JP4738346B2 - GlycoPEGylated factor IX - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a US Provisional Patent Application No. 60 / 527,089 filed Dec. 3, 2003, the entire contents of which are incorporated herein by reference for all purposes. US Provisional Patent Application No. 60 / 539,387, filed January 26, 2004; US Provisional Patent Application No. 60 / 592,744, filed July 29, 2004; filed September 29, 2004 U.S. Provisional Patent Application No. 60 / 614,518; and U.S. Provisional Patent Application No. 60 / 623,387, filed October 29, 2004, each of which is hereby incorporated by reference in its entirety for all purposes. Claim priority based on (incorporated in the description).

BACKGROUND OF THE INVENTION Vitamin K-dependent proteins (eg Factor IX) contain 9-13 gamma-carboxyglutamate residues (Gla) in their amino terminal 45 residues. Gla residues are produced by enzymes that carboxylate the side chains of glutamic acid residues in the protein precursor in the liver using vitamin K. Vitamin K-dependent proteins are involved in several biological processes, of which the best described is blood clotting (Nelststuen, “Vitam. Home.” 58: 355. 389 (2000)). Vitamin K-dependent proteins include protein Z, protein S, prothrombin (factor II) factor X, factor IX, protein C, factor VII, Gas6, and matrix GLA proteins. Factors VII, IX, X, and II work in the precoagulation process, whereas protein C, protein S, and protein Z work in an anticoagulant role. Gas6 is a growth-inhibitory hormone encoded by growth arrest specific gene 6 (gas6) and is related to protein S. See Manfioletti et al., “Mol. Cell. Biol.” 13: 4976-4985 (1993). Matrix GLA proteins are usually found in bones and are important in preventing soft tissue calcification in the circulation. Luo et al. “Nature” 386: 78-81 (1997).

  The regulation of blood clotting is a process that poses several major health problems, including both the inability to form a clot and the formation of thrombosis, an undesirable clot. Agents that prevent unwanted blood clots are used in many situations, and a variety of agents are available. Unfortunately, most current treatments have undesirable side effects. Orally administered anticoagulants such as warfarin act by inhibiting the action of vitamin K in the liver, thereby preventing the complete carboxylation of glutamate residues in vitamin K-dependent proteins. As a result, it causes a decrease in the concentration of active protein in the circulatory system and a decrease in the ability to form clots. Warfarin therapy is complicated by the competitive nature of drugs and targets. Dietary vitamin K fluctuations can result in warfarin overdose or underdose. Variation in clotting activity is an undesirable result of this therapy.

  Injectable substances such as heparin, including low molecular weight heparin, are also commonly used anticoagulants. These compounds are also prone to overdose and should be carefully monitored.

  A newer category of anticoagulants includes vitamin K-dependent clotting factors with altered active sites, such as Factor VIIa and Factor IXa. The active site is blocked by serine protease inhibitors such as amino acid or chloromethyl ketone derivatives of short peptides. Proteins with modified active sites retain their ability to form complexes with their respective factors, but are inactive, thereby producing no enzymatic activity, and each active enzyme and cofactor complex. Prevent body formation. In short, these proteins appear to provide anticoagulant therapy benefits without the deleterious side effects of other anticoagulants. Factor Xa with modified active sites is another possible anticoagulant within this group. The cofactor protein is Factor Va. Activated protein C (APC) with modified active sites can also form effective inhibitors of coagulation. See Sorensen et al., “J. Biol. Chem.” 272: 11863-11868 (1997). APC with an altered active site binds to factor Va and prevents factor Xa from binding.

  It is cost that mainly suppresses the use of vitamin K-dependent clotting factors. Vitamin K-dependent protein biosynthesis relies on the intact glutamate carboxylation system present in a few animal cell types. Overproduction of these proteins is limited by this enzyme system. Furthermore, the effective amount of these proteins is large. A common dosage is 1000 μg / kg (body weight) of peptide. See Harker et al. 1997, supra.

  Another phenomenon that hinders the use of therapeutic peptides is a well-known aspect of protein glycosylation, the relatively short in vivo half-life exhibited by these peptides. In general, the short half-life problem in vivo means that therapeutic glycopeptides often have to be administered at high dosages, which in turn, lasts longer, Higher health care costs are required than may be required when more efficient methods are available for effective glycoprotein therapy.

  Factor Vila, for example, illustrates this problem. Factor VII and Factor VIIa have a circulating half-life in humans of about 2-4 hours. That is, within 2-4 hours, the concentration of peptide in the serum is reduced by half. When Factor VIIa is used as a procoagulant to treat certain forms of hemophilia, the standard protocol is to increase VIIa at high dosages (45-90 μg / kg body weight) every 2 hours. Will be injected. See Hedner et al., “Transfus. Med. Rev.” 7: 78-83 (1993)). Thus, the use of these proteins as procoagulants or anticoagulants (in the case of Factor VIIa) requires that the proteins be administered at frequent intervals and at high dosages.

  One solution to providing cost effective glycopeptide therapy has been to provide peptides with a longer half-life in vivo. For example, glycopeptide therapeutics with improved pharmacokinetic properties have been produced by attaching synthetic polymers to the peptide backbone. A typical polymer that has been conjugated to peptides is poly (ethylene glycol) ("PEG"). The use of PEG to derivatize peptide therapeutics has been demonstrated to reduce peptide immunogenicity. For example, US Pat. No. 4,179,337 (Davis et al.) Discloses non-immunogenic polypeptides such as enzymes and peptide hormones conjugated with polyethylene glycol (PEG) or polypropylene glycol. Yes. In addition to reduced immunogenicity, clearance time in circulation is prolonged by increasing the size of the polypeptide's PEG-conjugate.

  The primary mode of attaching PEG and its derivatives to peptides is non-specific linkage via amino acid residues of the peptide (eg, US Pat. No. 4,088,538, US Pat. No. 4,496). No. 6,689, U.S. Pat. No. 4,414,147, U.S. Pat. No. 4,055,635, and WO 87/00056). Another mode of attaching PEG to peptides is via non-specific oxidation of glycosyl residues on glycopeptides (see, eg, WO 94/05332).

  In these non-specific methods, poly (ethylene glycol) is added in a random non-specific manner to reactive residues on the peptide backbone. Of course, random addition of PEG molecules has its drawbacks, including the lack of homogeneity of the final product and the potential for reduced biological or enzymatic activity of the peptide. Thus, derivatization strategies that produce specifically homogeneous products that are specifically labeled and easily characterized for the production of therapeutic peptides are superior. Such a method has been developed.

  Specifically labeled, homogeneous peptide therapeutics can be produced in vitro through the action of enzymes. Unlike typical non-specific methods for attaching synthetic polymers or other labels to peptides, enzyme-based synthesis has the advantage of regioselectivity and stereoselectivity. Two major classes of enzymes for use in the synthesis of labeled peptides are glycosyltransferases (eg, sialyltransferases, oligosaccharyltransferases, N-acetylglucosaminyltransferases), and glycosidases. These enzymes can be used for specific attachment of sugars, which may subsequently be modified to include a therapeutic moiety. Alternatively, glycosyltransferases and modified glycosidases can be used to transfer modified sugars directly to the peptide backbone (eg, US Pat. No. 6,399, each incorporated herein by reference). , 336, and U.S. Patent Application Publication Nos. 2003000370037, 200401132640, 200401367557, 20040126838, and 20040142856). . Methods that combine both chemical and enzymatic synthesis elements are also known (eg, Yamamoto et al., “Carbohydr. Res.” 305: 415-422 (1998), and are herein incorporated by reference. U.S. Patent Application Publication No. 20040137557, incorporated herein by reference).

  Factor IX is a highly beneficial therapeutic peptide. Although commercially available forms of Factor IX are used today, these peptides can be improved by modifications that enhance the pharmacokinetics of the resulting isolated glycoprotein product. Thus, there is still a need in the art for a more permanent Factor IX peptide with improved efficacy and better pharmacokinetics. In addition, it produces factor IX peptides on an industrial scale with improved therapeutic pharmacokinetics, with predictable and essentially homogeneous structures that can be easily regenerated many times, as would be effective for most people It must be possible.

  Fortunately, Factor IX peptides with improved pharmacokinetics and methods for making them have been discovered here. In addition to Factor IX peptides with improved pharmacokinetics, the present invention also provides an industrially practical and cost-effective method for the production of these Factor IX peptides. The Factor IX peptides of the present invention contain modifying groups such as PEG moieties, therapeutic moieties, biomolecules and the like. Thus, the present invention fulfills the need for Factor IX peptides with improved therapeutic efficacy and improved pharmacokinetics for the treatment of conditions and diseases where Factor IX provides an effective therapy. .

  It has now been discovered that controlled modification of Factor IX using one or more poly (ethylene glycol) moieties provides new Factor IX derivatives with improved pharmacokinetic properties. In addition, a cost-effective method for reliable production of the modified factor IX peptides of the present invention has been discovered and developed.

を含む第ＩＸ因子ペプチドを提供する。上式で、Ｄは、−ＯＨまたはＲ^１−Ｌ−ＨＮ−である。記号Ｇは、Ｒ^１−Ｌ−または−Ｃ（Ｏ）（Ｃ_１〜Ｃ_６）アルキルを表す。Ｒ^１は、直鎖または分枝ポリ（エチレングリコール）残基を含む部分であり、Ｌは、結合、置換または非置換アルキル、および置換または非置換ヘテロアルキルから選択されるメンバーであるリンカーである。通常、ＤがＯＨである場合、ＧはＲ^１−Ｌ−であり、Ｇが−Ｃ（Ｏ）（Ｃ_１〜Ｃ_６）アルキルである場合、ＤはＲ^１−Ｌ−ＮＨ−である。当分野の技術者であれば理解できる通り、本明細書に記述するシアル酸の類似体においては、ＣＯＯＨはまた、ＣＯＯ⁻またはその塩を表す。 In one aspect, the invention includes the following parts:

A Factor IX peptide comprising: 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 moiety that includes a linear or branched poly (ethylene glycol) residue, and L is a linker that is a member selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl . Usually, when D is OH, G is ^R 1-L-, when G is _{-C (O) (C 1 ~C} 6) alkyl, D is ^R 1 -L-NH-. As can be appreciated by those skilled in the art, in analogues of sialic acid described herein, COOH also, COO ^- represents a or a salt thereof.

In another aspect, the present invention provides a method for producing PEGylated Factor IX comprising a moiety as described above. The method of the invention comprises (a) transferring a substrate Factor IX peptide with a PEG-sialic acid donor and PEG-sialic acid to an amino acid or glycosyl residue of the Factor IX peptide under conditions suitable for transfer. Contacting with the enzyme to be treated. A typical PEG-sialic acid donor moiety has the formula:

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

  In another aspect, the invention provides a method of treating a condition in a subject in need thereof. However, this condition is characterized by impaired coagulation in the subject. 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. A typical disease treatable by this method is hemophilia.

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

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

Detailed Description of the Invention and Preferred Embodiment Abbreviations 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, xylosyl; and NeuAc, sialyl (N-acetylneuraminyl); M6P, mannose-6-phosphorus Acids; Sia, sialic acid, N-acetylneuraminyl, and derivatives and analogs thereof.

Definitions 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. Usually, the terminology used herein, and experimental methods in 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. These techniques and procedures are typically described in conventional methods and various general standards in the art (generally, Sambrook et al., “MOLECULAR CLONING: A LABORATORY MANUAL”, No. 1, incorporated herein by reference. 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (see NY)), which are documented in this document Provided throughout. The terminology used herein, and experimental methods in analytical chemistry, and the organic compounds described below are well known and commonly used in the art. Standard techniques, or modified versions thereof, are used for chemical synthesis and chemical analysis.

  All oligosaccharides described herein are named or abbreviated for non-reducing sugars (ie, Gal) followed by glycosidic bond (α or β) structure, ring bond (1 or 2), bond (2, 3, 4, 6 or 8) is described using the ring position of the reducing sugar, followed by the name or abbreviation of the reducing sugar (ie GlcNAc). Each saccharide is preferably pyranose. For a review of standard glycan biology terms, see “Essentials of Glycobiology”, Varki et al., CSHL Press (1999).

  Whether the reducing end saccharide is actually a reducing sugar or not, an oligosaccharide is considered to have a reducing end and a non-reducing end. In accordance with accepted terminology, oligosaccharides are represented herein with a non-reducing end on the left and a reducing end on the right.

  The term “sialic acid” refers to any member of the family of 9-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-1-acetamido-3,5-dideoxy-D-glycero-D-galactononuropyrano-1-son -1-onic) acid (often abbreviated as Neu5Ac, NeuAc, or NANA) The second member of this family is N-glycolyl-neuraminic acid (N-acetyl group of NeuAc hydroxylated). A third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) "J. Biol. Chem. "261: 11550-115. 7, Kanamori et al., “J. Biol. Chem.” 265: 21811-2121819 (1990)) 9-O—C1 such as 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac. Also included are 9-substituted sialic acids such as ~ C6 acyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac, and 9-azido-9-deoxy-Neu5Ac For a review of the sialic acid family, for example, Varki, “Glycobiology” 2: 25-40 (1992); “Sialic Acids: Chemistry, Metabolism and Function”, edited by R. Schauer (Springer-Ver, Springer-Ver) See New York (1992) .The synthesis and use of sialic acid compounds in sialylation procedures is disclosed in WO 92/16640 published Oct. 1, 1992. Has been.

  “Peptide” refers to a polymer in which the monomers are amino acids and are linked together through amide bonds, alternatively referred to as a polypeptide. In addition, unnatural amino acids such as β-alanine, phenylglycine, and homoarginine are also included. Amino acids that are not gene-encoded can also be used in the present invention. In addition, amino acids modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules, and the like can also be used in the present invention. All amino acids used in the present invention can be either the d- or l-isomer. The l-isomer is usually preferred. In addition, other peptidomimetics are also useful in the present invention. As used herein, “peptide” refers to both glycosylated and non-glycosylated peptides. Also included are peptides that are incompletely glycosylated by a system that expresses the peptide. For a general review, see Spatra, A. F. (Spatola, A.F.), “Chemistry and Biochemistry of Amino Acids”, “Peptides and Proteins”, B.C. See B. Weinstein, Marcel Dekker, New York, page 267 (1983).

  The term “peptide conjugate” refers to a species of the invention such that a peptide is conjugated with a modified sugar as described herein.

  The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that work in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified (eg, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). Amino acid analogs are compounds having the same basic chemical structure as a naturally occurring amino acid (ie, hydrogen, carboxyl group, amino group, and α carbon bonded to R group), such as homoserine, norleucine, methionine sulfoxide, methionine Refers to methylsulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. As used herein, “amino acid” refers to the D- and L-isomers of amino acids, and mixtures of these two isomers, regardless of whether it is a linker or component of a peptide sequence.

  As used herein, the term “modified sugar” refers to a naturally occurring or non-naturally occurring carbohydrate that is enzymatically added to an amino acid or glycosyl residue of a peptide within the process of the invention. Modified sugars include, but are not limited to, sugar nucleotides (mono, di, and triphosphates), activated sugars (eg, glycosyl halides, glycosyl mesylate), and unactivated , Selected from several enzyme substrates, including sugars that are not nucleotides. A “modified sugar” is covalently functionalized using a “modifying group”. Useful modifying groups include, but are not limited to, PEG moieties, therapeutic moieties, diagnostic moieties, biomolecules, and the like. The modifying group is preferably not a naturally occurring or unaltered carbohydrate. The position of functionalization using the modifying group is selected such that the “modified sugar” is not prevented from being enzymatically added to the peptide.

  The term “water-soluble” refers to a moiety that has some detectable degree of solubility in water. Methods for detecting and / or quantifying water solubility are well known in the art. Typical water soluble polymers include peptides, saccharides, poly (ethers), poly (amines), poly (carboxylic acids) and the like. Peptides can have a mixed sequence of single amino acids, such as poly (lysine). A typical polysaccharide is poly (sialic acid). A typical poly (ether) is poly (ethylene glycol). Poly (ethyleneimine) is a typical polyamine and poly (acrylic) acid is a typical poly (carboxylic acid).

  The polymer backbone of the water soluble polymer can be poly (ethylene glycol) (ie PEG). However, other related polymers are also suitable for use in the practice of the present invention, and the use of the term PEG or poly (ethylene glycol) is inclusive and not exclusive in this regard Should be understood as intended. The term PEG includes alkoxy PEG, bifunctional PEG, multi-armed PEG, bifurcated PEG, branched PEG, pendant PEG (i.e., PEG having one or more functional groups hanging on the polymer backbone or Related polymers), or any form of poly (ethylene glycol), including PEG containing degradable linkages therein.

The polymer main chain may be linear or branched. Branched polymer backbones are generally known in the art. Generally, a branched polymer has a central portion of a central branch and a plurality of linear polymer chains linked to the center of the central branch. PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyhydric alcohols such as glycerol, pentaerythritol and sorbitol. The central branch portion can also be obtained from several amino acids such as lysine. Branched poly (ethylene glycol) can be represented in general form as R (-PEG-OH) _m . Where R represents a central portion 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, the entire contents of which are incorporated herein by reference, can also be used as the polymer backbone. .

  Many other polymers are also suitable for the present invention. Polymer backbones that are non-peptidic and water-soluble and have 2 to about 300 termini are particularly useful in the present invention. Examples of suitable polymers include, but are not limited to, other poly (alkylene glycols) such as poly (propylene glycol) (“PPG”), copolymers such as ethylene glycol and propylene glycol, poly (oxyethylated polyols), Poly (olefin alcohol), poly (vinyl pyrrolidone), poly (hydroxypropyl methacrylamide), poly (α-hydroxy acid), poly (vinyl alcohol), polyphosphazene, polyoxazoline, poly (N-acryloylmorpholine) (contents) And those copolymers, terpolymers, and mixtures thereof, such as those described in US Pat. No. 5,629,384, which is incorporated herein by reference in its entirety. The molecular weight of each chain of the polymer backbone can vary but is generally in the range of about 100 Da to about 100,000 Da, often about 6,000 Da to about 80,000 Da.

  “Area under the curve” or “AUC” as used herein is a curve that indicates the concentration of a drug in the patient's systemic circulation as a function of time from zero to infinity in administering a peptide drug to the patient. It is defined as the total area below.

  The term “half-life” or “t1 / 2” is defined herein as the time required to reduce the plasma concentration of a drug in a patient by half in administering the peptide drug to the patient. The There may be more than one half-life associated with peptide drugs, depending on multiple clearance mechanisms, redistribution, and other mechanisms well known in the art. Usually alpha and beta half-lives are defined such that the alpha phase is related to redistribution and the beta phase is related to clearance. However, with protein drugs that are largely restricted to the bloodstream, there may be at least two clearance half-lives. For certain glycosylated peptides, rapid beta phase clearance is via acceptors on macrophages or endothelial cells that recognize terminal galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, or fucose. May be mediated. For molecules with an effective radius <2 nm (about 68 kD) and / or specific or non-specific uptake and metabolism in tissue, slower beta phase clearance via renal glomerular filtration may occur . GlycoPEGylation can cover terminal sugars (eg, galactose or N-acetylgalactosamine), thereby blocking rapid alpha phase clearance via acceptors that recognize these sugars. This can also give a larger effective radius, thereby reducing the volume of distribution and tissue uptake, thereby prolonging the late beta phase. Thus, as is known in the art, the exact impact of GlycoPEGylation on alpha and beta phase half-life varies depending on size, glycosylation status, and other parameters. A further description of “half-life” can be found in “Pharmaceutical Biotechnology” (1997, edited by DFA Crommelin and RD Cinderella, Harwood Publishers, Amsterdam 1 to Amsterda 120). Out.

The term “glycoconjugation” as used herein refers to an enzymatically mediated attachment of a modified sugar species to an amino acid or glycosyl residue of a polypeptide (eg, a Factor IX peptide substrate). . The genus under “sugar linkage” is “glycol-PEGylated”. However, modified sugar modifying groups are poly (ethylene glycol) and alkyl derivatives (eg, m-PEG) or reactive derivatives thereof (eg, H ₂ N-PEG, HOOC-PEG).

  The terms “large scale” and “industrial scale” are used interchangeably and at the completion of a single reaction cycle, at least about 250 mg, preferably at least about 500 mg, and preferably at least about 1 gram of glycoconjugate (glycoconjugate). ).

