GlcNAc-linked-Asn on the glycopeptide. UDP-Gal-PEG and appropriate galactosyltransferase are used to introduce PEG-galactose functional groups on the exposed GlcNAc.

In an alternative embodiment, the modified sugar is added directly to the peptide backbone using glycosyltransferases known to transfer sugar residues to the peptide backbone. This exemplary embodiment is described in Scheme 3. Examples of glycosyltransferases useful in practicing the present invention include GalNAc transferases (GalNAc T1-14), GlcNAc transferases, fucosyltransferases, glucosyltransferases, xyloyltransferases, mannosyltransferases, and the like. However, it is not limited to these. This method allows for the direct addition of modified sugars on peptides that are completely devoid of carbohydrates, or on existing glycopeptides instead. In both cases, the addition of modified sugars is not at random as occurs during modification of the peptide backbone of the protein using chemical methods, but at a specific location on the peptide backbone as defined by the substrate specificity of glycosyltransferase. Happens. An array of medicaments can be introduced into a protein or glycopeptide lacking a glycosyltransferase substrate peptide sequence by manipulating the appropriate amino acid sequence into a polypeptide chain.

In each of the exemplary embodiments described above, one or more additional chemical or enzymatic modification steps may be used after conjugation of the modified sugar to the peptide. In an exemplary embodiment, an enzyme (eg, fucosyltransferase) is used to attach a glycosyl unit (eg, fucose) onto the terminal modified sugar attached to the peptide. In another example, an enzymatic reaction is used to "cap" the site at which the modified sugar failed to conjugate. Instead, chemical reactions are used to alter the structure of the conjugated modified sugar. For example, the conjugated modified sugar is reacted with an agent that stabilizes or destabilizes its binding to the peptide component to which the modified sugar is attached. In another example, the components of the modified sugar are deprotected after its conjugation to the peptide. The skilled practitioner will recognize that there is an array of enzymatic and chemical procedures useful in the methods of the present invention at the stage after the modified sugar is conjugated to the peptide. Further manipulations of modified sugar-peptide conjugates are included within the scope of the present invention.

enzyme

In addition to the enzymes discussed above in connection with forming acyl-linked conjugates, the glycosylation patterns of conjugates and starting substrates (eg, peptides, lipids) can be manipulated, reversed, or otherwise employed by other enzymes. Can be modified in other ways. Methods for remodeling peptides and lipids using enzymes that transfer sugar donors to receptors are discussed in more detail in WO 03/031464 A2 (DeFrees, published 17 April 2003). A brief summary of selected enzymes useful in the methods of the present invention is mentioned below.

Glycosyltransferase

Glycosyltransferases promote the addition of activated sugars (donor NDP- or NMP-sugars) to proteins, glycopeptides, lipids or glycolipids or to non-reducing ends of growth oligosaccharides in a stepwise manner. N-linked glycopeptide is synthesized via transferase and lipid-linked oligosaccharide donor Dol-PP-NAG ₂ Glc ₃ Man ₉ upon en block transfer followed by trimming of the core. In this case, the nature of the "core" saccharide is slightly different from the subsequent attachment. Very many glycosyltransferases are known in the art.

The glycosyltransferase used in the present invention may be any as long as it can utilize a modified sugar as a sugar donor. Examples of such enzymes include galactosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, fucosyltransferases, sialyltransferases, mannosyltransferases, xyloyltransferases. Leloir pathway glycosyltransferases such as glucurononyltransferase and the like.

In the case of enzymatic saccharide synthesis involving a glycosyltransferase reaction, glycosyltransferase can be cloned or isolated from any source. Many cloned glycosyltransferases are known as well as their polynucleotide sequences (see, eg, "The WWW Guide To Cloned Glycosyltransferases" (http://www.vei.co.uk/TGN/gt_guide.htm)). . Glycosyltransferase amino acid sequences, and nucleotide sequences encoding glycosyltransferases from which the amino acid sequences can be deduced, are also publicly available, including GenBank, Swiss-Prot, EMBL, and the like. It is found in various databases.

Glycosyltransferases that may be used in the methods of the present invention include galactosyltransferases, fucosyltransferases, glucosyltransferases, N-acetylgalactosminyltransferases, N-acetylglucosaminyltransferases, glucousyltransferases. Ronyltransferases, sialyltransferases, mannosyltransferases, glucuronic acid transferases, galacturonic acid transferases, and oligosaccharyltransferases. Suitable glycosyltransferases include those obtained from prokaryotic cells as well as from eukaryotic cells.

DNA encoded glycosyltransferases can be obtained by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cell or cell line cultures, by screening genome libraries from appropriate cells, or by combining these procedures. Screening of mRNA or genome DNA can be performed using oligonucleotide probes generated from glycosyltransferase gene sequences. Probes can be labeled with detectable groups such as fluorescent groups, radioactive atoms or chemiluminescent groups used in conventional hybridization test methods according to known procedures. Alternatively, the glycosyltransferase gene sequence can be obtained using a polymerase chain reaction (PCR) procedure, wherein the PCR oligonucleotide primers are generated from the glycosyltransferase gene sequence. See US Pat. No. 4,683,195. (Mullis et al. ) And US Pat. No. 4,683,202 (Mullis).

Glycosyltransferases can be synthesized in a transformed host cell by a vector containing DNA encoding the glycosyltransferase enzyme. The vector is used to amplify the DNA encoding the glycosyltransferase enzyme and / or to express the DNA encoding the glycosyltransferase enzyme. Expression vectors are replicable DNA constructs in which a DNA sequence encoding a glycosyltransferase enzyme is operably linked to a suitable regulatory sequence capable of causing expression of the glycosyltransferase enzyme in a suitable host. The need for such regulatory sequences will vary depending upon the host selected and the transformation method chosen. In general, regulatory sequences include transcriptional promoters, any operator sequence that regulates transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation. Amplification vectors do not require expression control regions. All that is needed is a selection gene that normally is endowed by the origin of replication, the ability to replicate in the host, and the recognition of the transformant.

In an exemplary embodiment, the present invention utilizes prokaryotic enzymes. Such glycosyltransferases include enzymes involved in the synthesis of lipopolysaccharides (LOS) produced by a large number of Gram-negative bacteria [Preston et al., Critical Reviews in Microbiology 23 (3) : 139-180 ( 1996). Such enzymes include β1,6 galactosyltransferase and β1,3 galactosyltransferase [see, eg, EMBL Accession Nos. M80599 and M86935 (E. coli); EMBL Accession No. S56361 (S. typhimurium), Glucosyltransferase (Swiss-Prot Accession No. P25740 (E. coli)), β1,2-glucosyltransferase (rfaJ) (Swiss-Prot accession no. P27129 (E. coli) and Swiss-Prot Accession No. P19817 (S. typhimurium), and β1,2-N-acetylglucosaminyltransferase (rfaK) (EMBL Accession No. U00039). coli)), including. Proteins of the species rfa operon, such as, but not limited to, E. coli and Salmonella typhimurium. Other glycosyltransferases with known amino acid sequences therefor include Klebsiella pneumoniae, E. coli. In organisms such as E. coli, Salmonella typhimurium, Salmonella enterica, Yersinia enterocolitica, Mycobacterium leprosum Characterized rfaB, and those encoded by operons such as the rh1 operon of Pseudomonas aeruginosa.

