UA89364C2 - Granulocyte colony stimulating factor peptide conjugate (g-csf) - Google Patents

Abstract

The invention relates to a Granulocyte Colony Stimulating Factor peptide conjugate comprising the moiety, said moiety comprising a PEG residue:whereinD is a member selected from -OH and R-L-HN-; G is a member selected from R-L- and -C(O)(C-C)alkyl;Ris a moiety comprising a member selected from a moiety comprising a straight-chain or branched polyethylene glycol residue; and L is a linker which is a member selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, such that when D is OH, G is R-L-, and when G is -(O)(C-C)alkyl, D is R-L-NH-.The invention further relates to a method of making a G-CSF peptide conjugate, a method of stimulating inflammatory leukocyte production in a mammal, a method of treating infection in a patient, a pharmaceutical formulation comprising the G-CSF.

Description

This application claims priority to prior US patent application Moeb60/526796, filed December 2003; preliminary US patent application Moeb0/555813, filed March 23, 2004; preliminary US patent application Meb0/570282, filed May 11, 2004; preliminary US patent application No. 60/539387, filed on January 26, 2004; preliminary US patent application Mob0/592744, filed July 29, 2004; U.S. Provisional Patent Application No. 614518, filed September 29, 2004, and U.S. Provisional Patent Application No. 623387, filed October 29, 2004, each of which is hereby incorporated by reference in its entirety and for all purposes of the invention.

Granulocyte colony-stimulating factor (020-C5E) is a glycoprotein that stimulates the survival, proliferation, differentiation and performance of neutrophil progenitor cells - granulocytes and mature neutrophils. For clinical purposes, two forms of recombinant human a-C5E are used, which are strong stimulators of neutrophil granulopoiesis and have demonstrated effectiveness in preventing infectious complications in some neutropenias. They can be used to accelerate the recovery of neutrophils after treatment with myelosuppressants. 2-C5E helps to reduce the severity of complications caused by anticancer chemotherapy by reducing the incidence of febrile neutropenia and the severity of complications caused by high-dose chemotherapy administered in combination with bone marrow transplantation, and also helps to reduce the incidence and duration of infectious diseases in patients with severe chronic neutropenia. Recently, it has been demonstrated that 2-C5E has a therapeutic effect when administered after the onset of myocardial infarction.

The human form of 0-C5E was cloned by researchers in Japan and the United States in 1986 (see, for example, Madaiia et al.,

Mashte 319: 415-418, 1986). Natural human glycoprotein exists in two forms: one of them consists of 175 amino acids, and the other - of 178 amino acids. The preferred and more active form, consisting of 3,175 amino acids, was used in the production of pharmaceutical products by the recombinant DNA method.

Recombinant human 1-C5E, synthesized in the expression system of E. soy, was called filgrastim.

The structure of filgrastim is slightly different from the structure of the natural glycoprotein. Another form of recombinant human (3-C5E) is called lenograstim and was synthesized in Chinese hamster ovary (CHO) cells. pa-C5E is a monomeric protein that dimerizes the 2-C5E receptor by forming a 2:2 complex of 2 molecules (2 -C5E and two receptors (Nogap ei ai., Viospetivighu 35(15): 4886-96 (1996)).In studies conducted with the help of X-ray crystallography, the following pPO-SOE residues were identified as part of the contact regions that bind to the receptor, namely, such residues as (4, P5, Ab, 57, 58,19, P10, 011, 512,115, K16, E19, 020, 1108, 0109, 0112, T115, T116, 0119, E122, E123 and

I 124 (see, for example, Apyuopti ei ai. (1999) Mashtge 401: 713).

Commercially available forms of gps-C5E have a short-term pharmacological effect, and in most cases, they must be administered more than once a day during the entire course of treatment of leukopenia.

The use of a molecule with a longer half-life in the bloodstream allows to reduce the number of injections required to reduce leukopenia and prevent further possible infections. Another problem associated with the use of g3-COE products that exist at this time is the occurrence of dose-dependent bone pain. Since bone pain experienced by patients is a serious side effect caused by ha-C5E treatment, it would be desirable to obtain a ha-C5E product that does not cause bone pain, or to use a product that by its nature does not have such an effect or is effective in sufficiently small doses that do not cause pain in the bones. Thus, the need to improve the properties of recombinant molecules (3-C5E is absolutely obvious.

In the literature, it was reported about obtaining pS-C5E variants constructed on the basis of the protein (patents

USA Memo 5581476, 5214132, 5362853, 4904584 and Vivdnaag-Oizop ei a!., Biospetivigo 35: 9034-9041, 1996). Modification of pO-COE and other polypeptides was also described, which consists in the introduction of at least one additional carbohydrate chain compared to the natural polypeptide (US patent Me5218092). In addition, polymeric modifications of natural P2-C5E have been described and studied, including the addition of PEG groups (see, for example, Zaiake-IeNikamzha et al. (1992) Seiyi BZipisishte apa ERipsiop 17: 157; Vomzhmep et al. (1999)

Echreiptepia! Netaioidodu 27: 425; US patent Mo5824778; US patent Me5824784, UMO 96/11953, MO 95/21629 and UUO 94/20069)).

The addition of synthetic polymers to the peptide backbone in order to improve the pharmacokinetic properties of the therapeutic glycoprotein is known to specialists. A representative polymer that has been conjugated to peptides is polyethylene glycol ("PEG")M. The use of PEG for derivatization of therapeutic peptides to reduce the immunogenicity of these peptides was demonstrated. So, for example, in a patent

USA Me4179337 (Oamiz ei a!.) described non-immunogenic polypeptides, such as enzymes and peptide hormones, attached to polyethylene glycol (PEG) or to polypropylene glycol. An increase in the size of the PEG-conjugate of the considered polypeptides, in addition to a decrease in immunogenicity, also leads to an increase in the time of removal from the bloodstream.

The main method of attachment of PEG and its derivatives to peptides is nonspecific binding with the help of an amino acid residue of the peptide (see, for example, US patent Me4088538, US patent

Mo4496689, US patent Mo4414147, US patent Me4055635 and PCT UMO 87/00056). Another way to join

PEG to peptides is a non-specific oxidation of glycosyl residues on the glycopeptide (see, for example, UUO 94/05332).

These nonspecific methods involve the randomized, nonspecific attachment of polyethylene glycol to reactive residues on the peptide backbone. It is obvious that the random joining of PEG molecules has its drawbacks, including the lack of homogeneity of the final product and a possible decrease in the biological or enzymatic activity of such a peptide. Therefore, for the production of therapeutic peptides, the best strategy is derivatization, which leads to the formation of a specifically labeled essentially homogeneous product that is easily characterized. And such methods were developed.

Specifically labeled homogeneous peptides with therapeutic properties can be produced by enzymes. Unlike standard non-specific methods of attaching a synthetic polymer or other label to a peptide, enzymatic synthesis has the advantage of being regioselective and stereoselective. The two main classes of enzymes used in the synthesis of labeled peptides are glycosyltransferases (eg, sialyltransferases, oligosaccharyltransferases, M-acetylglucosaminyltransferases) and glycosidases. These enzymes can be used to specifically attach sugars that can then be modified to contain a therapeutic moiety.

Alternatively, glycosyltransferases and modified glycosidases can be used to directly transfer modified sugars to the peptide backbone (see, e.g., US Patent Mob399336 and US Patent Application Publications 20030040037, 20040132640, 20040137557, 20040126838, and 200406014, each of which is incorporated herein by reference). link). Methods of combining elements obtained by chemical and enzymatic synthesis are also known to those skilled in the art (see, for example, the work of Matatoyo et al. Sagropuag. Nev. 305: 415-422 (1998) and the publication of US patent application 20040137557, which are incorporated into this description by link).

To solve the problem associated with the need to create a more effective therapeutic (3-

C5E, this invention was aimed at obtaining glycopegylated C-C5E, which is therapeutically active and has improved pharmacokinetic parameters and properties in comparison with identical or closely related analogues of the non-glycopegylated peptide (3-C5E. In addition, this invention relates to a method of highly efficient and industrial synthesis 2-C5E peptides with improved properties.

It was found that regulated modification of granulocyte colony-stimulating factor (2-C5BE) with one or more polyethylene glycol groups allows obtaining new derivatives (3-C5E with improved pharmacokinetic properties compared to the corresponding natural (non-PEGylated) 1-C5E (Fig. 3).

In addition, the pharmacological activity of glycopegylated (3-C5E is approximately the same as the activity of commercially available mono-PEGylated filgrastim (Fig. 4).

In a representative variant of the invention, "glycopegylated" molecules (-C5E according to the invention are obtained by enzyme-mediated formation of a conjugate containing a glycosylated or non-glycosylated peptide (-C5E) and an enzymatically transferable sugar fragment, the structure of which includes a polyethylene glycol residue. The PEG residue is attached to sugar fragment directly (that is, with the help of one group formed as a result of the interaction of two reactive groups) or with the help of a linker, for example, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, etc.

An example of a transferable PEG-sugar structure is shown in Fig.5.

Thus, in one of its aspects, the present invention relates to a conjugate containing a PEG residue, e.g., PEG and a peptide having IP mimo activity similar or nearly similar to that of a known (-

SOR In the conjugate according to the invention, the PEG residue is covalently attached to the peptide using an intact glycosyl linker group. Representative intact glycosyl linker groups are sialic acid molecules derivatized with polyethylene glycol (PEG).

In one of its representative aspects, the present invention relates to the C2-COE peptide, which includes the fragment: it

Ge) o soon no 3 --NM on

In the above formula, O represents -OH or B!-I-NM-. The symbol C stands for B1-I or C(O)(C1-C6)alkyl.

B' represents a group containing a linear or branched polyethylene glycol residue, and I represents a linker selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. In general terms, if O is OH, then C is B!-I, and if C is -C(O)(C1-C1)alkyl, then

O represents В!-И-МН-. In the modified structures of sialic acid described in this application, COOH is also an SOC and/or its salt.

In another aspect, the present invention relates to a method of obtaining PEGylated C2-C5E containing the fragment described above. The method according to the invention includes (a) contacting the peptide substrate (-

C5E with a donor fragment "PEG-sialic acid" and with an enzyme that transfers PEG-sialic acid to an amino acid or glycosyl residue (3-C5E under conditions suitable for such transfer.

A representative donor fragment "PEG-sialic acid" has the formula: it (v

NN no o 7 n tr on that: Ge) hu o on v; Same thing

ZU chn;

In one of the variants of the invention, the specified host is a mammalian cell. In other embodiments of the invention, the host cell is an insect, plant, bacterial, or fungal cell.

The pharmacokinetic properties of the compounds according to the invention can be easily modified by changing the structure, number or positions of the glycosylation site(s) of the peptide. Thus, the scope of this invention includes a method of adding one or more mutations that introduce an O- or M-linked glycosylation site into the peptide (3-C5BE, and which are absent in the wild-type molecule. Antibodies against these mutants and their glycosylated final products and intermediate compounds are also included in the scope of this invention.

In another aspect, the present invention relates to a (3-C5BE-conjugate containing a set of PEG residues, for example, PEG covalently linked to (3-C5E by means of an intact glycosyl linker group. In the conjugate according to invention, in fact, each member of this set is connected by means of a glycosyl linker group to a glycosyl residue of the peptide, and each glycosyl residue has an identical structure.

In its representative version, the present invention relates to a (3-C5E-conjugate containing a set of PEG residues, for example, PEG covalently linked to 3-C5E by means of an intact glycosyl linker group. In the conjugate according to the invention , in fact, each member of this set is bound to an amino acid residue of the peptide, and each amino acid residue to which this polymer is attached has an identical structure. Thus, for example, if one of the peptides includes a glycosyl residue bound to Tpg, then at least approximately 7095, 8095, 9095, 9590, 9790, 99905, 99.2905, 99.490, 99.695, or, more preferably, 99.895 peptides in a given set will have the same number of glycosyl residues covalently linked to the same TNg residue.

The above discussion applies equally to O-glycosylation and M-glycosylation sites.

This invention also relates to a pharmaceutical composition. Such a composition includes a pharmaceutically acceptable carrier and a covalent conjugate containing an unnatural PEG residue and a glycosylated or non-glycosylated 2-COE peptide.

Other objects and advantages of the present invention will be apparent from the following detailed description of the invention.

Description of graphic material

Figure 1 shows the structure of C-C5BE, which illustrates the presence and localization of potential glycosylation in the TNg 133 residue (TNg 134, if methionine is present).

Figure 2 shows a diagram illustrating a representative variant of the present invention, in which the carbohydrate residue on peptide 5 is remodeled by enzymatic addition of the Ca!Mas group to the glycosidic residue in TIPg 133 (Tpg 134, if methionine is present) followed by the addition of a PEG-derivatized sugar fragment.

Fig. 3 presents graphs illustrating the comparison of the residence time of IP past non-glycosylated (-

C5E, Meshavatm and enzymatically glycopegylated (3-C5P.

Figure 4 shows graphs illustrating a comparison of the activity of various molecules shown in Figure 3.

Figure 5 shows a synthesis scheme for the production of a representative precursor "PEG-glycosylated linker group" (modified sugar) according to the invention in order to obtain conjugates according to the invention.

Figure 6 shows representative amino acid sequences of Y-COE. 5EO IUO MO:1 is a variant consisting of 175 amino acids, where the first amino acid is methionine, and where a threonine residue is present at position 134. 5BEO IUO MO:2 is a variant that contains 174 amino acids and has the same sequence as and a sequence consisting of 175 amino acids, except that it lacks the initiating methionine and therefore starts with a T and the threonine residue is at position 133.

Figure 7 illustrates some representative nucleotides containing a modified sugar used in the practice of this invention.

Figure 8 illustrates other representative modified sugar-containing nucleotides used in the practice of this invention.

Figure 9 shows the production of recombinant C2-CO5E in bacteria grown in different environments and induced by IRTa.

Figure 10 illustrates Western blot analysis of refolded 2-C5E after chromatography on 5pR-sepharose.

Figure 11 shows a table of sialyltransferases that are used to transfer the modified sialic acid and unmodified sialic acid molecules described here to the acceptor.

Abbreviation

PEG - polyethylene glycol; PPO (PPG) - polypropylene glycol; Aha - arabinosyl; Eggs - fructosyl; Rice - fucosyl; Sai - galactoside; Sa!Mas - M-acetylgalactosaminyl; Cis - glucosyl; SISMAs - M-acetylglucosaminyl;

Map - mannosyl; MapAs - mannosamine acetate; Hoo! - xylosyl; MecyAs - sialyl (Mm-acetylneuraminin); MBbR - mannose-b-phosphate; 5ia - sialic acid, M-acetylneuraminil and their derivatives and analogs.

Definition

Unless otherwise defined, all technical and scientific terms used herein have the generally accepted meaning known to those skilled in the art to which the present invention relates. In general terms, the nomenclature used herein and the laboratory procedures used in cell culture, molecular genetics, organic and nucleic acid synthesis chemistry, and the hybridization method are well known and described in detail in the literature. Standard methods are used for the synthesis of nucleic acids and peptides. Such methods and procedures are usually carried out in standard ways, described in the literature and in various general guidelines (in general terms, see the guide Zatogook ei ai., MoiIesshag

Siopipd: A I aroghaiogu Mapiai, 24 units. (1989) Soyd turnip Nagbog I aroghaiyugu Rgev5, Soyy brhipd Natbog, MU, which is incorporated herein by reference), and these methods are also described herein.

The nomenclature used here and the laboratory procedures used in analytical chemistry and organic synthesis chemistry and described below are well known and described in detail in the literature. Standard methods or their modifications are used for chemical synthesis and chemical analysis.

All oligosaccharides described here have the names or abbreviations adopted for the non-reducing saccharide (i.e., Cy) and the corresponding configuration of the glycosidic bond (4 or VD), the ring bond (1 or 2), and the position of the reducing saccharide in the ring involved in bond formation (2, C, 4, 6, or 8), as well as the name or abbreviation used for the reducing saccharide (i.e., CisSMas). Each of the saccharides is preferably pyranose. A description of the standard nomenclature used in the biology of glycosylation can be found in the Manual of Epidemiological Studies of Biochemistry, Ed. SONY. Rgezv (1999).

Oligosaccharides are believed to have a reducing end and a non-reducing end, regardless of whether the saccharide at the reducing end is actually a reducing sugar or not. According to conventional nomenclature, the oligosaccharides described here have a non-reducing end on the left and a reducing end on the right.

The term "sialic acid" means any member of the family of carboxylated sugars of nine carbon atoms. The most common member of the sialic acid family is M-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-O-glycero-O-galactononulopyrano-1-anoic acid (often abbreviated

MeibBAs, MetsAs or MAMA). The second member of this family is M-glycolyl-neuraminic acid (MeibOs or

Messs), in which the M-acetyl group of MesAs is hydroxylated. The third member of the family of sialic acids is 2-keto-3-deoxy-nonulosonic acid (KOM) (Madapo ei a/., (1986) y. Vioi. Spet. 261: 11550-11557; Kapatoii ey a. U. Visi. Spet. 265: 21811-21819 (1990)). The scope of this invention also includes 9-substituted sialic acids such as 9-O-C1-Svacyl-Mei5Ac, for example, 9-O-lactyl-Mei5Ac or 9-O-acetyl-Mei5Ac, 9-deoxy-9- fluorine-

Mey5Ac and 9-azido-9-deoxy-Men5Ac. A description of the family of sialic acids can be found, for example, in the publication of Maiki, Siusoriodu 2: 25-40 (1992) 5iaiis Acids: Spetivigu, Meishaboisit apa Ripsiop, A. 5spaceg Ei. (brgipdeg-Mepad, Mem Moik (1992)). The synthesis and use of sialic acid compounds in the sialylation procedure is described in International Application U/O 92/16640, published October 1, 1992.