  The term “glycosyl linking group” as used herein refers to a glycosyl residue to which a modifying group (eg, PEG moiety, therapeutic moiety, biomolecule) is covalently attached, A modifying group is linked to the remainder. In the methods of the present invention, a “glycosyl linking group” becomes covalently attached to a glycosylated or non-glycosylated peptide, whereby amino acids and / or glycosyl residues on the peptide. Link the drug to A “glycosyl linking group” is typically derived from a “modified sugar” by enzymatic attachment of a “modified sugar” to an amino acid and / or glycosyl residue of a peptide. The glycosyl linking group may be a structure derived from a saccharide that is cleaved during the formation of a sugar cassette that is modified with a modifying group (eg, oxidation → Schiff base formation → reduction), or the glycosyl linking group is impaired. It does not have to be. “Complete glycosyl linking group” refers to a linking group derived from a glycosyl moiety in which the saccharide monomer linking the modifying group to the rest of the conjugate is not degraded (eg, oxidized by, for example, sodium metaperiodate). “Complete glycosyl linking groups” of the present invention can be obtained from naturally occurring oligosaccharides by the addition of glycosyl units or the removal of one or more glycosyl units from the parent saccharide structure.

  The term “targeting moiety” as used herein refers to a species that will selectively concentrate on a particular tissue or region 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 directing drugs to specific tissues or areas are known to those skilled in the art. Typical targeting moieties include antibodies, antibody fragments, transferrin, HS-glycoprotein, clotting factors, serum proteins, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO, serum proteins (eg VII, Factor VIIa, Factor VIII, Factor IX, and Factor X).

  As used herein, “therapeutic moiety” means any agent useful in a therapeutic method, including but not limited to antibiotics, anti-inflammatory agents, anti-tumor agents, cytotoxins and radioactive agents. . A “therapeutic moiety” includes a prodrug of a bioactive agent, a construct in which two or more therapeutic moieties are attached to a carrier (eg, a multivalent agent). The therapeutic portion also includes proteins and constructs comprising proteins. Exemplary proteins include, but are not limited to, granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), interferon (eg, interferon-α, -β, -γ), interleukin ( Interleukin II), serum proteins (eg, factor VII, VIIa, VIII, IX, and factor X), human chorionic gonadotropin (HCG), follicle stimulating hormone (FSH) and luteinizing hormone (LH), As well as antibody fusion proteins such as tumor necrosis factor receptor ((TNFR) / Fc domain fusion protein).

  As used herein, “pharmaceutically acceptable carrier” includes any material that retains the activity of the conjugate when combined with the conjugate and does not react with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsions (such as oil / water emulsions), and various forms of wetting agents. Other carriers may also include sterile solutions, tablets including coated tablets, and capsules. In general, such carriers include starch, milk, sugar, certain types of clay, gelatin, stearic acid or its salts (magnesium or calcium stearate), talc, vegetable fats or oils, gums, glycols, or others Containing excipients such as known excipients. Such carriers may also include flavors and coloring additives or other ingredients. Compositions containing such carriers are prepared by well known conventional methods.

  As used herein, “administration” refers to oral administration, administration as a suppository, local contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, or sustained release device to a subject. Means, for example, the implantation of a mini-osmotic pump. Administration is by any route including parenteral and transmucosal (eg, oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Further, for example, where the infusion is to treat a tumor, eg, to induce apoptosis, administration can be direct to the tumor and / or to the tissue surrounding the tumor. Other modes of delivery include, but are not limited to, the use of liposome formulations, intravenous infusions, transdermal patches, and the like.

  The term “improving” or “improving” includes any objective or subjective parameter such as reduction, remission, or attenuation of symptoms, or improvement of a patient's physical or mental health. , Refers to any sign of success in treating a medical condition or condition. Symptom improvement can be based on objective or subjective parameters, including the results of physical examination and / or psychiatric evaluation.

  The term “therapy” is used to prevent a disease or condition from occurring (preventive therapy) in an animal that has a predisposition to the disease but has not yet experienced or presented symptoms of the disease (preventive therapy). "Treating" a disease or condition, including delaying or suppressing), reducing symptoms or side effects of the disease (including symptomatic therapy), and making the disease easier (causing regression of the disease) or Refers to “treatment”.

  The term “effective amount” or “effective amount to” or “therapeutically effective amount” or any lexically equivalent term is used to describe a disease when administered to an animal to treat the disease. By an amount that is sufficient to effect treatment.

  The term “isolated” refers to a material that is substantially or essentially free from components used to produce the material. For the peptide conjugates of the invention, the term “isolated” refers to a material that is substantially or essentially free from components that normally accompany the material in the mixture used to prepare the peptide conjugate. “Isolated” and “pure” are used interchangeably. In general, it is preferred that the isolated peptide conjugates of the present invention have a level of purity indicated as the following range. The lower end of the range of purity for the peptide conjugate is about 60%, about 70%, or about 80%, and the upper end of the range of purity is about 70%, about 80%, about 90%, or more than about 90% is there.

  If the peptide conjugate is more than about 90% pure, that purity is also preferably indicated as the following range. The lower end of the range of purity is about 90%, about 92%, about 94%, about 96%, or about 98%. The upper end of the range of purity is about 92%, about 94%, about 96%, about 98%, or about 100% purity.

  Purity is determined by any analytical method recognized in the art (eg, band intensity on a silver stained gel, polyacrylamide gel electrophoresis, HPLC, or similar means).

  “Essentially each member of a population” as used herein refers to the invention in which a selected proportion of modified sugars added to a peptide is added to a plurality of identical accepting sites on the peptide. Describe the characteristics of the population of peptide conjugates. “Essentially each member of the population” refers to the “homogeneity” of the site on the peptide that is attached to the modified sugar, at least about 80%, preferably at least about 90%, more preferably at least about 95. % Refers to a conjugate of the invention that is homogeneous.

  “Homogeneity” refers to the structural consistency of the entire population of acceptor moieties to which modified sugars are attached. Thus, in the peptide conjugates of the invention, each modified sugar moiety is bound to a receptor site having the same structure as the receptor site to which all other modified sugars are bound. The peptide conjugate is said to be about 100% homogeneous. Homogeneity is generally indicated as the following range. The lower end of the homogeneity range for the peptide conjugate is about 60%, about 70%, or about 80%, and the upper end of the purity range is about 70%, about 80%, about 90%, or about 90% It is super.

  If the peptide conjugate is about 90% or more homogeneous, its homogeneity is also preferably indicated as the following range. The lower end of the homogeneity range is about 90%, about 92%, about 94%, about 96%, or about 98%. The upper end of the range of purity is about 92%, about 94%, about 96%, about 98%, or about 100% homogeneous. The homogeneity of peptide conjugates can be determined by one or more methods known to those skilled in the art, such as liquid chromatography-mass spectrometry (LC-MS), matrix-assisted laser desorption time-of-flight mass spectrometry. Generally determined by the method (MALDITF), capillary electrophoresis and the like. The above discussion is equally relevant to other O-glycosylation and N-glycosylation sites.

  “Substantially uniform glycoform” or “substantially identical glycosylation pattern” refers to the percentage of an acceptor moiety that is glycosylated by a glycosyltransferase of interest (eg, fucosyltransferase) when referring to a glycopeptide species. Point to. For example, in the case of α1,2 fucosyltransferases, if substantially all (as defined below) Galβ1,4-GlcNAc-R and its sialic acid addition analogs are fucosylated in the peptide conjugates of the invention. For example, there is a substantially uniform fucosylation pattern. It will be appreciated by those skilled in the art that the starting material can contain a glycosylated acceptor moiety (eg, a fucosylated Galβ1,4-GlcNAc-R moiety). Thus, the calculated glycosylation (%) will include the acceptor moiety that is glycosylated by the method of the invention, as well as the acceptor moiety that was already glycosylated in the starting material.

  The term “substantially” in the above definition of “substantially uniform” is usually at least about 40%, at least about 70%, at least about 80%, or preferably at least about an acceptor moiety for a particular glycosyltransferase. It means that about 90%, more preferably at least about 95%, is glycosylated. For example, if the Factor IX peptide conjugate comprises a Ser-linked glycosyl residue, at least about 70%, 80%, 90%, 95%, 97%, 99%, 99.2%, 99 of the peptides in the population .4%, 99.6%, or preferably 99.8% have the same glycosyl residue covalently linked to the same Ser residue.

If a substituent is specified by its normal chemical formula (written from left to right), this is equivalent to chemically identical substituents that would be due to writing a right-to-left structure. Include. For example, —CH ₂ O— is also described as —OCH ₂ —.

The term “alkyl” means a straight-chain or branched-chain or cyclic hydrocarbon radical, or a combination thereof, by itself or as part of another substituent, unless otherwise stated. This can be fully saturated, monovalent or polyunsaturated, divalent and polyunsaturated with the specified number of carbon atoms (ie C ₁ -C ₁₀ means 1 to 10 carbons). Valent radicals can be included. Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, Groups such as homologues and isomers (eg n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.) are included. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups are vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1- and 3- This includes, but is not limited to, propynyl, 3-butynyl, and higher homologs and isomers. The term “alkyl” will also include derivatives of alkyl, as defined in more detail below, eg, “heteroalkyl”, unless otherwise stated. Alkyl groups that are limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent, but is not limited to, divalent from an alkane, as exemplified by —CH ₂ CH ₂ CH ₂ CH ₂ —. And further includes those described below as “heteroalkylene”. Generally, an alkyl (or alkylene) group will have 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, usually having eight or fewer carbon atoms.

  The terms “alkoxy”, “alkylamino”, and “alkylthio” (or thioalkoxy) are used in their conventional sense and are attached to the remainder of the molecule through an oxygen, amino, or sulfur atom, respectively. Refers to an alkyl group.

The term “heteroalkyl”, by itself or in combination with another term, unless otherwise specified, is at least one selected from the group consisting of a predetermined number of carbon atoms and O, N, Si, and S. It means a stable straight or branched chain or cyclic hydrocarbon radical consisting of heteroatoms, or a combination thereof. However, the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatoms may optionally be quaternized. The heteroatoms O, N, and S and Si can be placed at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Examples 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 are included, but are not limited thereto. Up to two heteroatoms may be consecutive, such as, for example, —CH ₂ —NH—OCH ₃ and —CH ₂ —O—Si (CH ₃ ) ₃ . Similarly, the term “heteroalkylene”, by itself or as part of another substituent, refers to a divalent radical derived from a heteroalkyl and is —CH ₂ —CH ₂ —S—CH ₂ — CH ₂ - and _{-CH 2 -S-CH 2 -CH 2} -NH-CH 2 - are exemplified by, but not limited to. For heteroalkylene groups, the heteroatom can also occupy one or both of the chain ends (eg, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Also, for alkylene and heteroalkylene linking groups, the direction of the linking group is not implied by the direction in which the formula of the linking group is written. For example, the formula —C (O) ₂ R′— corresponds to both —C (O) ₂ R′— and —R′C (O) ₂ —.

  The terms “cycloalkyl” and “heterocycloalkyl” by themselves or in combination with other terms represent cyclic forms of “alkyl” and “heteroalkyl”, respectively, unless otherwise specified. Further, for heterocycloalkyl, the heteroatom can occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl 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-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran Examples include, but are not limited to, -3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl and the like.

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

  The term “aryl”, unless stated otherwise, can be a single ring or multiple rings (preferably 1 to 3 rings) fused together or covalently linked together. Means a polyunsaturated aromatic substituent. The term “heteroaryl” refers to 1 to 4 selected from N, O, and S, in which the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. An aryl group (or ring) containing a heteroatom. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. 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 -Isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5 Quinoxalinyl, 3-quinolyl, tetrazolyl, benzo [b] furanyl, benzo [b] thienyl, 2,3-dihydrobenzo [1,4] dioxin-6-yl, benzo [1,3] dioxol-5-yl, and 6-quinolyl is included. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents set forth below.

  For brevity, the term “aryl” when used in combination with other terms (eg, aryloxy, arylthioxy, arylalkyl) is defined as aryl and heteroaryl rings as defined above. Including both. Thus, the term “arylalkyl” includes, for example, alkyl groups in which a carbon atom (eg, a methylene group) is replaced by an oxygen atom (eg, phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, etc. ), And a radical having an aryl group attached to an alkyl group (for example, benzyl, phenethyl, pyridylmethyl, etc.).

  Each of the above terms (eg, “alkyl”, “heteroalkyl”, “aryl”, and “heteroaryl”) will include substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

Substituents for alkyl and heteroalkyl radicals (often including groups called alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) are generally “alkyl Referred to as “group substituents”, which include, but are not limited to, a number in the range from zero to (2m ′ + 1), where m ′ is the sum of carbon atoms in such radicals. It can be one or more of various groups selected: -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 '', - NRSO 2 R ', - CN, and -NO _2. R ′, R ″, R ′ ″ and R ″ ″ are preferably each independently hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (eg 1 to 3 Halogen, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy group, or aryl substituted with an arylalkyl group). For example, if the compound of the present invention contains two or more R groups, each R ′, R ″, R ′ ″, and R ″ ″ group when two or more such groups are present, and Similarly, each R group is independently selected. When R ′ and R ″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5, 6 or 7 membered ring. For example, —NR′R ″ will include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above description of substituents, one of skill in the art will recognize that the term “alkyl” includes haloalkyl (eg, —CF ₃ and —CH ₂ CF ₃ ) and acyl (eg, —C (O) CH ₃ , It will be understood that groups containing carbon atoms bonded to groups other than hydrogen groups, such as —C (O) CF ₃ , —C (O) CH ₂ OCH ₃ will be included.

Similar to the described substituents for alkyl radicals, substituents for aryl and heteroaryl groups are commonly referred to as “aryl group substituents”. For example, these substituents are selected from: a number ranging from zero to the sum of open valences on the aromatic ring system, halogen, —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 ″, —NRSO ₂ R ′, —CN and —NOR ₂ , —R ′, —N ₃ , — CH _{(Ph) 2,} fluoro _(C 1 _{~C 4)} alkoxy, and fluoro _(C 1 -C ₄₎ alkyl. Where R ′, R ″, R ′ ″, and R ″ ″ are hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted Of heteroaryl are preferably independently selected. For example, if the compound of the present invention contains two or more R groups, each R ′, R ″, R ′ ″, and R ″ ″ group when two or more such groups are present, and Similarly, each R group is independently selected. In the scheme below, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ring can be optionally replaced with a substituent of the formula -TC (O)-(CRR ') q-U-. In the formula, T and U are independently —NR—, —O—, —CRR′—, or a single bond, and q is an integer of 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring, with the formula -A- (CH 2) _r-B- substituents can be replaced optionally. However, A and B are independently, -CRR '-, - O - , - NR -, - S -, - S (O) -, - S (O) 2 -, - S (O) 2 NR '-Or a single bond, and r is an integer of 1 to 4. One of the new ring single bonds thus formed is a double bond and can optionally be replaced. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring are optional substituents of the formula — (CRR ′) sX— (CR ″ R ′ ″) d— Can be replaced. In the formula, s and d are each independently an integer of 0 to 3, and X is —O—, —NR′—, —S—, —S (O) —, —S (O) ₂ —. Or —S (O) ₂ NR′—. The 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” is meant to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

Introduction As noted above, Factor IX is important in the blood coagulation cascade. The structure and sequence of Factor IX is provided in FIG. Factor IX deficiency in the body characterizes certain types of hemophilia (type B). Treatment of this disease is usually limited to intravenous transfusion of human plasma protein concentrates of factor IX. However, in addition to the practical disadvantages of time and cost, transfusion of blood concentrates carries the risk of transmission of viral hepatitis, acquired immune deficiency syndrome, or thromboembolic disease to recipients.

  While Factor IX has proven itself as an important and useful compound for therapeutic applications, current methods for producing Factor IX from recombinant cells (US Pat. No. 4,770,999) Results in a product with a rather short biological half-life and an incorrect glycosylation pattern, thereby reducing immunogenicity, loss of function, and more to achieve the same effect. This can potentially lead to the need for larger, more frequent doses.

  In order to improve the effectiveness of recombinant Factor IX used for therapeutic purposes, the present invention relates to polymers of glycosylated and non-glycosylated Factor IX peptides, such as PEG (m-PEG), PPG (m -PPG) etc.). Conjugates can additionally or alternatively be modified by further coupling with various species such as therapeutic moieties, diagnostic moieties, targeting moieties, and the like.

  The conjugates of the invention are formed by enzymatic attachment of modified sugars to glycosylated or non-glycosylated peptides. Glycosylation sites and glycosyl residues provide positions for attaching modifying groups to the peptide, for example by sugar linkage. A typical modifying group is a water soluble polymer such as poly (ethylene glycol) such as methoxy-poly (ethylene glycol). Modification of the factor IX peptide can 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 invention makes it possible to assemble peptides and glycopeptides having a substantially uniform derivatization pattern. Enzymes used in the present invention are usually selective for specific amino acid residues, combinations of amino acid residues, or specific glycosyl residues of a 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 large scale preparation of glycopeptides having a preselected uniform derivatization pattern.

  The present invention also contemplates glycosylated and non-glycosylated peptide conjugates that have an increased therapeutic half-life, for example, by reduced clearance rates or reduced rates of uptake by the immune or reticuloendothelial system (RES). I will provide a. Furthermore, the methods of the invention provide a means for masking antigenic determinants on peptides, thereby reducing or eliminating the host immune response to the peptide. Selective attachment of a target agent can also be used to direct the peptide to a specific tissue or cell surface acceptor that is specific for a particular target agent.

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

  The bond between the peptide and the modifying group includes a glycosyl linking group disposed between the peptide and the selected moiety. As described herein, the selected moiety is essentially a “modified sugar recognized by a suitable transferase enzyme that can attach to the sugar unit and add the modified sugar to the peptide. Any species that results in The sugar component of a modified sugar becomes a “glycosyl linking group”, eg, a “complete glycosyl linking group” when placed between a peptide and a selected moiety. Glycosyl linking groups are formed from any mono- or oligosaccharide that is a substrate for an enzyme that adds a modified sugar to an amino acid or glycosyl residue of a peptide after modification using the modifying group.

  The glycosyl linking group can be or can include a saccharide moiety that is degradably altered prior to or during the addition of the modifying group. For example, glycosyl linking groups can be obtained, for example, from saccharide residues generated by oxidative degradation of the complete saccharide to the corresponding aldehyde via the action of metaperiodate, and then the appropriate An amine is used to convert to a Schiff base, which is then subsequently reduced to the corresponding amine.

A typical conjugate of the invention corresponds to the following general structure:

  Where symbols b, c, d, and s represent positive integers other than zero, and t is 0 or a positive integer. “Agents” are therapeutic agents, bioactive agents, detectable labels, water-soluble moieties (eg, PEG, m-PEG, PPG and m-PPG) and the like. An “agent” can be a peptide (eg, enzyme, antibody, antigen, etc.). The linker can be any of a variety of linking groups below. 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 such as m-PEG. The water soluble polymer is covalently attached to the peptide via a glycosyl linking group. The glycosyl linking group is covalently attached to an amino acid residue or glycosyl residue of the peptide. The invention also provides conjugates in which amino acid residues and glycosyl residues are modified with glycosyl linking groups.

  A typical water soluble polymer is poly (ethylene glycol), for example, methoxy-poly (ethylene glycol). The poly (ethylene glycol) used in the present invention is not limited to any particular form or molecular weight range. For unbranched poly (ethylene glycol) molecules, the molecular weight is preferably between 500 and 100,000. A molecular weight of 2,000-60,000 daltons is preferably used, preferably from about 5,000 to about 30,000 daltons.

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

  In preferred embodiments, 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. More than 1,000 daltons.

  In addition to providing conjugates formed through enzymatically added glycosyl linking groups, the present invention provides conjugates that are very homogeneous in their substitution pattern. Using the methods of the invention, peptide conjugates in which essentially all of the modified sugar moieties of the entire population of conjugates of the invention are attached to multiple copies of structurally identical amino acids or glycosyl residues. Can be formed. Accordingly, in a second aspect, the present invention provides a peptide conjugate having a population of water soluble polymer moieties covalently attached to the peptide via a complete glycosyl linking group. In preferred conjugates of the invention, essentially each member of the population is linked to a glycosyl residue of the peptide via a glycosyl linking group and each glycosyl residue of the peptide to which the glycosyl linking group is attached is Have the same structure.

  Peptide conjugates having a population of water-soluble polymer moieties that are further covalently linked via a glycosyl linking group are also provided. In preferred embodiments, essentially every member of the population of water-soluble polymer moieties is attached to an amino acid residue of the peptide via a glycosyl linking group, and each amino acid residue having a glycosyl linking group attached thereto is identical. It has the following structure.