Suitable for use in the present invention are also the lacto-N-neotetraose, D-galactosyl, identified in the LOS of mucosal pathogens Neisseria gonnorhoeae and N. meningitidis. -β-1,4-N-acetyl-D-glucosaminyl-β-1,3-D-galactosyl-β-1,4-D-glucose and P ^k blood group trisaccharide sequences, D- Is a glycosyltransferase implicated in generating a structure containing galactosyl-α-1,4-D-galactosyl-β-1,4-D-glucose (Scholten et al., J. Med. Microbiol 41 : 236-243 (1994)). Genes from Neisseria meningitidis and Neisseria gonorrea, which encodes glycosyltransferases involved in the biosynthesis of these constructs, have been identified as Neisseria meningitidis immunotypes L3 and L1 (Jennings et al., Mol. Microbiol. 18 : 729-740 (1995)) and Neisseria gonorrea mutant F62 (Gotshlich, J. Exp. Med. 180 : 2181-2190 (1994)). In Neisseria meningitidis, the locus consisting of three genes lgtA, lgtB and lg E encodes the glycosyltransferase enzyme required for addition of the last three sugars in the lacto-N-neotetraose chain (Wakarchuk et al., J. Biol. Chem. 271 : 19166-73 (1996)). Recently, the enzymatic activity of the lgtB and lgtA gene products has been demonstrated, providing direct first evidence of their proposed glycosyltransferase function (Wakarchuk et al., J. Biol. Chem. 271 (45) : 28271- ) . 276 (1996)). In Neisseria gonorrea, lgtD adds β-D-GalNAc to position 3 of the terminal galactose of the lacto-N-neotetraose structure, and the terminal α to the lactose element of the truncated LOS. LgtC to which -D-Gal is added is present to generate P ^k blood group antigenic structures (Gotshlich (1994), supra). In Neisseria meningitidis, separate immunotype L1 also expresses the P ^k blood group antigen and has been shown to carry the lgtC gene (Jennings et al., (1995), supra). Neisseria glycosyltransferases and related genes are also described in USPN 5,545,553 (Gotschlich). Genes for α1,2-fucosyltransferase and α1,3-fucosyltransferase from Helicobacter pylori were specified (Martin et al., J. Biol. Chem. 272 : 21349-21356 (1997) ). Also useful in the present invention are glycosyltransferases of Campylobacter jejuni (see, eg, http://afmb.cnrs-mrs.fr/~pedro/CAZY/gtf_42.html).

Fucosyltransferase

In some embodiments, the glycosyltransferases used in the methods of the invention are fucosyltransferases. Fucosyltransferases are known to those skilled in the art. Examples of fucosyltransferases include enzymes that transfer L-fucose from GDP-fucose to the hydroxy site of the receptor sugar. Fucosyltransferases that transfer non-nucleotide sugars to the receptor are also useful in the present invention.

In some embodiments, the receptor sugar is, for example, GlcNAc in the Galβ (1 → 3,4) GlcNAcb- group in oligosaccharide glycosides. Fucosyltransferases suitable for this reaction include Galβ (1 → 3,4) GlcNAcβ1-α (1 → 3,4) fucosyltransferase (FTIII EC No. 2.4.1.65), first specified from human milk. , et al., Carbohydrate Res. 190: 1-11 (1989); Prieels, et al., J. Biol. Chem. 256: 10456-10463 (1981); And Nunez, et al., Can. J. Chem. 59: 2086-2095 (1981), and Galβ (1 → 4) GlcNAcβ-α fucosyltransferases (FTIV, FTV, FTVI) present in human serum. FTVII (EC No. 2.4.1.65), sialyl α (2 → 3) Galβ ((1 → 3) GlcNAcβ fucosyltransferases were also specified.Galβ (1 → 3,4) GlcNAcβ-α (1 → 3) 4) Fucosyltransferases have also been characterized [Dumas, et al., Bioorg.Med. Letters 1: 425-428 (1991) and Kukowska-Latallo, et al., Genes and Development 4: 1288-1303 (1990)] Other exemplary fucosyltransferases include, for example, α, 2 fucosyltransferases (EC No. 2.4.1.69) Enzymatic fucosylation is described in mollicone, et al. , Eur. J. Biochem. 191: 169-176 (1990) or US Pat. No. 5,374,655. The cells used to produce the fucosyltransferases are also GDP-fue. Enzymatic systems for synthesizing the cose.

Galactosyltransferase

In another group of embodiments, the glycosyltransferase is galactosyltransferase. Examples of galactosyltransferases include α (1,3) galactosyltransferases [E.C. No. 2.4.1.151, see, eg, Dabkowski et al., Transplant Proc. 25: 2921 (1993) and Yamamoto et al. Nature 345: 229-233 (1990), Bovine (GenBank j04989, Joziasse et al., J. Biol. Chem. 264: 14290-14297 (1989)), Rat (GenBank m26925; Larsen et al., Proc. Nat ' Acad. Sci. USA 86: 8227-8231 (1989)), pigs (GenBank L36152; Strahan et al., Immunogenetics 41: 101-105 (1995)). Another suitable α1,3 galactosyltransferase is involved in the synthesis of blood group B antigens [EC 2.4.1.37, Yamamoto et al., J. Biol. Chem. 265: 1146-1151 (1990) (human)]. Another example of a galactosyltransferase is the core Gal-T1.

Suitable for use in the methods of the invention are also described, for example, in EC 2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose synthetase) [bovine (D'Agostaro et al., Eur. 183: 211-217 (1989)), humans (Masri et al., Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), mice (Nakazawa et al., J. Biochem. 104: 165-168 (1988)) and EC Β (1,4) galactosyltransferase, including 2.4.1.38 and ceramide galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci. Res. 38: 234-242 (1994)) to be. Other suitable galactosyltransferases include, for example, α1,2 galactosyltransferases (eg, from Schizosaccharomyces pombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)). Is included).

Sialyltransferase

Sialyltransferases are another type of glycosyltransferase useful in the recombinant cells and reaction mixtures of the present invention. Cells producing recombinant sialyltransferase may also produce sialic acid donor CMP-sialic acid for sialyltransferase. Examples of sialyltransferases suitable for use in the present invention include ST3Gal III (eg, rat or human ST3Gal III), ST3Gal IV, ST3Gal I, ST3GalII, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II , And ST6GalNAc III (sialyltransferase nomenclature used in the present invention is described in Tsuji et al., Glycobiology 6: v-xiv (1996)). An exemplary α (2,3) sialyltransferase, called α (2,3) sialyltransferase (EC 2.4.99.6), may be used to pass sialic acid to the Galβ1 → 3Glc disaccharide or non-reducing terminal Gal of glycoside. Metastasis. See Van den Eijnden et al., J. Biol. Chem. 256: 3159 (1981), Weinstein et al., J. Biol. Chem. 257: 13845 (1982) and Wen et al., J. Biol. Chem. 267: 21011 (1992). Another example α2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid to the non-reducing terminal Gal of disaccharides or glycosides. See Rearick et al., J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J. Biol. Chem. 267: 21004 (1992). Examples of further enzymes include Gal-β-1,4-GlcNAc α-2,6 sialyltransferases. See Kurosawa et al. Eur. J. Biochem. 219: 375-381 (1994).

Preferably, for glycosylation of carbohydrates of glycopeptides, sialyltransferases sequence Galβ1,4GlcNAc, which is the second most common sequence at the end based on terminal sialic acid on the sialic acid carbohydrate structure completely sialic Can be transferred to-(see Table 2).

Sialyltransferase using Galβ1,4GlcNAc sequence as receptor substrate Sialyltransferase Source Formed sequence (s) references  ST6Gal I Mammal  NeuAcα2,6Galβ1,4GlcNAc- One  ST3Gal III Mammal NeuAcα2,3Galβ1,4GlcNAc-
NeuAcα2,3Galβ1,3GlcNAc- One  ST3Gal IV Mammal NeuAcα2,3Galβ1,4GlcNAc-
NeuAcα2,3Galβ1,3GlcNAc- One  ST6Gal II Mammal  NeuAcα2,6Galβ1,4GlcNAc-  ST6Gal II Luminous bacteria  NeuAcα2,6Galβ1,4GlcNAc- 2  ST3Gal V Neisseria Meningitidis
Neisseria Gonorrea  NeuAcα2,3Galβ1,4GlcNAc- 3 Goochee ey al., Bio / Technology 9 : 1347-1355 (1991)
2) Yamamoto et al., J. Biochem . 120 : 104-110 (1996)
3) Gilbert et al., J. Biol . Chem . 271 : 28271-28276 (1996)

Other sialyltransferases useful in the present invention include those described in the table of FIG. 4. Sialyltransferase transfers a phylated sialic acid site from a phenylated sialic acid donor species onto an N-linked glycosyl residue of the peptide (FIG. 2C) or an O-linked glycosyl residue of Factor IX (FIG. 2D). Can be used to

An example of a sialyltransferase useful in the claimed method is ST3Gal III, also called α (2,3) sialyltransferase (EC 2.4.99.6). This enzyme promotes the transfer of sialic acid to Gal of Galβ1,3GlcNAc or Galβ1,4GlcNAc glycosides. See, eg, Wen et al., J. Biol. Chem. 267 : 21011 (1992); Van den Eijnden et al., J. Biol. Chem. 256: 3159 (1991)], may be responsible for the silylation of asparagine-linked oligosaccharides in glycopeptides. Sialic acid is linked to Gal by the formation of an α-bond between two saccharides. The linkage between saccharides is between the 2-position of NeuAc and the 3-position of Gal. Such specific enzymes include rat liver (Weinstein et al., J. Biol. Chem. 257 : 13845 (1982)); Human cDNA (Sasaki et al. (1993) J. Biol. Chem. 268 : 22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269 : 1394-1401) et al. (1996) J. Biol. Chem. 271 : 931-938) DNA sequences are known and promote the production of this enzyme by recombinant expression. In a preferred embodiment, the claimed sialylation method uses rat ST3Gal III.