The term "sialic acid" refers to any member of the family of carboxylated sugars of nine carbon atoms. The most common member of the sialic acid family is M-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-O-glycero-O-galactononulopyrano-1-anoic acid (often abbreviated

MeibBAs, MetsAs or MAMA). The second member of this family is M-glycolyl-neuraminic acid (MeibOs or

Messs), in which the M-acetyl group of MesAs is hydroxylated. The third member of the family of sialic acids is 2-keto-3-deoxy-nonulosonic acid (KOM) (Madapo ei a/., (1986) y. Vioi. Spet. 261: 11550-11557; Kapatoii ey a. U. Visi. Spet. 265: 21811-21819 (1990)). The scope of this invention also includes 9-substituted sialic acids such as 9-O-C1-Svacyl-Meyl5Ac, for example, 9-O-lactyl-Meyl5Ac or 9-O-acetyl-Meyl5Ac, 9-deoxy-9- fluorine-

Mey5Ac and 9-azido-9-deoxy-Men5Ac. A description of the family of sialic acids can be found, for example, in the publication of Maiki, Siusoriodu 2: 25-40 (1992) 5iaiis Acids: Spetivigu, Meishaboisit apa Ripsiop, A. 5spaceg Ei. (Zrgipdeg-Mepad, Mem Moik (1992)). The synthesis and use of sialic acid compounds in the sialylation procedure is described in International Application U/O 92/16640, published October 1, 1992.

The term "granulocyte colony-stimulating factor" or "granulocyte colony-stimulating factor peptide" or "5-C5E" or "2-C5E peptide" refers to any wild-type or mutated peptide, recombinant or native C-C5E, or any - any of its fragments that have activity similar to the activity of native C-C5E or that mimics this activity. Of course, this term also covers non-peptide 2-C5E mimetics. In a representative embodiment of the invention, peptide 2-C5E has the amino acid sequence presented in 5EO I MO: 1. In other representative variants of the invention, peptide (1-C5E has a sequence selected from ZEO IU MO:3-11.

The term "granulocyte colony-stimulating factor activity" means any activity, including, but not limited to, receptor binding and receptor activation, inhibition of receptor binding, or any biochemical or physiological response that normally occurs under the action of a granulocyte wild-type colony-stimulating factor. Granulocyte colony-stimulating factor activity can be induced by any of the granulocyte colony-stimulating factor peptides defined above.

The term "peptide" means a polymer in which the monomers are amino acids and are linked to each other by amide bonds, and which, alternatively, is called a polypeptide. In addition, the term also covers non-natural amino acids such as β-alanine, phenylglycine and homoarginine. Amino acids that are not encoded by genes can also be used in the present invention. In addition, the present invention may also use amino acids that have been modified by incorporating reactive groups, glycosylation sites, polymers, therapeutic molecules, biomolecules, etc. All amino acids used in this invention can be either O-isomers or I-isomers. Of course, the I -isomer is preferred. In addition, other peptidomimetics can also be used in this invention. As used herein, the term "peptide" refers to glycosylated and non-glycosylated peptides. Peptides that are incompletely glycosylated in the system expressing this peptide can also be used. A general overview can be found in b5rayuia

A.R. Spetivigu apa Viospetivigu oi Atipo Asid5, Reriides apa Rgoieipv, V. MU/eipvieip, edv., Magse! Oekkeg, Mem hUogk, p. 267 (1983).

The term "peptide conjugate" means a molecule according to the invention in which a peptide is conjugated to a modified sugar described herein.

The term "amino acid" means natural and synthetic amino acids, as well as analogs and mimetics of amino acids, which have the same functions as natural amino acids. Natural amino acids are amino acids encoded by the genetic code, as well as amino acids that have been subsequently modified, for example, hydroxyproline, y-carboxyglutamine and O-phosphoserine. "Amino acid analogs" means compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., contain an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and a B group, for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium . These analogues have modified B-groups (for example, norleucine) or modified peptide backbones, but, at the same time, retain the basic chemical structure characteristic of the natural amino acid.

The term "amino acid mimetics" means chemical compounds that have a structure different from the general chemical structure of amino acids, but which have a function similar to that of a natural amino acid. As used herein, the term "amino acid", regardless of whether it is present in a linker or in a component of a peptide sequence, means the Y- and I-isomer of an amino acid, as well as mixtures of these two isomers.

As used herein, the term "modified sugar" means a natural or non-natural carbohydrate that is enzymatically attached to an amino acid or to a glycosyl residue of a peptide by a method according to the invention. The modified sugar is selected from a number of enzyme substrates, including, but not limited to, sugar-containing nucleotides (mono-, di-, and triphosphates), activated sugars (eg, glycosyl halides, glycosylmesylates), and sugars that are not activated and are not contained in nucleotides. A "modified sugar" is covalently linked to a functional "modifying" group. Suitable modifying groups include, but are not limited to, PEG molecules, therapeutic molecules, diagnostic molecules, biomolecules, and the like.

The modifying group is preferably not a natural or unmodified carbohydrate. The position of the functional modifying group is chosen so that this group does not interfere with the formation of a "modified sugar" due to the enzymatic addition of this group to the peptide.

The term "water-soluble" refers to molecules that have a determinable degree of solubility in water. Methods of detection and/or estimation of solubility in water are well known to specialists. Representative PEG parts are peptides, saccharides, polyesters, polyamines, polycarboxylic acids, etc. Peptides can have mixed sequences or can consist of a single amino acid, for example polylysine. Similarly, saccharides can have mixed sequences or can consist of a single saccharide subunit, for example, dextran, amylose, chitosan and polysialic acid. An example of polyester is polyethylene glycol.

An example of a polyamine is polyethyleneamine, and a representative polycarboxylic acid is polyacrylic acid.

As used herein, the term "glycosyl linker group" refers to a glycosyl residue to which an agent is covalently attached (eg, a PEG residue, a therapeutic molecule, a biomolecule). In the methods of the invention, a "glycosyl linker group" is covalently attached to a glycosylated or non-glycosylated peptide, thereby linking this agent to an amino acid and/or glycosylic residue of the peptide. A "glycosyl linker group" is usually formed from a "modified sugar" as a result of enzymatic attachment of a "modified sugar" to an amino acid and/or to a glycosyl residue of a given peptide. The term "Intact glycosyl linker group" means a linker group formed from a glycosyl moiety in which individual saccharide monomers, the binding elements of the conjugate, do not decompose, for example, are not oxidized, for example, under the action of sodium metaperiodate. "Intact glycosyl linker groups" according to the invention can be formed from a natural oligosaccharide by adding a glycosyl unit(s) or removing one or more glycosyl units from the initial saccharide structure.

As used herein, the term "targeting molecule" refers to a molecule that can be selectively localized to a specific tissue or region of the body. Such localization is mediated by the specific recognition of molecular determinants, the size of the molecule of the specified targeting agent or conjugate, ionic interactions, hydrophobic interactions, etc. Other mechanisms for delivering an agent to a specific tissue or area are known to those skilled in the art. Examples of targeting molecules are antibodies, antibody fragments, transferrin,

No5-glycoprotein, blood clotting factors, serum proteins, D-glycoprotein, 1-C5E, YAM-C5E, M-C5E, EPO, etc.

As used herein, the term "pharmaceutically acceptable carrier" includes any material that, when combined with a conjugate, retains the activity of the conjugate and does not react with the patient's immune system.

Examples of such carriers include, but are not limited to, any standard pharmaceutical carrier such as phosphate-buffered saline, water, emulsions such as oil-in-water emulsions, and various types of wetting agents. Other carriers can also be sterile solutions, tablets, including coated tablets and capsules. Of course, such carriers contain fillers such as starch, milk, sugar, clays of certain types, gelatin, stearic acid or its salts, magnesium or calcium stearate, talc, vegetable fats or oils, resins, glycols or other known fillers. Such carriers can also be flavorings, dyes or other ingredients. Compositions containing such carriers are prepared by well-known standard methods.

As used herein, the term "administration" refers to oral administration, suppository administration, topical administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal administration, or subcutaneous administration, or implantation in an individual of a sustained release device, e.g. , miniosmotic pump.

Administration can be by any route, including parenteral and transmucosal administration (eg, oral, intranasal, vaginal rectal or transdermal administration).

Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. In addition, if the injection is administered for the purpose of destroying the tumor, for example, for the purpose of inducing apoptosis, then such an injection can be made directly into the tumor and/or into the tissues surrounding the tumor. Other methods of administration include, but are not limited to, the use of liposomal preparations, intravenous infusion, use of transdermal patches, etc.

The term "relief" or "alleviate" means any sign of positive dynamics in the treatment of a pathology or condition, including objective or subjective parameters such as weakening, remission or reduction of disease symptoms, or improvement of the patient's physical or mental condition. Symptom relief may be based on objective or subjective parameters, including the results of an examination of the patient's physical and/or mental status.

The term "therapy" means the "treatment" or "elimination" of a disease or condition, including the prevention of a given disease or condition in an animal that may be predisposed to developing a given disease, but which does not yet have or has not yet shown symptoms of the disease, (prophylactic treatment) , suppression of the development of the disease (slowing down or stopping its development), weakening the symptoms or side effects of the disease (including palliative treatment), and improving the dynamics of the disease (which leads to regression, that is, to the reverse development of the disease).

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

The term "excluded" refers to material that is essentially or substantially free of the components used to produce such material. With respect to the peptide conjugates of the invention, the term "isolated" refers to a material that is substantially or substantially free of components that are normally present with this material in the mixture used to prepare the peptide conjugate. The terms "separated" and "pure" are interchangeable. Isolated peptide conjugates according to the invention usually have a level of purity, which is expressed, preferably, in the form of a range of values. The lower limit of the purity interval for the peptide conjugates is about 6095, about 7995, or about 8095, and the upper limit of the purity interval for the peptide conjugates is about 7095, about 8095, about 90905, or greater than about 9095.

If the peptide conjugates have a purity greater than about 9095, then their purity is also preferably expressed as a range of values. The lower limit of this range of purity values for peptide conjugates is about 9095, about 9295, about 9495, about 9695, or about 9895. The upper limit of the range of purity values for peptide conjugates is about 9295, about 9495, about 9695, about 9895 or approximately 100905.

Purity is determined by well-known analytical methods (for example, by band intensity on a silver-stained gel, polyacrylamide gel electrophoresis, HPLC, or similar methods).

As used herein, the term "substantially each member of the population" describes the properties of a population of peptide conjugates according to the invention in which a plurality of identical acceptor sites on a peptide contain a selected percentage of the modified sugar attached to the peptide. The term "substantially every member of the population" referred to in connection with the "homogeneity" of these sites on a peptide conjugated to a modified sugar refers to conjugates of the invention that are homogeneous by at least about 8095, preferably , by about at least 9095, and more preferably by about at least 9595.

The term "homogeneity" refers to the structural composition of the entire population of acceptor fragments with which modified sugars are conjugated. Thus, for example, a peptide conjugate of the invention in which each modified sugar moiety is conjugated to an acceptor site having the same structure as the acceptor site to which any other modified sugar is conjugated is said to , is homogeneous at approximately 10095. Homogeneity is usually expressed in the form of an interval of values. The lower limit of the homogeneity interval for the peptide conjugates is about 6095, about 7095, or about 8095, and the upper limit of the frequency interval for the peptide conjugates is about 7095, about 8095, about 9095, or greater than about 90905.

If the peptide conjugates have a homogeneity equal to or greater than approximately 90905, then their homogeneity is also preferably expressed in the form of a range of values. The lower limit of the homogeneity interval for the peptide conjugates is about 9095, about 92905, about 9495, about 9695, or about 9895. The upper limit of the homogeneity interval for the peptide conjugates is about 9295, about 9495, about 9695, about 9895, or about 10095. The purity of peptide conjugates is usually determined by one or more methods known to specialists, for example, using liquid chromatography-mass spectrometry (LC-MS), laser desorption time-of-flight mass spectrometry (MAG OITORE), capillary electrophoresis, etc.

The terms "substantially uniform glycosylated form" or "substantially uniform glycosylation pattern" when referring to glycopeptides refer to the percentage of acceptor moieties glycosylated by the glycosyltransferase of interest (eg, fucosyltransferase). So, for example, in the case of a-1,2-fucosyltransferase, in fact, a homogeneous pattern of fucosylation is observed in the case when almost all (as defined below) fragments of Cy!p1,4- are fucosylated in the peptide conjugate according to the invention

SisMas-B and their sialylated analogues. For specialists in this field, it is obvious that the starting material may contain glycosylated acceptor fragments (for example, fucrosylated Ca/p1,4-CysMas-B molecules). Thus, the calculated percentage of glycosylation means the number of acceptor fragments glycosylated by methods according to the invention, as well as the number of acceptor fragments that have already been glycosylated in the starting material.

The word "substantially" included in the above definition of "substantially homogeneous" generally means that the acceptor moieties for a particular glycosyltransferase are glycosylated at least about 4095, at least about 7095, at least about 8095, or preferably at least about 9095, and more more preferably by at least about 9595.

If the substituent groups are defined by their standard chemical formulas written from left to right, then they equally include chemically identical substituents having a structural formula that should be written from right to left, for example, -CHgO- also means -ОСН»-.

The term "alkyl", by itself or forming part of another substituent, unless otherwise specified, means a straight or branched chain radical or a cyclic hydrocarbon radical, or a combination thereof, which may be fully saturated, mono- or polyunsaturated and may include di- and polyvalent radicals having the specified number of carbon atoms (i.e. C1-SiO has from one to ten carbon atoms). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, t-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologues thereof and isomers, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. An unsaturated alkyl group is an alkyl group having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and Z-propynyl, Z-butynyl and higher homologues and isomers.

The term "alkyl", unless otherwise specified, also means derivatives of alkyl, as defined in more detail below, such as "heteroalkyl". Alkyl groups bounded by hydrocarbon groups are called "homoalkyls".

The term "alkylene" by itself or forming part of another substituent, unless otherwise specified,

means a divalent radical derived from an alkane, examples of which include, but are not limited to, -

CsnNsnnesnN»e-, and which, in addition, includes the groups defined below as "heteroalkylene". Of course, an alkyl (or alkylene) group has from 1 to 24 carbon atoms, however, according to the present invention, groups having 10 or less carbon atoms are preferred. "Lower alkyl" or "lower alkylene" is an alkyl or alkylene group with a shorter chain, usually having eight or fewer carbon atoms.

The terms "alkoxy", "alkylamino" or "alkylthio" (or thioalkoxy) are used in their conventional sense and mean alkyl groups attached to another part of the molecule by means of an oxygen atom, an amino group or a sulfur atom, respectively.

The term "heteroalkyl", by itself or in combination with another term, means, unless otherwise specified, a stable radical with a linear or branched chain or a cyclic hydrocarbon radical, or combinations thereof, consisting of the indicated number of carbon atoms and at least one heteroatom , selected from the group consisting of 0, M, 5i and 5, where the nitrogen and sulfur atoms can be, but not necessarily, oxidized, and the nitrogen heteroatom can be, but not necessarily, quaternized. The heteroatom(s) O, M, 5 and 5 can be at any internal position of the heteroalkyl group or at the position in which the alkyl group is attached to another part of the molecule. Examples of such groups are, but are not limited to, -СНа-СН2-О-СН3, -СНа-СНа-МН-СН3, -СН2-СНо-М(СН3)-СН3, -СН2-5-СН3-СН3, - СН2о-СН», -5(0)-СН3, -СНе-

Снг-5(0)2-СН3, -СН-СН-О-СН3, -51(СН3)3, -СНо-СН-М-ОСН3 and -СН-СН-М(СН3)-СН3. Two heteroatoms can be located one behind the other, for example, -СНо-МН-ОсСН3 and -СН2-О-51(СН3)3. Similarly, the term "heteroalkylene", by itself or as part of another substituent, means a divalent radical derived from a heteroalkyl, examples of which include, but are not limited to, -CH2-CHg-5-CH2-CHe- and -"CH »-5-СН2-СНо-МН-СНе-.

In the case of heteroalkylene groups, heteroatoms may also be present at either or both ends of the chain (eg, in alkyleneoxy-, alkylenedioxy-, alkyleneamino-, alkylenediamino groups, etc.). In addition, in the case of alkylene and heteroalkylene linker groups, the orientation of the linker groups does not necessarily correspond to the direction in which the formula of such linker group is written. So, for example, the formula -

C(O)2B! represents a structure that can be written by the formulas -C(O)2B: and -B'E(O) 2-.

The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, mean, unless otherwise specified, the cyclic variants of "alkyl" and "heteroalkyl", respectively. In addition, in the case of heterocycloalkyl, the heteroatom may be in a position in which said heterocycle is attached to another part 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, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran -Z-yl, tetrahydrothien-2-yl, tetrahydrothien-Z-yl, 1-piperazinyl, 2-piperazinyl, etc.

The terms "halogen" or "halogen" by themselves or as part of another substituent mean, unless otherwise specified, an atom of fluorine, chlorine, bromine or iodine. In addition, terms such as "haloalkyl" mean monohaloalkyl and polyhaloalkyl. Thus, for example, the term "halo(C1-Ca)alkyl" includes, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term "aryl" means, unless otherwise specified, a polyunsaturated aromatic substituent, which may be a single ring or a plurality of rings (preferably 1 to 3 rings) that are either fused or covalently bonded to each other. The term "heteroaryl" means aryl groups (or rings) containing from one to four heteroatoms selected from M, O and 5, where the nitrogen and sulfur atoms may, but need not, be oxidized and the nitrogen atom(s) are , but not necessarily quaternized(s).

A heteroaryl group can be attached to another part of the molecule using a heteroatom.