  The invention also provides conjugates similar to those described above, wherein the peptide is attached to the therapeutic moiety, diagnostic moiety, targeting moiety, toxin moiety, etc. via a complete glycosyl linking group. Each aforementioned moiety can be a small molecule, a natural polymer (eg, a polypeptide) or a synthetic polymer. The peptides of the invention comprise at least one N- or O-linked glycosylation site glycosylated with a glycosyl residue comprising a PEG moiety. PEG is covalently attached to the Factor IX peptide via a complete glycosyl linking group. The glycosyl linking group is covalently attached to an amino acid residue or glycosyl residue of the Factor IX peptide. Alternatively, the glycosyl linking group is attached to one or more glycosyl units of the glycopeptide. The invention also provides conjugates in which a glycosyl linking group is attached to an amino acid residue and a glycosyl residue.

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

Wherein D is a member selected from —OH and R ¹ —L—HN—, and G is selected from R ¹ —L— and —C (O) (C ₁ -C ₆ ) alkyl. R ¹ is a moiety comprising a member selected from moieties comprising linear or branched poly (ethylene glycol) residues, and L is a bond, substituted or unsubstituted alkyl, and substituted or non-substituted a linker which is a member selected from substituted heteroalkyl, provided that when D is OH, G is ^R 1-L-, is G is _{-C (O) (C 1 ~C} 6) alkyl In the case, D is R ¹ -L-NH-.

式中、ａは、０から２０の整数である。 In one embodiment, R ¹ -L has the following formula:

In the formula, a is an integer of 0 to 20.

式中、ｅおよびｆは、１から２５００からそれぞれ独立に選択される整数であり、ｑは、１から２０の整数である。他の実施形態では、Ｒ^１は、以下から選択されるメンバーである構造を有する：

式中、ｅ、ｆ、およびｆ’は、１から２５００からそれぞれ独立に選択される整数であり、ｑおよびｑ’は、１から２０からそれぞれ独立に選択される整数である。 In an exemplary embodiment, R ¹ has a structure that is a member selected from:

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

In the formula, e, f, and f ′ are integers independently selected from 1 to 2500, and q and q ′ are integers independently selected from 1 to 20.

式中、ｅ、ｆ、およびｆ’は、１から２５００からそれぞれ独立に選択される整数であり、ｑ、ｑ’、およびｑ’’は、１から２０の独立して選択される整数である。 In yet another embodiment, the invention provides a Factor IX peptide conjugate having a structure wherein R ¹ is a member selected from:

Wherein e, f, and f ′ are integers independently selected from 1 to 2500, and q, q ′, and q ″ are independently selected integers from 1 to 20. .

式中、ｅおよびｆは、１から２５００の独立して選択される整数である。 In other embodiments, R ¹ has a structure that is a member selected from:

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

Ｇａｌは、アミノ酸に、あるいは、直接的または間接的に（例えば、グリコシル残基を介して）アミノ酸に付着されたグリコシル残基に付着させることができる。 In another exemplary embodiment, the present invention provides a peptide comprising a moiety having the following formula:

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

Ｇａｌは、アミノ酸に、あるいは、直接的または間接的に（例えば、グリコシル残基を介して）アミノ酸に付着されたグリコシル残基に付着させることができる。 In other embodiments, this portion has the following formula:

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

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

式中、ＡＡは、前記ペプチドのアミノ酸残基であり、各々の上述の構造において、ＤおよびＧは本明細書に記述する通りである。 In a further exemplary embodiment, the peptide comprises a moiety according to the formula:

Where AA is an amino acid residue of the peptide and in each of the above structures D and G are as described herein.

  Exemplary amino acid residues of peptides to which one or more of the above species can bind include serine and threonine, such as serine 53 or 61 of SEQ ID NO: 1, or threonine 159, 162, or 172. It is.

式中、ａ、ｂ、ｃ、ｄ、ｉ、ｒ、ｓ、ｔ、およびｕは、０および１から独立して選択される整数である。指数ｑは、１である。指数ｅ、ｆ、ｇ、およびｈは、０から６の整数から独立して選択される。指数ｊ、ｋ、１、およびｍは、０から１００の整数から独立して選択される。指数ｖ、ｗ、ｘ、およびｙは、０および１から独立して選択され、ｖ、ｗ、ｘ、およびｙのうちの少なくとも１つは１である。記号ＡＡは、第ＩＸ因子ペプチドのアミノ酸残基を表す。 In another exemplary embodiment, the present invention provides a Factor IX conjugate comprising a glycosyl residue having the formula:

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

式中、Ｄは、−ＯＨおよびＲ^１−Ｌ−ＨＮ−から選択される。記号Ｇは、Ｒ^１−Ｌ−または−Ｃ（Ｏ）（Ｃ_１〜Ｃ_６）アルキルを表す。Ｒ^１は、直鎖のまたは分枝ポリ（エチレングリコール）残基を含む部分を表す。Ｌは、結合、置換または非置換アルキル、および置換または非置換ヘテロアルキルから選択されるメンバーであるリンカーである。一般に、ＤがＯＨである場合、ＧはＲ^１−Ｌ−であり、Ｇが−Ｃ（Ｏ）（Ｃ_１〜Ｃ_６）アルキルである場合、ＤはＲ^１−Ｌ−ＮＨ−である。 The symbol Sia- (R) represents a group having the following formula:

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 moiety containing a linear or branched poly (ethylene glycol) residue. L is a linker that is a member selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. In general, when D is OH, G is ^R 1-L-, when G is _{-C (O) (C 1 ~C} 6) alkyl, D is ^R 1 -L-NH-.

式中、指数「ｓ」は、０から２０の整数を表し、ｎは、１から２５００の整数である。選択された実施形態では、ｓは１であり、ＰＥＧは約２０ｋＤである。 In another exemplary embodiment, the PEG-modified sialic acid moiety in the conjugate of the invention has the formula:

In the formula, the index “s” 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.

式中、Ｌは、シアル酸部分とＰＥＧ部分を連結している、置換または非置換アルキルまたは置換または非置換ヘテロアルキルリンカー部分である。 In a further exemplary embodiment, the PEG modified sialic acid has the formula:

Where L is a substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl linker moiety linking the sialic acid moiety and the PEG moiety.

  In an exemplary embodiment where the glycosyl residue has the structure described above, it is attached to one or both of Asn 157 and Asn 167.

  Factor IX has been cloned and sequenced. Essentially any factor IX peptide having any sequence is what is used as the factor IX peptide component of the conjugate of the invention. In an exemplary embodiment, the peptide has the sequence set forth herein as SEQ ID NO: 1:

  The present invention is in no way limited to the sequences described herein. For example, U.S. Pat. Nos. 4,770,999, 5,521,070 (in the first position, tyrosine is replaced by alanine), U.S. Pat. No. 6,037,452. (Factor XI is linked to an alkylene oxide group), and US Pat. No. 6,046,380, where the DNA encoding Factor IX is modified to at least one splice site As such, Factor IX variants are well known in the art. As demonstrated herein, Factor IX variants are well known in the art, and the present disclosure encompasses known variants or variants that will be developed and discovered in the future.

  Methods for determining the activity of mutant or modified factor IX are described, for example, in Biggs (1972, “Human Blood Coagulation Haemostasis and Thrombosis” (1st edition), Oxford, Blackwell, Scientific Can be performed using methods described in the art, such as the one stage activated partial heel plastin time assay described in Fick (Oxford, Blackwell, Scientific, page 614). . Briefly, to analyze the biological activity of a Factor IX molecule developed in accordance with the method of the present invention, the assay consists of an equal volume of activated partial thromboplastin reagent, sterile phlebotomy well known in the art. The technique can be used to perform with factor IX-deficient plasma isolated from patients suffering from hemophilia B, and normal pooled plasma or samples as standards. In this assay, one unit of activity is defined as the amount present in 1 milliliter of normal pooled plasma. In addition, assays for biological activity based on Factor IX's ability to reduce the clotting time of plasma from patients who are deficient in Factor IX to normal include, for example, Proctor and Rapaport, " Amer. J. Clin. Path. "36: 212 (1961).

  The peptides of the present invention contain at least one N-linked or O-linked glycosylation site, at least one of which is attached to a glycosyl residue comprising a PEG moiety. PEG is covalently attached to the peptide via a complete glycosyl linking group. The glycosyl linking group is covalently attached to an amino acid residue or glycosyl residue of the peptide. Alternatively, the glycosyl linking group is attached to one or more glycosyl units of the glycopeptide. The invention also provides conjugates in which a glycosyl linking group is attached to an amino acid residue and a glycosyl residue.

  The PEG moiety is attached to the complete glycosyl linker directly or through an unsubstituted glycosyl linker, eg, a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl.

Modified Sugars The present invention uses modified sugars and modified sugar nucleotides to form modified sugar conjugates. In the modified sugar compound of the present invention, the sugar moiety is preferably a saccharide, deoxysaccharide, aminosaccharide or N-acylsaccharide. The terms “saccharide” and its equivalents, “saccharyl”, “sugar” and “glycosyl” refer to monomers, dimers, oligomers, and polymers. The sugar moiety can also be functionalized with a modifying group. The modifying group is generally attached to the sugar moiety via a conjugate with an amine, sulfhydryl, or hydroxyl (eg, the first hydroxyl moiety on the sugar). In an exemplary embodiment, the modifying group is attached via an amine moiety on the sugar, for example, via an amide, urethane, or urea formed via the reaction of a reactive derivative of the modifying group with an amine. Is done.

  Any sugar can be utilized as the sugar center of the conjugate of the invention. Exemplary sugar centers 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 (glucosamine, galactosamine, mannosamine, etc.), 5-amine analogs of sialic acid, and the like. The sugar center may be a structure found in nature or may be modified to provide a site for attachment to a modifying group. For example, in one embodiment, the present invention provides sialic acid derivatives in which the 9-hydroxy moiety is replaced with an amine. Amines are readily derivatized with activated analogs of selected modifying groups.

式中、Ｇは、グリコシル部分であり、Ｌは、結合またはリンカーであり、Ｒ^１は、修飾基である。典型的な結合は、グリコシル部分上のＮＨ_２と、修飾基上の相補的な反応性の基との間に形成されるものである。したがって、典型的な結合には、ＮＨＲ^１、ＯＲ^１、ＳＲ^１が含まれるが、これに限定されない。例えば、Ｒ^１が、カルボン酸部分を含む場合、この部分は、ＮＨＣ（Ｏ）Ｒ^１構造を有する結合を与えるグリコシル残基上のＮＨ_２部分を用いて、活性化させ、これと結びつけることができる。同様に、ＯＨおよびＳＨ基を、それぞれ、相当するエーテルまたはチオエーテル誘導体に転換させることができる。 In an exemplary embodiment, the present invention utilizes a modified sugar amine having the formula:

Where G is a glycosyl moiety, L is a bond or linker, and R ¹ is a modifying group. A typical linkage is that formed between NH ₂ on the glycosyl moiety and a complementary reactive group on the modifying group. Thus, exemplary bonds include but are not limited to NHR ¹ , OR ¹ , SR ¹ . For example, if R ¹ contains a carboxylic acid moiety, this moiety can be activated and associated with an NH ₂ moiety on a glycosyl residue that provides a bond with the NHC (O) R ¹ structure. it can. Similarly, OH and SH groups can be converted to the corresponding ether or thioether derivatives, respectively.

Typical linkers include alkyl and heteroalkyl moieties. Linkers include linking groups, such as acyl-based linking groups, such as -C (O) NH-, -OC (O) NH-, and the like. A linking group is a bond formed between components of the 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 reaction with an amine on the glycosyl residue, and the amine of glycine is converted to the corresponding amide by reaction with an activated carboxylic acid or carbonate ester of the modifying group. Or converted to urethane.

Another exemplary linker is a PEG moiety or a PEG moiety functionalized with amino acid residues. PEG is attached to the glycosyl group via an amino acid residue at one PEG terminus and to R ¹ via the other PEG terminus. Alternatively, the amino acid residue is attached to R ¹ and the PEG terminus not attached to the amino acid is attached to the glycosyl group.

A typical species for NH-LR ¹ has the following formula:
_{-NH {C (O) (CH} 2) a NH} s {C (O) (CH 2) b (OCH 2 CH 2) O (CH 2) d NH} t R 1
In the formula, 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, eg, —S, —O, or —CH ₂ .

More particularly, the present invention utilizes compounds wherein NH-LR ¹ is:
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 NHR 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 NHR ¹ , NHC (O) (CH ₂ ) _a NHR ¹ , NH (CH ₂ ) _a NHR ¹ , and NHR ¹ . In these formulas, the indices, a, b and d are independently selected from integers from 0 to 20, preferably from 1 to 5. The index c is an integer from 1 to 2500.

  In the discussion that follows, the present invention is illustrated by reference to the use of selected derivatives of sialic acid. For those skilled in the art, the focus of the discussion is for clarity of explanation, and the structures and compositions described are typically saccharide groups, modified saccharide groups, activated It will be appreciated that the present invention is applicable to all types of modified saccharide groups and conjugates of modified saccharide groups.

In an exemplary embodiment, G is sialic acid and the selected compound used in the present invention has the formula:

  As will be appreciated by those skilled in the art, the sialic acid moiety in the above exemplary compounds includes, but is not limited to, glucosamine, galactosamine, mannosamine, its N-acetyl derivative, and the like. It can be replaced with any other amino sugar.

In another exemplary embodiment, the primary hydroxyl moiety of the sugar is functionalized with a modifying group. For example, the 9-hydroxyl of sialic acid can be converted to the corresponding amine and can be functionalized to provide a compound according to the invention. Formulas according to this embodiment include the following:

式中、Ｒ^３〜Ｒ^５およびＲ^７は、Ｈ、ＯＨ、Ｃ（Ｏ）ＣＨ_３、ＮＨ、およびＮＨＣ（Ｏ）ＣＨ_３から独立して選択されるメンバーであり、Ｒ^６は、ＯＲ^１、ＮＨＲ^１、またはＮＨ−Ｌ−Ｒ^１であり、これは、上記の通りである。 In a further exemplary embodiment, the present invention utilizes a modified sugar having a 6-hydroxyl position converted to the corresponding amine moiety with a linker-modifying group cassette such as those described above. Typical saccharyl groups that can be used as centers of these modified sugars include Gal, GalNAc, Glc, GlcNAc, Fuc, Xyl, Man, and the like. A typical modified sugar according to this embodiment has the formula:

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

The selected conjugates for use in the present invention are based on mannose, galactose, or glucose, or a species having the stereochemistry of mannose, galactose, or glucose. The general formula of these conjugates is as follows:

  In another exemplary embodiment, the present invention utilizes a compound as described above that is activated as the corresponding nucleotide sugar. In their modified form, typical sugar nucleotides used in the present invention include nucleotide mono-, di-, or triphosphates or analogs thereof. In preferred embodiments, the modified sugar nucleotide is selected from UDP-glycoside, CMP-glycoside or GDP-glycoside. More preferably, the sugar nucleotide portion of the modified sugar nucleotide is selected from UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, or CMP-NeuAc. Is done. In an exemplary embodiment, the nucleotide phosphate is attached to C-1.

式中、Ｌ−Ｒ^１は上で述べた通りであり、Ｌ^１−Ｒ^１は修飾基に結合されたリンカーを表す。Ｌと同様に、Ｌ^１に従う典型的なリンカー種には、結合、アルキル、またはヘテロアルキル部分が含まれる。これらの実施形態による典型的な改変された糖ヌクレオチド化合物は、図７および図８に記述される。 Thus, in an exemplary embodiment where the glycosyl moiety is sialic acid, the present invention utilizes a compound having the formula:

Wherein, L-R ¹ is as stated above, L ¹ -R ¹ represents a coupled to the modifying group linker. As with L, typical linker species according to L ¹ include a bond, alkyl, or heteroalkyl moiety. Exemplary modified sugar nucleotide compounds according to these embodiments are described in FIGS.

In another exemplary embodiment, the present invention provides a conjugate formed between a modified sugar of the present invention and a substrate Factor IX peptide. In this embodiment, the sugar moiety of the modified sugar becomes a glycosyl linking group disposed between the substrate and the modifying group. A typical glycosyl linking group is a complete glycosyl linking group in which the glycosyl moieties forming the linking group are not weakened by chemicals (eg, sodium metaperiodate) or enzymatic processes (eg, oxidase). is there. Selected conjugates of the invention include a modifying group attached to the amine moiety of an amino sugar (eg, mannosamine, glucosamine, galactosamine, sialic acid, etc.). A typical modifying group-complete glycosyl linking group cassette in accordance with this spirit is based on a sialic acid structure, such as one having the following formula:

In the above formula, R ¹ and L ¹ are as described above.

式中、ラジカルは、上に述べた通りである。当分野の技術者であれば、上で述べた改変されたサッカリル部分はまた、２、３、４、または５炭素原子で、酸素または窒素原子を介して基質と結合させることができることを理解するであろう。 In a further exemplary embodiment, the conjugate is formed between the substrate Factor IX and the saccharyl moiety (the modifying group is attached via a linker at the 6-carbon position of the saccharyl moiety). Thus, an exemplary conjugate according to this embodiment has the following formula:

In the formula, the radical is as described above. One skilled in the art will appreciate that the modified saccharyl moiety described above can also be attached to the substrate via an oxygen or nitrogen atom at 2, 3, 4, or 5 carbon atoms. Will.

式中、Ｒ基および指数は、上記の通りである。 Exemplary compounds used in the present invention include compounds having the following formula:

In the formula, the R group and the index are as described above.

式中、Ｒ基およびＬは、上で示した通りの部分を表す。指数「ｙ」は、０、１または２である。 The present invention also provides for the use of sugar nucleotides that are modified with LR ¹ at the 6-carbon position. Exemplary species according to this embodiment include:

In the formula, R group and L represent a moiety as shown above. The index “y” is 0, 1 or 2.

Further exemplary nucleotide sugars used in the present invention are based on species having the stereochemistry of GDP mannose. A typical species according to this embodiment has the following structure:

In a further exemplary embodiment, the present invention provides a conjugate wherein the modified sugar is based on the stereochemistry of UDP galactose. A typical nucleotide sugar used in the present invention has the following structure:

In another exemplary embodiment, the nucleotide sugar is based on the stereochemistry of glucose. A typical species according to this embodiment has the following formula:

The modifying group R ¹ is any of several species including, but not limited to, water soluble polymers, water insoluble polymers, therapeutic agents, diagnostic agents, and the like. The nature of typical modifying groups is described in more detail below.

Modifying Group Water-Soluble Polymers Many water-soluble polymers are known to those skilled in the art and are useful for practicing the present invention. The term “water-soluble polymer” refers to 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 (ethers), eg, poly (ethylene glycol); peptides, proteins, etc. The present invention provides that the polymer is the remainder of the conjugate. It can be practiced with any water-soluble polymer with the only restriction that it must include a location where it can be attached.

  Methods for polymer activation are described in WO 94/17039, US Pat. No. 5,324,844, WO 94/18247, WO 94/04193, US No. 5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No. 5,281,698, and even WO 93. The methods for conjugation between activated polymers and peptides can be found in, for example, coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027). Pamphlet), oxygen-carrying molecules (US Pat. No. 4,412,989), ribonuclease Over zero and superoxide dismutase: it can be seen in (Veronese (Veronese), et al., 11 "App.Biochem.Biotech." 141-45 (1985)).

  Preferred water-soluble polymers are those in which a significant proportion of polymer molecules in the polymer sample are of approximately the same molecular weight. Such polymers are “homodisperse”.

  The invention is further illustrated by reference to poly (ethylene glycol) conjugates. Several reviews and research books on PEG functionalization and conjugation are available. For example, 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 (p. 1992); Chem. "6: 150-165 (1995); and Bhadra et al.," Pharmacizie ", 57: To 29 of the reference that (2002). Routes for preparing reactive PEG molecules, and using reactive molecules to form conjugates are known in the art. For example, US Pat. No. 5,672,662 is from linear or branched poly (alkylene oxide), poly (oxyethylated polyol), poly (olefin alcohol), and poly (acrylomorpholine). Disclosed are water-soluble and isolatable conjugates of active esters of selected polymeric acids.

  US Pat. No. 6,376,604 discloses the reaction of water-soluble and non-peptidic polymers by reacting di (1-benzotriazolyl) carbonate with the terminal hydroxyl of the polymer in an organic solvent. A process for preparing water-soluble 1-benzotriazolyl carbonate is described. The active ester is used to form a conjugate with a biologically active agent (such as a protein or peptide).