Other examples of sialyltransferases useful in the present invention include those isolated from Campylobacter jejuni, including α (2,3) (see, eg, WO99 / 49051).

Sialyltransferases, not listed in Table 2, are also useful for economical and efficient large scale methods for sialylation of commercially important glycopeptides. As a simple test to confirm the utility of these other enzymes, various amounts of each enzyme (1-100 mU / mg protein) were reacted with asialo-α ₁ AGP (1-10 mg / ml) to ensure that cattle ST6Gal I The ability of the corresponding sialyltransferases to sialylate glycopeptides is compared to both ST3Gal III or sialyltransferases. Alternatively, glycopeptides or N-linked oligosaccharides that are enzymatically released from other glycopeptides or peptide backbones can be used in place of asialo-α ₁ AGP for this assessment. Sialyltransferases, which have the ability to sialylate N-linked oligosaccharides of glycopeptides more efficiently than ST6Gal I, are useful in practical large-scale methods for peptide sialylation.

GalNAc Transferase

N-acetylgalactosaminyltransferases are useful in practicing the present invention, particularly for binding GalNAc sites to amino acids of O-linked glycosylation sites of peptides. Suitable N-acetylgalactosaminyltransferases include α (1,3) N-acetylgalactosaminyltransferase, β (1,4) N-acetylgalactosaminyltransferase (Nagata et al., J. Biol. Chem. 267 : 12082-12089 (1992) and Smith et al., J. Biol Chem. 269 : 15162 (1994)) and polypeptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol. Chem. 268 : 12609 (1993)), but is not limited to these.

Enzyme GalNAc T _I from the gene cloned by genetic manipulation _- of protein production, such as _XX are well known [see, for example, U.S. Patent No. 4,761,371]. One method is to collect sufficient samples and then determine the amino acid sequence of the enzyme by N-terminal sequencing. This information is then used to isolate cDNA clones encoding full-length (membrane bound) transferases that, when expressed in insect seroline Sf9, provide synthesis of a fully active enzyme. Receptor specificity of the enzyme is then determined using semi-quantitative analysis of amino acids surrounding known glycosylation sites in 16 different proteins followed by in vitro glycosylation testing of the synthetic peptides. This study demonstrates that certain amino acid residues are excessive in glycosylated peptide fragments, and that residues at specific positions surrounding glycosylated serine and threonine residues may have a more significant effect on receptor efficiency than other amino acid residues. It was.

cell- Combined Glycosyltransferase

In another embodiment, the enzyme used in the methods of the invention is cell-bound glycosyltransferase. Although many soluble glycosyltransferases are known (see, eg, US Pat. No. 5,032,519), glycosyltransferases generally exist in membrane-bound form when bound to cells. Many membrane-bound enzymes tested thus far are considered to be endogenous proteins, ie they are not liberated from the membrane by sonication and require detergent for solubilization. Surface glycosyltransferases have been identified on the surface of vertebrate and invertebrate cells, and these surface transferases are also recognized to maintain catalytic activity under physiological conditions. However, a better recognized function of cell surface glycosyltransferases is for intercellular recognition (Roth, Molecular Approaches to Supracellular Phenomena, 1990).

A method for altering glycosyltransferases expressed by cells has been developed. See, eg, Larsen et al., Proc. Natl. Acad. Sci. USA 86: 8227-8231 (1989) reported a genetic method to isolate cloned cDNA sequences that determine cell surface oligosaccharide structures and the expression of their homologous glycosyltransferases. UDP-galactose: Isolation from mouse cell lines known to express β.-D-galactosyl-1,4-N-acetyl-D-glucosaminide α-1,3-galactosyltransferase CDNA libraries generated from the generated mRNAs were transfected into COS-1 cells. The transfected cells were then cultured and tested for α1-3 galactosyltransferase activity.

Francisco et al., Proc. Natl. Acad. Sci. USA 89: 2713-2717 (1992) describe a method of immobilizing β-lactamase on the outer surface of Escherichia coli. A three-part fusion consisting of (i) the signal sequence of the outer membrane protein, (ii) the membrane-spanning fragment of the outer membrane protein, and (iii) the fully mature β-lactamase sequence is generated to produce an active surface-bound β -Provides a lactase molecule. However, the Francisco method is limited only to prokaryotic systems and, as recognized by the authors, requires a complete three-part fusion for proper functioning.

Sulfurtransferase

The invention also provides a method of producing peptides comprising sulfated molecules including sulfated polysaccharides such as, for example, heparin, heparin sulfate, carrageenene and related compounds. Suitable sulfotransferases include, for example, the cDNA of chickens described in chondroitin-6-sulfotransferase (Fukuta et al., J. Biol. Chem. 270 : 18575-18580 (1995)); Genbank Accession No. D49915], glycosaminoglycan N-acetylglucosamine N-deacetylase / N-sulfurtransferase 1 (Dixon et al., Genomics 26 : 239-241 (1995); UL18918), and Glycosaminoglycan N-acetylglucosamine N-deacetylase / N-sulfurtransferase 2 [Orellana et al., J. Biol. Chem. 269 : 2270-2276 (1994) and Eriksson et al., J Rat cDNA as described in Biol.Chem. 269 : 10438-10443 (1994)); Human cDNA described by GenBank Accession No. U2304.

Glycosidase

The invention also encompasses the use of wild type and mutant glycosidase. Mutant β-galactosidase enzymes have been demonstrated to promote the formation of disaccharides via coupling of α-glycosyl fluoride to galactosyl receptor molecules (Withers, US Pat. No. 6,284,494; 2001 Granted on September 4). Other glycosidases useful in the present invention include, for example, β-glucosidase, β-galactosidase, β-mannosidase, β-acetyl glucosaminidase, β-N-acetyl galactosami Nidase, β-xylosidase, β-fucosidase, cellulase, xylanase, galactanase, mannase, hemicellulase, amylase, glucosamylase, α-glucosidase, α-gal Lactosidase, α-mannosidase, α-N-acetyl glucosaminidase, α-N-acetyl galactosaminidase, α-xylosidase, α-fucosidase and neuraminidase / sial Lidases are included. In an exemplary embodiment, sialic acid is used to remove sialic acid from the N-glycans of factor IX prior to glycofezilation (FIG. 2A). The present invention also provides a method in which there is no need to remove sialic acid in advance. Thus, methods incorporating a sialic acid exchange reaction using a modified sialic acid moiety and ST3Gal3 are useful in the present invention.

Immobilized enzyme

The invention also provides for the use of enzymes immobilized on solid and / or soluble supports. In an exemplary embodiment there is provided a glycosyltransferase conjugated to PEG via an intact glycosyl linker according to the methods of the invention. The PEG-linker-enzyme conjugate is optionally attached to a solid support. The use of a solid supported enzyme in the process of the present invention simplifies the workup of the reaction mixture and purification of the reaction product, and also allows for easy recovery of the enzyme. Glycosyltransferase conjugates are used in the methods of the invention. Still other combinations of enzymes and supports will be apparent to those skilled in the art.