Non-limiting examples of aryl and heteroaryl groups are 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, Z-quinolyl, tetrazolyl, benzo(Hb|furanyl, benzoi|p|hyenyl, 2,3-dihydrobenzo|1,4|dioxin-b-yl, benzo(1, 3|dioxol-b-yl and 6-quinolyl Substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

Briefly, the term "aryl" when used in combination with other terms (eg, aryloxy, arylthiooxy, arylalkyl) includes aryl and heteroaryl rings as defined above. Thus, for example, the term "arylalkyl" means radicals in which an aryl group is attached to an alkyl group (eg, benzyl, phenethyl, pyridylmethyl, etc.), including alkyl groups in which a carbon atom (eg, a methylene group) is substituted, for example, on an oxygen atom (for example, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

Each of the above terms (eg, "alkyl", "heteroalkyl", "aryl" and "heteroaryl") means both substituted and unsubstituted forms of the specified radical. Preferred substituents for each type of radical are described below.

Substituents for alkyl and heteroalkyl radicals (including groups often called alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) have the general generic name "alkyl substituents" and these substituents may be one or more various groups selected from, but not limited to, -Ov", -0, -MA -M-OB", -MA'B", -58", halogen, -5IV'B"B", -OS (ОВ, -С(О)В, -СО2в, -СОМА'В", -ОС(ОМАВ"-,

MAE), -MA-S(OMA", -MA"S(O)2A, -MA-S(MA'VYA")-MA"", -MA-S(MA'YA")-MA" , -5(0)A -50)228, -

З(О)2MA'B", -MABO»B", -SM and -MO", where the number of substituents ranges from O to 2t'-1, and t means the total number of carbon atoms in such a radical. Each of B", B", A" and A" independently and preferably means hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, for example, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkyl groups, or arylalkyl in groups If a compound according to the invention, for example, includes more than one group B, then each group A is independently selected from the groups B", B", A" and A"", if more than one of these groups is present. If A and B" are attached to the same nitrogen atom, then these groups, together with the nitrogen atom, form a 5-, 6- or 7-membered ring. For example, -MA'B" includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the foregoing discussion of substituents, it is apparent that the term "alkyl" refers to groups comprising carbon atoms bonded to groups, which are not hydrogen groups, such as haloalkyl (for example, -СЕ3 and -СНеСЕ3) and acyl (for example, -С(О)СН3, -С(О)СЕ", -

С(О)СНгОСН, etc.).

Similar to the substituents described for the alkyl radical, the substituents for aryl and heteroaryl radicals have the general generic name "aryl substituents". Such substituents are chosen, for example, from halogen, -ОВ!, -0, -MA, -М-ОВ', -МВА'В", -58", -halogen, -5IV'В"В", -ОС(О )В, -С(О)В", -СО»В", -СОМВ'В", -

ОоС(О)МАВ"-, MA'Е()В -MA-С(ОМА'В", -МА"С(О)2А -MA-С(MA'ВЯ") MA", -MA- C(MA'YA")-MA", -5(OA, - (028, -5(0)2МВ'В", -MABO»В", -SM and -MO", -В", -Mz, - "CH(РНЙ)", fluorine (Ci-Ca) alkoxy and fluorine (C1i-Ca) alkyl, where the number of substituents ranges from 0 to the total number of free valences on the aromatic system, where B", B",

A" and A" are preferably and independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

If the compound according to the invention, for example, includes more than one group НА, then each group A is independently selected from the groups B", A", A" and A", if more than one of these groups is present. In the diagrams below, the symbol X represents the "B" defined above.

Two substituents on adjacent aryl or heteroaryl atoms can be, but not necessarily, replaced by a substituent of the formula -Т-С(0)-(САА 4-0), where T and 0 independently represent -МА-, -О, -САА' or a single bond, and 4 is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may, but are not required, be replaced by a substituent of the formula A-(CHg)-B-, dei and B independently represent -СВА'-, -О-, -МВ-, -5-, -5(0)-, -5(0)2-, -5(00)2MA! or a simple bond, and is an integer from 1 to 4. One of the single bonds of the new ring thus formed may, but need not, be replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may, but need not, be necessarily, replaced by the substitute of the formula (SBA)5-X-(B'YAV "a where 5 and a are independently equal to a whole number from 0 to 3, and X represents -O-, -МВ'-, -5-, -5 (0)-, -5(0)2-or

З(О)2МА"-. Substituents PH, B", B" and A", are preferably independently selected from hydrogen or substituted or unsubstituted (C1i-Cv)alkyl.

As used herein, the term "heteroatom" means oxygen (C), nitrogen (M), sulfur (5), and silicon (51).

Introduction

The present invention relates to a method of modifying the glycan structure by 2-С5Р. (3-C5E is well known in the art as a cytokine produced by activated T cells, macrophages, endothelial cells, and stromal fibroblasts. 2-C5BE acts primarily on the bone marrow and increases the production of inflammatory leukocytes, and in addition, this cytokine functions as an endocrine hormone, initiating the replenishment of neutrophils spent in the process of inflammatory reactions.(3-C5E also finds clinical application in bone marrow recovery after chemotherapy.

This invention relates to the conjugate of granulocyte colony-stimulating factor (0-C5NE). The present invention also relates to conjugates of glycosylated and non-glycosylated peptides having granulocyte colony-stimulating activity. Such conjugates can also be modified by additional conjugation with other molecules, such as therapeutic molecules, diagnostic molecules, molecules for targeted delivery, etc.

The present invention also relates to a method of remodeling and/or modification of (3-C5E. (13-C5E is a valuable agent for the treatment of various diseases, but as indicated above, its clinical effectiveness is limited by its relatively poor pharmacokinetics.

In representative embodiments of the invention, the peptide (-SOE) according to the invention can be administered to prevent infection in cancer patients undergoing certain types of radiation therapy, chemotherapy and bone marrow transplantation, in order to mobilize progenitor cells to collect transplanted peripheral blood progenitor cells, for the treatment of severe chronic or relative leukopenia, regardless of the cause, and for the maintenance therapy of patients with acute myeloid leukemia. In addition, the polypeptide conjugate or composition according to the invention can be used for the treatment of AIDS or other immunodeficiency diseases , as well as bacterial infections. a2-C5E has been cloned and sequenced. In a representative embodiment of the invention, 1-C5E has the amino acid sequence represented in 5EO IU MO:1. It is absolutely obvious to any person skilled in the art that the present invention is not limited to the sequences described here , as well as the 2-C5E options discussed above.

Thus, for example, the present invention also covers variants (3-C5E, well known to those skilled in the art. As an example, which does not limit the present invention, the variant 2-C5E can be considered in US patent Mob166183, which describes 2-C5E containing the natural complement of lysine residues and attached to one or two molecules of polyethylene glycol.In addition, US Patent Nos. 6,004,548, 5,580,755, 5,582,823, and 5,676,941 describe a 2-C5E variant in which one or more cysteine residues are at positions 17, 36, 42, 64 and 74 are replaced by alanine or, alternatively, by serine.US Patent Me5416195 describes a C-C5BE molecule in which cysteine at position 17, aspartic acid at position 27, and serines at positions 65 and 66 are replaced by serine, serine, proline, and proline , respectively. Other variants are well known to those skilled in the art, and these variants are described, for example, in US Patent No. 5,399,345. Other variants have an amino acid sequence selected from BEO IU MO:3-11.

The expression and activity of the modified molecule (24-C5E according to the invention can be analyzed by methods well known to those skilled in the art and described, for example, in US patent Me4810643. So, for example, activity can be measured using radiolabeled thymidine absorption assays. Briefly, bone marrow, taken from a healthy person, is cut in a ficol-hippac density gradient (1.077g/ml,

Rpagtasia Rizsaiamzhau, MU), and cells with a low density are suspended in Iskov medium (SIVSO, Ia doiIa,

SA), containing 1095 fetal bovine serum, glutamine and antibiotics. Approximately 2x107 human bone marrow cells are incubated with either control medium or (1-C5E according to the invention in a 96-well flat-bottomed plate at approximately 37"C in an air atmosphere with 595 CO" for approximately 2 days. Then, the cultures for approximately For 4 hours, pulses of 0.5 μCi/well of ZN-thymidine are applied (Mem Epdiapa Mysivag,

Vozyup, Mazz.) and measure the level of absorption, as described, for example, in the work of Mepish ei ai. (1983, Viosa 61: 781). An increase in the level of incorporation of ZH-thymidine into human bone marrow cells, in comparison with bone marrow cells treated with the control compound, is an indicator of the activity and stability of the compound (-SOE.

As described above, conjugates according to the invention are formed by enzymatic addition of a modified sugar to a glycosylated or non-glycosylated 2-C5E peptide. A modified sugar, if it is between the C2-C5E peptide and a modifying group on the sugar, will form a group that may be referred to herein as, for example, an "Intact Glycosyl Linker Group." With the application of the method according to the invention, which involves the use of highly selective enzymes, such as glycosyltransferases, it is possible to obtain peptides bearing the desired group in one or more specific positions. In addition, according to the present invention, the modified sugar is attached directly to the selected site of the peptide chain (1-C5E, or, alternatively, the modified sugar is attached to the carbohydrate part of the glycopeptide. Peptides in which the modified sugars are linked to the carbohydrate of the glycopeptide and are directly linked with the amino acid residue of the peptide backbone (-C5E, are also included in the scope of this invention.

In contrast to the known strategies of chemical and enzymatic synthesis of peptides, methods according to the invention make it possible to obtain peptides and glycopeptides that have, in fact, a homogeneous nature of derivatization; moreover, the enzymes used in the present invention are, in fact, selective for a specific amino acid residue or for a combination of amino acid residues of the C2-C5E peptide. Such methods also allow large-scale production of modified peptides and glycopeptides. Thus, the methods according to the invention can find practical application in the large-scale synthesis of glycopeptides having a preselected homogeneous nature of derivatization. These methods are particularly suitable for modifying therapeutic peptides, including, but not limited to, glycopeptides that are not fully glycosylated during their synthesis in cell cultures (eg, in mammalian, insect, plant, fungal, yeast, or prokaryotic cells) or in transgenic plants or animals.

The present invention also relates to conjugates of glycosylated and non-glycosylated peptides (3-COE) with an increased therapeutic half-life due to, for example, a reduced rate of clearance or a reduced rate of absorption by the immune or reticuloendothelial system (RES). In addition, the methods according to the invention allow to mask antigenic determinants on peptides, leading to a reduction or prevention of the host's immune response against that peptide Selective attachment of targeted delivery agents can also be used to deliver a peptide to a specific tissue or to a cell surface receptor that is specific for a specific targeting agent

Conjugates

In its first aspect, the present invention relates to a conjugate consisting of a selective modifying group and C-C5E peptide.

The linkage between the (-C5E) peptide and the selected moiety includes an intact glycosyl linker group located between the peptide and the selected moiety. As discussed herein, the selected moiety can be essentially any moiety that is capable of attaching to a saccharide moiety, resulting in the formation of a "modified sugar", recognized by the appropriate transferase enzyme, which attaches the modified sugar to the peptide (3-C5E. The saccharide component of the modified sugar, if it is located between the 2-C5E peptide and the selected fragment, becomes the "Intact Glycosyl Linker Group".

Such a glycosyl linker group is formed from any mono- or oligosaccharide, which, after modification with a selected fragment, is a substrate for the corresponding transferase.

Conjugates according to the invention usually correspond to the general structure: a v/suv in which the symbols a, b, c, a and 5 mean a positive integer that is not equal to zero, and Her is equal to 0 or is a positive integer. The term "agent" usually means a water-soluble moiety, such as a residue

PEG. The term "linker" can include a wide range of any linking groups, see lower. Alternatively, the linker may be a single bond or "zero-order linker".

In a representative embodiment of the invention, the selected modifying group is a water-soluble polymer, for example, m-PEG. Such a water-soluble polymer is covalently linked to the 2-C5E peptide by means of a glycosyl linker group, which is covalently attached to an amino acid residue or to a glycosyl residue of the 2-C5E peptide. The present invention also relates to conjugates in which the amino acid residue and the glycosyl residue are modified with a glycosyl linker group.

A representative water-soluble polymer is polyethylene glycol, for example, methoxy polyethylene glycol.

The polyethylene glycol used in the present invention is not limited to any particular form or molecular weight. For unbranched polyethylene glycol molecules, the molecular weight is preferably 500-100,000. A preferred molecule has a molecular weight of 2,000 to 60,000, and more preferably from about 5,000 to 30,000.

In another embodiment of the invention, said polyethylene glycol is a branched PEG having more than one attached PEG residue. Examples of branched PEGs are described in US patents MoMe5932462, 5342940, 5643575, 5919455, 6113906, 5183660; in UUO 02/09766 and in Kodega U, Viosopiidaye Spetivigu 5:283-288 (1994) ii Matavaki ei ai., Adgis. Vioi Spet., 52:2125-2127, 1998. Other relevant branched structures of PEG are described in this application.

In representative embodiments of the invention, the molecular weight of each branched polyethylene glycol

(PEG) is equal to or greater than about 2000, 5000, 10000, 15000, 20000, 40000 or 60000 daltons.

Peptides according to the invention include at least one M- or O-linked glycosylation site. In addition to conjugates that are formed with the help of a glycosyl linker group that is attached enzymatically, the present invention relates to conjugates that are highly homogeneous in their nature of substitution. With the use of methods according to the invention, peptide conjugates can be obtained in which almost all modified sugar groups throughout the population of conjugates according to the invention are attached to multiple copies of structurally identical amino acid or glycosyl residues. Thus, in its second aspect, the present invention relates to a peptide conjugate having a set of water-soluble polymer fragments that are covalently linked to the peptide (-C5E by means of an intact glycosyl linker group. In a preferred conjugate according to the invention, in fact, each member of such a group is connected through a glycosyl linker group to a glycosyl residue of the peptide (3-COE, and each glycosyl residue of the 2-C5E peptide, to which a glycosyl linker group is attached, has an identical structure.

The present invention also relates to a peptide conjugate having a group of water-soluble polymer fragments covalently linked to each other by means of a glycosyl linker group. In the preferred version of the invention, essentially each member of the group of water-soluble polymer fragments is connected to an amino acid residue of the peptide (3-C5E by means of a glycosyl linker group, and each amino acid residue containing an attached glycosyl linker group has an identical structure.

The present invention also relates to conjugates similar to the conjugates described above, in which the peptide (-

C5E is conjugated to a therapeutic molecule, a diagnostic molecule, a targeting molecule, a toxin molecule, or the like. using an intact glycosyl linker group. Each of the aforementioned molecules can be a small molecule, a natural polymer (for example, a polypeptide) or a synthetic polymer.

In fact, any granulocyte colony-stimulating factor peptide or agent having any sequence can be used as a peptide component of conjugates according to the invention. Granulocyte colony-stimulating factor was cloned and sequenced. In a representative variant of the invention, the peptide (1-C5E has the sequence presented in 5ХЕО ЮЮ MO:1:

MTRISRAZBI ROZA KSGEOUKKIOYUOSRSAAGOVKI,

SATUKI SNREBILID.ONBI IRR ARY Z5SRZOAGOH

SOSI5OINZOTESCOSIOACEOYIBRESORTOTSOGO

UAPREATIMOOMEBI SMARAGHORTOSAMRAKAZAE

OKKASSUG MAZNISOVEGEMUKMIANI.AOR (560

IR MO: 1).

In another representative embodiment of the invention, the 2-C5E peptide has the sequence presented in ZEO IU

MO:2:

THREE-FOLLOWED ROBY KSTEOMAKIOSPOAAGOEKT SA

TUKISNREEGLLI.ONVGOIRZHARIZ5SREOASOGA OS

ZONES EMO OAGESIZRESSRT OT OGOMA

REATTGUOOOMEEGOMARAGORTOSAMRABABAROK.

KASSUBUABNI OATS EMUZUKUT ANIAOR (5BEO TO

MO: 2).

In another representative embodiment of the invention, the 2-C5E peptide has the sequence presented below in

ZEO IU MO:3-11:

MTRIOZRAZBZCROZEKSGEOUAKIOSVOSAAGOVKI,

U5VESATUKISPREEG MI ONZSOTRMARIZ5SRBOMI,

OBAOSI BONN ETO OAGESIBREGORTGHOTI,

OHOMAPEATTRMOOMEE SMARAGHORTOSAMRAVA

5AgOKKASOMTSMAZNIO5ETSEUBUV MUTNI A OR (5BEO TO MO:3)

MASRATOZBRMKI MAO LUNZAGMUTUOVATRIOR

AVZVIROVBEY ML KSGEOUVKYUSOSAAOEKI SATUKI,

SNREESUSSONO STRMARI55SRZOAGOGASOSI ZOYA,

NISTESTOSIGOAIESISZREMORTOMOGOMAREAT

TIGMOOMEBIOMARASOVTOSAMRABAZAVOVKA SS

UGMA5NIOVESEUZUKUTAKNIAOR (5EO 10 MO:4)

MASRATOBRMKI MAOTLILUNZALUTMOVATRI OR

AZZIGROZEILKSGEOUEKYUSOSAAGOVKIMZESAT

HKISNREEG UP ONZISIRJUARY 55SRBOAIS JSC,

BOSNZOGE OST OAESIVRESSRT OT OBOMA

EATTGMOOMEBISMARAGHORTOSAMRABABAKOVE

ASOUGLAZNIOSE EUMVUKUTKNIAOR (5BEO

MO:5)

MUTRISRAZVGROZEL KSTEOUVKOSOSASASOEK

TSATUKONREVESUSTSONT OF OSIV TARIAVSRZOATOY,

AOSIKOSNeOT ED UOSGOAGESVRESORTOTTOI,

PVUAOBATTKOOMEK OMARABORTOSAMRAKAVA

KEOKWAVSOI MAVNYOVESEUVUVUGENSAORIVEO

TU MO:6);

MIRA ROB KOSGEOUVKIOYUSOSASAASOEKI,

SATUKESNREESU M ONTIOIRZHARGBVSRVOATOVA

OSI DON OASEOIVVESOR TOYU CHAVRATTUOOMEESOMARAGHORTOVAMRAKABAE

OVKASOUSUAZNIOBESEKHUVUVUSVNSAORKO TO

MO:t);

MUTRISRAVBSROZVI KSTVOUVKYUOSOSAATSOVE

SSATUKISNREVES HONEY LOBSTER YAVSRZOATOY,

AOSTISTSNIOBESKOSISOACEOIZRESO RTV.