  WO 99/45964 is a conjugate comprising a biologically active agent comprising a polymer backbone having at least one terminus linked to the polymer backbone via a stable bond and an activated water-soluble polymer. Describe the gate. Wherein the at least one terminus includes a branching portion having a proximal reactive group linked to the branching portion, and the biologically active agent comprises a proximal reactive group Connected to at least one. Other branched poly (ethylene glycols) are described in WO 96/21469, US Pat. No. 5,932,462 includes branched ends containing reactive functional groups. Describes conjugates formed using branched PEG molecules. Free reactive groups can be used to react with biologically active species such as proteins or peptides to form conjugates of poly (ethylene glycol) with biologically active species . US Pat. No. 5,446,090 describes a bifunctional PEG linker and its use in forming conjugates with peptides at each PEG linker end.

  Conjugates containing degradable PEG linkages are described in WO 99/34833; and WO 99/14259, and US Pat. No. 6,348,558. Such degradable bonds are applicable to the present invention.

  The art-recognized methods of activating the polymers described above are those in the formation of the branched polymers described herein, and their separation into other species such as sugars, sugar nucleotides, and the like. Useful in the present invention for the attachment of branch polymers.

式中、Ｒ^８は、Ｈ、ＯＨ、ＮＨ_２、置換または非置換アルキル、置換または非置換のアリール、置換または非置換のヘテロアリール、置換または非置換のヘテロシクロアルキル、置換または非置換ヘテロアルキル、例えば、アセタール、ＯＨＣ−、Ｈ_２Ｎ−（ＣＨ_２）_ｑ−、ＨＳ−（ＣＨ_２）_ｑ、または−（ＣＨ_２）_ｑＣ（Ｙ）Ｚ^１である。指数「ｅ」は、１から２５００の整数を表す。指数ｂ、ｄ、およびｑは、独立して、０から２０の整数を表す。記号ＺおよびＺ^１は、独立して、ＯＨ、ＮＨ_２、脱離基、例えば、イミダゾール、ｐ−ニトロフェニル、ＨＯＢＴ、テトラゾール、ハライド、Ｓ−Ｒ^９、活性化エステル類のアルコール部分、−（ＣＨ_２）_ｐＣ（Ｙ^１）Ｖ、または−（ＣＨ_２）_ｐＵ（ＣＨ_２）_ｓＣ（Ｙ^１）_Ｖを表す。記号Ｙは、Ｈ（２）、＝Ｏ、＝Ｓ、＝Ｎ−Ｒ^１０を表す。記号Ｘ、Ｙ、Ｙ^１、Ａ^１、およびＵは、独立して、部分Ｏ、Ｓ、Ｎ−Ｒ^１１を表す。記号Ｖは、ＯＨ、ＮＨ_２、ハロゲン、Ｓ−Ｒ^１２、活性化エステルのアルコール成分、活性化アミドのアミン成分、糖ヌクレオチド、およびタンパク質を表す。指数ｐ、ｑ、ｓ、およびｖは、０から２０の整数から独立して選択されるメンバーである。記号Ｒ^９、Ｒ^１０、Ｒ^１ｌ、およびＲ^ｌ２は、独立して、Ｈ、置換または非置換アルキル、置換または非置換ヘテロアルキル、置換または非置換のアリール、置換または非置換のヘテロシクロアルキルおよび置換または非置換のヘテロアリールを表す。 Exemplary poly (ethylene glycol) molecules used in the present invention include, but are not limited to, those having the following 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) a q C (Y) ^{Z 1.} The index “e” represents an integer from 1 to 2500. The indices b, d, and q independently represent an integer from 0 to 20. The symbols Z and Z ¹ independently represent OH, NH ₂ , a leaving group such as imidazole, p-nitrophenyl, HOBT, tetrazole, halide, S—R ⁹ , the alcohol moiety of activated esters, — ( ^{_{CH 2) p C (Y 1}} ) V _{or, - (CH 2) p U} (CH 2) s C (Y 1) represents the _V. Symbol Y, H (2), = O , represents a ^{= S, = N-R 10} . The symbols X, Y, Y ¹ , A ¹ , and U independently represent the moieties O, S, N—R ¹¹ . Symbol V represents OH, NH _2, halogen, ^{S-R 12,} the alcohol component of activated esters, the amine component of activated amides, sugar nucleotides, and proteins. The indices p, q, s, and v are members selected independently from integers from 0 to 20. Symbols ^{R 9, R 10, R 1l} , and ^{R l2} are, independently, H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl and Represents a substituted or unsubstituted heteroaryl.

In another exemplary embodiment, the poly (ethylene glycol) molecule is selected from:

式中、Ｒ^８およびＲ^８’は、独立して、上の、Ｒ^８について定義された基から選択されるメンバーである。Ａ^１およびＡ^２は、独立して、上の、Ａ^１について定義された基から選択されるメンバーである。指数ｅ、ｆ、ｏおよびｑは、上記の通りである。ＺおよびＹは、上記の通りである。Ｘ^１およびＸ^１’は、Ｓ、ＳＣ（Ｏ）ＮＨ、ＨＮＣ（Ｏ）Ｓ、ＳＣ（Ｏ）Ｏ、Ｏ、ＮＨ、ＮＨＣ（Ｏ）、（Ｏ）ＣＮＨおよびＮＨＣ（Ｏ）Ｏ、ＯＣ（Ｏ）ＮＨから独立して選択されるメンバーである。 Poly (ethylene glycol) useful for forming the conjugates of the invention is linear or branched. Suitable branched poly (ethylene glycol) molecules for use in the present invention include, but are not limited to, those represented by the following formula:

Wherein R ⁸ and R ^{8 ′} are independently members selected from the groups defined for R ⁸ above. A ¹ and A ² are independently members selected from the groups 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) Member selected independently from NH.

In other exemplary embodiments, the branched PEG is based on a cysteine, serine, or di-lysine center. Thus, more typical branched PEGs include:

式中、ｅ、ｆおよびｆ’は、１から２５００の独立して選択された整数であり、およびｑ、ｑ’、およびｑ’’は、１から２０の独立して選択された整数である。 In yet another embodiment, the branched PEG moiety is based on a tri-lysine peptide. Tri-lysine can be mono-, di-, tri- or tetra-PEGylated. A typical species according to this embodiment has the following formula:

Where e, f and f ′ are independently selected integers from 1 to 2500, and q, q ′, and q ″ are independently selected integers from 1 to 20. .

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

  As will be appreciated by those skilled in the art, the branched polymers used in the present invention include variations on the issues discussed above. For example, the di-lysine-PEG conjugate described above may contain three macromolecular subunits, the third bound to the α-amine shown to be unmodified in the above structure. Is done. Similarly, the use of tri-lysine functionalized with three or four polymeric subunits is also within the scope of the present invention.

および、例えば、これらの種の炭酸エステルおよび活性なエステル類：

Additional exemplary species used in the present invention include:

And, for example, carbonates and active esters of these species:

Other activating or leaving groups that are suitable for activating the linear PEG used to prepare the compounds described herein include, but are not limited to the following species: Is included.

  PEG molecules activated using these and other species, and methods for producing activated PEG are described in WO 04/083259.

Those of skill in the art, one or more of m-PEG arms of the branched polymer, different terminal, for _{example, OH, COOH, NH 2,} C 2 ~C 10 - replaced by a PEG moiety with alkyl and You will understand that you can. Furthermore, the above structure is easily modified by inserting an alkyl linker (or removing a carbon atom) between the α-carbon atom of the side chain of the “amino acid” and the functional group. Thus, “homo” derivatives and higher homologues, as well as lower homologues, are useful “amino acid” centers for the branched PEGs used in the present invention.

式中、Ｘ^ａは、ＯまたはＳであり、ｒは１から５の整数である。指数ｅおよびｆは、１から２５００の、独立して選択される整数である。 The branched PEG species described herein are readily prepared by methods such as those described in the scheme below.

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

Therefore, according to this scheme, a natural or unnatural amino acid, activated m-PEG derivative, in this case in contact with the tosylate, by alkylating the side-chain heteroatom X ^a, to form a 1. A mono-functionalized m-PEG amino acid is subjected to N-acylation conditions with a reactive m-PEG derivative, thereby assembling a branched m-PEG2. One skilled in the art will appreciate that the tosylate leaving group can be replaced with any suitable leaving group, such as halogen, mesylate, triflate, and the like. Similarly, the reactive carbonate utilized to acylate the amine can be replaced with an active ester such as N-hydroxysuccinimide. Alternatively, the acid can be activated in situ using a dehydrating agent (dicyclohexylcarbodiimide, carbonyldiimidazole, etc.).

  In an exemplary embodiment, the modifying group is a PEG moiety, but any modifying group (eg, a water soluble polymer, a water insoluble polymer, a therapeutic moiety, etc.) is incorporated into the glycosyl moiety via a suitable linkage. be able to. Modified sugars are formed by enzymatic means, chemical means, or combinations thereof, thereby producing modified sugars. In an exemplary embodiment, the saccharide is an active amine (allowing attachment of the modifying moiety at any position, and the saccharide functions as a substrate for an enzyme that can bind the modified saccharide to a peptide. To be replaced). In an exemplary embodiment, when galactosamine is a modified sugar, the amine moiety is attached to the carbon atom at the 6-position.

Seeds modified with water-soluble polymers Nucleotide sugar species modified with water-soluble polymers in which the sugar moiety is modified with a water-soluble polymer are useful in the present invention. A typical modified sugar nucleotide has a modified sugar group through an amine moiety on the sugar. Modified sugar nucleotides, such as saccharyl-amine derivatives of sugar nucleotides, are also used in the methods of the invention. For example, a saccharylamine (without a modifying group) can be enzymatically coupled to a peptide (or other species) and the free saccharylamine moiety can then be coupled to the desired modifying group. Alternatively, the modified sugar nucleotide can function as a substrate for an enzyme that transfers the modified sugar to a saccharyl acceptor on a substrate (eg, a peptide, glycopeptide, lipid, aglycone, glycolipid, etc.).

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

In an exemplary embodiment, the modified sugar is based on a 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, preferably 10 to 1200. The symbol “A” represents an activating group such as halogen, a component of an activated ester (for example, N-hydroxysuccinimide ester), a component of a carbonate ester (for example, p-nitrophenyl carbonate), and the like. One skilled in the art will appreciate that other PEG-amide nucleotide sugars are readily prepared by this and similar methods.

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

式中、Ｘ^４は、結合またはＯである。 In a further embodiment, R ¹ is a branched PEG, eg, one of the species mentioned above. Exemplary compounds according to this embodiment include:

In the formula, X ⁴ is a bond or O.

式中、Ｘ^４は、Ｏまたは結合である。 Furthermore, as indicated above, the present invention provides nucleotide sugars that are modified using linear or branched water-soluble polymers. For example, compounds having the formulas shown below are within the scope of the invention.

In the formula, X ⁴ is O or a bond.

式中、Ｘ^４は、結合またはＯである。 Similarly, the present invention provides nucleotide sugars of modified sugar species in which the 6-position carbon is modified:

In the formula, X ⁴ is a bond or O.

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

Water Insoluble Polymer In another embodiment similar to that described above, the modified sugar comprises a water insoluble polymer rather than a water soluble polymer. The conjugates of the invention can also include one or more water-insoluble polymers. This embodiment of the invention is illustrated by using the conjugate as a vehicle for delivering therapeutic peptides in a controlled manner. Polymeric drug delivery systems are known in the art. For example, edited by Dunn et al., "POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS", ACS Symposium Series Vol. 469, American Chemical Society, Washington City, 91. . Those skilled in the art will appreciate that virtually any known drug delivery system is applicable to the conjugates of the present invention.

  Representative water-insoluble polymers include, but are not limited to, polyphosphazine, poly (vinyl alcohol), polyamide, polycarbonate, polyalkylene, polyacrylamide, polyalkylene glycol, polyalkylene oxide, polyalkylene terephthalate, polyvinyl Ether, polyvinyl ester, polyvinyl halide, polyvinylpyrrolidone, polyglycolide, polysiloxane, polyurethane, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (methacrylic) Acid hexyl), 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 oxide), poly (ethylene terephthalate), poly (vinyl acetate), polyvinyl chloride , Polystyrene, polyvinylpyrrolidone, pluronic, and polyvinylphenol, and copolymers thereof.

  Synthetically modified natural polymers used in the conjugates of the invention include, but are not limited to, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters and nitrocellulose. A particularly preferred member of the broad class of synthetically modified natural polymers includes, but is not limited to, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, butyric acetate Cellulose, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, and polymers of acrylic and methacrylic acid esters and alginic acid are included.

  These and other polymers described herein are available from Sigma Chemical Co. (St. Louis, Missouri), Polysciences (Warrenton, Pa.), Aldrich (Aldrich) Can be readily obtained from commercial sources such as Milwaukee, Wisconsin, Fluka (Ronkonkoma, NY), and BioRad (Richmond, CA), or from these sources From the resulting monomer, it is synthesized using standard techniques.

  Representative biodegradable polymers used in the conjugates of the present invention include, but are not limited to, polylactic acid, 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. Particularly useful are compositions that form gels, such as those including collagen, pluronics, and the like.

  The polymers used in the present invention include “hybrid” polymers that include water-insoluble materials that have bioresorbable molecules in at least a portion of their structure. Examples of such polymers are those that comprise a water-insoluble copolymer with a bioresorbable region, a hydrophilic region, and a plurality of crosslinkable functional groups for each polymer chain.

  For the purposes of the present invention, a “water-insoluble material” includes a material that is substantially insoluble in water or a hydrous environment. Thus, even if certain regions or segments of the copolymer are hydrophilic or water soluble, the polymer molecule as a whole does not dissolve in water for any substantial measurement.

  For the purposes of the present invention, the term “bioresorbable molecule” includes a region that can be metabolized or degraded and reabsorbed and / or eliminated by the body via normal excretory pathways. Such metabolites or degraded products are preferably substantially non-toxic to the body.

  The bioresorbable region may be hydrophobic or hydrophilic so long as the copolymer composition as a whole is not water soluble. Thus, the bioresorbable region is selected based on the priority that the polymer as a whole remains water insoluble. Thus, the relative properties, i.e. the type of functional groups contained, and the relative proportion of the bioresorbable region, and the hydrophilic region, ensure that the useful bioresorbable composition is water insoluble. Selected to remain.

  Typical resorbable polymers include, for example, synthetically produced resorbable block copolymers of poly (α-hydroxy-carboxylic acid) / poly (oxyalkylene) (Cohn). Et al., U.S. Pat. No. 4,826,945). These copolymers are not crosslinked and are water soluble so that the body can expel 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). checking ...

  Presently preferred bioresorbable polymers include poly (ester), poly (hydroxy acid), poly (lactone), poly (amide), poly (ester-amide), poly (amino acid), poly (anhydride). , Poly (orthoester), poly (carbonate ester), poly (phosphadine), poly (phosphate ester), poly (thioester), polysaccharide, and mixtures thereof. . More preferably, the bioresorbable polymer includes a poly (hydroxy) acid component. Of 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 in vivo ("bioresorbed"), preferred polymeric coatings for use in the methods of the present invention are also excretable and / or Alternatively, metabolic fragments can be formed.

  Higher degrees of copolymer can also be used in the present invention. For example, Casey et al., US Pat. No. 4,438,253, issued March 20, 1984, is a transesterification reaction of poly (glycolic acid) and hydroxyl-terminated poly (alkylene glycol). Discloses tri-block copolymers. Such compositions are disclosed for use as resorbable monofilament sutures. The flexibility of such compositions is controlled by the incorporation of aromatic orthocarbonates (such as tetra-p-tolyl orthocarbonate) into the copolymer structure.

  Other polymers based on lactic acid and / or glycolic acid can also be utilized. For example, Spinu, US Pat. No. 5,202,413, issued April 13, 1993, describes the opening of lactide and / or glycolide to either oligomeric diol or diamine residues. Having sequentially ordered blocks of polylactic acid and / or polyglycolide produced by ring polymerization followed by chain extension using a bifunctional compound (e.g. diisocyanate, diacyl chloride, or dichlorosilane), Biodegradable multi-block copolymers are disclosed.

  The bioresorbable region of the coating useful in the present invention can be designed to be hydrolytically and / or enzymatically cleavable. For the purposes of the present invention, “hydrolytically cleavable” refers to the copolymer, especially the bioresorbable region, being sensitive to hydrolysis in water or a hydrous environment. Similarly, as used herein, “enzymatically cleavable” refers to a copolymer, particularly a bioresorbable region, that is sensitive to cleavage by endogenous or exogenous enzymes.

  When placed in the body, the hydrophilic region may be processed into excretable and / or metabolic fragments. Thus, hydrophilic regions include, for example, polyethers, polyalkylene oxides, polyols, poly (vinyl pyrrolidines), poly (vinyl alcohols), poly (alkyloxazolines), polysaccharides, carbohydrates, peptides, proteins, and copolymers thereof. And mixtures may be included. In addition, the hydrophilic region can also be, for example, a poly (alkylene) oxide. Such poly (alkylene) oxides can 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 compounds that form hydrogels include, but are not limited to, polyacrylic acid, sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidine, gelatin, carrageenan and other polysaccharides, hydroxyethylene methacrylic acid (HEMA), and their Derivatives and the like are included. A stable, biodegradable, bioresorbable hydrogel can be produced. In addition, the hydrogel composition can include one or more subunits that exhibit such properties.

  Biocompatible hydrogel compositions are known whose integrity can be controlled via cross-linking, which is presently preferred for use in the methods of the present invention. For example, Hubbell et al., US Pat. No. 5,410,016 issued April 25, 1995, US Pat. No. 5,529,914 issued June 25, 1996. Discloses a water soluble system which is a cross-linked block copolymer having a water soluble central block segment sandwiched between two hydrolytically variable extensions. Such copolymers are further end-capped with photopolymerizable acrylic acid functional groups. When crosslinked, these systems become hydrogels. The water-soluble central block of such copolymers can include poly (ethylene glycol), but the hydrolytically variable extension is poly (α-hydroxy acid) (such as polyglycolic acid or polylactic acid). possible. See Sawney et al., “Macromolecules” 26: 581-587 (1993).

  In another preferred embodiment, the gel is a thermoreversible gel. Thermoreversible gels containing components such as pluronic, collagen, gelatin, hyaluronic acid, polysaccharides, polyurethane hydrogels, polyurethane-urea hydrogels, and combinations thereof are presently preferred.

  In yet another exemplary embodiment, the conjugate of the invention comprises a component of a liposome. Liposomes can be prepared according to well-known methods, for example, as described in Epstein et al., US Pat. No. 4,522,811, issued Jun. 11, 1985. For example, liposomal formulations may be prepared by dissolving suitable lipids (such as stearoylphosphatidylethanolamine, stearoylphosphatidylcholine, arachadoylphosphatidylcholine, and cholesterol) in an inorganic solvent, which is then evaporated and dried on the surface of the container It can be prepared by leaving a lipid film. An aqueous solution of the active compound or pharmaceutically acceptable salt thereof is then introduced into the container. The container is then turned by hand to peel the lipid material from the sides of the container and disperse the lipid aggregates, thereby forming a liposome suspension.

  The foregoing microparticles and methods for preparing such microparticles are provided as examples, but this is not intended to define the range of microparticles used in the present invention. It will be apparent to those skilled in the art that a series of microparticles made by different methods are useful in the present invention.

  In water-soluble polymers (both linear and branched), the structural forms described above are generally applicable as well for water-insoluble polymers. Thus, for example, cysteine, serine, dilysine, and trilysine branch centers can be functionalized using two water-insoluble polymer moieties. The methods used to generate such species are usually closely similar to the methods used to generate water-soluble polymers.

  The degree of PEG substitution on the conjugate can be controlled by choice of stoichiometry, number of available glycosylation sites, selection of enzymes that are selective for a particular site, etc. (FIG. 2F). GlycoPEGylated Factor IX species show an increase in circulating half-life when compared to labeled Factor IX (FIGS. 3 and 6).

Methods In addition to the conjugates described above, the present invention provides methods for preparing such conjugates and other conjugates. Furthermore, the present invention provides a method for preventing, treating or ameliorating a disease state by administering a conjugate of the present invention to a subject at risk of developing a disease or a subject suffering from a disease.

  Accordingly, the present invention provides a method of forming a covalent conjugate of a selected moiety and a factor IX peptide.