Fusion protein

In another exemplary embodiment, the methods of the present invention utilize fusion proteins having one or more enzymatic activity associated with the synthesis of the desired glycopeptide conjugate. The fusion polypeptide may, for example, consist of a catalytically active region of glycosyltransferase bound to the catalytically active region of the accessory enzyme. The accessory enzyme catalytic region can, for example, facilitate the step in the formation of a nucleotide sugar that is a donor for glycosyltransferase or can promote a reaction involved in the glycosyltransferase cycle. For example, a polynucleotide encoding a glycosyltransferase may be bound to a polynucleotide encoding an enzyme included in an in-frame nucleotide sugar system. The resulting fusion protein can then promote the synthesis of nucleotide sugars as well as the transfer of sugar sites to receptor molecules. The fusion protein may be two or more cycle enzymes linked by one expressable nucleotide sequence. In another embodiment, the fusion protein comprises a catalytically active region of two or more glycosyltransferases (see, eg, 5,641,668). Modified glycopeptides of the present invention can be readily designed and prepared using a variety of suitable fusion proteins (see, eg, PCT Patent Application PCT / CA98 / 01180, published as WO 99/31224 on June 24, 1999). .

Preparation of Modified Sugars

In general, sugar sites or sugar site-linker cassettes and PEG or PEG-linker cassette groups are generally linked together by using reactive groups transformed into new organic functional groups or unreacted species by linkage methods. The sugar reactive functional group (s) are placed at any position on the sugar site. Reactive groups and classes of reactions useful in practicing the present invention are generally well known in the art of bioconjugate chemistry. A presently preferred class of reactions available by reactive sugar moieties is to proceed under relatively mild conditions. These include nucleophilic substitution reactions (eg, amines and alcohols with acyl halides, active esters), electrophilic substitution reactions (eg, enamine reactions) and carbon-carbon and carbon-heteroatomic multiple bonds. Addition reactions to (eg, Michael reaction, Diels-Alder addition reaction) are included, but are not limited to these. These and other useful reactions are described, for example, in March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et al. , Modification of Proteins; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982).

Useful reactive functional groups pendant from sugar nuclei or modifying groups include, but are not limited to, the following groups (a) to (j):

(a) including N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters ( Carboxyl groups and various derivatives thereof;

(b) hydroxyl groups which can be converted, for example, to esters, ethers, aldehydes and the like;

(c) haloalkyl groups, wherein the halides are later replaced by nucleophilic groups such as, for example, amines, carboxylate anions, thiol anions, carbanions or alkoxide ions, thereby creating new Provide shared attachment of groups);

(d) a dienophilic group capable of participating in the Diels-Alder reaction, eg, a maleimido group;

(e) Subsequent derivatization reactions can be made, for example, through the formation of carbonyl derivatives such as imines, hydrazones, semicarbazones or oximes, or through mechanisms such as Grignard addition reactions or alkyllithium addition reactions. Aldehyde or ketone groups;

(f) sulfonyl halide groups for subsequent reaction with amines, for example to form sulfonamides;

(g) thiol groups that can be converted, for example, to disulfides or reacted with acyl halides;

(h) amine or sulfhydryl groups, which may be, for example, acylated, alkylated or oxidized;

(i) alkenes, for example, capable of carrying out cycloaddition, acylation, Michael addition, etc .; And

(j) epoxides that can react with, for example, amines and hydroxyl compounds.

Reactive functional groups can be chosen so that they do not participate in or inhibit the reactions required to assemble reactive sugar nuclei or modifying groups. Alternatively, reactive functional groups can be protected from participating in the reaction by the presence of a protecting group. Those skilled in the art will understand how to protect a particular functional group so as not to inhibit a selected set of reaction conditions. For examples of useful protecting groups, see, for example, Greene et al., Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

In the following description, numerous specific examples of modified sugars useful in practicing the present invention are described. In an exemplary embodiment, a sialic acid derivative is used as the sugar nucleus to which the modifying group is attached. The focus of the description for sialic acid derivatives is merely to clarify the description and should not be understood as limiting the scope of the invention. Those skilled in the art will appreciate that various other sugar moieties can be activated and derivatized in a manner similar to that described using sialic acid as an example. For example, a number of methods can be used to modify galactose, glucose, N-acetylgalactosamine and fucose to refer to several sugar substrates that are readily modified by methods recognized in the art [ See, eg, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); And Schafer et al., J. Org. Chem. 65: 24 (2000).

In an exemplary embodiment, the peptides modified by the methods of the invention are glycopeptides produced in mammalian cells (eg, CHO cells), or in transgenic animals, and thus incompletely sialylated N- and / Or O-linked oligosaccharide chains. Oligosaccharide chains of glycopeptides that lack sialic acid and contain terminal galactose residues may be modified PEGylated, PPGylated, or otherwise modified by modified sialic acid.

In Scheme 4, amino glycoside 1 is treated with an active ester of a protected amino acid (eg glycine) derivative to convert the sugar amine residues into the corresponding protected amino acid amide adducts. The adduct is treated with aldolase to form α-hydroxy carboxylate 2. Compound 2 is converted to the corresponding CMP derivative by the action of CMP-SA synthetase, followed by the catalytic hydrogenation of the CMP derivative to give compound 3. The amines introduced through the formation of glycine adducts reacted with compound 3 with activated PEG or PPG derivatives (eg PEG-C (O) NHS, PEG-OC (O) Op-nitrophenyl), respectively 4 or 5 It is used as the site of PEG attachment by creating a kind such as

Table 3 describes representative examples of sugar monophosphates derivatized by PEG moieties. Certain of the compounds in Table 3 are prepared by the method of Scheme 4. Other derivatives are prepared by methods recognized in the art. See, eg, Keppler et al., Glycobiology 11 : 11R (2001); And Charter et al., Glycobiology 10 : 1049 (2000)]. Other amine reactive PEG and PPG homologues are available commercially, or they can be prepared by methods readily available to those skilled in the art.

example To practice the invention  Useful modified sugar Phosphate is  In addition to the positions described above, they may be substituted in other positions. Currently preferred substitutions of sialic acid are described by the formula:

Wherein X is preferably -O-, -N (H)-, -S-, -CH _2- , and -N (R) ₂ , wherein each R is independent from R ¹ -R ⁵ Is the selected member). The symbols Y, Z, A and B each represent a group selected from the group described above for the main body of X. X, Y, Z, A and B are each independently selected, so they may be the same or different. The symbols R ¹ , R ² , R ³ , R ⁴ and R ⁵ represent H, PEG moieties, therapeutic moieties, biomolecules or other moieties. Instead, these symbols represent linkers bound to PEG sites, therapeutic sites, biomolecules or other sites.

Examples of sites attached to the conjugates described herein include PEG derivatives (eg, acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-PEG, aryl-PEG), PPG derivatives (eg For example, acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPG carbamoyl-PPG, aryl-PPG), therapeutic sites, diagnostic sites, mannose-6-phosphate, heparin, SLe _x , mannose , Mannose-6-phosphate, sialyl lewis X, FGF, VFGF, protein, chondroitin, keratan, dermatan, albumin, integrin, antennae oligosaccharides, peptides and the like. Does not. Methods of conjugating various modifying groups to saccharide sites are readily available to those skilled in the art. [Poly (Ethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; Poly (Ethylene Glycol) Chemical and Biological Applications, J. Milton Harris, Ed., ACS Symposium Series No. 680, American Chemical Society, 1997; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; And Dunn et al. , Eds. Polymeric Drugs And Drug Delivery Systems, ACS Symposium Series Vol. 469, American Chemical Society, Washington, DC 1991].

Linker Group (Crosslinkable Group)

Methods of preparing modified sugars for use in the methods of the present invention include the attachment of PEG moieties to sugar moieties and the formation of stable adducts, preferably substrates for glycosyltransferases. Thus, it is often desirable to use linkers, for example, those formed by reacting PEG and sugar moieties with crosslinkers to conjugate PEG and sugars. Examples of bifunctional compounds that can be used to attach the modifying group to the carbohydrate moiety include, but are not limited to, bifunctional poly (ethylene glycol), polyamides, polyethers, polyesters, and the like. General methods of linking carbohydrates to other molecules are known in the literature. See, eg, Lee et al., Biochemistry 28: 1856 (1989); Bhatia et al., Anal. Biochem. 178: 408 (1989); Janda et al., J. Am. Chem. Soc. 112: 8886 (1990) and Bednarski et al., WO 92/18135. In the description below, reactive groups are treated positive on the sugar moiety of the modified sugar early in development. The focus of the explanation is to clarify the explanation. Those skilled in the art will understand that this description applies equally to reactive groups on deformable groups.