ОХАОГАТПХУООМЕБ ОМАРАСОРТОВАМРАКАВА кЕОККАСОУСМАЗНИОФЕ EUВХВУТКНИАОРИЗЕО p» MOV);

MOTRISRAZVGROVKI KSO KKYUVOSAASOVK.

HOSATUKSNREVS IN ON OT ZHTARSZZSRZOAKOY, dISSVOGNVSY EE UOOGOAGESYAREBORTSOTTSOY,

OUABEATTTMOOMEESOMARABORTOBAMRAKAWA

EOKWASOUSMAVNIOVBE EUBUKU TAN AORIZKO

TO MO:9u;

MTRIDRABZYASROBRLKSVOMEKIOSOSAAGOVKI,

SATUKOSNREBESALL ON HR UARIZBSRZOA SOVA

OSTLOSNUSTI KOT OAGVOIZRESOR TS OT OO

CHAREATT ZHY OOMEBIOMARASORTOSAMRAVACHAE

OBLASOUGUAYANOVE EMV OF ENTHUSIASTIC AORTOSAM

RIBEOIV MY)

MTRIOSRAZZI ROBY KSIEOUKKYUSOS ALI OEKI,

SATUKIOSNREESMI. 55 MARIVVSRVOASOG

OSIZOSNVa g ESTOSTL OA HESIZRESORTOTGOI

MHAOEATIGIOOMEEEIOMARTTTTRTOTAMRABABE

OKKASSUTMAVNIOVESEUVUKUTANGAOR (ZEO I

MO1)

The present invention is not limited to the sequences presented herein.

In a representative variant of the invention, the 2-C5E peptides according to the invention include at least one site of O-linked glycosylation, which is glycosylated by a glycosyl residue that includes a PEG fragment.

PEG is covalently linked to the peptide (2-C5E via an intact glycosyl linker group. Such a glycosyl linker group is covalently linked to either an amino acid residue or a glycosyl residue of the 1-C5E peptide. Alternatively, the glycosyl linker group is attached to one or more The present invention also relates to conjugates in which a glycosyl linker group is attached to an amino acid residue and to a glycosyl residue.

The PEG moiety is attached to an intact glycosyl linker directly or via a non-glycosyl linker, for example, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

In a preferred embodiment of the invention, the peptide (1-C5E contains a group having the formula I.

Formula 1

He

Ge) o soon no 0-3 --H on de O selected from -OH and B!-I-NM; C is selected from B'-Y or C(O)(Ci-Cv)alkyl; B' represents a group containing a fragment selected from linear or branched residues of polyethylene glycol, and | means a linker which is selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, provided that if O is OH, then Si is B'!-I, and if Si is C(O)( Ci-Cv)alkyl, then Y represents B"-I-MH-. In the modified structures of sialic acid described here, COOH also represents

SOO.- and/or its salt.

In one of the variants of the invention, B'"-Y has the formula: o where a is equal to a whole number from 0 to 20.

In a representative version of the invention, V! has a structure selected from:

M in pyay; M fe ono mne(oinisnyosnIisndosn, kne(oyusn,snhosnIsnDdosN, o 2 ryu--nnenaeh gi "Hu neah

Mmie(Fsn,snosnsndOSsN, chne(o0osn,snuosn,snuOsn, where are and y are independently equal to integers selected from 1-2500, and 4 is equal to an integer from 1 to 20. In other variants of the invention, B! has a structure that is a member selected from: z nysSioyusnynuosnasNnI Osn" 4 : nn neoyusnenaosnisnayusn, o " o mysoyusnyosnsNn Osn.

With

Mn; we

In neryunyunosnonnaosn, o ch o mie(oyusnnyosn, sni Osn" i; sh kne(snisnyosnsnIOsn, not

In Ts epensniosnonassya 0 t

MneeoyusnIssnosnIsneosn,

Z : nneshoyusnisnhosnisnaosn" nya (In eorosnescosnsnoyv o and where e, they are equal to integers independently selected from 1-2500, and d and d mean integers independently selected from 1-20.

In yet another variant, the present invention relates to a factor IX peptide conjugate, where B' has a structure selected from: ; University of Uvna Ch

Same to. dnaoyusnesniosnnoyus chn nm Mn U (HC ooksnenyusnenauyus , o " ; 1 o nne(snIsnhosnIsnI Osn" : m nn AODU dit u orisnisncosnisnayusn" nm vne t i ne(osn,snkosNasna asn" h where there are, and 7 are independently equal to integers selected from 1-2500, and d, 4, and a" are equal to integers independently chosen from 1-20.

In other variants of the invention, V! has a structure selected from: 5-s(o)snosnuOSNosnadeO CH, :and 8--s(oyusnosnHosNsH Osn» where are and are independently equal to integers selected from 1-2500.

In another representative embodiment, the present invention relates to a peptide containing a group having the formula: on o o soon o--sa-- v--nk on de Sa! can be attached to an amino acid or to a glycosyl residue that is directly or indirectly (eg, through a glycosyl residue) attached to an amino acid.

In other variants of the invention, the molecule has the formula: on 0 son no

O--Phan--Obamds--5 8-H on de CyMas can be attached to an amino acid or to a glycosyl residue that is directly or indirectly (for example, through a glycosyl residue) attached to an amino acid.

In another representative variant of the invention, the peptide contains a group of the formula:

where AA is an amino acid residue of the specified peptide, and in each of the above-mentioned structures, ОІs are as they were defined in the description of this invention.

A representative amino acid residue of the 2-C5E peptide, in which one or more of the aforementioned molecules can be conjugated, is serine and threonine, for example, threonine 133 5EO IU MO:1.

In another representative variant, the present invention relates to the conjugate (2-СО9Е, which includes a glycosyl residue having the formula: te | nosa vn KM

Z--AA--TOiSMAs- OM As Mai (gviemAen vi S ivu- (m)

Y "mas ((lemAs-(Oa de (Biau, - (К), are independently equal to the integers chosen from 0 and 1. The index d is equal to 1. The indices e, Я, д and п are independently equal to the integers chosen from the integers from 0 to 6. The indices |, К, И and т are equal to integers, independently selected from integers from 0 to 100. The indices m, mu, x and y are independently equal to a number selected from 0 and 1, and at least one of m, m, x and y is equal to 1. The symbol AA means the amino acid residue of the peptide C- SELF

The symbol 5ia-(P) means a group having the formula: no

AND". noas o on 5-9 chn-o on de O selected from -OH and V!-I-NM. The symbol C means B'-I- or -C(O)(C1-C6)alkyl. B' is a group that includes a linear or branched residue of polyethylene glycol. It is a linker selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl. In general terms, if O means OH, then C means B'-I, and if C means -C(O)(C1-Cv)alkyl, then O means B!-I -MH-.

In another representative embodiment of the invention, the PEG-modified fragment of sialic acid in the conjugate according to the invention has the formula: he coni b d ce; (0. n v sh leti " where the index "5" means an integer from 0 to 20, and n equals an integer from 1 to 2500. In the preferred version of the invention, 5 is equal to 1, and the m-PEG part has a molecular weight of approximately 20 kD.

In yet another representative embodiment of the invention, PEG-modified sialic acid has the formula: on non ne /

Isch g") tire, p where Y is a substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl linker group connecting a sialic acid molecule and a PEG molecule.

In a preferred embodiment of the invention, at least two, more preferably, three, and even more preferably, four of the above-mentioned asparagine residues are functionalized with the above-mentioned M-linked glycan chain.

Conjugates according to the invention include intact glycosyl linker groups that are monovalent or polyvalent (for example, "antenna" structures). So, for example, conjugates according to the invention include both molecules in which the selected group is attached to the peptide by means of a monovalent glycosyl linker group and a polyvalent linker group. The present invention also includes conjugates in which more than one selected molecule is attached to the peptide by means of a polyvalent linker group.

Modified sugar

This invention relates to modified sugar, to modified sugar-containing nucleotides and to conjugates of modified sugar. In modified sugar-containing compounds according to the invention, the sugar group is preferably a saccharide, deoxysaccharide, aminosaccharide or M-acylsaccharide. The term "saccharide" and its equivalents, "saccharyl", "sugar" and "glycosyl" mean monomers, dimers, oligomers and polymers. Such a sugar group is also functionalized by a modifying group. This modifying group is conjugated to the sugar group, of course, by conjugation with an amine, sulfhydryl, or hydroxyl, for example, with the primary hydroxyl on said sugar. In a representative embodiment of the invention, the modifying group is attached to the sugar by means of an amino group, for example, by means of an amide, urethane or urea, which is formed by the reaction of an amine with a reactive derivative of the modifying group.

Any sugar can be used as the sugar backbone of the conjugates according to the invention.

Representative sugars that can be used to prepare the compositions of the invention include, but are not limited to, glucose, galactose, mannose, fucose, and sialic acid. Other suitable sugars are amino sugars such as glucosamine, galactosamine, mannosamine, the 5-amino analog of sialic acid, and the like. Said sugar backbone may have a naturally occurring structure, or it may be modified to form a site for conjugation with a modifying group. So, for example, in one of its variants, the present invention relates to a peptide conjugate containing a derivative of sialic acid, in which the 9-hydroxy group is replaced by an amine. This amine is readily derivatized with an activated analog of the selected modifying group.

The discussion of this invention illustrates the use of selected sialic acid derivatives.

Those skilled in the art will appreciate that this discussion is for illustrative purposes only, and that the structures and compositions described herein are essentially applicable to all types of saccharide moieties, modified saccharide moieties, activated modified saccharide moieties, and conjugates of modified saccharide moieties. .

In its representative version, the present invention relates to a peptide conjugate containing an amino-modified sugar having the formula: "MmNA--- I where Ci is a glycosyl group, Y is a bond or linker, and B' represents a modifying group. A representative bond is a bond formed between МН" on the glycosyl moiety and a group with corresponding reactivity on the modifying group. Thus, representative bonds include, but are not limited to, MNEA"', ОВ ', 5B! etc. So, for example, if B! includes a carboxylic acid group, then this group can be activated and linked to the MIM group on the glycosyl residue with the formation of a bond having the structure MHC(O)B!. Similarly, the OH and 5H groups can be converted into the corresponding ether or thioester derivatives, respectively.

Representative linkers are alkyl and heteroalkyl groups. Such linkers are binding groups, for example, acyl binding groups, for example, -С(О)МН-, -ОС(О)МН-, etc. Linker groups are bonds formed between the components of the molecule according to the invention, for example, between a glycosyl group and a linker (I), or between a linker and a modifying group (B). Other linker groups are ethers, thioethers and amines. So, for example, in one of the variants of the invention, the specified linker is an amino acid residue, such as a glycine residue. The glycine carboxylic acid group is converted to the corresponding amide by reaction with an amine on the glycosyl residue, and the glycine amine is converted to the corresponding amide or urethane by reaction with an activated carboxylic acid or carbonate modifying group.

Another representative linker is a PEG molecule or a PEG molecule functionalized with an amino acid residue. Such PEG is linked to a glycosyl group by means of an amino acid residue at one end

PEG and with B! at the other end of the PEG. Alternatively, this amino acid residue is linked to B', and the end of PEG, not linked to the amino acid, is attached to the glycosyl group.

Representative molecule МН-И-В! has the formula: -MmNES(ОХСнНИаамнь с(ОХсСНаОсSNегсСНг)е(СНг)аМНньВ!, where the indices 5 and Her are independently equal to 0 or 1.

Indices a, B and a are independently equal to integers from 0 to 20, and c is equal to a whole number from 1 to 2500. Other similar linkers are obtained on the basis of molecules in which the -МН group is replaced, for example, by -5, -О and - SN".

More specifically, the present invention relates to a peptide conjugate containing compounds in which МН-И -В! represents: чне(Фу(сСнНааМне(охснагоосСнНегсСнНг)(СНг)аМнА",

MNesoFusSNav(OsSnegsSnNg)(SNg)aMNV,

MNeusooF(сСнайОснесСнНг)(СНг)аМНВ!, чнНеохсСнНгаМне(охснагОосСнНгсСНг)еSNгамняА!, МНОС(ХСНг)аМмнв,

МН(СНг)аМНе! and ME!. In these formulas, the indices a, b and a are independently selected from the integers from 0 to 20, and preferably from 1 to 5. The index c is equal to the whole number from 1 to 2500.

In an illustrative embodiment of the invention, c represents sialic acid, and selected compounds according to the invention have the formulas:

sshchonenomsnM "mysoKsnu nya! N and NN Hsnvnnoo sno sno:

B: nose. isnonsnin snkonenonnon . o mo "mnesnsnu,nnohnsn yus Osn noos, o. oschonskonsnon ni o echatetettetttilen knehohsnomosnsn neni op stonisconisnion nos. nenennonsnyon no nov roya "nnerknsnanosnonyusnannia b nos. at. snomsnonsnIyun approx. neither 1

As will be apparent to those skilled in the art, the sialic acid molecule in the representative compounds described above may be replaced by any other amino saccharide, including, but not limited to, glucosamine, galactosamine, mannosamine, their M-acetyl derivatives, etc. .

In another illustrative embodiment of the invention, the primary hydroxyl group of the sugar is functionalized with a modifying group. Thus, for example, the 9-hydroxyl of sialic acid can be converted into the corresponding amine and functionalized to obtain compounds according to the invention. Compounds according to this variant of the invention have the formulas: noos. konusnnneyudsn), NSS snu sn o(snchnia! n, p nos onusnionIsnunNe osn "Neon osn sno o sndeNya!

Gde) nysyuuna ich noos. onyunusn(onIsn,nnsni nn YuhsnosN.sno(snInya" but nobe. onisnonIsnchne ah sny stonyuonienuchnostnNya not

In n ny lstonsnonIsnno(osn sn s; ne dosh soneniyunusnnnya! schu onzhnkonknyano oh sn Osn sosna Anya "nnogyusn, yu:

OST on

In another representative embodiment, the present invention relates to a peptide conjugate containing modified sugars in which the hydroxy group in the b-position has been converted into a corresponding amino group carrying a "linker - modifying group" cluster, such as the cluster described above. Representative sugar groups that can be used as the backbone of this modified sugar are SaI!, SaIMas, Sis, SisMas, Rys,

HUI, Map, etc. A representative modified sugar according to this variant of the invention has the formula: where vk t v vi where BZ-B5 and B" are members independently selected from H, OH, C(O)CH3, MH and MHC(OSH), and B? is ОВ', МНВ! or МН-И-В', defined above.

The selected conjugates according to the invention are obtained on the basis of mannose, galactose or glucose, or on the basis of molecules having the stereochemical structure of mannose, galactose or glucose.

These conjugates have general formulas: in tk there is z; in, 1 "ov .

In another representative embodiment, the present invention relates to the compounds described above, which have been activated in the form of a corresponding sugar contained in nucleotides. Representative sugar-containing nucleotides used in the present invention in their modified form are mono-, di- or triphosphates of nucleotides or their analogues. In a preferred embodiment of the invention, the modified sugar-containing nucleotide is selected from UDF-glucose, CMF-glycoside or GDFf-glycoside. Even more preferably, the sugar portion of the modified sugar-containing nucleotide is selected from UDF-galactose, UDF-galactosamine, UDF-glucose, UDF-glucosamine, GDF-mannose, GDF-fucose, CiMF-sialic acid or CMFf-MenAs. In a representative embodiment of the invention, the nucleotide phosphate is attached to C-1.

Thus, in its illustrative version, where the glycosyl group is sialic acid, the present invention relates to peptide conjugates that were obtained using compounds having the formula: syayunsnyyusnyum chu that, , in sh-y ii 2 nonisshOonunn la 8

From pu nnskisn, n. he n he de I-V! has the values defined above, and II-B! is a linker connected to a modifying group. As in the case of Y, a representative linker molecule in accordance with 1" includes a bond, alkyl or heteroalkyl groups. Representative nucleotide compounds containing a modified sugar according to these variants of the invention are shown in Fig. 1 and Fig. 2.

In another variant, the present invention relates to a conjugate formed by a modified sugar according to the invention and a substrate, for example, a peptide, a lipid, an aglycon, etc., and more specifically, a modified sugar and a glycosyl residue of a glycopeptide or glycolipid. In this variant of the invention, the sugar part of the specified modified sugar becomes a glycosyl linker group located between the substrate and the modifying group. A representative glycosyl linker group is an intact glycosyl linker group in which the glycosyl moiety or moieties forming the linker group are not cleaved by chemical (eg, sodium metaperiodate) or enzymatic (eg, oxidase) reactions. Selected conjugates according to the invention include a modifying group that is attached to the amino group of an amino-saccharide, for example, mannosamine, glucosamine, galactosamine, sialic acid, etc. A representative cluster "modifying group - intact glycosyl linker group" according to this variant is obtained on the basis of the structure of sialic acid, such as the structure having the formulas: no on vi-y--nn o o. UN by o. ugon no 2-5 0-5 kA-y--kn and snusyumn dn on where, in the above-mentioned formula, B", I! and I? have the values specified above.

In another representative embodiment of the invention, the conjugate was formed by the substrate and the sugar group in the 1-position, and in this conjugate the modifying group is attached by means of a linker near the carbon atom in the 6-position of the sugar group. Thus, illustrative conjugates according to this variant of the invention have the formulas: o. 0. 87 days; M o m i t ct i i v

Wherein the indicated radicals are as described above. It is obvious to those skilled in the art that the modified sugar groups presented above can also be conjugated to the substrate near the carbon atoms in positions 2, 3, 4 or 5.

Illustrative compounds according to this variant of the invention have the formulas:

Neon

The basic principles of NC groups and indices are as defined above.