  In an exemplary embodiment, a conjugate of a water soluble polymer, therapeutic moiety, targeting moiety, or biomolecule and a glycosylated or non-glycosylated Factor IX peptide is formed. The polymer, therapeutic moiety, or biomolecule is attached to the peptide via a glycosyl linking group that is positioned between the peptide and the modifying group (eg, a water soluble polymer) and covalently linked to both. The method includes contacting the peptide with a mixture containing a modified sugar and enzyme, eg, a glycosyltransferase that binds the modified sugar to a substrate (eg, peptide, aglycone, glycolipid). This reaction is performed under conditions suitable to form a covalent bond between the modified sugar and the Factor IX peptide.

  Acceptor factor IX peptides are generally synthesized de novo or in prokaryotic cells (eg, bacterial cells such as E. coli) or in eukaryotic cells (mammalian, yeast, insect, fungal, or plant cells). Etc.) are expressed recombinantly. The peptide can be a full-length protein or fragment. Furthermore, the peptides may be wild type or mutated peptides. In an exemplary embodiment, the peptide includes mutations such that one or more N- or O-linked glycosylation sites are added to or removed from the peptide sequence.

In an exemplary embodiment, Factor IX is O-glycosylated and functionalized with a water soluble polymer as follows. Peptides are generated using available amino acid glycosylation sites, or when glycosylated, the glycosyl moiety is removed and exposed to amino acids. For example, serine or threonine is aminogalactosylated α-1N-acetyl (GalNAc), and NAc-galactosylated peptides are sialylated with ST6GalNAcT1 using a sialic acid-modifying group cassette. Alternatively, the NAc-galactosylated peptide is galactosylated using Core-1-GalT-1 and the product is sialylated with ST3GalT1 using a sialic acid-modifying group cassette. A typical conjugate according to this method has the following bonds:
Thr-α-1-GalNAc-β-1,3-Gal-α2,3-Sia *,
In the formula, Sia * is a sialic acid-modifying group cassette.

  In the methods of the invention, such as those described above, using multiple enzymes and saccharyl donors, the individual glycosylation steps may be performed separately or “single pot”. You may combine with reaction. For example, in the three enzymatic reactions described above, GalNAc transferase, GalT and SiaT, and their donors can be combined in a single container. Alternatively, the GalNAc reaction can be performed alone and both GalT and SiaT and the appropriate saccharyl donor can be added as a single step. Another way to carry out this reaction is to add each enzyme and the appropriate donor in succession and carry out the reaction on the “single pot” basis. Each method combination described above is useful for preparing the compounds of the invention.

  In the conjugate of the present invention, in particular, a GlycoPEGylated N-linked glycan, Sia-modifying group cassette can be linked to Gal with an α-2,6 or α-2,3 bond.

  The methods of the invention also provide for modification of incompletely glycosylated Factor IX peptides produced recombinantly. In the methods of the present invention, peptides can be further glycosylated and derivatized simultaneously with, for example, water soluble polymers, therapeutic agents, etc., using modified sugars. The sugar moiety of the modified sugar can be a residue that will be properly attached to an acceptor in a fully glycosylated peptide, or another sugar moiety with desirable properties.

  Typical methods for modifying the peptides used in the present invention are described in WO 04/099231, WO 03/031464, and references described therein.

式中、Ｄは、−ＯＨまたはＲ^１−Ｌ−ＨＮ−である。記号Ｇは、Ｒ^１−Ｌ−または−Ｃ（Ｏ）（Ｃ_１〜Ｃ_６）アルキルを表す。Ｒ^１は、直鎖または分枝ポリ（エチレングリコール）残基を含む部分である。記号Ｌは、結合、置換または非置換アルキル、および置換または非置換ヘテロアルキルから選択されるリンカーを表す。一般に、ＤがＯＨである場合、ＧはＲ^１−Ｌ−であり、Ｇが−Ｃ（Ｏ）（Ｃ_１〜Ｃ_６）アルキルである場合、ＤはＲ^１−Ｌ−ＮＨ−である。本発明の方法は、（ａ）ＰＥＧ−シアル酸供与体をもつ基質第ＩＸ因子ペプチドと、供与体のＰＥＧ−シアル酸部分を、基質第ＩＸ因子ペプチドに転移することができる酵素とを接触させることを含む。 In an exemplary embodiment, the present invention provides a method for producing PEGylated Factor IX comprising the following moieties:

In the 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 moiety containing a linear or branched poly (ethylene glycol) residue. The symbol L represents a linker selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. In general, when D is OH, G is ^R 1-L-, when G is _{-C (O) (C 1 ~C} 6) alkyl, D is ^R 1 -L-NH-. The method of the invention comprises (a) contacting a substrate Factor IX peptide with a PEG-sialic acid donor and an enzyme capable of transferring the PEG-sialic acid moiety of the donor to the substrate Factor IX peptide. Including that.

および転移に適切な状態下で、第ＩＸ因子ペプチドのアミノ酸またはグリコシル残基にＰＥＧ−シアル酸を転移する酵素である。 Exemplary PEG-sialic acid donors are nucleotide sugars such as those having the formula:

And an enzyme that transfers PEG-sialic acid to an amino acid or glycosyl residue of a Factor IX peptide under conditions suitable for transfer.

  In one embodiment, the substrate Factor IX peptide is expressed in the host cell prior to formation of the conjugate of the invention. A typical host cell is a mammalian cell. In other embodiments, the host cell is an insect cell, a plant cell, a bacterium, or a fungus.

  The methods presented herein are applicable to each Factor IX conjugate described in the above section.

  The Factor IX peptide modified by the method of the present invention may be a synthetic or wild-type peptide or may be mutated, generated by methods known in the art such as site-directed mutagenesis. Or a peptide. Peptide glycosylation is generally N-linked or O-linked. A typical N-link 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 is any amino acid except proline) are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. . Thus, the presence of any such tripeptide sequence in the polypeptide provides a potential glycosylation site. O-linked glycosylation is the hydroxyamino acid (preferably serine or threonine, but not unusual or unnatural) of one sugar (eg N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose, or xylose). It refers to the attachment of amino acids such as 5-hydroxyproline or 5-hydroxylysine) to the hydroxy side chain.

  Addition of glycosylation sites to a peptide or other structure is conveniently accomplished by altering the amino acid sequence such that it contains one or more glycosylation sites. The addition can also be done by incorporation of one or more species corresponding to the —OH group (for O-linked glycosylation sites), preferably serine or threonine residues, into the sequence of the peptide. The addition can be done by mutation or by complete chemical synthesis of the peptide. The peptide amino acid sequence is mediated by changes in the DNA level, in particular by mutating the DNA encoding the peptide at a preselected base to produce a codon that will be translated into the desired amino acid. Preferably, it can be changed. The DNA mutation (s) are preferably made using methods known in the art.

  In an exemplary embodiment, glycosylation sites are added by shuffling the polynucleotide. A polynucleotide encoding a candidate peptide can be regulated using a DNA shuffling protocol. DNA shuffling is a recursive recombination and mutation process performed by random fragmentation of a pool of related genes followed by reassembly of the fragments by a process such as the polymerase chain reaction. Stemmer, “Proc. Natl. Acad. Sci. USA” 91: 10747-10751 (1994), Stemmer “Nature” 370: 389-391 (1994), and US Pat. No. 5,605. No. 5,793, No. 5,837,458, No. 5,830,721, and No. 5,811,238,

  Exemplary methods for adding or removing glycosylation sites and adding or removing glycosyl structures or substructures are described in WO 04/099231, WO 03/031464, and Detailed in relevant US and PCT applications.

  The present invention also adds (or removes) one or more selected glycosyl residues to the Factor IX peptide, and then modifies the modified sugar to at least one of the selected glycosyl residues of the peptide. Utilize means to combine them into one. Such techniques are useful, for example, when it is desired to attach the modified sugar to a selected glycosyl residue that is not present on the Factor IX peptide or not present in the desired amount. Thus, prior to attaching the modified sugar to the peptide, the selected glycosyl residue is attached to the peptide by enzymatic or chemical attachment. In another embodiment, the glycosylation pattern of the glycopeptide is altered prior to conjugation of the modified sugar by removing carbohydrate residues from the glycopeptide. For example, see the pamphlet of WO 98/31826. For example, sialic acid groups can be removed from factor IX to form asialo factor IX prior to GlycoPEGylation using PEG-modified sialic acid (FIG. 2E).

  Exemplary attachment positions for selected glycosyl residues include, but are not limited to: (a) a consensus site for N-linked glycosylation, and O-linked glycosylation. A site for (b) a terminal glycosyl moiety that is an acceptor for a glycosyltransferase, (c) arginine, asparagine, and histidine, (d) a free carboxyl group, (e) a free sulfhydryl group such as that of cysteine, (F) a free hydroxyl group such as that of serine, threonine, or hydroxyproline, (g) an aromatic residue such as that of phenylalanine, tyrosine or tryptophan, or (h) an amide group of glutamine. Typical methods used in the present invention are described in WO 87/05330 published September 11, 1987, and in Aplin and Wriston, “CRC CRIT. REV. BIOCHEM. ”, Pages 259-306 (1981).

  PEG-modified sugars are conjugated to glycosylated or non-glycosylated peptides using appropriate enzymes to mediate conjugation. Preferably, the concentration of the modified donor sugar (s), enzyme (s), and acceptor peptide (s) is such that glycosylation proceeds before the acceptor is consumed. Selected to do. The discussion below is described for sialyltransferases, but is generally applicable to other glycosyltransferase reactions.

  Several methods of using glycosyltransferases to synthesize desired oligosaccharide structures are known and are generally applicable to the present invention. Typical methods are described, for example, in WO 96/32491, Ito et al., “Pure Appl. Chem.” 65: 753 (1993), US Pat. No. 5,352,670, US Pat. Nos. 5,374,541, 5,545,553, and commonly owned US Pat. Nos. 6,399,336 and 6,440,703 ( These are described in (incorporated herein by reference).

  The present invention is practiced using a single glycosyltransferase or a combination of glycosyltransferases. For example, a combination of sialyltransferase and galactosyltransferase can be used. In embodiments using more than one enzyme, the enzyme and substrate are combined in the initial reaction mixture, or when the first enzymatic reaction is complete or nearly complete, the second enzymatic Enzymes and reagents for the reaction are preferably added to the reaction medium. By performing two enzymatic reactions in succession in a single vessel, the total yield is improved over procedures in which intermediate species are isolated. In addition, there is less purification and disposal of excess solvent and by-products.

  In a preferred embodiment, each of the first and second enzymes is a glycosyltransferase. In another preferred embodiment, one enzyme is an endoglycosidase. In a further preferred embodiment, more than two enzymes are used to assemble the modified glycoprotein of the invention. These enzymes are used to change the saccharide structure on the peptide at any point before or after the addition of the modified sugar to the peptide.

  In another embodiment, the method uses one or more exo- or endoglycosidases. Glycosidases are generally variants that have been engineered such that glycosyl bonds are formed rather than broken. Mutant glycanases generally comprise a substitution of an active site acidic amino acid residue for an amino acid residue. For example, if the endoglycanase is endo-H, the active site residue that is replaced is typically Asp at position 130, Glu at position 132, or a combination thereof. Amino acids are usually replaced with serine, alanine, asparagine, or glutamine.

  The mutant enzyme catalyzes the reaction, usually 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 proceeds with the addition of the donor molecule to a GlcNAc residue on the protein. . For example, the leaving group can be a halogen (eg, fluoride). In other embodiments, the leaving group is Asn, or an Asn-peptide moiety. In yet other embodiments, the GlcNAc residue on the glycosyl donor molecule is modified. For example, the GlcNAc residue can include a 1,2 oxazoline moiety.

  In a preferred embodiment, each enzyme utilized to produce the conjugates of the invention is present in a catalytic amount. The catalytic amount of a particular enzyme varies depending on the concentration of the enzyme substrate and the reaction conditions (temperature, time, pH value, etc.). Means for determining the amount of catalyst 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 above process is performed can range from just above the freezing temperature to the temperature at which the most sensitive enzyme denatures. A preferred temperature range is from about 0 ° C. to about 55 ° C., preferably from about 20 ° C. to about 37 ° C. In another exemplary embodiment, one or more components of the methods of the invention are performed at an elevated temperature using a thermophilic enzyme.

  The reaction mixture is maintained for a time sufficient for the acceptor to be glycosylated, thereby forming the desired conjugate. Some conjugates can often be detected after a few hours, and usually recoverable amounts are obtained within 24 hours. For those skilled in the art, the rate of reaction depends on a number of different factors (eg, enzyme concentration, donor concentration, acceptor concentration, temperature, solvent volume), which depends on the selected system. You will understand that it is optimized for.

  The present invention also provides for industrial scale production of modified peptides. As used herein, the industrial scale usually produces a finished, purified conjugate of at least 250 mg, preferably at least 500 mg, preferably at least 1 gram.

  In the discussion that follows, the invention is illustrated by attaching a modified sialic acid moiety to a glycosylated peptide. A typical modified sialic acid is labeled with PEG. The focus of the following description on the use of PEG-modified sialic acid and glycosylated peptides is for clarity of illustration and to imply limiting the binding of these two partners It is not a thing. Those skilled in the art will appreciate that this discussion is usually applicable to the addition of modified glycosyl moieties other than sialic acid. Furthermore, this discussion is equally applicable to glycosyl unit modifications using agents other than PEG, including other PEG moieties, therapeutic moieties, and biomolecules.

  Enzymatic techniques can be used for the selective introduction of PEGylated or PPGylated carbohydrates onto peptides or glycopeptides. This method utilizes modified sugars containing PEG, PPG, or masked reactive functional groups, and is combined with an appropriate glycosyltransferase or glycosynthase. By selecting a glycosyltransferase that creates the desired carbohydrate linkage and utilizing the modified sugar as a donor substrate, the PEG or PPG can be on the peptide backbone, on an existing sugar residue of the glycopeptide, or Can be introduced directly onto the sugar residue added to the peptide.

  Acceptors for sialyltransferases are present on the peptides to be modified by the methods of the invention, either as naturally occurring structures, or as recombinantly, enzymatically, or chemically disposed therein. Exists. Suitable acceptors include, for example, galactosyl acceptors such as Galβ1,4GlcNAc, Galβ1,4GalNAc, Galβ1,3GalNAc, lacto-N-4 sugar, Galβ1,3GlcNAc, Galβ1,3Ara, Galβ1,6GlcNAc, Galβ1,4Glc (lactose). , And other acceptors known to those skilled in the art. (See Paulson et al., “J. Biol. Chem.” 253: 5617-5624 (1978)).

  In one embodiment, the acceptor for the sialyltransferase is present on the glycopeptide to be modified in response to in vivo synthesis of the glycopeptide. Such glycopeptides can be sialylated using claimed methods that do not involve conventional modifications of glycopeptide glycosylation patterns. Alternatively, the method of the present invention can be used to sialylate a peptide that does not contain a suitable acceptor, which will first contain the acceptor by methods known to those skilled in the art. To modify the peptide. In an exemplary embodiment, GalNAc residues are added by the action of GalNAc transferase.

  In an exemplary embodiment, a galactosyl acceptor is assembled by attaching a galactose residue to an appropriate acceptor that is linked to a peptide (eg, GlcNAc). This method involves incubating the peptide to be modified with a reaction mixture containing an appropriate amount of galactosyltransferase (eg galβ1,3 or galβ1,4) and an appropriate galactosyl donor (eg UDP-galactose). . The reaction is allowed to proceed until substantially complete, or the reaction is terminated once a preselected amount of galactose residues has been added. Other methods of assembling the selected saccharide acceptor will be apparent to those skilled in the art.

  In yet another embodiment, the glycopeptide-linked oligosaccharide is first “trimmed” in whole or in part to accept an acceptor for sialyltransferase, or one or more suitable residues. Expose the parts that can be added to obtain the appropriate acceptor. Enzymes such as glycosyltransferases and endoglycosidases (see, eg, US Pat. No. 5,716,812) are useful for attachment and trimming reactions.

  In the discussion that follows, the method of the present invention is exemplified by using a modified sugar having a PEG moiety attached thereto. The focus of the discussion is to clarify specific examples. Those skilled in the art will appreciate that this discussion is equally relevant to embodiments where modified sugars have therapeutic moieties, biomolecules, and the like.

  In an exemplary embodiment of the invention where the carbohydrate residues are “trimmed” prior to the addition of the modified sugar, the high mannose is trimmed back to the first generation biantennary structure. Is done. The modified sugar with a PEG moiety is coupled to one or more of the sugar residues that are exposed by “trimming back”. In one example, the PEG moiety is added via a GlcNAc moiety attached to the PEG moiety. The modified GlcNAc is attached to one or both of the bifurcated terminal mannose residues. Alternatively, unmodified GlcNAc can be added to one or both ends of the branched species.

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

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

  In another exemplary embodiment, the high grade mannose structure is “trimmed back” from there to a mannose from which the bifurcated structure branches. In one example, the PEG moiety is added via a GlcNAc modified with a polymer. Alternatively, unmodified GlcNAc is added to mannose followed by Gal with an attached PEG moiety. In yet another embodiment, unmodified GlcNAc and Gal residues are added sequentially to mannose followed by a sialic acid moiety modified with a PEG moiety.

  In a further exemplary embodiment, the high grade mannose is “trimmed back” to the GlcNAc to which the first mannose is attached. GlcNAc is attached to a Gal residue with a PEG moiety. Alternatively, unmodified Gal is added to GlcNAc followed by the addition of modified sialic acid using a water soluble sugar. In a further example, the terminal GlcNAc is conjugated with Gal and the GlcNAc is then fucosylated using a modified fucose with a PEG moiety.

Higher mannose can also be trimmed back to the first GlcNAc attached to the peptide Asn. In one example, the GlcNAc of the GlcNAc- (Fuc) _a residue is combined with a GlcNAc with a water soluble polymer. In another example, the GlcNAc of the GlcNAc- (Fuc) _a residue is modified with Gal with a water soluble polymer. In a further embodiment, GlcNAc is modified with Gal, followed by conjugation of the modified sialic acid to Gal with a PEG moiety.

  Other exemplary embodiments include commonly owned U.S. Patent Application Publication Nos. 20040132640, 20040063911, 20040137557, U.S. Patent Application No. 10 / 369,979, Nos. 10 / 410,913, 10 / 360,770, 10 / 410,945, and PCT / US02 / 32263, each of which is incorporated herein by reference. (Incorporated in the specification).

  The embodiments described above provide examples of the capabilities of the methods described herein. Using the methods described herein, it is possible to "trim back" and build up carbohydrate residues of virtually any desired structure. The modified sugar may be added to the end of the carbohydrate moiety as described above, or may be intermediate between the peptide center and the end of the carbohydrate.

In an exemplary embodiment, existing sialic acid is removed from the Factor IX glycopeptide using sialidase, thereby unmasking all or most of the underlying galactosyl residues. Alternatively, the peptide or glycopeptide is labeled with galactose residues, or oligosaccharide residues that terminate in galactose units. Following exposure or addition of galactose residues, a suitable sialyltransferase is used to add the modified sialic acid. This approach is outlined in Scheme 1.

In a further approach outlined in Scheme 2, a masked reactive functional group is present on the sialic acid. The masked reactive group is preferably unaffected by the conditions used to attach the modified sialic acid to Factor IX. After covalent attachment of the modified sialic acid to the peptide, the mask is removed and the peptide is conjugated with an agent such as PEG. This drug binds to the peptide in a specific manner by reaction with a masked reactive group on the modified sugar residue.

  Any modified sugar described herein can be used with its appropriate glycosyltransferase, depending on the terminal sugar of the oligosaccharide side chain of the glycopeptide (Table 1). As described above, the terminal sugar of the glycopeptide required for the introduction of the PEGylated structure may be introduced naturally during expression, or may be a suitable glycosidase (s), glycosyltransferase ( One or more), or a mixture of glycosidase (s) and glycosyltransferase (s) may be used after expression.

  In a further exemplary embodiment, UDP-galactose-PEG is reacted with milk β1,4-galactosyltransferase, thereby transferring the modified galactose to the appropriate terminal N-acetylglucosamine structure. Terminal GlcNAc residues on glycopeptides may be generated during expression, as may occur in such expression systems as mammals, insects, plants or fungi, but if desired, sialidases and It can also be produced by treating glycopeptides with glycosidases and / or glycosyltransferases.

  In another exemplary embodiment, GlcNAc transferase (eg, GNT1-5) is utilized to transfer PEGylated GlcN to a terminal mannose residue on a glycopeptide. In a further exemplary embodiment, N-linked and / or O-linked glycan structures are enzymatically removed from the glycopeptide to expose amino acids or terminal glycosyl residues that are subsequently coupled to the modified sugar. Is done. For example, endoglycanase is used to remove the N-linked structure of the glycopeptide, exposing the terminal GlcNAc as a GlcNAc-linked-Asn on the glycopeptide. The PEG-galactose functional group is introduced onto the exposed GlcNAc using UDP-Gal-PEG and a suitable galactosyltransferase.