A variety of reagents are used to modify the components of the modified sugars with intramolecular chemical crosslinking (for review of crosslinking reagents and crosslinking methods see: Wold, F., Meth. Enzymol. 25: 623- 651, 1972; Weetall, HH, and Cooney, DA, In: Enzymes as Drugs. (Holcenberg, and Roberts, eds.) Pp. 395-442, Wiley, New York, 1981; Ji, TH, Meth.Enzymol. 91 : 580-609, 1983; Mattson et al., Mol. Biol. Rep. 17: 167-183, 1993, all of which are incorporated herein by reference). Preferred crosslinking reagents are derived from various zero-length, homo-bifunctional, and hetero-bifunctional crosslinking reagents. Zero-length crosslinking reagents include direct conjugation of two endogenous chemical groups without the introduction of exogenous materials. Reagents that promote the formation of disulfide bonds fall into this category. Another example is a reagent which induces a condensation reaction between a carboxyl and a primary amino group to form an amide bond such as carbodiimide, ethylchloroformate, Woodward's reagent K (2-ethyl-5-phenylisox Sazolium-3'-sulfonate) and carbonyldiimidazole. In addition to these chemical reagents, enzyme transglutaminase (glutamyl-peptide-γ-glutamyltransferase; EC 2.3.2.13) can be used as the zero-length crosslinking reagent. This enzyme typically promotes an acyl transfer reaction in the carboxamide group of a protein-linked glutaminyl residue having a primary amino group as a substrate. Preferred homo- and hetero-bifunctional reagents contain two identical or two different sites each, which can be reactive to amino, sulfhydryl, guanidino, indole or non-specific groups.

Argument IX Conjugate  refine

The product produced by this method can be used without purification. However, it is generally desirable to recover the product. Well-known standard methods for the recovery of glycosylated saccharides can be used, such as thin or thick layer chromatography, column chromatography, ion exchange chromatography, or membrane filtration. Preference is given to using membrane filtration, or one or more column chromatography techniques, more preferably with reverse osmosis membranes, for recovery as described below and in the literature cited herein. For example, membrane filtration (where the membrane has a molecular weight cutoff of about 3000 to about 10,000) can be used to remove proteins such as glycosyl transferase. Subsequently, nanofiltration or reverse osmosis can be used to remove salts and / or to purify product saccharides (see for example WO 98/15581). Nanofiltration membranes are a type of reverse osmosis membrane that allows monovalent salts to pass, depending on the membrane used, but retains polyvalent salts and uncharged solutes greater than about 100 to about 2,000 Daltons. Thus, in typical applications saccharides prepared by the process of the present invention will remain in the membrane and contaminating salts will pass through it.

If modified glycoproteins are produced intracellularly, particulate fragments, either host cells or lysed fragments, are removed by centrifugation or ultrafiltration as a step; The protein is optionally concentrated using a commercially available protein enrichment filter, followed by immunoaffinity chromatography, ion exchange column fractionation (eg, diethylaminoethyl (DEAE), or containing carboxymethyl or sulfopropyl groups) On the matrix), Blue-Sepharose, CM Blue-Sepharose, Mono-Q, Mono-S, Lentil Lectin-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether Toyopearl ( Toyopearl), Butyl Toyopearl, Phenyl Toyopearl, or Chromatography on Protein A Sepharose, SDS-PAGE Chromatography, Silica Chromatography, Chromatofocusing, Reversed Phase HPLC (e.g., silica gel with suspended aliphatic groups) ), Eg gel filtration or size-exclusion chromatography with Sephadex molecular sieves, chromatography on columns that specifically bind polypeptides. By one or more of the steps selected from the blood, and ethanol or ammonium sulfate precipitation can be separated the polypeptide variant from other impurities.

Modified glycopeptides produced in culture are typically separated by initial extraction from cells, enzymes and the like, followed by one or more concentration, salting out, aqueous ion-exchange, or size-exclusion chromatography steps. In addition, the modified glycoprotein can be purified by affinity chromatography. Finally, HPLC can be used for the final purification step.

Protease inhibitors such as methylsulfonylfluoride (PMSF) can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of accidental contaminants.

In a further scope of embodiments, the supernatant from the system for producing modified glycopeptides of the present invention may be prepared using a commercially available protein enrichment filter such as Amicon or Millipore Pellicon ultrafiltration. Concentrate using a filtration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. For example, suitable affinity matrices can include ligands for peptides, lectins or antibody molecules bound to a suitable support. Instead, an anion-exchange resin, for example a matrix or substrate with pendant DEAE groups can be used. Suitable matrices include acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation-exchange step may be used. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are particularly preferred.

Finally, one or more RP-HPLC steps using hydrophobic RP-HPLC media, such as silica gel with pendant methyl or other aliphatic groups, can be used to further purify the polypeptide variant composition. In addition, some or all of the aforementioned purification steps may be used in various combinations to provide homogeneously modified glycoproteins.

Modified glycopeptides of the invention provided from large scale fermentation can be purified by methods similar to those described in Urdal et al., J. Chromatog . 296: 171 (1984). This document describes two sequential RP-HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column. Alternatively, modified glycoproteins may be purified using techniques such as affinity chromatography.

Pharmaceutical composition

In another aspect, the present invention provides a pharmaceutical composition. Pharmaceutical compositions include pharmaceutically acceptable diluents and covalent conjugates between naturally occurring PEG sites, therapeutic sites or biomolecules and glycosylated or non-glycosylated factor IX peptides. The polymer, therapeutic moiety or biomolecule is conjugated to the peptide via an intact glycosyl linking group disposed between and covalently linked to the peptide and the polymer, therapeutic moiety or biomolecule.

The pharmaceutical compositions of the present invention are suitable for use in various drug delivery systems. Suitable formulations for use in the present invention are described in Remington's Pharmaceutical Sciences , Mace Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of methods for drug delivery, see Langer, Science 249: 1527-1533 (1990).

The pharmaceutical composition may be formulated in any suitable mode of administration, including, for example, topical, oral, intranasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration such as subcutaneous injection, the carrier comprises water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate can be used. Biodegradable microspheres (eg polylactate polyglycolate) may also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are described, for example, in US Pat. Nos. 4,897,268 and 5,075,109.

Typically, the pharmaceutical composition is administered parenterally, for example intravenously. Accordingly, the present invention provides compositions for parenteral administration comprising a compound dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, such as water, buffered water, saline, PBS, and the like. The composition may contain pharmaceutically acceptable auxiliary ingredients such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, etc., as necessary to bring them closer to physiological conditions.

These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solution may be packaged as such or lyophilized, and the lyophilized formulation is combined with a sterile aqueous carrier prior to administration. The pH of the formulations may generally be 3 to 11, more preferably 5 to 9, most preferably 7 and 8.

In some embodiments, glycopeptides of the invention may be incorporated into liposomes formed from standard vesicle-forming lipids. Various methods, such as described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), US Pat. Nos. 4,235,871, 4,501,728 and 4,837,028, are used to prepare liposomes. Can be. Targeting liposomes with various targeting agents (eg, sialyl galactoside of the present invention) is well known in the art (see, eg, US Pat. Nos. 4,957,773 and 4,603,044).

Standard methods of coupling targeting agents to liposomes can be used. These methods generally involve incorporation of lipid components, such as phosphatidylethanolamine, or derivatized lipophilic compounds, such as lipid-derived glycopeptides of the invention, into liposomes, which may be activated for attachment of targeting agents. .

Targeting mechanisms generally require the placement of a targeting agent on the surface of the liposome in a manner that makes the target site available for interaction with a target, eg, a cell surface receptor. Carbohydrates of the invention can be prepared by liposomes using methods known to those skilled in the art (e.g., alkylation or acylation reactions of hydroxyl groups present on liposomes with long-chain alkyl halides or fatty acids, respectively). It may be attached to the lipid molecule before it is formed. Alternatively, liposomes can be made by first incorporating the linker moiety into the membrane at the time of forming the membrane. The linker moiety should have a lipophilic moiety that is tightly embedded and immobilized in the membrane. It must also have a reactive moiety that is chemically available on the aqueous surface of the liposome. The reactive moiety is chosen such that it is chemically suitable to form a stable chemical bond with the targeting agent or carbohydrate added later. In some cases, the targeting agent may attach directly to the linker molecule, but in most cases it is a third agent that acts as a chemical bridge to link the linker molecule in the membrane with the targeting agent or carbohydrate that extends three-dimensionally out of the vesicle surface. It is more suitable to use molecules.