This invention also relates to sugar-containing nucleotides modified by group I-B'! near the carbon atom in the b-position. An example of such a molecule according to this variant of the invention is: "me-me"

He is the basis of shi Chi; cha s rogo

God Go

However, groups B and Y are the groups discussed above. The index "y" is equal to 0, 1 or 2.

Another representative sugar-containing nucleotide according to the invention has at its base a stereochemical structure of GDF-mannose. A representative molecule according to this variant of the invention has the structure: "Zv; and I we what ke nn shi che yi 1 ' z ,, ih ch rt choi i nn; I'm talking about

With her he

In another representative variant, the present invention relates to a conjugate based on the stereochemical structure of UDfF-galactose. A representative compound according to this variant of the invention has the structure:

Lot vk a v Y - o» v u

Zon hi no

WITH

Bo U chi o A

Go in Y "in d) M in i i

Sk.

Cannes « she nan.

In another representative variant of the invention, the sugar-containing nucleotide has the stereochemical structure of glucose as its basis. A representative molecule according to this variant of the invention has the formula: nn y y khre p o . pH and

Not: and re: t

KO" AH. with

Hn n n.

Modifying group B! represents any group, including, but not limited to, water-soluble polymers, water-insoluble polymers, therapeutic agents, diagnostic agents, etc. The nature of representative modifying groups is discussed in more detail below.

Modifying groups

Water-soluble polymers

Many water-soluble polymers are known to those skilled in the art and can be used to implement this invention. The term "water-soluble" polymer includes molecules such as saccharides (eg, dextran, amylose, hyaluronic acid, polysialic acid, heparans, heparins, etc.); polyamino acids, for example, polyaspartic acid and polyglutamic acid; nucleic acids; synthetic polymers, for example, polyacrylic acid, polyesters, for example, polyethylene glycol; peptides, proteins, etc. The present invention can be carried out using any water-soluble polymer, however, with the only condition that such a polymer must have a position in which the other part of the conjugate can be attached.

A description of polymer activation methods can also be found in UUO 94/17039, in US patent Me5324844, in MO 94/18247, in UMO 94/04193, in US patent Me5219564, in US patent Me5122614, in UUO 90/13540, in US patent

Me5281698, as well as in UUO 93/15189, and the literature also describes methods of conjugating activated polymers with peptides, for example, the coagulation factor of MI! (MO 94/15625), hemoglobin (UMO 94/09027), an oxygen-carrying molecule (US patent Moe4412989), ribonuclease and superoxide dismutase (Megopeze ei ai., Arr.

Viospet. Vioyesn. 11: 141-45 (1985)).

Preferred water-soluble polymers are polymers in which the bulk of the polymer molecules in the polymer sample have approximately identical molecular weight, and such polymers are called "homodisperse."

The present invention is also illustrated on a polyethylene glycol conjugate. There are a number of articles and monographs describing the functionalization and conjugation of PEG. See for example, Naitis, Masgopoi. Spent Rpuz. C25: 325-373 (1985); bsociep, MeiSshoav" ip Eplutoiodo 135: 30-65 (1987); UUopd ei aI., Eplute Misgor. Thespians 14: 866-874 (1992); OyeIdado ei ai., Spysa! Nemieme ip TPpegareshis YuOgpid Satiyei buvziet5 9: 249-304 (1992); 7aiireKu,

Viosopishdaie Spet. 6: 150-165 (1995) apa Vnaiga eyi aiI., Rpaptaliye, 57: 5-29 (2002). Methods of obtaining reactive PEG molecules and conjugates using such reactive molecules are known to specialists.

So, for example, US patent Mo5672662 describes a water-soluble and secreted active ester conjugate of a polymeric acid selected from linear or branched polyalkylene oxides, polyoxyethylated polyols, polyolefin alcohols, and polyacrylomorpholine.

US patent Mob376604 describes a method of obtaining a water-soluble complex ester of 1-benzotriazolylcarboxylic acid of a water-soluble and non-peptide polymer by reacting the terminal hydroxyl of this polymer with di(1-benzotriazolyl)carbonate in an organic solvent. The active ester is used to obtain conjugates with a biologically active agent, such as a protein or peptide.

MO 99/45964 describes a conjugate containing a biologically active agent and an activated water-soluble polymer, which includes a polymer backbone, which at least one end is connected to another polymer backbone by means of a stable bond, where at least one end contains a branching group having proximal reactive groups attached to such a branching group, and where the indicated biologically active agent is linked to at least one of the proximal reactive groups. Other branched polyethylene glycols are described in MO 96/21469. US patent Mo5932462 describes a conjugate formed by a branched PEG molecule that has a branched end containing reactive functional groups.

Free reactive groups can react with a biologically active molecule, such as a protein or peptide, to form conjugates of polyethylene glycol and the biologically active molecule. US patent Meo5446090 describes a bifunctional PEG linker and describes its use to obtain conjugates containing a peptide at each end of the PEG linker.

Conjugates including degradable PEG bonds are described in U/O 99/34883 and UUO 99/14259, as well as in US patent Mob348558. Such degradable linkers can be used in the present invention.

Well-known methods of polymer activation, described above, are used in the context of the invention to obtain the branched polymers described here, as well as to conjugate these branched polymers with other molecules, for example, sugars, sugar-containing nucleotides, etc.

Representative polyethylene glycol molecules used in the present invention include, but are not limited to, molecules having the formula: aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroalkyl, e.g. acetal, -OHS-, H2M-(CHg)a-, HO-(CHg)d or -(CHg)aC(M)2" The index "e" is equal to a whole number from 1 to 2500. The indices b, a and d are independently equal to integers from 0 to 20. Symbols 7 12 independently represent OH, MNae, leaving groups, for example, imidazole, p-nitrophenyl . 5, -M-B!Y. The symbols X, U, Y", A! and O independently represent O, 5, M-B'Y. The symbol M represents OH, MH", halogen, 5-B!2 , the alcohol component of the activated ester, the amine k component of activated amides, sugar-containing nucleotides and proteins. Indices p, 43, 51 m are equal to integers independently selected from 0-20. The symbols VU, BC, B"! and D" independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl.

In other representative embodiments of the invention, the polyethylene glycol molecule is selected from:

Mme-(snosnoyto. 7 Me-(oSsnasnov-O ih te 2SNv ve o me-(osnisnato. M, mechosnisnya M. rotu o o ke) me-iosnisndito. and Me-(osnosni. I Y and I o

Me-(OSsnosnadyu-2 iy

Mme-(osnasaya.-K- no yy

The polyethylene glycol used to obtain the conjugate according to the invention is either linear or branched. Branched polyethylene glycol molecules suitable for use in the present invention include, but are not limited to, molecules of the formula: A? independently selected from groups,

defined above for A!. The indices are, Her, o and d are defined above. 72 and M are also defined above. X! and X" are independently selected from 5, С(О)МН, НМС(О)5, 5С(О0, 0, МН, МНС(О), (ОО0СМН, МНОС(ОО) and ОС(О)МН.

In other representative variants of the invention, branched PEG is obtained on the basis of a cysteine, serine or di-lysine backbone. So, for example, other representative branched PEGs are: n nneoyusnusnuosnusna osn, n; 1 nn. r tenenetetecht of the lady. ru npeetetchns "Ko soyu ; "M pen

MyeohsnisnuosNnIsndyusna knefusnisnyosninayuenu "Hol taya ; "Mation of many (oyunsnuosN, snuOSN kneoyusnsnostunayuesn" HM pnia "HC ochne" nysohenanyusn, Y nneoyusn and yen cheno

Me)"

In another variant of the invention, the branched PEG molecule is based on a trilysine-containing peptide. Such trilysine can be mono-, di-, tri- or tetra-PEGylated. Representative molecules according to this variant of the invention have the formulas: o nneoyusn,snyOsn,sNI Osn no h po um «n yak. nn. , 4 and d" are independently selected from integers from 1 to 20.

In representative embodiments of the invention, the PEG is m-PEG (5kD, 10kD or 20kD). A representative branched PEG is serine- or cysteine-(m-PEG)”, where m-PEG has a mass of 20 kD.

As will be apparent to those skilled in the art, the branched polymers used in the present invention include variants of the molecules described above. So, for example, the "di-lysine - PEG" conjugate described above can include three polymer subunits, where the third subunit, connected to an a-amine, is shown in the above structure as unmodified. Similarly, the use of three lysines functionalized with three or four polymer subunits in this polymer is within the scope of this invention.

Specific variants of this invention are:

with code) e nya 9; me" e on nn ri ma e ny n neob o) toy 1 - with and their carbonates and active ester, such as:

M E e

E neo) og cho E

And the method is 8 nm

Me o chan) oo E

AND

Other activating or leaving groups suitable for activating the linear PEGs used to prepare the compounds described herein include, but are not limited to, the following groups:

With Fr

K. about; in, b fo de

Kk I- Zh

X, g - ii

IS -

PEG molecules, which are activated by these and other molecules, and methods of obtaining activated PEGs are described in

MO 04/083259.

It is obvious to those skilled in the art that one or more m-PEG branches of a branched polymer can be replaced by a PEG part with different ends, for example, with OH, COOH, MH", C2-Sioalkyl, etc. In addition, the above-mentioned structures can be easily modified by inserting alkyl linkers (or removing carbon atoms) between the α-carbon atom and the functional group of the side chain. Thus, "homo"-derivatives and higher homologues, as well as lower homologues, are included in the backbone volume for the branched PEGs used in this invention.

The branched PEG molecules presented here can be easily prepared by the methods illustrated in the scheme below:

on the "corner", the owner of it is 1 and trunk, ucha au, and with . where Xe represents O or 5, and r is an integer from 1 to 5. The indices are and i are independently selected from integers from 1 to 2500.

Thus, according to this scheme, a natural or unnatural amino acid is brought into contact with an activated derivative of m-PEG, in this case, with a tosylate, as a result of which, by alkylation of the heteroatom

H of the side chain, compound 1 is obtained. by any appropriate leaving group, for example, halogen, mesylate, triflate, etc. Similarly, the reactive carbonate used to acylate the amine can be replaced by an active ester such as M-hydroxysuccinimide, etc., or the acid can be activated by using a dehydrating agent such as dicyclohexylcarbodiimide, carbonyldiimidazole, etc. .p.

In a representative embodiment of the invention, said modifying group is a PEG group, however, any modifying group, e.g., a water-soluble polymer, a water-insoluble polymer, a therapeutic molecule, etc., may be incorporated into the glycosyl moiety by means of an appropriate connection Modified sugar is obtained by enzymatic methods, chemical methods or a combination thereof. In a representative variant of the invention, sugars are subjected to a substitution reaction with an active amine in any position, which allows the attachment of a modifying group, and also allows the use of sugar as a substrate for an enzyme capable of attaching a modified sugar to a peptide (-C5E. In a representative variant of the invention, if galactosamine is a modified sugar, the amine part is attached near the carbon atom in the 6-position.

Molecules modified with a water-soluble polymer

The present invention uses nucleotide sugar molecules modified with a water-soluble polymer, in which the sugar group is modified with the indicated water-soluble polymer. A representative modified sugar-containing nucleotide carries a sugar group modified by an amino group on said sugar. In the methods according to the invention, modified sugar-containing nucleotides can also be used, for example, saccharylamine derivatives of a sugar-containing nucleotide. So, for example, saccharylamine (without a modifying group) can be enzymatically conjugated with a peptide (or with another molecule), and then the free saccharylamino group can be conjugated with the desired modifying group. Alternatively, the modified sugar-containing nucleotide can function as a substrate for an enzyme that transfers the modified sugar to a sugar receptor on a substrate, for example, a peptide, glycopeptide, lipid, aglycone, glycolipid, etc.

In one of the variants of the invention, in which the saccharide backbone is galactose or glucose, and B? is

by me

In a representative variant of the invention, the modified sugar is obtained on the basis of the b-amino-M-acetylglycosyl molecule. As shown below for M-acetylgalactosamine, the b-amino sugar molecule can be easily prepared by standard methods. it's fine.

A A, in od yu n k»on

Soshe se Kofrrtchev g.

GI "He ieya ch p a. galactooxidaga; MN.SAe, MaVNuSM; B. "Zhenya 1" e Kto p

In the above scheme, the index n means an integer from 1 to 2500, preferably from 10 to 1500, and more preferably from 10 to 1200. The symbol "A" means an activating group, for example, a halogen, a component of an activated ester (for example, M-hydroxysuccinimidoether), carbonate component (for example, p-nitrophenyl carbonate), etc. For specialists in this field, it is obvious that other nucleotide sugars modified with PEG-amide can be easily obtained by these and similar methods.

In other representative embodiments of the invention, the amide group is replaced by a group such as urethane or urea.

In other variants of the invention, B' is a branched PEG, one of which, for example, is described above.

Illustrative compounds according to this embodiment of the invention are noos. chonyunenunyun - nne(khsna ns yutssnmyusnsn osn ny: -smonoisn,

He mysiomesn, snhosnsndOsn, noos. at. nOonsschonen,n no kneyunenan 8-(snonyusn, : he nys(oresnn(OsNsNOSsN, noos. sleeponannen rya escape: no mnys(oussn,snyosnondyusn, kN, he rtotechenanerttreneniennyi Uchena nns(oresn,sndosnusniOSn, carry, he p o zknenenectemnektlechovotyai ny echenaya kneoresnsnyosnndosn, neon on noos. poteteenkttkndittetalnnan chena no kne(rssn,snIOosn,sn osn, mne(o)sn; on where X" means connection or 0.

In addition, as discussed above, the present invention relates to peptide conjugates obtained using sugar-containing nucleotides modified with a water-soluble polymer having either a linear or branched chain. So, for example, the scope of this invention includes compounds having the formulas presented below: noos. new ra

From Oshna; ий занцохоснсниоснонден, о вн ресненаоснстюей, -3 is, Я.Я mogsn, op where X" means O or connection.

Similarly, the present invention relates to peptide conjugates obtained using sugar-containing nucleotides having a modified sugar molecule in which the carbon atom in the b-position is modified: in! nnekenilny 7 -snovoms, kneurysnIsnaosnsnamosn» t, bla ; huh

AND

Kk o. th Mn Mn; I

And more often than not X" means connection or 0.

The present invention also relates to conjugates of peptides and glycopeptides, lipids and glycolipids, which include the compositions of the present invention. So, for example, this invention relates to conjugates having the following formulas:

onusnonsnIon mooschnyu oai on nysozhhnsnyosn,sydOsn, onsnionIsnIn start: neitsoyunisnkosnIsndOsn, noyus. poketochnetya r e-snsno and rya nneoryun, snlosn, snkosn, eyes

He is a nose. according to Z-psnoksm, ov nieojjen, isnuosnisnosn, chanesn,

Polymers insoluble in water

In another variant of the invention, similar to the variants discussed above, the modified sugar includes instead of a water-soluble polymer, a polymer that does not dissolve in water. Conjugates according to the invention may also include one or more water-insoluble polymers. This variant of the invention is illustrated by the example of the use of a conjugate as a carrier that provides regulated delivery of a therapeutic peptide. Polymer-conjugated drug delivery systems are known to those skilled in the art.

See, for example, Eupp ei ai!., Eaz. Roiutegis Ogadv apa Ogad Oeiiimegu bZuviet5, AS5 butrozisht bepevz Mo!i.469,

Ategisap Spetis! bosieiu, UMazpipdiop, Yu.S. 1991. It is obvious to those skilled in the art that essentially any known drug delivery system can be used for the conjugates of the invention.

Representative non-water soluble polymers include, but are not limited to, polyphosphazines, polyvinyl alcohols, polyamides, polycarbonates, polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycols, polysiloxanes, polyurethanes, поліметилметакрилат, поліетилметакрилат, полібутилметакрилат, поліїізобутилметакрилат, полігексилметакрилат, полізодецилметакрилат, полілаурилметакрилат, поліфенілметакрилат, поліметилакрилат, поліззопропілакрилат, поліїзобутилакрилат, поліоктадецилакрилат, поліетилен, поліпропілен, поліетиленгліколь, поліетиленоксид, поліетилентерефталат, полівінілацетат, полівінілхлорид, полістирол, полівінілпіролідон, плюроніки і полівінілфенол і їх співполімери.

Synthetically modified natural polymers suitable for use in conjugates according to the invention include, but are not limited to, alkylcelluloses, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, and nitrocelluloses. Particularly preferred members of the broad class of synthetically modified natural polymers include, but are not limited to, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethylcellulose, cellulose triacetate, sodium sulfate cellulose and polymers of complex ester of acrylic and methacrylic acid and alginic acid.

These and other polymers discussed herein can be readily obtained from commercially available sources such as bBidta SPpetisa! Co. (5 Gotsiv, MO), Roiuzsieepsez (Uuaitepiop, RA), AIdpsp (Mim"akee, UM), Rika (Wopkopkota, MU), and Viobay (Visptopa, SA), or they can be synthesized by standard methods from monomers purchased from these suppliers.

Representative biodegradable polymers used in the conjugates of the invention include, but are not limited to, polylactides, polyglycolides and their copolymers, polyethylene terephthalate, polybutyric acid, polyvaleric acid, lactide-caprolactone copolymer, lactide-glycolide copolymer, polyanhydrides , polyorotoethers, their mixtures and copolymers. Especially acceptable are compositions that form gels, such as gels that include collagen, pluronics, etc.

The polymers used in the present invention are "hybrid" polymers that include non-water-soluble materials that contain at least part of their structure a bioresorbable molecule.

An example of such a polymer is a polymer that is a non-water-soluble copolymer having a bioresorbable region, a hydrophilic region, and multiple cross-linkable functional groups on one polymer chain.

According to the present invention, the term "water-insoluble materials" includes materials that are essentially insoluble in water or in aqueous media. Thus, although some regions or segments of a copolymer may be hydrophilic or even water-soluble, in general, such a polymer molecule is essentially insoluble in water.