In another embodiment, the modified sugar is added directly to the peptide backbone using a glycosyltransferase known to transfer sugar residues to the peptide backbone. This exemplary embodiment is described in Scheme 3. Exemplary glycosyltransferases useful for practicing the present invention include, but are not limited to, GalNAc transferase (GalNAc T1-14), GlcNAc transferase, fucosyltransferase, glucosyltransferase, xylosyltransferase, mannosyltransferase, and the like. Is included. Use of this approach also allows for the direct addition of modified sugars on peptides lacking any carbohydrate or on existing glycopeptides. In either case, the addition of the modified sugar occurs at a specific position on the peptide backbone, as defined by the substrate specificity of the glycosyltransferase, but the peptide of the protein using chemical methods. It does not occur in a random manner as occurs during backbone modification. A series of agents can be introduced into a protein or glycopeptide lacking a glycosyltransferase substrate peptide sequence by manipulating the appropriate amino acid sequence in the polypeptide chain.

  In each exemplary embodiment described above, one or more additional chemical or enzymatic modification steps can be utilized after conjugation of the modified sugar to the peptide. In an exemplary embodiment, an enzyme (eg, fucosyltransferase) is used to add a glycosyl unit (eg, fucose) onto a terminally modified sugar attached to a peptide. In another example, an enzymatic reaction is utilized at the “cap” site where the modified sugar failed to bind. Alternatively, chemical reactions are utilized to alter the structure of the attached modified sugar. For example, the conjugated modified sugar is reacted with an agent that stabilizes or destabilizes the binding with the peptide component to which the modified sugar is attached. In another example, the modified sugar component is deprotected after its conjugation to the peptide. Those skilled in the art will understand that there is a range of enzymatic and chemical procedures that are useful in the methods of the invention at a stage after the modified sugar is attached to the peptide. Let's go. Further finishing of the modified sugar-peptide conjugate is within the scope of the present invention.

Enzyme In forming acyl-linked conjugates, trimming back, refines glycosylation patterns of conjugates and starting substrates (eg peptides, lipids) in addition to the enzymes mentioned above Alternatively, it can be modified by a method using another enzyme. A method for reconstituting peptides and lipids using an enzyme that transfers a sugar donor to an acceptor is described in DeFreees, WO 03/031464 A2, published April 17, 2003. Has been described in great detail. A summary of the selected enzymes used in the method of the invention is set forth below.

Glycosyltransferases Glycosyltransferases add activated sugars (donor NDP- or NMP-sugars) to proteins, glycopeptides, lipids, or glycolipids, or to the non-reducing ends of growing oligosaccharides. Is catalyzed in a stepwise fashion. N-linked glycopeptides are synthesized via the oligosaccharide donor Dol-PP-NAG ₂ Glc ₃ Man ₉ linked to a transferase and lipids in a batch transfer followed by central trimming. In this case, the nature of the “central” saccharide is somewhat different from subsequent attachment. A large number of glycosyltransferases are known in the art.

  The glycosyltransferase used in the present invention may be any as long as it is likely to utilize a modified sugar as a sugar donor. Examples of such enzymes include luloal pathway glycosyltransferases such as galactosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, fucosyltransferase, sialyltransferase, mannosyltransferase, xylosyltransferase, glucurononyltransferase, and the like. (Leiloir pathway glycosyltransferase) is included.

  For enzymatic saccharide synthesis involved in glycosyltransferase reactions, glycosyltransferases can be cloned or isolated from any source. Many cloned glycosyltransferases are known for their polynucleotide sequences. For example, see “The WWW Guide To Clone Glycosyltransferases” (http://www.vei.co.uk/TGN/gt_guide.htm). Glycosyltransferase amino acid sequences, and nucleotide sequences encoding glycosyltransferases (from which amino acid sequences can be deduced) are also found in various publicly available databases, including GenBank, Swiss-Prot, EMBL, etc. .

  The glycosyltransferase that can be used in the method of the present invention includes, but is not limited to, galactosyltransferase, fucosyltransferase, glucosyltransferase, N-acetylgalactosaminyltransferase, N-acetylglucosaminyltransferase, glucuronyltransferase, Sialyltransferases, mannosyltransferases, glucuronic acid transferases, galacturonic acid transferases, and oligosaccharyltransferases are included. Suitable glycosyltransferases include those obtained from eukaryotes as well as prokaryotes.

  DNA encoding glycosyltransferases can be obtained by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by It can be obtained by a combination of procedures. Screening for mRNA or genomic DNA can be performed using oligonucleotide probes produced from glycosyltransferase gene sequences. The probe can be labeled with a detectable group such as a fluorescent group, radioactive atom or chemiluminescent group according to known procedures and can be used in conventional hybridization assays. In a variation, the glycosyltransferase gene sequence can be obtained using PCR oligonucleotide primers produced from the glycosyltransferase gene sequence by using a polymerase chain reaction (PCR) procedure. See U.S. Pat. No. 4,683,195 to Mullis et al. And U.S. Pat. No. 4,683,202 to Mullis et al.

  Glycosyltransferases can be synthesized in host cells transformed with a vector containing DNA encoding the glycosyltransferase enzyme. Vectors are used to amplify DNA encoding a glycosyltransferase enzyme and / or to express DNA encoding a glycosyltransferase enzyme. An expression vector is a replicable DNA construct in which a DNA sequence encoding a glycosyltransferase enzyme is operably linked to appropriate control sequences capable of effecting expression of the glycosyltransferase enzyme in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Control sequences typically include a transcription promoter, any operator sequence to control transcription, a sequence encoding an appropriate mRNA ribosome binding site, and sequences that control the termination of transcription and translation. An amplification vector does not require an expression control domain. All that is needed is a selection gene that facilitates recognition of the transformant, as well as the ability to replicate in the host, usually given by the origin of replication.

  In an exemplary embodiment, the present invention utilizes prokaryotic enzymes. Such glycosyltransferases include enzymes involved in the synthesis of lipooligosaccharide (LOS), produced by many Gram-negative bacteria (Preston et al., “Critical Reviews in Microbiology” 23 (3): 139- 180 (1996)). Such enzymes include, but are not limited to, proteins of the rfa operon of species such as E. coli and Salmonella typhimurium (including β1,6 galactosyltransferase and β1,3 galactosyltransferase) (eg, EMBL contracts). Nos. M80599 and M86935 (see E. coli); EMBL accession number S56361 (S. typhimurium), glucosyltransferase (Swiss-Prot accession number P25740 (E. coli)), β1,2-glucosyltransferase (rfaJ) (Swiss-Prot accession number P27129 (E. coli)) and Swiss- rot accession number P19817 (S. typhimurium)), and β1,2-N-acetylglucosaminyltransferase (rfaK) (EMBL accession number U00039 (E. coli)). Other known glycosyltransferases include Klebsiella pneumoniae, E. coli, Salmonella typhimurium, Salmonella enterica, Yersinia enterocoli ), Mycobacterium leprosum, and Pseudomonas aeruginosa Included are those encoded by operons such as rfaB, which are characterized in organisms such as the rhl operon of Pseudomonas aeruginosa.

Also suitable for use in the present invention is the lacto-N-neotetraose identified in the LOS of the mucosal pathogens Neisseria gonnorhoeae and N. meningitidis, D-galactosyl-β-1,4-N-acetyl-D-glucosaminyl-β-1,3-D-galactosyl-β-1,4-D-glucose, and P ^k blood group trisaccharide sequence (P ^k blood group trisaccharide sequence), a glycosyltransferase involved in the production of a structure containing D-galactosyl-α-1,4-D-galactosyl-β-1,4-D-glucose (Scholten et al., “J Med. Microbiol. "41:23 ～243 (1994)). The genes from N. meningitidis and N. gonorrhoeae encoding glycosyltransferases involved in the biosynthesis of these structures are N. meningitidis immunotypes L3 and L1 ( Jennings et al. “Mol. Microbiol.” 18: 729-740 (1995)), and N. gonorrhoeae mutant F62 (Gottshrich), “J Exp. Med.” 180: 2181 2190 (1994)). In N. meningitidis, a locus consisting of three genes (lgtA, lgtB, and lgE) is required for the last three additions of sugars in the lacto-N-neotetraose chain It encodes a glycosyltransferase enzyme (Wakarchuk et al. “J. Biol. Chem.” 271: 19166-73 (1996)). Recently, the enzymatic activity of the lgtB and lgtA gene products has been demonstrated and provided the first direct evidence for their proposed glycosyltransferase function (Wakarchuk et al., “J. Biol. Chem.” 271 ( 45): 28271-276 (1996)). In N. gonorrhoeae, two additional genes, lgtD, which adds β-D-GalNAc at position 3 of the terminal galactose of the lacto-N-neotetraose structure, and terminal to the cleaved LOS lactose element There is lgtC that adds α-D-Gal, which results in a ^Pk blood group antigen structure (Gotshrich (1994), supra). In Neisseria meningitidis (N. meningitidis), separate immunotype L1 Kamata, express ^{p k} blood group antigen, was shown to carry the lgtC gene (Jennings (Jennings) et al, (1995) supra) . Neisseria glycosyltransferases and related genes are also described in US Pat. No. 5,545,553 (Gotschlich). The genes for α1,2-fucosyltransferase and α1,3-fucosyltransferase from Helicobacter pylori have also been characterized (Martin et al. “J. Biol. Chem.” 272: 21349. 21356 (1997)). Also used in the present invention is Campylobacter jejuni glycosyltransferase (see, eg, http://afmb.cnrs-mrs.fr/~pedro/CAZY/gtf_42.html). .

Fucosyltransferase In certain embodiments, the glycosyltransferase used in the methods of the invention is a fucosyltransferase. Fucosyltransferases are known to those skilled in the art. Typical fucosyltransferases include enzymes that transfer L-fucose from GDP-fucose to the hydroxy position of the acceptor sugar. Fucosyltransferases that transfer non-nucleotide sugars to acceptors are also those used in the present invention.

  In certain embodiments, the acceptor sugar is, for example, GlcNAc in a Galβ (1 → 3,4) GIcNAcβ-group in an oligosaccharide glycoside. Suitable fucosyltransferases for this reaction include Galβ (1 → 3,4) GlcNAcβ1-α (1 → 3,4) fucosyltransferase (FTIII EC No. 2 initially characterized from human breast milk. 4.1.65) (Paltic et al., “Carbohydrate Res.” 190: 1-11 (1989); Prieels et al., “J. Biol. Chem.” 256: 10456-10463 (1981). Nunez et al., “Can. J. Chem.” 59: 2086-2095 (1981)), and Galβ (1 → 4) GlcNAcβ-α found in human serum. Fucosyltransferases (FTIV, FTV, FTVI) are included. FTVII (EC number 2.4.1.65), sialyl α (2 → 3) Galβ (1 → 3) GlcNAcβfucosyltransferase has also been characterized. The recombinant form of Galβ (1 → 3,4) GlcNAcβ-α (1 → 3,4) fucosyltransferase has also been characterized (Dumas et al., “Bioorg. Med. Letters” 1: 425-428 (1991), and Kukovska-Latallo et al., “Genes and Development” 4: 1288-1303 (1990)). Other exemplary fucosyltransferases include, for example, α1,2 fucosyltransferase (EC number 2.4.4.19). Enzymatic fucosylation can be performed by the method described in Mollicone et al., “Eur. J. Biochem.” 191: 169-176 (1990) or US Pat. No. 5,374,655. . The cells used to produce fucosyltransferase will also include an enzymatic system that synthesizes GDP-fucose.

Galactosyltransferase In another group of embodiments, the glycosyltransferase is a galactosyltransferase. Exemplary galactosyltransferases include α (1,3) galactosyltransferase (EC No. 2.4.1.11, for example, Dabkowski et al., “Transplant Proc.” 25: 2921 (1993). ) And Yamamoto et al., “Nature” 345: 229-233 (1990)), GenBank j04989, Joziasse et al., “J. Biol. Chem.” 264: 14290-14297 (1989). ), Mice (GenBank m26925, Larsen et al., “Proc. Nat'l. Acad. Sci. USA” 86: 8227-8231 (1989)), pigs (GenBank L36152, Strahan (St. rahan) et al., "Immunogenetics" 41: 101-105 (1995)). Another suitable α1,3 galactosyltransferase is involved in the synthesis of blood group B antigen (EC 2.4.4.1.37, Yamamoto et al., “J. Biol. Chem.” 265: 1146. 1151 (1990) (human)). A more typical galactosyltransferase is core Gal-T1.

  Also suitable for use in the methods of the invention are β (1,4) galactosyltransferases, including, for example, EC 2.4.4.10 (LacNAc synthase) and EC 2.4. 1.22 (lactose synthase) (D'Agostaro et al., “Eur. J. Biochem.” 183: 211-217 (1989)), human (Masri et al. (Biochem. Biophys.Res.Commun.157: 657-663 (1988)), mice (Nakazawa et al., "J. Biochem." 104: 165-168 (1988)), and EC 2.4. 1.38, and ceramide galactosyltransferase (EC 2.4.4.1.45, Stutter (Stahl) et al., “J. Neurosci. Res.” 38: 234-242 (1994)) Other suitable galactosyltransferases include, for example, α1,2 galactosyltransferases (eg, Schizosaccharomyces pombe). (Schizosaccharomyces pombe), Chapel et al., “Mol. Biol. Cell” 5: 519-528 (1994)).

Sialyltransferases Sialyltransferases are another type of glycosyltransferase that are useful in the recombinant cells and reaction mixtures of the present invention. Cells that produce recombinant sialyltransferases will also produce CMP-sialic acid, which is a sialic acid donor for sialyltransferases. Examples of sialyltransferases suitable for use in the present invention include ST3Gal III (eg, rat or human ST3Gal III), ST3Gal IV, ST3Gal I, ST3Gal II, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II And ST6GalNAc III (the sialyltransferase nomenclature used herein is as described in Tsuji et al., “Glycobiology” 6: v-xiv (1996)). A typical α (2,3) sialyltransferase called α (2,3) sialyltransferase (EC 2.4.99.6) transfers sialic acid to the Galβ1 → 3Glc disaccharide or the non-reducing terminal Gal of the glycoside. To do. Van den Eijdenden 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 exemplary α2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid to the non-reducing terminal Gal of a disaccharide or glycoside. See Realick et al., “J. Biol. Chem.” 254: 4444 (1979)), and Gillespie et al., “J. Biol. Chem.” 267: 21004 (1992). Further typical enzymes include Gal-β-1,4-GlcNAcα-2,6 sialyltransferase. (See Kurosawa et al., “Eur. J. Biochem.” 219: 375-381 (1994)).

  Preferably, for glycopeptide glycosylation of glycopeptides, the sialyltransferase is the most common last that forms the basis of terminal sialic acid on the sequence Galβ1,4GlcNAc—that is, a fully sialylated carbohydrate structure. It is possible to transfer sialic acid from the first to the second sequence (see Table 2).

  Other sialyltransferases used in the present invention include those described in the table of FIG. Sialyltransferases are PEGylated sialyl from PEGylated sialic acid donor species to peptide N-linked glycosyl residues (FIG. 2C) or factor IX O-linked glycosyl residues (FIG. 2D). Can be used to transfer the acid moiety.

  An example of a sialyltransferase that is useful in the claimed method is ST3Gal III, also referred to as α (2,3) sialyltransferase (EC 2.4.99.6). This enzyme catalyzes the transfer of sialic acid to Gal β1,3GlcNAc or Galβ1,4GlcNAc glycoside to Gal (Wen et al., “J. Biol. Chem.” 267: 21011 (1992), (See Van den Eijnden) et al., “J. Biol. Chem.” 256: 3159 (1991)), responsible for sialylation of asparagine-linked oligosaccharides in glycopeptides. Sialic acid is linked to Gal with the formation of an α-bond between the two sugars. The bond (linkage) between saccharides is between the 2-position of NeuAc and the 3-position of Gal. This particular enzyme has been described in 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, and the genome (Kitagawa et al. (1996)" J. Biol. Chem. "271: 931-938). DNA sequences that facilitate the production of this enzyme by recombinant expression are known. In a preferred embodiment, the claimed sialylation method uses rat ST3Gal III.

  Other exemplary sialyltransferases used in the present invention include those isolated from Campylobacter jejuni, including α (2,3). For example, see WO99 / 49051 pamphlet.

Others of the sialyltransferases listed in Table 2 are also useful for economical and efficient large-scale processes for the sialylation of commercially important glycopeptides. As a simple test to discover the usefulness of these other enzymes, various amounts (1-100 mU / mg protein) of each enzyme were reacted with (1-10 mg / ml) asialo-α ₁ AGP, The ability of the sialyltransferases to sialylate glycopeptides was compared to either bovine ST6Gal I, ST3Gal III, or both sialyltransferases. Alternatively, other glycopeptides or glycopeptides, or N-linked oligosaccharides enzymatically released from the peptide backbone can be used in place of asialo-α ₁ AGP for this evaluation. Sialyltransferases with the ability to sialylate N-linked oligosaccharides of glycopeptides more efficiently than ST6Gal I are useful in practical large-scale processes for peptide sialylation.

GalNAc transferase N-acetylgalactosaminyltransferase is used in practicing the present invention, in particular to attach a GalNAc moiety to an amino acid at the O-linked glycosylation site of a peptide. 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 the polypeptide N-acetylgalactosaminyltransferase (Homa). ) Et al., But not limited to, “J. Biol. Chem.” 268: 12609 (1993).

The production of proteins such as the enzyme GalNAc T _I-XX from genes cloned by genetic engineering is well known. See, for example, US Pat. No. 4,761,371. One method involves a sufficient collection of samples, after which the amino acid sequence of the enzyme is determined by N-terminal sequencing. This information is then used to isolate a cDNA clone encoding a full-length (membrane-bound) transferase that, when expressed in the insect cell line Sf9, synthesizes a fully active enzyme. . The acceptor specificity of the enzyme is then determined using a semi-quantitative analysis of the amino acids surrounding known glycosylation sites in 16 different proteins, followed by in vitro glycosylation studies of synthetic polypeptides. . This study shows that certain amino acid residues are too many in glycosylated peptide segments, and residues at specific positions around glycosylated serine and threonine residues Thus, it has been demonstrated that it may have a significant influence than other amino acid moieties.

Cell-associated glycosyltransferases In another embodiment, the enzyme utilized in the methods of the invention is a cell-associated glycosyltransferase. Although many soluble glycosyltransferases are known (see, eg, US Pat. No. 5,032,519), glycosyltransferases are usually in a membrane-bound form when bound to cells. . Many membrane-bound enzymes studied so far are thought to be endogenous proteins, ie they are not released from the membrane by sonication and require a surfactant 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, the more recognized function of cell surface glycosyltransferases is for intercellular recognition. (Roth, “MOLECULAR APPROACHES to SUPRACELLULAR PHENOMENA”, 1990).

  This method was developed to alter glycosyltransferases expressed by cells. For example, Larsen et al., “Proc. Natl. Acad. Sci. USA” 86: 8227-8231 (1989) have been cloned to determine cell surface oligosaccharide structures and the expression of their cognate glycosyltransferases. Reported a genetic approach to isolating the cDNA sequence. CDNA library produced from mRNA isolated from a murine cell line known to express UDP-galactose: β-D-galactosyl-1,4-N-acetyl-D-glucosaminide α-1,3 -Galactosyltransferase was transfected into COS-1 cells. Transfected cells were then cultured and assayed for α1-3 galactosyltransferase activity.

  Francisco, et al., “Proc. Natl. Acad. Sci. USA” 89: 2713-2717 (1992)) discloses a method of immobilizing β-lactamase on the outer surface of E. coli. A tripartite fusion consisting of (i) a signal sequence of the outer membrane protein, (ii) a transmembrane section of the outer membrane protein, and (iii) a fully mature β-lactamase sequence is produced and active surface binding A β-lactamase molecule is provided. However, the Francis method is limited to prokaryotic cell systems only and, as recognized by the authors, requires a complete tripartite fusion for proper functionalization.