Compounds prepared by the methods of the invention may also have use as diagnostic reagents. For example, labeled compounds can be used to locate inflammation or tumor metastasis regions in patients suspected of having inflammation. For this use, the compound may be labeled with ¹²⁵ I, ¹⁴ C, or tritium.

The active ingredient used in the pharmaceutical compositions of the present invention is glycofezilated factor IX and derivatives thereof which have biological properties that participate in blood coagulation steps. Liposomal dispersions of the present invention are useful as parenteral preparations in the treatment of coagulation disorders characterized by low or deficient coagulation, such as various forms of hemophilia. Preferably, the Factor IX composition of the present invention is administered parenterally (eg IV, IM, SC or IP). Effective dosages are expected to vary significantly depending on the condition to be treated and the route of administration, but is expected to range from about 0.1 to 000 μg of active substance per kg of body weight. Preferred doses for the treatment of coagulation disorders are from about 50 to about 3000 μg / kg three times a week, more preferably from three times a week, from about 500 to about 2000 μg / kg, more preferably one week At about 750 to about 1500 μg / kg, more preferably at about 1000 μg / kg three times a week. Since the present invention provides Factor IX with enhanced in vivo residence time, the dosages mentioned when the compositions of the present invention are administered are optionally lowered.

The following examples are presented to illustrate the conjugates and methods of the present invention, but are not intended to limit the claimed invention.

Example 1

Preparation of UDP-GalNAc-6'-CHO

UDP-GalNAc (200 mg, 0.30 mmoles) was dissolved in 1 mM CuSO ₄ solution (20 mL) and 25 mM NaH ₂ PO ₄ solution (pH 6.0; 20 mL). Then, galactose oxidase (240 U; 240 μl) and catalase (13000 U; 130 μl) were added, and the reaction system was equipped with an oxygen-filled balloon and stirred at room temperature for 7 days. The reaction mixture was then filtered (spin cartridge; MWCO 5K) and the filtrate (˜40 mL) was stored at 4 ° C. until needed. TLC (silica; EtOH / water (7/2); R _f = 0.77; visualized by anisealdehyde staining).

Example 2

Preparation of UDP-GalNAc-6'-NH ₂

Ammonium acetate (15 mg, 0.194 mmoles) and NaBH ₃ CN (1M THF solution; 0.17 mL, 0.17 mmoles) were added to the UDP-GalNAc-6'-CHO solution (2 mL or ˜20 mg) obtained above at 0 ° C. Add and warm to rt overnight. The reaction solution was filtered through a G-10 column with water and the product was collected. Appropriate fractions were freeze-dried and stored frozen. TLC (silica; ethanol / water (7/2); R _f = 0.72; visualized with ninhydrin reagent).

Example 3

Preparation of UDP-GalNAc-6-NHCO (CH ₂ ) ₂ -O-PEG-OMe (1 KDa)

Galactosaminyl-1-phosphate-2-NHCO (CH ₂ ) ₂ -O-PEG-OMe (1 KDa) (58 mg, 0.045 mmoles) was dissolved in DMF (6 mL) and pyridine (1.2 mL). Then UMP-morpholidate (60 mg, 0.15 mmoles) was added and the resulting mixture was stirred at 70 ° C. for 48 hours. The solvent was removed by bubbling nitrogen through the reaction mixture and the residue was purified by reverse phase chromatography (C-18 silica, gradient between 10 and 80%, methanol / water). The desired fractions were collected and dried at reduced pressure to yield 50 mg (70%) of a white solid. TLC (silica, propanol / H ₂ O / NH ₄ OH, (30/20/2), Rf = 0.54). MS (MALDI): observed, 1485, 1529, 1618, 1706.

Example 4

Preparation of Cysteine-PEG ₂ (2)

4.1 Synthesis of (1)

Potassium hydroxide (84.2 mg, 1.5 mmol, as powder) was added to a solution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous methanol (20 mL) under argon. The mixture was stirred at room temperature for 30 minutes and then 20 kilodaltons of mPEG-O-tosylate (Ts; 1.0 g, 0.05 mmol) was added in several portions over 2 hours. The mixture was stirred at rt for 5 days and concentrated by rotary evaporation. The residue was diluted with water (30 mL) and stirred at room temperature for 2 hours to decompose excess 20 kilodalton mPEG-O-tosylate. The solution was then neutralized with acetic acid, the pH was adjusted to pH 5.0 and loaded on a reverse phase chromatography (C-18 silica) column. The column was eluted with a gradient of metaol / water (product eluted in about 70% methanol) and product elution was monitored by evaporative light scattering and the appropriate fractions collected and diluted with water (500 mL). This solution was chromatographed (ion exchange, XK 50 Q, BIG Beads, 300 mL, hydroxide form; gradient from water to water / acetic acid-0.75 N) and the pH of the appropriate fractions was lowered to 6.0 using acetic acid. . This solution was then captured on a reversed phase column (C-18 silica) and eluted with a methanol / water gradient as described above. The product fractions were combined, concentrated, redissolved in water and freeze-dried to give 453 mg (44%) of a white solid (1). Structural data for this compound were as follows: ¹ H-NMR (500 MHz; D ₂ O) δ 2.83 (t, 2H, OCC H ₂ -S), 3.05 (q, 1H, SC H H-CHN), 3.18 (q, 1H, (q, 1H, SC H H-CHN), 3.38 (s, 3H, C H ₃ O), 3.7 (t, OC H ₂ C H ₂ O), 3.95 (q, 1H, C H N) The purity of the product was confirmed by SDS PAGE.

4.2. Synthesis of (2)

Triethylamine (˜0.5 mL) was added dropwise to a solution of 1 (440 mg, 22 μmol) dissolved in anhydrous CH ₂ Cl ₂ (30 mL) until the solution became basic. A solution of 20 kilodalton mPEG-Op-nitrophenyl carbonate (660 mg, 33 μmol) and N-hydroxysuccinimide (3.6 mg, 30.8 μmol) in CH ₂ Cl ₂ (20 mL) over 1 hour at room temperature Add in several portions. The reaction mixture was stirred at rt for 24 h. After this time the solvent was removed by rotary evaporation, the residue was dissolved in water (100 mL) and the pH was adjusted to 9.5 with 1.0 N NaOH. The basic solution was stirred at room temperature for 2 hours and then neutralized to pH 7.0 with acetic acid. The solution was then loaded onto a reverse phase chromatography (C-18 silica) column. The column was eluted with a methanol / water gradient (product elutes in about 70% methanol) and product elution was monitored by evaporative light scattering and the appropriate fractions collected and diluted with water (500 mL). This solution was chromatographed (ion exchange, XK 50 Q, BIG Beads, 300 mL, hydroxide form; gradient of water to water / acetic acid-0.75 N) and the pH of the appropriate fraction was lowered to 6.0 with acetic acid. This solution was then captured on a reversed phase column (C-18 silica) and eluted with a methanol / water gradient as described above. The product fractions were combined, concentrated, redissolved in water and freeze-dried to give 575 mg (70%) of a white solid (2). Structural data for the compound are as follows: ¹ H-NMR (500 MHz; D ₂ O) δ 2.83 (t, 2H, OCC H ₂ -S), 2.95 (t, 2H, OCC H ₂ -S), 3.12 (q, 1H, SC H H-CHN), 3.39 (s, 3H C H ₃ O), 3.71 (t, OC H ₂ C H ₂ O). The purity of the product was confirmed by SDS PAGE.

Example 5

Preparation of UDP-GalNAc-6-NHCO (CH ₂ ) ₂ -O-PEG-OMe (1 KDa)

Galactosaminyl-1-phosphate-2-NHCO (CH ₂ ) ₂ -O-PEG-OMe (1 kilodalton) (58 mg, 0.045 mmoles) was dissolved in DMF (6 mL) and pyridine (1.2 mL). . Then UMP-Mor polydate (60 mg, 0.15 mmoles) was added and the resulting mixture was stirred at 70 ° C. for 48 hours. The solvent was removed by bubbling nitrogen through the reaction mixture and the residue was purified by reverse phase chromatography (C-18 silica. Gradient gradient between 10 and 80%, methanol / water). The desired fractions were collected and dried at reduced pressure to yield 50 mg (70%) of a white solid. TLC (silica, propanol / H ₂ O / NH ₄ OH, (30/20/2), R _f = 0.54). MS (MALDI): observed, 1485, 1529, 1618, 1706.