According to the present invention, the term "bioresorbable molecule" includes a region capable of undergoing metabolism or decomposition in the body and being resorbed and/or eliminated by the body through normal excretory pathways. It is preferable that such metabolites or breakdown products are essentially non-toxic to the body.

The bioresorbable region can be either hydrophobic or hydrophilic, provided that the copolymer composition is generally not water soluble. So, for example, the area that is biologically resorbed is chosen, preferably, so that this polymer, in general, remains insoluble in water.

Accordingly, the relative properties, i.e., the type of functional groups contained in the specified bioresorbable region, and the relative content of the bioresorbable region and the hydrophilic region are selected so that the bioresorbable compositions used remain insoluble in water

Representative resorbable polymers are, for example, synthetically produced resorbable poly(α-hydroxycarboxylic acid)/poly(oxyalkylene) block copolymers (see, Sopp et al., US Patent No. 4,826,945). These copolymers are not cross-linked, but are water-soluble, and therefore, split block copolymer compositions can be excreted from the body. See Moipez ei ai.,.). Viotea. Maieg. Nev. 21: 1301-1316 (1987) and Sonpei a)., 9). Vioted. Maghieg. Nev. 22: 993-1009 (1988).

Preferred bioresorbable polymers according to the invention are one or more components selected from polyesters, polyoxyacids, polylactones, polyamides, polyetheramides, polyamino acids, polyanhydrides, polyorthoesters, polycarbonates, polyphosphazines, polyphosphoesters, polythioethers, polysaccharides, and mixtures thereof. Even more preferably, the bioresorbable polymer includes a polyoxyacid component. Of these polyoxyacids, polylactic acid, polyglycolic acid, polycaproic acid, polybutyric acid, polyvaleric acid and their copolymers and mixtures are preferred.

Preferred polymer coatings used in the methods of the invention, in addition to forming fragments that are absorbed by the body ("biologically resorbed"), may also form fragments that are excreted and/or metabolized.

Higher order copolymers may also be used in the present invention. So, for example, in a patent

USA Me4438253, issued March 20, 1984, Sazeu et al., described three-component block copolymers obtained from polyglycolic acid and hydroxy-terminated polyalkylene glycol by transesterification. These described compositions are intended for use as monofilament resorbable suture materials. The elasticity of such compositions is regulated by introducing an aromatic orthocarbonate, such as tetra-p-tolylorthocarbonate, into the structure of the copolymer.

Other polymers obtained on the basis of lactic and/or glycolic acids can also be used. Thus, for example, US patent No. 5,202,413, issued April 13, 1993, describes biodegradable multicomponent block copolymers containing sequentially arranged blocks of polylactide and/or polyglycolide produced by ring-opening polymerization of lactide and/or glycolide to form or of an oligomeric diol, or diamine residue, and subsequent chain elongation with a bifunctional compound such as a diisocyanate, diacyl chloride, or dichlorosilane.

The regions of the bioresorbable polymer coatings used in the present invention can be designed to be hydrolytically and/or enzymatically degradable. According to the present invention, the term "hydrolytically cleavable" refers to the susceptibility of a given copolymer, and particularly the bioresorbable region, to hydrolysis in water or an aqueous medium. Likewise, the term "enzymatically cleavable" as used herein refers to the susceptibility of the copolymer, and particularly the bioresorbable region, to cleavage by endogenous or exogenous enzymes.

When introduced into the body, the hydrophilic region can be processed to form fragments that are excreted and/or metabolized. Thus, the hydrophilic region may include, for example, polyesters, polyalkylene oxides, polyols, polyvinylpyrrolidine, polyvinyl alcohol, polyalkyloxazolines, polysaccharides, carbohydrates, peptides, proteins and their copolymers and mixtures. In addition, the hydrophilic region can also be, for example, polyalkylene oxide. Such polyalkylene oxides can be, for example, polyethylene oxide, polypropylene oxide and their mixtures and copolymers.

Polymers that are components of hydrogels can also be used in this invention. Hydrogels are polymeric materials capable of absorbing a relatively large amount of water. Examples of compounds that form hydrogels are, but are not limited to, polyacrylic acids, sodium-containing carboxymethylcellulose, polyvinyl alcohol, polyvinylpyrrolidine, gelatin, carrageenan and other polysaccharides, hydroxyethyl methacrylic acid (HEMA), as well as their derivatives, etc. Hydrogels that are stable, biodegradable, and bioresorbable can be obtained. In addition, hydrogel compositions may include subunits having one or more of the specified properties.

Biologically compatible hydrogel compositions, the integrity of which can be regulated by the formation of cross-links (cross-linking), are known and preferred for use in the methods according to the invention.

Thus, for example, US Patent No. 5,410,016, issued April 25, 1995, and US Patent No. 5,529,914, issued June 25, 1996, to Nibrbei et al., describe water-soluble systems that are cross-linked block copolymers having a water-soluble central block segment arranged in a "sandwich" between two hydrolytically labile extension segments. Such copolymers are "capped" at the end with photopolymerizable acrylate functional groups. When crosslinked, such systems form hydrogels.

The water-soluble central block of such copolymers may include polyethylene glycol, while the hydrolytically active extension fragments may be a poly-α-hydroxy acid such as polyglycolic or polylactic acid. See Zam/ppeu ei a!., Masgotoietschiev 26: 581-587 (1993).

In another preferred embodiment of the invention, the specified gel is a thermoreversible gel. According to the present invention, thermoreversible gels are preferred, including such components as pluronics, collagen, gelatin, hyaluronic acid, polysaccharides, polyurethane hydrogel, polyurethane-urea hydrogel, and combinations thereof.

In another representative variant of the invention, the conjugate according to the invention includes such a component as liposomes. Liposomes can be obtained by methods known to those skilled in the art, for example, as described in Errziwiep et al. in U.S. Patent No. 4,522,811, issued June 11, 1985. For example, liposomal compositions can be prepared by dissolving the appropriate lipid(s) (such as stearoylphosphotidylethanolamine, stearoylphosphatidylcholine, arachodoylphosphatidylcholine, and cholesterol) in an inorganic solvent, which is then evaporated, and then on the surface of the vessel a thin film of dry lipid remains. Then an aqueous solution of the active compound or its pharmaceutically acceptable salt is introduced into this vessel. After that, the contents of the vessel are shaken manually to separate the lipid material from the side walls of the vessel and to disperse the lipid aggregates, as a result of which a liposomal suspension is obtained.

The above-mentioned microparticles and methods of obtaining such microparticles are described here as examples and should not be considered as limiting the volume of microparticles used in the present invention. It is obvious to those skilled in the art that a set of microparticles obtained by visible methods can be used in this invention.

The structural forms discussed above in connection with water-soluble polymers having both linear and branched chains, in general terms, may also be applicable to polymers that do not dissolve in water. So, for example, branched backbones consisting of cysteine, serine, two lysines, and three lysines can be functionalized with two water-insoluble polymer molecules. The methods used to produce such polymers are essentially similar to the methods used to produce water-soluble polymers.

The half-life of therapeutic glycopeptides can also be increased using PEG molecules such as polyethylene glycol (PEG). So, for example, chemical modification of proteins using PEG (PEGylation) leads to an increase in the size of molecules and a decrease in the accessibility of their surface and functional groups, where each of these properties depends on the size of PEG attached to the protein. This helps to increase the half-life of these proteins in plasma and resistance to proteolysis, as well as to reduce the levels of immunogenicity and absorption by the liver (Speyee ei ai., 9. Siip. Ipmevzi. 89: 1643-1651 (1992); Ruaiak ei ai., Vev.

Sottype Spent RaipoII Rpaptosai 29: 113-127 (1980)). It has been reported that PEGylation of interleukin-2 increases its antitumor efficacy by mimo (Kaige et al., Rgos. Mai). Asad 5 OBA, 84: 1487-1491 (1987)), and PEGylation of the E(ar)g fragment derived from the monoclonal antibody A7 helps to improve its localization in the tumor (Kiatyga ei ai., Viospet. Viorpuz. Nez. Sottyp. 28: 1387-1394 (1990)). Thus, in another preferred variant of the invention, the half-life of the ip mimo peptide, derivatized with a PEG molecule by the method according to the invention, increases compared to the half-life of the ip mimo non-derivatized peptide.

The increase in the half-life of the ip mimo peptide is best expressed as an interval of the percentage increase in its amount. The lower end of said range of percent increase is about 4095, about 60905, about 8095, about 10095, about 15095, or about 20095. The upper end of such range is about 6095, about 8095, about 10095, about 15095, or greater than about 25095.

In its representative version, this invention relates to PEGylated E5H (Fig. 1, Fig. 2 and

Fig. 5).

Methods

In addition to the conjugates discussed above, the present invention relates to methods of obtaining these and other conjugates. So, for example, in another aspect, the present invention relates to a method of obtaining a covalent conjugate consisting of a selected fragment and a 2-C5P peptide. In addition, the present invention relates to methods of delivery of conjugates according to the invention to a specific tissue or area of the body.

In representative embodiments of the invention, said conjugate is formed by a PEG molecule (or a glycosylated part that is enzymatically transferred containing a PEG molecule) and a glycosylated or non-glycosylated peptide. PEG is conjugated to the peptide (2-C5E with the help of an intact glycosyl linker group located between them, and is covalently linked to the C2-C5E peptide and to the PEG group, or to the construction "PEG-non-glycosyl linker" (such a linker is , for example, substituted or unsubstituted alkyl, substituted or unsubstituted alkyl.) This method involves contacting the 2-C5E peptide with a mixture containing a modified sugar and a glycosyltransferase for which said modified sugar is a substrate.

This reaction is carried out under conditions sufficient for the formation of a covalent bond between the modified sugar and the C2-C5E peptide. The sugar part of the modified sugar is preferably selected from sugar-containing nucleotides, activated sugars and sugars that are not components of nucleotides and are not activated.

The acceptor peptide (glycosylated or non-glycosylated) is usually synthesized by or recombinantly expressed in prokaryotic cells (eg, bacterial cells such as E. soya) or eukaryotic cells such as mammalian, yeast, insect, fungal or plant cells. Peptide -SOE can be either a full-length protein or its fragment. In addition, the 24-C5E peptide can be a wild-type peptide or a mutated peptide. In a representative embodiment of the invention, the -C5E peptide includes a mutation that adds one or more M- or O-linked glycosylation sites to the peptide sequence.

In a representative variant of the invention, factor IX is subjected to O-glycosylation and functionalization with a water-soluble polymer according to the method described below. The peptide is produced either using an available amino acid glycosylation site or, if it is glycosylated, the glycosyl moiety is "trimmed" to ensure amino acid availability. So, for example, serine or threonine is subjected to a-1-M-acetylamino-galactosylation (CaiYMAc), and the MAc-galactosylated peptide is subjected to sialylation by interaction with the "sialic acid - modifying group" cluster using 5T6cCaiMAst1. Alternatively,

The MAs-galactosylated peptide is subjected to galactosylation using the "backbone - 1-saiT-17" moiety, and the resulting product is subjected to sialylation by interaction with the "sialic acid - modifying group" cluster using 5TZSai!11. A representative conjugate obtained in this way has the following connections:

ТНг-а-1-саИ!Мас-рВ-1,3-СаИ-с2,3-5ia", where bia" means the cluster "sialic acid - modifying group".

In the methods according to the invention, for example, described above and carried out using a variety of enzymes and sugar donors, individual stages of glycosylation can be carried out separately or in combination with reactions "in one vessel". So, for example, in the reaction involving the three enzymes described above, SaiYIMAs-transferase, Ca! and 5iaT and their donors can be combined in one vessel. Alternatively, the Ca!Mas reaction can be carried out separately followed by the addition of asacite and 5ate and the corresponding sugar donors in one step. Another method of carrying out the reaction involves the sequential addition of the enzyme and the appropriate donor and carrying out the reaction in one vessel. Combinations of each of the above-described methods can be used to obtain compounds according to the invention.

In the conjugates according to the invention, and in particular, glycopegylated M-linked glycans, the "bio-modifying group" cluster can be attached to Cya in the α-2,6- or α-2,3Z-bond position.

The method according to the invention also includes the modification of incompletely glycosylated peptides, which are produced by recombinant methods. In the method according to the invention, carried out using a modified sugar, the C2-C5E peptide can then be simultaneously glycosylated and derivatized, for example, with a PEG molecule, a therapeutic agent, or the like. The sugar part of the modified sugar can be a residue that must be appropriately conjugated with an acceptor in a fully glycosylated peptide, or another sugar part with the desired properties.

Peptides 2-C5E modified by methods according to the invention can be synthetic peptides or wild-type peptides produced by known methods, such as site-directed mutagenesis.

Glycosylation of peptides is usually either M-linked or O-linked. An example of M-bonding is the addition 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 sequences recognized by the enzyme to attach the carbohydrate part to the side chain of asparagine. Thus, the presence of any of these tripeptide sequences in the polypeptide leads to the formation of a potential glycosylation site. O-linked glycosylation refers to the addition of one sugar (e.g., M-acetylgalactosamine, galactose, mannose, SISMAs, glucose, fucose, or xylose) to the hydroxy side chain of a hydroxyamino acid, preferably serine or threonine, although 5- hydroxyproline or 5-hydroxylysine.

So, for example, in one of the variants of the invention, 1-C5E is expressed in a mammalian system and modified by treatment with sialidase to remove the final residues of sialic acid followed by PEGylation using 57ZSaiZ and donor PEG-sialic acid.

In another representative embodiment of the invention, 2-C5E expressed in mammalian cells is first treated with sialidase to remove the terminal sialic acid residues, and then subjected to PEGylation using 53CsaI3 and donor PEG sialic acid and sialylation using 573cSai!3 and donor sialic acid . α-C5HE expressed in a mammalian system can also be treated with sialidase and galactosidase to remove sialic acid and galactose residues, and then subjected to galactosylation using a galactose donor and galactosyltransferase, followed by PEGylation using 57Z3CaiZ and a PEG-sialic donor acid

In yet another representative embodiment of the invention, 1-C5E is not first subjected to sialidase treatment, but is subjected to glycoPppPEGylation by carrying out a sialic acid transfer reaction using a "modifying group-sialic acid" cluster and an enzyme such as 5TZCaiZ.

In another representative embodiment of the invention, 2-C5E is expressed in insect cells and modified according to the following procedure: M-acetylglucosamine is first attached to (-C5E using the appropriate donor M-acetylglucosamine and one or more of (pt-II, II, IM and M, and then exposed

PEGylation using donor PEG-galactose and galactosyltransferase. 2-C5E produced in yeast can also be glycopegylated. So, for example, 2-C5E is first treated with endoglycanase to remove glycosyl groups, subjected to galactosylation using donor galactose and galactosyltransferase, and then PEGylated using 5TZ(zaIZ) and donor PEG-sialic acid.

Attachment of glycosylation sites to a peptide or to another structure is usually carried out by introducing a modification into the amino acid sequence so that this sequence contains one or more glycosylation sites. Such attachment can also be carried out by including in the sequence of the peptide (2-C5E one or more molecules presenting the -OH group, and preferably, serine or threonine residues (for sites of O-linked glycosylation). Such attachment can be carried out by introducing a mutation or by carrying out a complete chemical synthesis of the peptide (-C5BE. The amino acid sequence of the C-C5E peptide is preferably modified by making changes at the DNA level, and in particular, by mutating the DNA encoding this peptide in preselected nucleotides to create codons that will to be translated into the required amino acids.Mutation() of DNA is mainly carried out by methods known to specialists.

In a representative embodiment of the invention, the glycosylation site is attached by "shuffling" the polynucleotides. Polynucleotides encoding a candidate peptide can be modulated according to DNA shuffling protocols. DNA shuffling is a process of recursive recombination and mutation carried out using randomized fragmentation of a pool of related genes with subsequent assembly of fragments in a manner similar to the polymerase chain reaction. See for example bietteg,

Rgos. Mai). Asad 5si., PERSON, 91: 10747-10751 (1994); biettegg, Maishte 370: 389-391 (1994) and US Pat.

MoMme5605793, 5837458, 5830721 and 5811238.

The present invention also relates to a method of attaching (or removing) one or more selected glycosyl residues to a peptide followed by conjugation of a modified sugar with at least one of the selected glycosyl residues of this peptide. This variant of the present invention can be used, for example, if it is necessary to conjugate a modified sugar with a selected glycosyl residue, which is either not present in the peptide, or is present in an insufficient amount. So, for example, before attaching the modified sugar to the peptide, the selected glycosyl residue is conjugated to the peptide a-

C5E by enzymatic or chemical addition. In another variant of the invention, before conjugation of the modified sugar, the nature of glycosylation of the glycopeptide is changed by removing the carbohydrate residue from the glycopeptide. See for example, UUO 98/31826.

Addition or removal of any carbohydrate molecules present on the glycopeptide is carried out either chemically or enzymatically. Chemical deglycosylation is carried out, preferably, by treating the pol and peptide variant with a compound of trifluoromethanesulfonic acid or an equivalent compound. Such processing leads to the cleavage of most or all of the sugar, except for the binding sugar (M-acetylglucosamine or M-acetylgalactosamine), but, at the same time, the peptide itself remains intact. Chemical deglycosylation is described in Nakityadaiip eyi ai., Agsy. Viospet. Viorpuz. 259: 52 (1987) and in Edde ei ai., Apai.

Viospet. 118: 131 (1998). Enzymatic cleavage of carbohydrate molecules on polypeptide variants can be carried out with the use of various endo- and exoglycosidases, as described in the work of Tpoiakiga, ai., Meij.

Epgutoi 138: 350 (1987).

Chemical attachment of glycosyl residues is carried out by any known method. Enzymatic addition of sugar molecules is preferably carried out by the methods described here, modified by replacing the modified sugar used in this invention with natural glycosyl links.