Sulfotransferases The present invention also provides methods for producing peptides comprising sulfated molecules, including, for example, sulfated polysaccharides such as heparin, heparan sulfate, carrageenan, and related compounds. provide. Suitable sulfotransferases include, for example, the chicken cDNA described by chondroitin-6-sulfotransferase (Fukuta et al., “J. Biol. Chem.” 270: 18575-18580 (1995)); No. D49915), glycosaminoglycan N-acetylglucosamine N-deacetylase / N-sulfotransferase 1 (Dixon et al., “Genomics” 26: 239-241 (1995), UL 18918), and glycosaminoglycan N Acetylglucosamine N-deacetylase / N-sulfotransferase 2 (Orellana et al., “J. Biol. Chem.” 269: 2270-2276 (1994) ), And Ericsson (Eriksson), et al., 269: 10438-10443 (murine cDNA described in 1994); includes human cDNA described in GenBank Accession No. U2304) "J Biol. Chem."

Glycosidases The present invention also encompasses the use of wild-type and mutant glycosidases. Mutant β-galactosidase enzymes have been demonstrated to catalyze the formation of disaccharides through the binding of α-fluorinated sugars to galactosyl acceptor molecules. (Withers, US Pat. No. 6,284,494, issued September 4, 2001). Other glycosidases used in the present invention include, for example, β-glucosidase, β-galactosidase, β-mannosidase, β-acetylglucosaminidase, β-N-acetylgalactosaminidase, β-xylosidase, β-fucosidase, cellulase, Xylanase, galactanase, mannanase, hemicellulase, amylase, glucoamylase, α-glucosidase, α-galactosidase, α-mannosidase, α-N-acetylglucosaminidase, α-N-acetylgalactose-aminidase, α-xylosidase, α-fucosidase And neuraminidase / sialidase. In an exemplary embodiment, sialidase is used to remove sialic acid from Factor IX N-glycans (FIG. 2A) prior to GlycoPEGylation. The present invention also provides a process that does not require conventional removal of sialic acid. Thus, methods that incorporate a sialic acid exchange reaction using a modified sialic acid moiety and ST3Gal3 are useful in the present invention.

Immobilized enzyme The present invention also provides the use of an enzyme immobilized on a solid and / or soluble support. In an exemplary embodiment, a glycosyltransferase is provided that is attached to PEG via a complete 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 method of the present invention simplifies work-up of the reaction mixture and purification of the reaction product, and further allows easy recovery of the enzyme. Glycosyltransferase conjugates are utilized in the methods of the present invention. Other combinations of enzyme and support will be apparent to those skilled in the art.

Fusion Proteins In other exemplary embodiments, the methods of the invention utilize a fusion protein having two or more enzymatic activities involved in the synthesis of the desired glycopeptide conjugate. For example, a fusion polypeptide can consist of a catalytically active domain of a glycosyltransferase linked to a catalytically active domain of an auxiliary enzyme. The coenzyme catalytic domain may, for example, catalyze a step in the formation of a nucleotide sugar that is a donor for a glycosyltransferase, or catalyze a reaction involved in a glycosyltransferase cycle. For example, a polynucleotide encoding a glycosyltransferase can be linked in-frame to a polynucleotide encoding an enzyme involved in nucleotide sugar synthesis. The resulting fusion protein can then catalyze not only the synthesis of the nucleotide sugar, but also the transfer of the sugar moiety to the acceptor molecule. A fusion protein can be two or more cycle enzymes linked to one expressible nucleotide sequence. In other embodiments, the fusion protein comprises catalytically active domains of two or more glycosyltransferases. See, for example, 5,641,668. The modified glycopeptides of the present invention can be easily designed and manufactured using a variety of suitable fusion proteins (PCT patent published as WO 99/31224 on June 24, 1999). See application PCT / CA98 / 01180).

Preparation of modified sugars In general, a sugar moiety or sugar moiety-linker cassette and a PEG or PEG-linker cassette group are linked together by using a reactive group, which is linked to a novel organic functional group by a linking process. Generally converted to a group or reactive species. The sugar reactive functional group (s) are located at any position on the sugar moiety. The reactive groups and classes of reactions useful in practicing the present invention are generally those well known in the field of bioconjugate chemistry. The currently convenient class of reactions available with reactive sugar moieties are those that proceed under relatively mild conditions. This includes nucleophilic substitution (eg reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (eg enamine reaction), and addition to carbon-carbon and carbon-heteroatom multiple bonds. However, the present invention is not limited to this (for example, Michael reaction, Diels-Alder addition). These and other useful reactions are described, for example, in March, “ADVANCED ORGANIC CHEMISTRY”, 3rd edition, John Wiley & Sons, New York, New York, 1985. Year: Hermanson, “BIOCONJUGATE TECHNIQUES”, Academic Press, San Diego, 1996; and Feney et al. 198, American Chemical Society, Wa. Cantonal City (Washington, D.C.), Has been described in 1982.

Useful reactive functional groups that hang from a sugar center or modifying group include, but are not limited to:
(A) including but not limited to N-hydroxysuccinimide ester, N-hydroxybenztriazole ester, acid halide, acylimidazole, thioester, p-nitrophenyl ester, alkyl, alkenyl, alkynyl, and aromatic ester, A carboxyl group and various derivatives thereof;
(B) a hydroxyl group that can be converted into, for example, an ester, ether, aldehyde, etc .;
(C) a haloalkyl group (wherein the halide can later be replaced by a nucleophilic group such as, for example, an amine, carboxylate anion, thiol anion, carbanion, or alkoxide ion; Resulting in the covalent attachment of a new group at the group);
(D) a dienophile group capable of participating in a Diels-Alder reaction (such as a maleimide group);
(E) Aldehyde or ketone groups (where subsequent derivatization is through such formation of carbonyl derivatives such as imines, hydrazones, semicarbazones, or oximes, or as a Grignard or alkyllithium addition) Is possible);
(F) a sulfonyl halide group for subsequent reactions with amines, for example to form sulfonamides;
(G) a thiol group that can be converted, for example, to a disulfide or reacted with an acyl halide;
(H) amine or sulfhydryl groups that can be acylated, alkylated, or oxidized;
(I) alkenes capable of undergoing, for example, cycloaddition, acylation, Michael addition; and (j) epoxides capable of reacting with, for example, amines and hydroxyl compounds.

  The reactive functional groups can be selected such that they do not participate in or interfere with the reactions necessary to assemble the reactive sugar center or modifying group. Alternatively, reactive functional groups can be protected from participating in the reaction by the presence of protecting groups. One skilled in the art will understand how to protect a particular functional group such that it does not interfere with the chosen 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 discussion that follows, some specific examples of modified sugars that are useful in practicing the present invention are described. In an exemplary embodiment, the sialic acid derivative is utilized as the sugar center to which the modifying group is attached. The focus of the discussion on sialic acid derivatives is for clarity of illustration only and should not be construed to limit 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 are available to modify galactose, glucose, N-acetylgalactosamine, and fucose (to name a few sugar substrates) that are readily modified by methods recognized in the art. is there. See Elhalabi et al., “Curr. Med. Chem.” 6:93 (1999); and Schaffer et al., “J. Org. Chem.” 65:24 (2000).

  In an exemplary embodiment, the peptide modified by the method of the invention is a glycopeptide produced in mammalian cells (eg CHO cells) or in transgenic animals, and thus incompletely sialic acid Contains added N-linked and / or O-linked oligosaccharide chains. Oligosaccharide chains of glycopeptides lacking sialic acid and containing terminal galactose residues can be PEGylated, PPGylated, or otherwise modified with sialic acid Can be modified.

In Scheme 4, aminoglycoside 1 is treated with an active ester of a protected amino acid (eg, glycine) derivative to convert the sugar amine residue to the corresponding protected amino acid amide adduct. This adduct is treated with aldolase to form α-hydroxycarboxylic acid ester 2. Compound 2 is converted to the corresponding CMP derivative by the action of CMP-SA synthetase, followed by catalytic hydrogenation of the CMP derivative to produce compound 3. Reaction of an amine introduced via formation of a glycine adduct with compound 3 with an activated PEG or PPG derivative (eg PEG-C (O) NHS, PEG-OC (O) Op-nitrophenyl) By using it, it is used as a position of PEG attachment, and a species such as 4 or 5 is generated, respectively.

  Table 3 describes representative examples of sugar monophosphates derivatized with a PEG moiety. Certain compounds in Table 3 are prepared by the method of Scheme 4. Other derivatives are prepared by methods recognized in the art. See, for example, Kepler et al., “Glycobiology” 11: 11R (2001); and Charter et al., “Glycobiology” 10: 1049 (2000). Other amine-reactive PEG and PPG analogs are commercially available, or they can be prepared by methods readily available to those skilled in the art.

式中、Ｘは、好ましくは−Ｏ−、−Ｎ（Ｈ）−、−Ｓ、ＣＨ_２−、および−Ｎ（Ｒ）_２から選択される連結基であり、上式で、各Ｒは、Ｒ^１〜Ｒ^５から独立して選択されるメンバーである。記号Ｙ、Ｚ、Ａ、およびＢは、それぞれ、Ｘの識別特性について上で述べた群から選択される基を表す。Ｘ、Ｙ、Ｚ、ＡおよびＢは、それぞれ独立して選択され、したがって、これらは同じであっても異なってもよい。記号Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４、およびＲ^５は、Ｈ、ＰＥＧ部分、治療的部分、生体分子、または他の部分を表す。あるいは、これらの記号は、ＰＥＧ部分、治療的部分、生体分子、または他の部分に結合されるリンカーを表す。 The modified sugar phosphate used in practicing the present invention can be substituted not only at the positions mentioned above, but also at other positions. The presently preferred substitution of sialic acid is described in the following formula:

Wherein X is preferably a linking group selected from —O—, —N (H) —, —S, CH ₂ —, and —N (R) ₂ , wherein each R is It is a member selected independently from R ^{1 to} R ⁵ . The symbols Y, Z, A, and B each represent a group selected from the group described above for the distinguishing properties of X. X, Y, Z, A and B are each independently selected, and therefore 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. Alternatively, these symbols represent a linker that is attached to a PEG moiety, therapeutic moiety, biomolecule, or other moiety.

Exemplary moieties attached to the conjugates disclosed herein include PEG derivatives (eg, acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEGcarbamoyl-PEG, aryl PEG), PPG derivatives ( For example, acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPG carbamoyl-PPG, aryl-PPG), therapeutic moiety, diagnostic moiety, mannose-6-phosphate, heparin, heparan, SLe _x , mannose, Examples include, but are not limited to, mannose-6-phosphate, Sialyl Lewis X, FGF, VFGF, protein, chondroitin, keratan, dermatan, albumin, integrin, branched oligosaccharide, peptide, and the like. Methods for attaching various modifying groups to the saccharide moiety are readily available to those skilled in the art ("POLY (ETHYLENE GLYCOL CHEMISTRY: BIOTECHNICAL AND BIOMODICAL APPLICATIONS", edited by J. Milton Harris). Plenum Pub. Corp., 1992; “POLY (ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS”, edited by J. Milton Harris (ACS No. 6 Symposium Series (ACS Symos 80). , American Chemical Society, 1997; -Hermanson, “BIOCONJUGATE TECHNIQUES”, Academic Press, San Diego, 1996; and Dunn et al. 469, American Chemical Society, Washington, Washington, 1991).

Linker group (crosslinking group)
Preparation of modified sugars for use in the methods of the invention includes attachment of a PEG moiety to a sugar residue, and formation of a stable adduct, preferably a substrate for a glycosyltransferase. . Therefore, it is often preferred to use a linker (eg, one formed by the reaction of PEG and a sugar moiety using a cross-linking agent to link PEG and sugar). Exemplary 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), polyamide, polyether, polyester, and the like. General techniques for linking carbohydrates to other molecules are known in the literature. For example, 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 discussion that follows, reactive groups are treated as benign on the sugar moiety of the nascent modified sugar. The focus of the discussion is to clarify specific examples. Those skilled in the art will appreciate that this discussion also relates to reactive groups on the modifying group.

  Various reagents are used to modify the modified sugar components using intramolecular chemical cross-linking (for review of cross-linking reagents and cross-linking procedures, see: Waldo, F. (Wold, F.), “Meth. Enzynol.” 25: 623-651, 1972; Wetal, H. H. (Weetall, H. H.) and Cooney, D. A. (Cooney, D.). A.) “ENZYMES AS DRUGS.” (Edited by Holcenberg and Roberts) pages 395-442, Wiley, New York, 1981; Ji, TH (Ji, T H.), “Meth. Enzymol.” 91: 580-609, 1983; ) Et al., “Mol. Biol. Rep.” 17: 167-183, 1993, all of which are incorporated herein by reference. Preferred cross-linking reagents include various zero-lengths, Obtained from homobifunctional and heterobifunctional cross-linking reagents Zero-length cross-linking reagents involve the direct attachment of two endogenous chemical groups without the introduction of external materials. Agents that catalyze are in this category.Another example is the induction of the condensation of a carboxyl group with a first amino group, carbodiimide, ethyl chloroformate, Woodward's reagent K ( 2-ethyl-5-phenylisoxazolium-3′-sulfonic acid), and amide bonds such as carbonyldiimidazole In addition to these reagents, the enzyme transglutaminase (Glutamyl-Peptide γ-Glutamyltransferase, EC 2.3.2.13) can also be used as a zero-length cross-linking reagent. Catalyzes acyl transfer reactions at the carboxamide group of protein-bound glutaminyl residues using an amino group as a substrate, each of which preferred homo- and hetero-bifunctional reagents are either two identical sites or two different Contain moieties, which can be reactive with amino, sulfhydryl, guanidino, indole, or non-specific groups.

Purification of Factor IX conjugates The product produced by the above process can be used without purification. However, it is usually preferred to recover the product. Standard, well-known techniques for the recovery of glycosylated saccharides such as thin or thick layer chromatography, column chromatography, ion exchange chromatography, or membrane filtration can be used. One or more column chromatography techniques for recovery using membrane filtration, more preferably a reverse osmotic membrane, or as described in the references cited below and cited herein. It is preferable to use it. For example, membrane filtration having a molecular weight fraction of about 3000 to about 10,000 can be used to remove proteins such as glycosyltransferases. Thereafter, nanofiltration or reverse osmosis can be used to remove salts and / or purify the product saccharide. (See, for example, WO 98/15581 pamphlet). Nanofilter membranes are a class of reverse osmosis membranes that allow monovalent salts to pass through, but retain polyvalent salts greater than about 100 to about 2,000 daltons and uncharged solutes, depending on the membrane used. It is. Thus, in typical applications, sugars prepared by the method of the present invention will be retained in the membrane and impure salts will pass through.

  As a first step, if a modified glycoprotein is produced intracellularly, microparticle fragments (either host cells or lysed fragments) are removed, eg, by centrifugation or ultrafiltration, and optionally The protein can then be concentrated using a commercially available protein concentration filter followed by separation of the polypeptide variant from other impurities by one or more steps selected from: : Immunoaffinity chromatography, ion exchange column fractionation (eg on diethylaminoethyl (DEAE) or on a matrix containing carboxymethyl or sulfopropyl groups), Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-S Pharose, WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl, or Protein A Sepharose chromatography, SDS-PAGE chromatography, silica chromatography, silica chromatography, For example, silica gel to which aliphatic groups have been added), eg, gel filtration using Sephadex molecular sieves, or size exclusion chromatography, chromatography on columns that selectively bind polypeptides, and ethanol or ammonium sulfate precipitation.

  Modified glycopeptides produced during culture are typically first extracted from cells, enzymes, etc., followed by one or more enrichment, salting-out, aqueous ion exchange, or size exclusion chromatography steps. Isolated by Furthermore, the modified glycoprotein can be purified by affinity chromatography. Finally, HPLC may be used for the final purification step.

  To inhibit protein hydrolysis, a protease inhibitor, such as methylsulfonyl fluoride (PMSF), may be included in any of the foregoing steps, and antibiotics may be used to prevent accidental contaminant growth. May be included.

  Within another embodiment, the supernatant from the modified glycopeptide producing system of the present invention is obtained from commercially available protein concentration filters such as Amicon or Millipore Pellicon It is first concentrated using a filtration unit. After the concentration step, the concentrate can be applied to a suitable purification matrix. For example, a suitable affinity matrix can include a ligand for a peptide, lectin, or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin such as a matrix or substrate having pendant DEAE groups may be used. Suitable matrices include acrylamide, agarose, dextran, cellulose, or other types commonly used for protein purification. Alternatively, a cation exchange step may be used. Suitable cation exchangers include a variety of insoluble matrices containing sulfopropyl or carboxymethyl groups. A sulfopropyl group is particularly preferred.

  Finally, to further purify the polypeptide variant composition, using one or more RP-HPLC steps using hydrophobic RP-HPLC media (eg, silica gel with pendant methyl or other aliphatic groups). Also good. Some or all of the foregoing purification steps can be used in various combinations to provide a uniform modified glycoprotein.

  The modified glycopeptides of the present invention obtained from large scale fermentations are purified by a method similar to that disclosed by Urdal et al., “J. Chromatog.” 296: 171 (1984)). Also good. This reference describes two sequential RP-HPLC steps for the purification of recombinant human IL-2 on a preparative HPLC column. Alternatively, techniques such as affinity chromatography may be used to purify the modified glycoprotein.

Pharmaceutical Composition In another aspect, the present invention provides a pharmaceutical composition. The pharmaceutical composition includes a pharmaceutically acceptable diluent and a covalent conjugate of a non-naturally occurring PEG moiety, therapeutic moiety, or biomolecule with a glycosylated or non-glycosylated Factor IX peptide. . The polymer, therapeutic moiety, or biomolecule is through a complete glycosyl linking group (located between the peptide and the polymer, therapeutic moiety, or biomolecule and covalently linked to both). Bound to the peptide.

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

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

  In general, the pharmaceutical composition is administered parenterally, for example, intravenously. Accordingly, the present invention provides a composition for parenteral administration comprising a compound dissolved or suspended in an acceptable carrier, preferably an aqueous carrier (eg, water, buffered water, saline, PBS, etc.). provide. The composition can contain pharmaceutically acceptable auxiliary substances (pH adjusters and buffers, isotonic agents, wetting agents, surfactants, etc.) required for approximate physiological conditions.

  These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation will generally be between 3 and 11, preferably 5 to 9, most preferably 7 to 8.

  In certain embodiments, glycopeptides of the invention can be incorporated into liposomes formed from standard vesicle-forming lipids. To prepare liposomes, see, for example, Szoka et al., “Ann. Rev. Biophys. Bioeng.” 9: 467 (1980), US Pat. No. 4,235,871, US Pat. No. 4,501. , 728, and 4,837,028, various methods are available. Targeting liposomes using various targeting agents (eg, sialylgalactosides of the present invention) is well known in the art (eg, US Pat. Nos. 4,957,773 and 4,603,044). See the specification).

  Standard methods for attachment of targeting agents to liposomes can be used. These methods are usually activated for the attachment of targeting agents, or of lipid components such as phosphatidylethanolamine, which can derivatize lipophilic compounds such as glycopeptides derivatized with lipids of the present invention. Use incorporation into liposomes.

  The targeting mechanism usually requires that the targeting agent be placed on the surface of the liposome so that the targeting moiety is available for interaction with the target (eg, a cell surface acceptor). The carbohydrates of the present invention are attached to lipid molecules and then present on the carbohydrate in a manner known to those skilled in the art (eg, using long chain alkyl halides or using fatty acids, respectively). It can be formed using alkylation or acylation of hydroxyl groups). Alternatively, liposomes can be shaped in such a way that at the same time the membrane is formed, the connector portion is first incorporated into the membrane. The connector portion must have a lipophilic portion that is securely embedded and secured to the membrane. The connector portion must also have a reactive portion that is chemically available on the aqueous surface of the liposome. The reactive moiety is selected to be chemically suitable to form a stable chemical bond with the targeting agent or carbohydrate (which is added later). In certain cases, it is possible to attach the target agent directly to the connector molecule, but in most cases it acts as a chemical bridge, thereby causing the connector molecule to be in the membrane with the target agent, Alternatively, it is more appropriate to use a third molecule for linking carbohydrates that are extended three-dimensionally from the vesicle surface.

The compounds prepared by the methods of the present invention have also found use as diagnostic reagents. For example, a labeled compound can be used for positioning areas of inflammation or tumor metastasis in patients suspected of having inflammation. For this use, the compound can be labeled with ¹²⁵ I, ¹⁴ C, or tritium.