Example 6

Glycopezylation of Factor IX Produced in CHO Cells

This example describes the preparation of asialo factor IX and its sialylation by CMP-sialic acid-PEG.

6.1. Desialylation of r Factor IX

Recombinant form of aggregate factor IX (rFactor IX) was prepared in CHO cells. 6000 IU of rfactor IX was dissolved in a total of 12 ml USP H ₂ O. This solution was transferred to another Centricon Plus 20, PL-10 centrifugal filter by another 6 ml USP H ₂ O. The solution was concentrated to 2 mL, then diluted with 15 mL 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl ₂ , 0.05% NaN ₃ and then reconcentrated. Dilution / concentration was repeated four times to effectively change the buffer to a final volume of 3.0 ml. Transfer 2.9 mL (about 20 mg rfactor IX) in this solution to a small plastic tube and add 530 mU α2- 3,6,8-neuramidase-agarose conjugate (Vibrio cholerae). , Calbiochem, 450 μl) was added. The reaction mixture was gently rotated at 32 ° C. for 6.5 h. The mixture was centrifuged at 10,000 rpm for 2 minutes and the supernatant collected. Agarose beads (containing neuraminidase) were washed six times with 0.5 ml of 50 mM Tris-HCl pH 7.12, 1 M NaCl, 0.05% NaN ₃ . The wash and supernatant were collected and centrifuged again at 10,000 rpm for 2 minutes to remove any residual agarose resin. The desialylated protein solution was collected and diluted to 19 ml with the same buffer and concentrated to ˜2 ml in a Centricon plus 20 PL-10 centrifugal filter. The solution was diluted twice with 15 mL of 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 0.05% NaN ₃ and reconcentrated to 2 mL. The final desialylated rFactor IX solution was diluted with Tris buffer to 3 ml final volume (˜10 mg / ml). Natural and desialylated rfactor IX samples were analyzed by IEF-electrophoresis. Isoelectric focusing gels (pH 3-7) were run using 1.5 μl (15 μg) of sample diluted first with 10 μl of Tris buffer and mixed with 12 μl of sample load buffer. Loaded, run and fixed using the canon method The gel was stained with Colloidal Blue Stain (Figure 154) showing a band for desialylated factor IX.

Example 7

Preparation of PEG (1 kDa and 10 kDa) -SA-Factor IX

Desialylated rfactor-IX (29 mg, 3 ml) was divided into two 1.5 ml (14.5 mg) samples in two 15 ml centrifuge tubes. Each solution was diluted with 12.67 mL of 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 0.05% NaN ₃ and CMP-SA-PEG-1k or 10k (7.25 μmol) was added. The tubes were gently inverted and mixed and 2.9 U ST3Gal3 (326 μl) was added (total volume 14.5 mL). The tube was inverted again and gently rotated at 32 ° C. for 65 hours. The reaction was stopped by freezing at -20 ° C. 10 μg sample of the reaction solution was analyzed by SDS-PAGE. Toxo Haas Biosep G3000SW (21.5 × 30 cm, 13 μm) HPLC column using phenylated protein with Dulbecco's phosphate buffered saline, pH 7.1 (Gibco) at 6 ml / min Purification on phase. Reactions and purification were monitored using SDS PAGE and IEF gels. Dilute 2 μl of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NaN ₃ buffer in Novex Tris-Glycine 4-20% 1 mm gel, 12 μl of sample load buffer and 1 μl. 10 μl (10 μg) of sample after loading with 0.5 M DTT was loaded and heated at 85 ° C. for 6 minutes. Gels were stained with a colloidal blue dye (Figure 155) showing bands for PEG (1 kDa and 10 kDa) -SA-Factor IX.

Example 8

Direct Sialyl-Glycopegylation of Factor IX

This example describes a method for sialyl-pezylation of factor IX without prior sialidase treatment.

8.1. Sialyl-pegylation of Factor-IX by CMP-SA-PEG- (10 KDa)

Factor IX (1100 IU) expressed in CHO cells or fully sialylation was dissolved in 5 ml of 20 mM histidine, 520 mM glycine, 2% sucrose, 0.05% NaN ₃ and 0.01% polysorbate 80, pH 5.0. . CMP-SA-PEG- (10 kDa) (27 mg, 2.5 mmol) was then dissolved in the solution and 1 U of ST3Gal3 was added. The reaction was completed after gentle mixing at 32 ° C. for 28 hours. The reaction was analyzed by SDS-PAGE as described by Invitrogen. The product protein was economyed on an Amersham Superdex 200 (10 × 300 mm, 13 μm) HPLC column at 1 mL / min using phosphate buffered saline, pH 7.0 (PBS). R _t = 9.5 min.

Example 9

Sialyl-pegylation of Factor-IX by CMP-SA-PEG- (20 kDa)

Factor IX (1100 IU) expressed and fully sialylated in CHO cells was dissolved in 5 ml of 20 mM histidine, 520 mM glycine, 2% sucrose, 0.05% NaN ₃ and 0.01% polysorbate 80, pH 5.0. Then CMP-SA-PEG- (20 kDa) (50 mg, 2.3 μmol) was dissolved in the solution and CST-II was added. The reaction mixture was mixed after gentle mixing at 32 ° C. for 42 hours. The reaction was analyzed by SDS-PAGE as described by Invitrogen.

The product protein was purified on an Amersham Superdex 200 (10 × 300 mm, 13 μm) HPLC column at 1 mL / min using phosphate buffered saline, pH 7.0 (Fisher). R _t = 8.6 min.

Example 10

Sialic acid capping of glycofezilated factor IX

This example describes a method for sialic acid capping of sialyl-glycopezylated peptides. Wherein Factor-IX is an exemplary peptide.

10.1. Sialic acid capping of N-linked and O-linked glycans of factor-IX-SA-PEG (10 kDa)

Purified r-Factor-IX-PEG (10 kDa) (2.4 mg) was concentrated in a Centricon plus 20 PL-10 (Millipore Corp., Bedford, Mass.) Centrifugal filter and the buffer was 50 mM to a final volume of 1.85 ml. Tris-HCl pH 7.2, 0.15 M NaCl, 0.05% NaN ₃ . The protein solution was diluted with 372 μl of the same Tris buffer and 7.4 mg CMP-SA (12 μmol) was added as a solid. The solution was mixed inverted gently and 0.1 U ST3Gal1 and 0.1 U ST3Gal3 were added. The reaction mixture was gently rotated at 32 ° C. for 42 hours.

10 μg sample of the reaction solution was analyzed by SDS-PAGE. Novex Tris-Glycine 4-12% 1 mm gel was treated and stained using colloidal blue dye as described by Invitrogen. Briefly, 10 μl (10 μg) of sample was mixed with 12 μl of sample load buffer and 1 μl of 0.5 M DTT and heated at 85 ° C. for 6 minutes (Figure 156, lane 4).

Example 11

Glycopezylated Factor IX Pharmacodynamic Test

Four glycofezilated FIX variants (PEG-9 variants) were tested by PK test in normal mice. The activity of the four compounds was previously demonstrated in vitro by blood clots, endogenous thrombin potential (ETP), and thromboelastograph (TEG) tests. Activity results are summarized in Table I below.

compound Blood clot activity
(% Of plasma) ETP
(Relatively inactive) TEG
(Relatively inactive) Benepix 45% 1.0 1.0 PEG-9-2K (LS) 27% 0.3 0.2 PEG-9-2K (HS) 20% 0.2 0.1 PEG-9-10K 11% 0.6 0.3 PEG-9-30K 14% 0.9 0.4

 PK tests were designed and performed to evaluate the prolongation of the activity of the four PEG-9 compounds in the circulation. Non-hemophilia mice were used as two samples per time point and three samples per animal. Sampling time points were 0, 0.08, 0.17, 0.33, 1, 3, 5, 8, 16, 24, 30, 48, 64, 72, and 96 hours after compound administration. Blood samples were centrifuged and stored in two aliquots, one for blood clotting and one for ELISA. Due to material limitations the PEG-9 compound was administered in amounts: 250 U / kg Benepix; 2K (low substitution: "LS" (1-2 PEG substitutions per peptide molecule) 200 U / kg; 2K (high substitution: "HS" (3-4 PEG substitutions per peptide molecule) 200 U / kg; 10K 100 U / kg 30K 100 U / kg All doses were based on the calculated blood clotting test unit.