Other methods of attaching sugar groups are described in US patents MeMe 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Representative attachment positions for selected glycosyl residues include, but are not limited to, (a) consensus sites for M- and O-glycosylation; (b) terminal glycosyl groups, which are acceptors for glycosyltransferase; (c) arginine, asparagine and histidine; (4) free carboxyl groups; (c) free sulfhydryl groups, such as cysteine groups; (Y) free hydroxyl groups, such as serine, threonine or hydroxyproline groups; (4) aromatic residues such as phenylalanine, tyrosine or tryptophan or (n) the amide group of glutamine.

Representative methods used in the present invention are described in UMO application 87/05330, published September 11, 1987, and in Ariip 8 M/givup, SAS Si. Wow Viospet., pp. 259-306 (1981).

Methods

In addition to the conjugates discussed above, the present invention relates to methods of obtaining these and other conjugates. In addition, the present invention relates to methods of preventing, treating or alleviating a pathological condition by administering a conjugate according to the invention to a patient at risk of developing such a disease or to a patient already suffering from the specified disease.

For example, this invention relates to a method of obtaining a covalent conjugate consisting of a selected molecule and a C-C5E peptide.

In representative embodiments of the invention, the specified conjugate is formed by a water-soluble polymer, a therapeutic molecule, a targeted delivery molecule, or a biomolecule, and a glycosylated or non-glycosylated peptide (3-C5E. The polymer, therapeutic molecule, or biomolecule is conjugated to a peptide (-C5E using a glycosylated of the linker group, which is located between them, and covalently linked to the indicated peptide and to the modifying group (for example, with a water-soluble polymer). This method includes contacting the C2-C5E peptide with a mixture containing a modified sugar and an enzyme, for example, a glycosyltransferase, which mediates the conjugation of the modified sugar with a substrate (eg, peptide, aglycone, glycolipid).This reaction is carried out under conditions suitable for the formation of a covalent bond between the modified sugar and the 1-C5E peptide.

The acceptor peptide (3-C5E is usually synthesized de pomo or recombinantly expressed in prokaryotic cells (for example, in bacterial cells such as E. soy) or in eukaryotic cells such as mammalian, yeast, insect, fungal or plant cells. Peptide 1- C5E can be either a full-length protein or a fragment thereof. In addition, the 2-C5E peptide can be a wild-type peptide or a mutated peptide. In a representative embodiment of the invention, the peptide (3-C5E includes a mutation that adds one or more M- or O- of linked glycosylation in the peptide sequence.

The method according to the invention also provides for the modification of incompletely glycosylated peptides (3-C5E, which are produced by recombinant methods. Many recombinantly produced glycoproteins are incompletely glycosylated, and have available carbohydrate residues that may have undesirable properties, for example, immunogenicity, that is, they can be recognized by RES. In a method according to the invention, in which a modified sugar is used, this peptide can then be simultaneously glycosylated and derivatized, for example, with a water-soluble polymer, a therapeutic agent, or the like. The sugar moiety of the modified sugar can be a residue that must be appropriately conjugated with an acceptor in a fully glycosylated peptide, or another sugar moiety with the required properties.

Representative methods of obtaining modifying peptides used in the present invention are described in applications MO 04/099231, UMO 03/031464 and works cited therein.

In its representative version, the present invention relates to a method of obtaining PEGylated a-C5E containing the molecule: no o nos o

He is 8-9 mn-v where O is -OH and B'-I-NM. The symbol C means B1-I- or -C(O)(C1-C6)alkyl. IN! is a group containing a linear or branched polyethylene glycol residue. Symbol | means a linker selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl.

In general terms, if О represents OH, then b represents B!-I, and if C represents -С(О)(С1-

C) alkyl, then O represents B'-I -MH-. The method according to the invention includes: (a) contacting the C-C5E peptide, which is a substrate, with the PEG-sialic acid donor and an enzyme capable of transferring a PEG-sialic acid molecule from the donor to the P-C5E peptide-substrate.

A representative PEG-sialic acid donor is a nucleotide sugar with the formula:

he would be 5-1. z-ya and I on Fi kn; and an enzyme that transfers PEG-sialic acid to the amino acid or glycosyl residue of the peptide C-

C5E under conditions conducive to such transfer.

In one of the variants of the invention, the peptide substrate 1-C5E is expressed in the host cell before the formation of the conjugate according to the invention. A representative host cell is a mammalian cell. In other embodiments of the invention, the host cell is an insect, plant, bacterial or fungal cell.

The method described here can be applied to each of the Y4-SOE conjugates described in the previous sections.

Peptides 2-C5E modified by methods according to the invention can be synthetic peptides or wild-type peptides produced by known methods, such as site-directed mutagenesis.

Glycosylation of peptides is usually either M-linked or O-linked. An example of M-bonding is the addition 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 sequences recognized by an enzyme that mediates the attachment of a carbohydrate part to the side chain of asparagine. Thus, the presence of any of these tripeptide sequences in the polypeptide leads to the formation of a potential glycosylation site. O-linked glycosylation refers to the addition of one sugar (e.g., M-acetylgalactosamine, galactose, mannose, (3IcSMAs, glucose, fucose, or xylose)) to the hydroxy side chain of a hydroxyamino acid, preferably serine or threonine, although rare or unnatural amino acids, such as 5-hydroxyproline or 5-hydroxylysine.

Attachment of glycosylation sites to a peptide or to another structure is usually carried out by introducing a modification into the amino acid sequence so that this sequence contains one or more glycosylation sites. Such attachment can also be carried out by including in the sequence of the peptide (2-C5E one or more molecules presenting the -OH group, and preferably, serine or threonine residues (for sites of O-linked glycosylation). This attachment can be carried out by by introducing a mutation or by carrying out a complete chemical synthesis of the peptide. will be translated into the required amino acids.DNA mutation(s) are carried out, preferably, by methods known to specialists.

In a representative embodiment of the invention, the glycosylation site is attached by "shuffling" the polynucleotides. Polynucleotides encoding a candidate peptide can be modulated according to DNA shuffling protocols. DNA shuffling is a process of recursive recombination and mutation, carried out with the help of randomized fragmentation of a pool of related genes with subsequent assembly of fragments in a manner similar to the polymerase chain reaction. See for example, bietteg", Rhos.

Mai! Asad 5si., BOTH, 91: 10747-10751 (1994); biettegg, Mayte 370: 389-391 (1994) and US Patents 5,605,793, 5,837,458, 5,830,721, and 5,811,238.

Representative methods of attaching or removing glycosylation sites and adding or removing glycosylated structures or substructures are described in detail in UMO applications 04/099231, UYU 03/031464 and related US applications and PCT applications.

The present invention also relates to a method of attaching (or removing) one or more selected glycosyl residues to a peptide (14-C5E with subsequent conjugation of a modified sugar with at least one of the selected glycosyl residues of this peptide. Such methods can be used, for example, in in the event that it is necessary to conjugate a modified sugar with a selected glycosyl residue, which is either not present in the peptide, or is present in an insufficient amount. So, for example, before attaching the modified sugar to the peptide, the selected glycosyl residue is conjugated to the 2-C5E peptide by enzymatic or chemical attachment. In another variant of the invention, before conjugation of the modified sugar, the nature of glycosylation of the glycopeptide is changed by removing a carbohydrate residue from the glycopeptide. See, for example, UUO 98/31826.

Representative positions of attachment for selected glycosyl residues are, but are not limited to, (a) consensus sites for M-linked glycosylation and sites for O-linked glycosylation; (B) terminal glycosyl groups, which are acceptors for glycosyltransferase; (c) arginine, asparagine and histidine; (4) free carboxyl groups; (c) free sulfhydryl groups, such as cysteine groups; (Y) free hydroxyl groups, such as serine, threonine or hydroxyproline groups; (94) aromatic residues such as phenylalanine, tyrosine or tryptophan or (GP) the amide group of glutamine. Representative methods used in the present invention are described in UUO 87/05330, published September 11, 1987, and in Ariip 5 Ugivop, SAC Art. Neu. Viospet., pp. 259-306 (1981).

PEG-modified sugars are conjugated with a glycosylated or non-glycosylated peptide using an appropriate enzyme that mediates such conjugation. At the same time, it is preferable that the concentrations of the modified donor sugar, enzyme and acceptor peptide are chosen so that the glycosylation reaction proceeds until the desired degree of acceptor modification is achieved. Although the discussion below is specific to sialyltransferase, it may in fact also apply to other glycosyltransferase reactions.

Some methods using glycosyltransferases for the synthesis of the required oligonucleotide structures are well known and, in fact, can be used in this invention. Such methods are described, for example, in UMO 96/32491, Mo ei ai., Riga Arri. Spent 65: 753 (1993), in US patents MoMe 5352670, 5374541, 5545553 | in U.S. Patent Nos. 3,993,336 and 6,440,703, which are incorporated herein by reference.

This invention is carried out using one glycosyltransferase or a combination of glycosyltransferases. For example, a combination of sialyltransferase and galactosyltransferase can be used. In these variants using more than one enzyme, the indicated enzymes and substrate are preferably combined to obtain the initial reaction mixture, or the indicated enzymes and reagents for the second enzymatic reaction are added to the reaction medium immediately after the first enzymatic reaction is completely completed or almost completed . When two enzymatic reactions are carried out sequentially in one vessel, the overall yield can be improved compared to the yield achieved by procedures in which intermediate molecules are isolated. In addition, it simplifies the removal and disposal of excess solvents and byproducts.

In a preferred embodiment of the invention, the first and second enzymes are glycosyltransferase. In another preferred embodiment of the invention, one of such enzymes is endoglycosidase. In another preferred embodiment of the invention, more than two enzymes are used to assemble the modified glycoprotein according to the invention. These enzymes are used to modify the saccharide structure on the C-C5E peptide in any position before or after the addition of the modified sugar to this peptide.

In another variant of the invention, the specified method includes the use of one or more exo- or endoglycosidases. A glycosidase is usually a mutant, which is designed so that glycosyl bonds are formed in it, rather than destroyed. A mutant glycanase usually involves replacing an acidic amino acid residue of the active site with another amino acid residue. For example, if the endoglyconase is endo-H, then the replaced residues of the active center are usually Avr in position 130, sy in position 132, or their combination. Such amino acids are usually replaced by serine, alanine, asparagine or glutamine.

The mutant enzyme catalyzes a reaction, of course, in the synthesis stage, which is analogous to the reverse reaction of the hydrolysis stage by endogluconase. In these embodiments of the invention, the glycosyl donor molecule (for example, the desired oligo- or monosaccharide structure) contains a leaving group, and this reaction proceeds with the attachment of the donor molecule to the SISMAc residue on the protein. For example, the leaving group may be a halogen such as fluoride. In other variants of the invention, the group to be removed is an Aβ or an Aβ peptide molecule. In other variants of the invention, the SISMAc residue on the glycosyl donor molecule is modified. So, for example, the SISMAs residue may contain a 1,2-oxacholine group.

In a preferred embodiment of the invention, each of the enzymes used to obtain the conjugate according to the invention is present in a catalytic amount. The catalytic amount of a specific enzyme varies depending on the concentration of the enzyme substrate, as well as on the reaction conditions, such as temperature, reaction time, and pH value. Methods of determining the catalytic amount of this enzyme for pre-selected substrate concentrations and reaction conditions are well known to specialists.

The temperature at which the above-mentioned reaction is carried out can vary from a temperature slightly above the freezing point to the denaturation temperature of most sensitive enzymes. The preferred temperature ranges from about 0"C to 55"C, and more preferably from about 207C to 37"C. In another representative embodiment of the invention, one or more stages of the method according to the invention are carried out at an elevated temperature using a thermophilic enzyme.

The reaction mixture is maintained for a period of time sufficient for glycosylation of the acceptor, as a result of which the desired conjugate is formed. In most cases, some of these conjugates can be detected after several hours; moreover, the amounts released are usually obtained in 24 hours or less. It is obvious to those skilled in the art that the reaction rate depends on a number of different factors (for example, enzyme concentration, donor concentration, acceptor concentration, temperature, solvent volume) that can be optimized for the selected system.

The present invention also relates to the large-scale production of modified peptides.

As used herein, the term "large-scale" refers to obtaining at least one gram of the final purified conjugate.

As discussed below, the present invention is illustrated by the example of conjugation of modified sialic acid molecules with a glycosylated peptide. A representative modified sialic acid is labeled with PEG. The following discussion regarding the use of PEG-modified sialic acid and glycosylated peptides is provided by way of illustration only, and is not intended to limit the present invention to the conjugation of these two components. Those skilled in the art will understand that such a discussion, in fact, also applies to the addition of modified glycosyl groups that are not sialic acid.

In addition, this discussion applies equally to the modification of the glycosyl link by agents other than PEG, for example, including other PEG molecules, therapeutic molecules and biomolecules.

An enzymatic method can be used to selectively introduce PEGylated or PPGylated carbohydrates into a peptide or glycopeptide. This method involves the use of a modified sugar containing PEG, PPG or a masked reactive functional group and their combination with the appropriate glycosyltransferase or glycosynthase. By selecting a glycosyltransferase that forms the desired carbohydrate bond and using a modified sugar as a donor substrate, PEG or PPG can be introduced directly into the peptide backbone of 2-COE, into sugar residues present in the glycopeptide, or into sugar residues that have been attached to the peptide.

On the peptide 2-C5E, which is modified by methods according to the invention, there is an acceptor for sialyltransferase either in the form of a natural structure or in the form of a structure obtained by recombinant, enzymatic or chemical methods. Suitable acceptors are, for example, galactosyl acceptors such as szaIr1,4c3IisMAS, aiIr1,4cSamMas, saIr1,ZsaMmAs, lacto-M-tetraose, Cyrv1,Z(IsMAs, sba!B1,ZaGa, sa!r1,bsisMAS, sa!r1 .

In one of the variants of the invention, the acceptor for sialyltransferase is present on the glycopeptide modified after ip mimo synthesis of the glycopeptide. Such glycopeptides can be sialylated using the claimed methods without prior modification of the nature of glycosylation of the glycopeptide. Alternatively, the methods according to the invention can be used to sialylate a peptide that does not include a suitable acceptor, while first the peptide (2-C5E is modified by known methods so that it includes an acceptor. In a representative variant of the invention, the residue (za!Mas is attached under action (SzamMAs - transferases.

In a representative variant of the invention, the assembly of the galactosyl acceptor is carried out by attaching the galactose residue to the corresponding receptor bound to the C2-C9E peptide, for example, cIsMas. This method involves incubating the modified peptide, 2-COE, with a reaction mixture containing an appropriate amount of galactosyltransferase (eg, daIri,3 or da!/B1,4) and an appropriate galactosyl donor (eg, UDP-galactose). The reaction mixture is left to undergo the reaction, basically until its completion, or alternatively, this reaction is completed by adding a preselected amount of galactose residue. It is obvious to those skilled in the art that there are other methods of collecting the selected saccharide acceptor.

In yet another variant of the invention, the glycopeptide-linked oligosaccharides are first "cut off" either completely or partially, thereby exposing the sialyltransferase acceptor or part to which one or more suitable residues can be attached, resulting in a suitable acceptor. Enzymes such as glycosyltransferases and endoglycosidases can be used to carry out attachment and cleavage reactions (see, for example, US patent Me5716812).

As discussed below, the method according to the invention is illustrated by the use of modified sugars containing a PEG molecule attached to them. This discussion is provided to better illustrate the present invention. Those skilled in the art will appreciate that this discussion applies equally to those embodiments of the invention in which the modified sugar contains a therapeutic molecule, biomolecule, or the like.

In a representative variant of the invention, in which the carbohydrate residue is "cut off" before the addition of the modified sugar, the high molecular weight mannose is "cut off" to obtain the biantennary structure of the first generation. A modified sugar carrying a PEG molecule is conjugated to one or more sugar residues exposed as a result of "uTruncation". In one example, the PEG moiety is added using

of the SiISMAs-part conjugated with this PEG-part. The modified SISMAs are attached to one or two terminal mannose residues of the biantennary structure. Alternatively, unmodified CSMAs can be attached to one or both ends of branched molecules.

In another embodiment of the invention, the PEG moiety is attached to one or both terminal mannose residues of the biantenna structure using a modified sugar having a galactose residue that is conjugated to a SISMAc residue attached to the terminal mannose residues. Alternatively, unmodified Sai can be attached to one or both terminal residues of SISMAs.

In another version of the invention, the PEG part is attached to the remaining Ca! using modified sialic acid.

In another variant of the invention, the high-molecular mannose structure is "cut" to the mannose from which the biantennary structure branches. In one of the examples, the PEG molecule is attached with the help of SiISMAs modified by the polymer. Alternatively, the unmodified (3ISMAc) is attached to the mannose, followed by the attachment of a C1 with a bound PEG molecule. In another embodiment of the invention, the unmodified c1IcSMAc and Ca! residues are sequentially attached to the mannose, and then a sialic acid molecule modified by the group

PEG.

In another representative embodiment, the high molecular weight mannose structure is "trimmed" to the SisMAs to which the first mannose is attached. SisMAs is conjugated to the Sai residue carrying a PEG molecule. Alternatively, unmodified (za!) is attached to (c3ISMAs) and then a water-soluble sugar-modified sialic acid is attached. In yet another example, the terminal (ISMAs) is conjugated to Cya, and then the CisSMAs is subjected to fucosylation with a modified fucose bearing a PEG group.

High-molecular-weight mannose can also be cut to the first CisSMAs attached to Avhp of this peptide. In one example, the SisSMAs residue of SisMas-(Pys)a is conjugated to (3ISMAs) carrying a water-soluble polymer. In another example, the SisMas residue of SisMas-(Pys)a is modified with Sai carrying a water-soluble polymer.

In another variant of the invention, SISMAs are modified with Sai, with subsequent conjugation of sialic acid modified with a PEG molecule to Sai.

Other representative variants are described in the publications of joint US patent applications: 20040132640; 20040063911; 20040137557; in US patent applications: MoMe10/369079, 20/410913, 10/360770, 10/410945 and

PCT/O502/32263, each of which is included in this description by reference.