The active ingredients used in the pharmaceutical composition of the present invention are GlycoPEGylated Factor IX and its derivatives with the biological properties of participating in the blood coagulation cascade. The liposome dispersions of the present invention are useful for parenteral formulations in treating coagulation abnormalities characterized by reduced or defective clotting, such as various forms of hemophilia. Preferably, the Factor IX composition of the invention is administered parenterally (eg, IV, IM, SC, or IP). Effective dosages are expected to vary considerably depending on the condition being treated and the route of administration, but are expected to be in the range of about 0.1 to 000 μg of active substance per kg body weight. A preferred dose for the treatment of coagulopathy is about 50 to about 3000 μg / kg three times per week. Preferably, from about 500 to about 2000 μg / kg three times per week. More preferably, from about 750 to about 1500 μg / kg, 3 times per week, and preferably about 1000 μg / kg, 3 times per week. The present invention therefore provides a Factor IX residence time is improved in in vivo, when the composition of the present invention is administered, the predetermined dosage Ru is reduced optionally.

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

Example 1
Preparation of UDP-GalNAc-6′-CHO UDP-GalNAc (200 mg, 0.30 mmole) was dissolved in 1 mM CuSO ₄ solution (20 ml) and 25 mM NaH ₂ PO ₄ solution (pH 6.0; 20 mL). Galactose oxidase (240 U; 240 μL) and catalase (13000 U; 130 μL) were then added and the reaction system with balloons was charged with oxygen and stirred for 7 days at room temperature. The reaction mixture was then filtered (spin cartridge; MWCO 5K) and the filtrate (about 40 ml) was stored at 4 ° C. until needed. TLC (silica; EtOH / water (7/2); R _f = 0.77; visualized with anisaldehyde dye).

Example 2
Preparation of UDP-GalNAc-6′-NH ₂ A solution of ammonium acetate (15 mg, 0.194 mmole) and NaBH ₃ CN (1M in THF; 0.17 mL (0.17 mmole)) was added at 0 ° C. (2 ml or about 20 mg). ) From UDP-GalNAc-6′-CHO and warmed to room temperature overnight. The reaction was filtered through a G-10 column with water and the collected product. Appropriate fractions were lyophilized and stored frozen. TLC (silica; ethanol / water (7/2); R _f = 0.72; visualized using ninhydrin reagent).

Example 3
_{UDP-GalNAc-6-NHCO (} CH 2) 2 -O-PEG-OMe Preparation of (1 kDa) galactosaminyl-1-phosphate _{-2-NHCO (CH 2) 2} -O-PEG-OMe (1kDa) (58mg , 0.045 mmole) was dissolved in DMF (6 mL) and pyridine (1.2 mL). UMP-morpholide (60 mg, 0.15 mmole) was then 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, step gradient between 10 and 80%, methanol / water). The desired fractions were collected and dried in vacuo to yield 50 mg of white solid (70%). TLC (silica, propanol / H ₂ O / NH ₄ OH, (30/20/2), R _f = 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 a powder) was added to a solution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous methanol (20 mL) under argon. added. The mixture was stirred for 30 minutes at room temperature, after which mPEG-O-tosylate (Ts; 1.0 g, 0.05 mmol) with a molecular weight of 20 kilodalton was added in several portions over 2 hours. The mixture was stirred for 5 days at room temperature and concentrated by rotary evaporation. The residue was diluted with water (30 mL) and stirred for 2 hours at room temperature to destroy some excess 20 kilodalton mPEG-O-tosylate. The solution was then neutralized with acetic acid and the pH was adjusted to pH 5.0 and loaded onto a reverse phase chromatography (C-18 silica) column. The column is eluted using a methanol / water gradient (the product elutes at approximately 70% methanol) and product elution is monitored by evaporative light scattering, collecting the appropriate fractions and collecting with water (500 ml). Diluted. This solution was color layer analyzed (ion exchange, XK 50 Q, Big Beads, 300 mL, hydroxide form; water to water / acetic acid-0.75N gradient) and the pH of the appropriate fractions adjusted. To 6.0 using acetic acid. This solution was then captured on a reverse phase column (C-18 silica) and eluted with a methanol / water gradient as described above. The product fractions were pooled, concentrated, redissolved in water and lyophilized to give 453 mg (44%) of a white solid (1). The structural data for the compound were as follows: ¹ H-NMR (500 MHz; D ₂ O) δ 2.83 (t, 2H, O—C—CH ₂ —S), 3.05 ( q, 1H, S-CHH- CHN), 3.18 (q, 1H, (q, 1H, S-CHH-CHN), 3.38 (s, 3H, CH 3 O), 3.7 (t, OCH ₂ CH ₂ O), 3.95 (q, 1H, CHN) The purity of the product was confirmed by SDS PAGE.

4.2 Synthesis of (2) Triethylamine (about 0.5 mL) was added dropwise to a solution of 1 (440 mg, 22 μmol) in anhydrous CH ₂ Cl ₂ (30 ml) until the solution was basic. A solution of 20 kilodaltons of mPEG-Op-nitrophenyl carbonate (660 mg, 33 μmol) and N-hydroxysuccinimide (3.6 mg, 30.8 μmol) in CH ₂ Cl ₂ (20 ml) was divided into several portions at room temperature. And added over 1 hour. The reaction mixture was stirred for 24 hours at room temperature. The solvent was then removed by rotary evaporation, the residue was dissolved in water (100 mL) and the pH was adjusted to 9.5 using 1.0 N NaOH. The basic solution was stirred for 2 hours at room temperature and then neutralized to pH 7.0 with acetic acid. This solution was then loaded onto a reverse phase chromatography (C-18 silica) column. The column is eluted with a gradient of methanol / water (product elutes at about 70% methanol), product elution is monitored by evaporative light scattering, appropriate fractions collected and water (500 mL ). This solution was color layer analyzed (ion exchange, XK 50 Q, Big Beads, 300 mL, hydroxide form; water to water / acetic acid-0.75N gradient) and the pH of the appropriate fractions adjusted. To 6.0 using acetic acid. This solution was then captured on a reverse phase column (C-18 silica) and eluted using a methanol / water gradient as described above. The product fractions were pooled, concentrated, redissolved in water and lyophilized to give 575 mg of a white solid (2) (70%). The structural data for this compound were as follows: ¹ H-NMR (500 MHz; D ₂ O) δ 2.83 (t, 2H, O—C—CH ₂ —S), 2.95 (t , 2H, O—C—CH ₂ —S), 3.12 (q, 1H, S—CHH—CHN), 3.39 (s, 3H CH ₃ O), 3.71 (t, OCH ₂ CH ₂ O). The purity of the product was confirmed by SDS PAGE.

Example 5
_{UDP-GalNAc-6-NHCO (} CH 2) 2 -O-PEG-OMe Preparation of (1 kDa) galactosaminyl-1-phosphate _{-2-NHCO (CH 2) 2} -O-PEG-OMe (1 kilodalton) ( 58 mg, 0.045 mmole) was dissolved in DMF (6 mL) and pyridine (1.2 mL). UMP-morpholidate (60 mg, 0.15 mmole) was then 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, step gradient between 10 to 80%, methanol / water). The desired fractions were collected and dried under reduced pressure to give 50 mg of white solid (70%). 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
GlycoPEGylation of Factor IX produced in CHO cells This example describes the preparation of asialo Factor IX and its sialylation using CMP-sialic acid-PEG.

6.1 r Desialylation of Factor IX A recombinant form of coagulation Factor IX (Factor IX) was generated in CHO cells. 6000 IU r Factor IX was dissolved in a total of 12 mL USP H ₂ O. This solution was transferred to a Centricon Plus 20, PL-10 centrifugal filter with another 6 ml of USP H ₂ O. The solution was concentrated to 2 mL and 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 re-concentrated. Dilution / concentration was repeated 4 times, and the buffer was effectively changed to a final volume of 3.0 mL. Of this solution, 2.9 mL (approximately 29 mg of r Factor IX) was transferred to a small plastic tube to which 530 mU α2-3,6,8-neuraminidase-agarose conjugate was added (Vibrio). cholerae), Calbiochem, 450 μL). The reaction mixture was gently rotated at 32 ° C. for 26.5 hours. The mixture was centrifuged for 2 minutes at 10,000 rpm and the supernatant was collected. Agarose beads (containing neuraminidase) were washed 6 times with 0.5 ml 50 mM Tris HCl pH 7.12, 1 M NaCl, 0.05% NaN ₃ . The pooled washings and supernatants were centrifuged again at 10,000 rpm for 2 minutes to remove any remaining agarose resin. The pooled, desialylated protein solution was diluted to 19 mL with the same buffer and concentrated to about 2 mL with a Centricon Plus 20 PL-10 centrifugal filter. This 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 r Factor IX solution was diluted with Tris buffer to a final volume of 3 mL (approximately 10 mg / ml). Native and desialylated r Factor IX samples were analyzed by IEF-electrophoresis. Isoelectric Focusing Gels (pH 3-7) were performed using 1.5 μL (15 μg) sample, first diluted with 10 μL Tris buffer and mixed with 12 μL sample loading buffer. . The gel was loaded, run and fixed using standard procedures. The gel was colored with Colloidal Blue Stain (FIG. 154) and showed a band for desialylated Factor IX.

Example 7
Preparation of PEG (1 kDa and 10 kDa) -SA-Factor IX Desialylated r Factor IX (29 mg, 3 ml) was sampled in two 15 ml centrifuge tubes in two 1.5 ml (14.5 mg) samples. Divided into. Each solution was diluted with 12.67 ml 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 tube was gently inverted to mix and 2.9 U ST3Gal3 (326 μL) was added (total volume 14.5 ml). The tube was turned over again and gently rotated at 32 ° C. for 65 hours. The reaction was interrupted by freezing at −20 ° C. A 10 μg sample of the reaction was analyzed by SDS-PAGE. PEGylated protein was transferred to a Toso Haas Biosep G3000SW (21.5 × 30 cm, 13 um) using Dulbecco's Phosphate Buffered Saline (pH 7.1 (Gibco)), 6 mL / min. ) Purified on an HPLC column. Reaction and purification were monitored using SDS pages and isoelectric focusing gels. Novex Tris-Glycine 4-20% 1 mm gel is diluted with 2 μL of 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.05% NaN ₃ buffer, and 12 μL sample loading buffer And mixed with 1 μL 0.5 M DTT and loaded with 10 μL (10 μg) of sample and heated at 85 ° C. for 6 minutes. Gels were colored with colloidal blue stain (Figure 155) and showed bands for PEG (1 kDa and 10 kDa) -SA-factor IX.

Example 8
Direct Sialyl-GlycoPEGylation of Factor IX This example describes the preparation of sialyl-PEGylation of Factor IX without conventional sialidase treatment.

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

Example 9
Factor IX sialyl-PEGylation using CMP-SA-PEG (20 kDa) 5 ml of 20 mM histidine, 520 mM glycine, 2% Dissolved in sucrose, 0.05% NaN ₃ and 0.01% polysorbate 80, pH 5.0. Thereafter, CMP-SA-PEG- (20 kDa) (50 mg, 2.3 μmol) was dissolved in this solution and CST-II was added. After gently mixing at 32 ° C. for 42 hours, the reaction was terminated. The reaction was analyzed by SDS-PAGE as described by Invitrogen.

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

Example 10
Sialic acid covering GlycoPEGylated Factor IX This example describes a procedure for sialic acid coating of sialyl-GlycoPEGylated peptides. Here, Factor IX is a typical peptide.

10.1 Sialic acid coating 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 added to Centricon ( Concentrated on a Plus 20 PL-10 (Millipore Corp., Bedford, Mass.) Centrifugal filter and the buffer was 50 mM Tris HCl pH 7.2, 0.15 M. Change to NaCl, 0.05% NaN ₃ to a final volume of 1.85 mL. The protein solution was diluted with 372 μL of the same Tris buffer and 7.4 mg of CMP-SA (12 μmol) was added as a solid. The solution was gently inverted to mix and 0.1 U ST3Gal1 and 0.1 U ST3Gal3 were added. The reaction mixture was gently rotated at 32 ° C. for 42 hours.

  A 10 μg sample of the reaction was analyzed by SDS-PAGE. Novex Tris-Glycine 4-12% 1 mm gels were run and stained using colloidal blue as described by Invitrogen. Briefly, 10 μL (10 μg) of sample was mixed with 12 μL sample loading buffer and 1 μL 0.5M DTT and heated at 85 ° C. for 6 minutes (FIG. 156, lane 4).

Example 11
Pharmacokinetic Studies of GlycoPEGylated Factor IX Four GlycoPEGylated FIX variants (PEG-9 variants) were tested in normal mouse pharmacokinetic studies. The activity of the four compounds has already been established in vitro by blood clots, endogenous thrombin potential (ETP) and thromboelastograph (TEG) assays. The activity results are summarized in Table I.

  A pharmacokinetic study was designed and conducted to evaluate the prolongation of activity of the four PEG-9 compounds in the circulation. Nonhemophilic mice were used in 2 animals per time point and 3 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. The blood samples were centrifuged and stored in two aliquots (one for clot analysis and one for ELISA). Due to material limitations, PEG-9 was administered in different amounts: BeneFIX 250 U / kg; 2K (low substitution: “LS” (1-2 PEG substitution 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 measured clotting assay units.

  These results are outlined in FIG. 6 and Table II.

  The results demonstrate a trend towards extension for all PEG-9 compounds. AUC and Cmax values were not directly compared. However, clearance (CL) is compared and CL is lower for the PEG-9 compound compared to BeneFIX, suggesting a longer residence time in the mouse. The time for the last detectable clot activity is increased with the PEG-9 compound compared to BeneFIX, even if BeneFIX is administered at the highest dose.

Example 12
Preparation of LS and HSGlycoPEGylated Factor IX Low degree of substitution with PEG, GlycoPEGylated Factor IX was prepared from native Factor IX by an exchange reaction catalyzed by ST3Gal-III. The reaction was performed in a buffer of 10 mM histidine, 260 mM glycine, 1% sucrose, and 0.02% Tween 80 (pH 7.2). For PEGylation using CMPSA-PEG (2 kD and 10 kD), factor IX (0.5 mg / mL) was mixed with ST3GalIII (50 mU / mL) and CMP-SA-PEG (0.5 mM) at 32 ° C. for 16 hours. And kept warm. For PEGylation using CMP-SA-PEG 30 kD, the Factor IX concentration was increased to 1.0 mg / mL and the CMP-SA-PEG concentration was reduced to 0.17 mM. Under these conditions, over 90% of the Factor IX molecule was replaced with at least one PEG moiety.

GlycoPEGylated Factor IX highly substituted with PEG was prepared by enzymatic desialylation of native Factor IX. Factor IX peptide was buffer exchanged to 50 mM mES (pH 6.0) using a PD10 column, adjusted to a 0.66 mg / ml concentrate, and AUS sialidase (5 mU / mL for 16 hours at 32 ° C. ) Is processed. Desialylation was verified by SDS-PAGE, HPLC, and MALDI glycan analysis. Asialo Factor IX was purified on Q Sepharose FF to remove sialidase. The CaCl ₂ fraction was concentrated using an Ultra 15 concentrator and buffer exchanged to MES, pH 6.0 using a PD10 column.

  Incubation with ST3Gal-III (50 mU / mL) and CMP-SA-PEG (0.5 mM) for 16 hours at 32 ° C. resulted in 2 kD and 10 kD PEGylation of asialo factor IX (0.5 mg / ml). Carried out. For PEGylation using CMPSA-PEG-30 kD, the Factor IX concentration was increased to 1.0 mg / mL and the CMP-SA-PEG concentration was reduced to 0.17 mM. After 16 hours of PEGylation, glycans with terminal galactose were covered with sialic acid by adding 1 mM CMP-SA and continuing an additional 8 hours of incubation at 32 ° C. Under these conditions, over 90% of the Factor IX molecule was replaced with at least one PEG moiety. Factor IX produced by this method has a higher apparent molecular weight in SDS-PAGE.

Example 13
Preparation of O-GlycoPEGylated Factor IX By incubating GalNAcT-II (25 mU / mL) and 1 mM UDP-GalNAc and peptide at 32 ° C., the O-glycan chain was denatured into the newly native factor IX ( 1 mg / ml). After 4 hours of incubation, PEGylation reaction was initiated by adding CMPSA-PEG (0.5 mM 2Kd or 10Kd or 0.17 mM 30 kDd) and ST6GalNAc-I (25 mU / mL) and incubating for another 20 hours did.

  The examples and embodiments described herein are for illustrative purposes only, and various modifications or alterations in view thereof are suggested to those skilled in the art, and the present application and attached patents. It will be understood that it will fall within the spirit and scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Description of drawings
FIG. 4 is the structure of Factor IX showing the presence and location of potential glycosylation sites at Asn 157, Asn 167, Ser 53, Ser 61, Thr 159, Thr 169, and Thr 172. 1 is a scheme illustrating an exemplary embodiment of the present invention where carbohydrate residues on a Factor IX peptide are remodeled and GlycoPEGylated: (A) the galactose residue obtained by removing the sialic acid moiety by sialidase Is GlycoPEGylated using the sialic acid derivative of FIG. 5; (B) the mannose residue is GlycoPEGylated using sialic acid PEG; (C) the sialic acid moiety of the N-glycan is PEGylated (D) The sialic acid moiety of the O-glycan is GlycoPEGylated using sialic acid PEG; (E) Factor IX SDS PAGE gel from 2 (A); ) SDS PAGE gel of Factor IX from the reaction producing 2 (C) and 2 (D). FIG. 6 is a plot comparing the in vivo residual lifetime of non-glycosylated Factor IX and enzymatically GlycoPEGylated Factor IX. 4 is a table comparing the activities of the species shown in FIG. Amino acid sequence of Factor IX. FIG. 5 is a graphical representation of the pharmacokinetic properties of various GlycoPEGylated Factor IX molecules compared to non-PEGylated Factor IX. 1 is a table of representative modified sugar species used in the present invention. 1 is a table of representative modified sugar species used in the present invention. FIG. 5 is a table of sialyltransferases used to transfer modified sialic acid moieties, such as those described herein, and unmodified sialic acid moieties to acceptors.

Claims (11)

Formula (I) :

(I)
(Where
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 selected from linear or branched poly (ethylene glycol) residues;
L is a linker that is a member selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
When D is OH, G is ^R 1-L-, when G is _{-C (O) (C 1 ~C} 6) alkyl, D is Ri ^R 1 -L-NH- der,
LR ¹ is represented by the following formula:

[Wherein a is an integer from 0 to 20]
A Factor IX peptide comprising at least one moiety having,
The moiety represented by the formula (I) binds to a glycosyl residue bound to a residue selected from the Asn residue at position 157, the Asn residue at position 167, and combinations thereof of the Factor IX peptide. A Factor IX peptide . R ¹ is

(Where
e and f are integers independently selected from 1 to 2500,
2. The Factor IX peptide of claim 1 having a structure that is a member selected from q is an integer from 0 to 20. R ¹ is

(Where
e and f are integers independently selected from 1 to 2500)
2. The Factor IX peptide of claim 1 having a structure that is a member selected from. 4. The Factor IX peptide according to any one of claims 1 to 3 , wherein the peptide has the amino acid sequence of SEQ ID NO: 1. A pharmaceutical preparation comprising the factor IX peptide according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier. A blood coagulation stimulant comprising the factor IX peptide according to any one of claims 1 to 4. A therapeutic agent for hemophilia comprising the factor IX peptide according to any one of claims 1 to 4. Formula (I):

(I)
(Where
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 selected from linear or branched poly (ethylene glycol) residues;
L is a linker that is a member selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
When D is OH, G is ^R 1-L-, when G is _{-C (O) (C 1 ~C} 6) alkyl, D is Ri ^R 1 -L-NH- der,
LR ¹ represents the following formula:

[Wherein a is an integer from 0 to 20]
A Factor IX peptide conjugate comprising,
The moiety represented by the formula (I) binds to a glycosyl residue bound to a residue selected from the Asn residue at position 157, the Asn residue at position 167, and combinations thereof of the Factor IX peptide. Wherein the first IX peptide conjugate is
(A) The following formula:

A PEG-sialic acid donor moiety having a substrate , a substrate Factor IX peptide, and an enzyme that transfers the PEG-sialic acid to an amino acid or glycosyl residue of the Factor IX peptide under conditions suitable for transfer A method comprising. R ¹ is:

(Where
e and f are integers independently selected from 1 to 2500,
q is an integer from 0 to 20)
9. The method of claim 8 , having a structure that is a member selected from. R ¹ is:

(Where
e and f are integers independently selected from 1 to 2500)
9. The method of claim 8 , having a structure that is a member selected from. 11. The method according to any one of claims 8 to 10, wherein the Factor IX substrate peptide has the amino acid sequence set forth in SEQ ID NO: 1.

Images