The results are shown schematically in FIG. 6 and in Table II below.

compound Volume
(U / kg) Cmax
(U / ml) AUC
(hU / ml) CL
(Ml / h / kg) Benepix 250 0.745 1.34 187 PEG-9-2K (LS) 200 0.953 4.69 42.7 PEG-9-2K (HS) 200 0.960 9.05 22.1 PEG-9-10K 100 0.350 2.80 35.7 PEG-9-30K 100 1.40 8.83 11.3

This result showed a tendency to extend for all PEG-9 compounds. The values of AUC and Cmax were not compared directly. However, clearance rates (CL) were compared and CL was lower for PEG-9 compounds compared to Benepix, suggesting a longer residence time in mice. Although Benepix was administered at the highest dose, the time for the last detectable blood clot activity was increased for the PEG-9 compound compared to Benefix.

Example 12

Preparation of LS and HS Glycopezylated Factor IX

Glycopezylated Factor IX with low degree of substitution by PEG was prepared from native Factor IX by an exchange reaction promoted by ST3Gal-III. The reaction was carried out in a buffer of 10 mM histidine, 260 mM glycine, 1% sucrose and 0.02% Tween 80, pH 7.2. For glycofezilation by CMPSA-PEG (2 kD and 10 kD), Factor IX (0.5 mg / mL) was added to ST3GalIII (50 mU / mL) and CMP-SA-PEG (0.5 mM) for 16 hours at 32 ° C. Incubated with For PEGylation by CMP-SA-PEG 30 kD, the concentration of Factor IX was raised to 1.0 mg / ml and the concentration of CMP-SA-PEG was reduced to 0.17 mM. Under these conditions at least 90% of the Factor IX molecules were substituted with at least one PEG moiety.

Glycopezylated Factor IX with high degree of substitution by PEG was prepared by enzymatic desialylation of native Factor IX. Factor IX peptides were buffer exchanged to 50 mM mES, pH 6.0 using a PD10 column and adjusted to a concentration of 0.66 mg / ml with AUS sialidase (5 mU / ml) at 32 ° C. for 16 hours. Desialylation was confirmed by SDS-PAGE, HPLC and MALDI glycan analysis. Asialo Factor IX was purified on Q Sepharose FF to remove sialidase. CaCl ₂ fractions were concentrated using an Ultra15 concentrator and buffer exchanged to MES, pH 6.0 using a PD10 column.

2kD and 10 kD pegylation of asialo-factor IX (0.5 mg / ml) was performed by incubating with ST3Gal-III (50 mU / ml) and CMP-SA-PEG (0.5 mM) for 16 hours at 32 ° C. It became. For PEGylation by CMPSA-PEG-30kD, the concentration of Factor IX was raised to 1.0 mg / ml and the concentration of CMP-SA-PEG was reduced to 0.17 mM. After 16 hours of pegylation, glycans with terminal galactose were capped by sialic acid by adding 1 mM CMP-SA and continuing incubation at 32 ° C. for an additional 8 hours. Under these conditions at least 90% of the Factor IX molecules were substituted with at least one PEG moiety. Factor IX produced by this method has a larger apparent molecular weight in SDS-PAGE.

Example 13

Preparation of O-Glycopezylated Factor IX

O-glycan chains were newly introduced in native factor IX (1 mg / ml) by incubating peptides with GalNAcT-II (25mU / ml) and 1 mM UDP-GalNAc at 32 ° C. After incubation for 4 hours, the phylation reaction was followed by addition of CMPSA-PEG (2Kd or 10Kd at 0.5 mM, or 0.5 mM or 30 kDd at 0.17 mM) and ST6GalNAc-I (25 mU / ml), and an additional 20 hours. It was initiated by incubating for a while.

The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes may be suggested to those skilled in the art in light of the above, and the meanings and scope of the present application, and appended It is understood that it is included within the scope of the claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

<110> NEOSE TECHNOLOGIES INC. <120> GLYCOPEGYLATED FACTOR IX <130> 40853-01-5144WO <140> 10-2006-7010603 <141> 2006-05-30 <150> US 60 / 527,089 <151> 2003-12-03 <150> US 60 / 539,387 <151> 2004-01-26 <150> US 60 / 592,744 <151> 2004-07-29 <150> US 60 / 614,518 <151> 2004-09-29 <150> US 60 / 623,387 <151> 2004-10-29 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 415 <212> PRT <213> Human <400> 1 Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg   1 5 10 15 Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe              20 25 30 Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly          35 40 45 Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp      50 55 60 Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys  65 70 75 80 Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu                  85 90 95 Glu Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr             100 105 110 Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val         115 120 125 Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr     130 135 140 Arg Ala Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu 145 150 155 160 Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn                 165 170 175 Asp Phe Thr Arg Val Val Gly Glu Asp Ala Lys Pro Gly Gln Phe             180 185 190 Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly         195 200 205 Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu     210 215 220 Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu 225 230 235 240 Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His                 245 250 255 His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu             260 265 270 Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile         275 280 285 Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser     290 295 300 Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala 305 310 315 320 Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys                 325 330 335 Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly             340 345 350 Phe His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro         355 360 365 His Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser     370 375 380 Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys 385 390 395 400 Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr                 405 410 415  

Claims (33)

Formula (I)

         (I) [In the above formula, D is a member selected from -OH and R ¹ -L-HN-; G is a member selected from R ¹ -L- and -C (O) (C ₁ -C ₆ ) alkyl; R ¹ is a site comprising a member selected from straight or branched poly (ethylene glycol) residues; L is a linker which is a member selected from bonded, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkal, Provided that when D is OH, G is R ¹ -L-, and when G is -C (O) (C ₁ -C ₆ ) alkyl, D is R ¹ -L-NH-, LR ¹ is represented by the following formula:

(Wherein a is an integer of 0 to 20) A Factor IX peptide comprising at least one site having R ¹ is a member selected from the group of formula

[Wherein e and f are independently integers selected from 1 to 2500; q is an integer from 0 to 20; A Factor IX peptide wherein the site represented by Formula (I) is attached to a glycosyl residue attached to a member selected from Asn 157, Asn 167, and a combination thereof of Factor IX peptide. delete delete The method of claim 1, wherein the peptide is SEQ. ID. Factor IX peptide with amino acid sequence of NO: 1. A coagulation promoter or hemophilia therapeutic agent comprising a Factor IX peptide according to claim 1 and a pharmaceutically acceptable carrier. A coagulation promoter comprising the Factor IX peptide according to claim 1. A hemophilia therapeutic agent comprising the Factor IX peptide according to claim 1. Formula (I)

        (I) [In the above formula, D is a member selected from -OH and R ¹ -L-HN-; G is a member selected from R ¹ -L- and -C (O) (C ₁ -C ₆ ) alkyl; R ¹ is a site comprising a member selected from straight or branched poly (ethylene glycol) residues; L is a linker which is a member selected from bonded, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkal, Provided that when D is OH, G is R ¹ -L-, and when G is -C (O) (C ₁ -C ₆ ) alkyl, D is R ¹ -L-NH-, LR ¹ is represented by the following formula:

(Wherein a is an integer of 0 to 20) A factor IX peptide conjugate comprising: R ¹ is a member selected from the group of formula

[Wherein e and f are independently integers selected from 1 to 2500; q is an integer from 0 to 20] A method for preparing a Factor IX peptide conjugate wherein a site represented by Formula (I) is attached to a glycosyl residue attached to a member selected from Asn 157, Asn 167, and a combination thereof, of the Factor IX peptide, (a) the formula below:

A method for preparing a Factor IX peptide conjugate comprising contacting each other with a PEG-sialic acid donor, a substrate Factor IX peptide, and an enzyme that transfers the PEG-sialic acid onto an amino acid or glycosyl residue of the Factor IX peptide. delete delete The method of claim 8, wherein said Factor IX substrate peptide is SEQ. ID. A method having an amino acid sequence of NO: 1. A coagulation promoter or hemophilia therapeutic agent comprising the Factor IX peptide according to claim 4 and a pharmaceutically acceptable carrier. A coagulation promoter comprising the Factor IX peptide according to claim 4. A hemophilia therapeutic agent comprising the Factor IX peptide according to claim 4. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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