These examples presented above are provided only for the purpose of illustrating the effectiveness of the methods described herein.

According to the methods described here, it is possible to "cut" and build up carbohydrate residues to obtain, in fact, any desired structure. The modified sugar can be attached to the ends of the carbohydrate molecule described above, or it can occupy an intermediate position between the peptide backbone and the end of the carbohydrate.

In a representative embodiment of the invention, the present sialic acid is cleaved from the -C5E glycopeptide using sialidase, thereby unmasking all or most of the downstream galactosyl residues. Alternatively, the peptide or glycopeptide is labeled with galactose residues or an oligosaccharide residue that is terminal in the galactose chain. For the addition of modified sialic acid after unmasking or addition of galactose residues, a suitable sialyltransferase is used. This method is illustrated in diagram I.

Scheme with I Glycoprotein and moba! / Y

Sialyltransferase was used

SMR-5A-5-NNSOSNIchNA-RES(PRO)

ZA-5-NNSOSNOaMN-REO

Glycoprotein with - AI-5A-B-MNSOSNoMN-REO ba!

Gvmnsosnunntes

In yet another method, systematized in Scheme 2, a masked reactive functional group is present on sialic acid. Preferably, this masked reactive group is not affected by the conditions used to attach the modified sialic acid to C-SOBE.

After covalent attachment of the modified sialic acid to the peptide (C-C5E, the masking is removed and the 2-C5E peptide is conjugated with an agent such as PEG. This agent is specifically conjugated to the peptide by its reaction with an unmasked reactive group on the modified sugar residue .

Scheme 2

I 0 Glycoprotein p «fo | in ABMNSOSN, V-YAEI

Her. sne Y y I no. o on u Sialyltransferase Fo vne» vovni On

M vainsosnov-ek

Y M ZA-5-MNSOSNV-RES

Glycoprotein a 1. dithiothretoyl

Father Mch. 2. PEG-halide or PPG-halide AI-ZA-5-MNSOSN28Z-RES t

BA-5-NNSOSN25-REV

Any modified sugar described herein can be used with its corresponding glycosyltransferase, depending on the terminal sugar of the oligosaccharide side chains of the glycopeptide (Table 1). As discussed above, the terminal sugar of a given glycopeptide is required for administration

PEGylated structure, can be introduced in the process of natural expression, or it can be produced after expression under the action of a suitable glycosidase, glycosyltransferase or a mixture of glycosidase and glycosyltransferase.

Table I - x. 37 h-v, Az still at Vgh

You nn | be "Ah! I i s I I -E. tuesday

Ro ka fu na na s i

Derivatives of UDF-galactose h Derivatives of UDF-galactosamine (if A-MH, B, can be acetyl) o i. min, fluff is y z a a b:2 nn sa yan ve ; 4 no kA) -Ya, no ro ki Ba o n

UDF-galactose derivatives UDF-galactosamic derivatives (if A-MH, Ki can be acetyl)

Кк хк; "skh r. in 5 o-i Mona ion 9 oo i kn, "on mo u in dv, "

Hey 2 but he is in the village.

Derivatives of UDF-galactose Derivatives of UDF-frucose х Оу МН, 5, sno Me.

Me HE X AH; VH, o-n.O, 8, My, K-K.

M ya-a I SUPER ticker M, M,

M "RES, paprika,, pRES

In another representative variant of the invention, the UDP-galactose-PEG conjugate is subjected to interaction with B1,4-galactosyltransferase of cow's milk, which allows to transfer the modified galactose to the corresponding final M-acetylglucosamine structure. The terminal CIsMAS residues on the glycopeptide can be produced by expression, which can be carried out in expression systems such as mammalian, insect, plant or fungal cells, but, if necessary, they can also be produced by treating the glycopeptide with sialidase and/or glycosidase and / or glycosyltransferase.

In another embodiment of the invention, a CisMAs transferase, such as CMT1-5, is used to transfer

PEGylated SiISM on the terminal mannose residue of the glycopeptide. In another representative embodiment of the invention, M- and/or O-linked glycan structures are enzymatically removed from the glycopeptide, as a result of which an amino acid or terminal glycosyl residue becomes available, which is then conjugated with a modified sugar. So, for example, in order to ensure access to the final (3ISMAs in the form of (IsMAs-Avp) on the glycopeptide, endoglycanase is used to remove the M-linked structures of the glycopeptide. To introduce functional PEG-galactose into the available (3ISMAs) UDP-Ca-PEG and the appropriate galactosyltransferase.

In an alternative variant of the invention, the modified sugar is attached directly to the peptide backbone (-C5E using a glycosyltransferase, which is known to transfer sugar residues to the peptide backbone. Such a representative variant is presented in Scheme 3. Examples of glycosyltransferases used in the implementation of this invention are , but not limited to,

Ca!Mas-transferases (CaIMas TI-14), (cMAs-transferases, fucosyltransferases, glucosyltransferases, xylosyltransferases, mannosyltransferases, etc. The use of this method allows directly attaching a modified sugar to peptides that do not contain any carbohydrates, or alternatively , to the glycopeptides present. In both cases, the attachment of the modified sugar occurs in specific positions on the peptide backbone, as determined by the specificity of the glycosyltransferase for the substrate, but not randomly, as occurs in the process of modifying the peptide backbone of a protein by chemical methods. In proteins or glycopeptides, in which there is no peptide sequence that is a substrate for glycosyltransferase, a number of agents can be introduced by constructing the appropriate amino acid sequence in the polypeptide chain.

Scheme C o Protein or glycoprotein in m), in 2 A zymn-Co(snU,chN-REE and otv in) bagchAs transferase mon (bvimAs TZ) IMNA-CO(SNgMN-REO rve

In each of the representative embodiments of the invention described above, after the conjugation of the modified sugar with the peptide, one or more additional stages of chemical or enzymatic modification may be carried out. In a representative embodiment of the invention, an enzyme (for example, fucosyltransferase) is used to attach a glycosyl link (for example, fucose) to the final modified sugar attached to the peptide (1-C5P. In another example, an enzymatic reaction is used to "cap" sites to which the modified sugar cannot be conjugated. Alternatively, a chemical reaction is performed to change the structure of the conjugated modified sugar. Thus, for example, the conjugated modified sugar is subjected to interaction with agents that stabilize or destabilize its bond with the peptide component to which the modified sugar is attached. Those skilled in the art know that that there are a number of enzymatic and chemical procedures that can be used in the methods according to the invention at the stage carried out after the conjugation of the modified sugar with the 2-C5E peptide. Further improvement of the "modified sugar - peptide" conjugate is within the scope of this invention.

Enzymes

In addition to the enzymes discussed above in connection with obtaining an acyl-linked conjugate, the nature of glycosylation of the conjugate and the initial substrates (eg, peptides, lipids) can be improved, adjusted, or otherwise modified by methods using other enzymes. Peptide and lipid remodeling methods using enzymes that transfer a donor sugar to an acceptor are discussed in great detail in OeRgeev, UMO 03/031464 A2, published April 17, 2003. A brief description of selected enzymes used in the method of the invention is provided below.

Glycosyltransferases

Glycosyltransferases catalyze the stepwise attachment of an activated sugar (donor MOP or MMP sugars) to a protein, glycopeptide, lipid, or to a glycolipid or to the non-reducing end of a growing oligosaccharide. M-linked glycopeptides are synthesized with the help of transferase and the lipid-bound donor oligosaccharide YuoI-PP-MASiegSyszMapo by block transfer followed by "sub-alignment" of the backbone. In this case, the nature of the "bone" saccharide will be somewhat different from the nature of the subsequently attached groups.

A very large number of glycosyltransferases are known to those skilled in the art.

The glycosyltransferase used in the present invention can be any glycosyltransferase, provided that it is capable of utilizing the modified sugar as a donor sugar.

Examples of such enzymes are glycosyltransferase of the Leloir pathway, such as galactosyltransferase, M-acetylglucosaminyltransferase, M-acetylgalactosaminyltransferase, fucosyltransferase, sialyltransferase, mannosyltransferase, xylosyltransferase, glucurononyltransferase, etc.

For the enzymatic synthesis of saccharides, which involves glycosyltransferase reactions, glycosyltransferase can be cloned or isolated from any source. Many cloned glycosyltransferases, as well as the polynucleotide sequences encoding them, are known. See, for example, "pe

MUMUMU Sbcide To Siopei Siusozuigapeieegaze5" / (peer//Lumliu.mei. so. iK/GOeM/di. diide.pit). Amino acid sequences of glycosyltransferases and nucleotide sequences encoding glycosyltransferases, from which these amino acid sequences may be derived, are also in various available databases, including

Seprapk, Zumz5-Rhoi, EMVI. etc.

Glycosyltransferases that can be used in the methods of the invention include, but are not limited to, galactosyltransferases, fucosyltransferases, glucosyltransferases, M-acetylgalactosaminyltransferases, M-acetylglucosaminyltransferases, glucuronyltransferases,

sialyltransferases, mannosyltransferases, (glucuronic acid)transferases, (galacturonic acid)transferases and oligosaccharyltransferases. Suitable glycosyltransferases are glycosyltransferases derived from eukaryotes as well as from prokaryotes.

DNA encoding glycosyltransferases can be obtained by chemical synthesis, by screening mRNA reverse transcripts from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by performing combined procedures. Screening of mRNA or genomic

DNA can be carried out using oligonucleotide probes generated from the gene sequence of glycosyltransferases. Probes can be labeled with a detectable group, such as a fluorescent group, a radioactive atom, or a chemiluminescent group according to known procedures, and used in standard hybridization assays. Alternatively, gene sequences of glycosyltransferases can be obtained by polymerase chain reaction (PCR) using oligonucleotide PCR primers produced from the gene sequence of glycosyltransferases. See US patent Me4683195, Miiii5 ei ai. and a patent

USA Me4683202, Myiiv.

Glycosyltransferase can be synthesized in host cells transformed with vectors 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 replicating DNA construct in which the DNA sequence encoding the glycosyltransferase enzyme is operably linked to appropriate regulatory sequences capable of expressing the glycosyltransferase enzyme in the appropriate host. The need for the presence of such regulatory sequences varies depending on the selected host and the selected method of transformation. In general, regulatory sequences include a transcriptional promoter; optional operator sequence for regulation of transcription; the sequence encoding the corresponding mRNA ribosome binding sites; and sequences regulating the termination of transcription and translation. Vectors do not need an expression regulatory domain for amplification. All that is required for a given vector is the ability to replicate in the host, usually communicated by the replication region, and the presence of a selective gene to facilitate identification of transformants.

In a representative embodiment of the invention, a prokaryotic enzyme is used.

Glycosyltransferases are enzymes involved in the synthesis of lipooligosaccharides (VOCs), which are produced by many gram-negative bacteria (Rhesiop ei ai., Spiisa! Nemiemuv ip Misgobioiyuodo 23(3): 139-180 (1996)).

Such enzymes include, but are not limited to, the proteins of the ela operon of such species as E. soii and Zaitopeii

Urpitigitis, which include p1,6-galactosyltransferase and B1,3-galactosyltransferase (see, for example, EMVI.

Asseviop MeM80599 and M86935 (E. soii), EMVI. Asseyop Me556361 (5. turpitipit)), glucosyltransferase (Zm/iv8-

Rgoi Asseviop MeR25470 (E. soybean), B1,2-glucosyltransferase (pa) (Zm/i585-Rgoi Asseviop MeR27129 (E. soybean) apa

Zmi55-Rgoi Asseviop MeR 19817 (5. (uUrpitigisht)) and B81,2-M-acetylglucosaminyltransferase (Ppak) (EMVI. Asseviop

Me000039 (E. soyii). Other glycosyltransferases with known amino acid sequences are enzymes that are encoded by operons such as pav and have been characterized in microorganisms such as KierziePa rpeitopiae, E. soii, ZaiItopeiPa iurpitipita, ZaiItopepapa epiegisa, Uegzipia epiegosoiiisa, Musobasieepyi t Iergozit, and the PI operon of Rzeidotopavz aedygis .

An enzyme suitable for use in this invention are also glycosyltransferases involved in the formation of structures containing lacto-M-neotetraose, O-galactosyl-B-1,4-M-acetyl-O-glucosaminyl-B-1 ,3-O-galactosyl-VD-1,4-ХО-glucose and the trisaccharide sequence of blood group RK O-galactosyl-p-1,4-0-galactosyl-p-1,4-O-glucose, and which were identified in the VOCs of the pathogens Meyzepa dopogpoeae and M. tepidiknaiv mucosa (5spogep eyi a!., U. Med. Mysgobio!. 41: 236-243 (1994)). The genes of M. tepipoyaiv and M. dopogpoyae, which encode glycosyltransferases involved in the biosynthesis of these structures, were identified in immunotypes! Zi11 M. tepipdiinaiv (deppipdv ei ai., Moi. Misgobio!. 18: 729-740 (1995)) and in mutant Eb2 M. dopogpoyeae (SoivpisN, 9. Ehr.

Mea. 180: 2181-2190 (1994)). In M. tepidiaiiv, a locus consisting of three genes, DA, IdIV and CHE, encodes glycosyltransferases necessary for the addition of the last three sugars in the lacto-M-neotetraose chain (Uuakagspik ei ai., 9. Vioi. Spet. 271: 19166 -73 (1996)). Recently, the enzymatic activity of the IdB and dDiA gene products was demonstrated, which is the first direct evidence of their putative glycosyltransferase function (U/akagsnpik ei ai, 9. Vioi. Snet. 271(45): 28271-276 (1996)). In M. dopoitpoeae there are two additional genes: IDIO, which attaches B-O-(CaIMAs) in the C-position of the terminal galactose of the lacto-"neotetraose structure, and IDS, which attaches the terminal α-0-Ca| to the lactose element of the truncated VOC, forming , thereby, the antigenic structure of the RK blood group (SoivnNiisn (1994), see above). In M. tepipdiaiv, a separate immunotype 1 1 also expresses the antigen of the RK blood group, and it was shown that it carries the IDS gene (Cheppipov ei a! ., (1995) see above).

Meizv5eia glycosyltransferases and genes associated with them are also described in US patent Me5545553 (Szoi5sPIISP).

Genes of α1,2-fucosyltransferase and α1,3-fucosyltransferase of Neussobasieg ruogti were also characterized (Mapip eii ai., 9. Wioi. Speth. 272: 21349-21356 (1997)). In this invention, glycosyltransferases Satrupobasieg |e)ipi can also be used (see, for example, per:/aito.spgv-tgv.p/"redgo/SA?U/diyi 42. piti).

Fucosyltransferases

In some embodiments of the invention, the glycosyltransferase used in the method according to the invention is a fucosyltransferase. Fucosyltransferases are known to specialists. Representative fucosyltransferases are enzymes that transfer I-fucose from GDF-fucose to the hydroxy position of the acceptor sugar. The present invention also uses fucosyltransferases that transfer non-nucleotide sugars to an acceptor.

In some embodiments of the invention, the acceptor sugar is, for example, CisSMas in the sa!1p(1-53,4)siSSMasvr group in the oligosaccharide glycoside. Fucosyltransferases suitable for this reaction are sai (1-3,4)cIcSMAsV1-4(1-»3,4)fucosyltransferase (ET ES Me2.4.1.65), which was first isolated from human milk and characterized (see . Vioi Spent 256: 10456-10463 (1981) and Mypel ei ai,, Sap. 9). Spent 59: 2086-2095 (1981)) and Ca1Ir(1-c4)SisSMAsB-a-fucosyltransferases (ETIM, ETU, ETMI), which were detected in human serum. It was also characterized

ETMI (ES Mo2.4.1.65), sialyl-F(2-y3)CyIr(1-»3)SISMAsrB-fucosyltransferase. In addition, the recombinant form of cSaI!r(1--53,4)cIsSMAsr-o(1-»3,4)-fucosyltransferase was characterized (see Oitav eyi a!., Viogd. Med. I etsTseiv 1: 425- 428 (1991).

Other examples of fucosyltransferases are, for example, (1,2-fucosyltransferase (ES Me2.4.1.69).

Enzymatic fucosylation can be carried out by the methods described in the work of Moissope et al., Eig. .).

Viospet. 191: 169-176 (1990) or in US patent Me5374655. Cells used to produce fucosyltransferase also include a system of enzymatic synthesis of GDF-Ffucose.

Galactosyltransferases

In another group of variants of the invention, the indicated glycosyltransferase is galactosyltransferase. Examples of galactosyltransferases are a(1,3)-galactosyltransferase (EC Me2.4.1.151, see, for example, RBarkomugki ei ai.

Tgaperiapi Rgos. 25: 2921 (1993) and Matatoi et al. Mashge 345: 229-233 (1990), cow galactosyltransferase (SepVapk 04989, udogiazze ei ai. 9. Vioi. Speth. 264: 14290-14297 (1989)), mouse galactosyltransferase (SepVapk ot26925; Iagzep ei a)., Rgos. May Asai. si BA 86: 8227-8231 (1995)) and porcine galactosyltransferase (SepVapk 136152; Zschtsmapap ei ai., Ittipodepeiiysv 41: 101-105 (1995)). Another acceptable a1,3-galactosyltransferase is galactosyltransferase, which is involved in the synthesis of antigens of the human blood group B (EC 2.4.1.37, Matatoiyo ei ai., 9. Vioi. Speth. 265: 1146-1151 (1990)). Another example of galactosyltransferase is Ca!-TI skeletons.

In the methods according to the invention, B(1,4)-galactosyltransferases, such as, for example, E.S. 2.4.1.90 (I asMmAs-synthetase) and E.S.2.4.1.22 (lactose-synthetase) (cow (S'Adoziago ei ai., Eig. y. Viospet. 183: 211-217 (1989)), human (Mavigni et al., Viospet. Viorpuz. Nev5. Sottyp. 157: 657-663 (1998)) and mouse galactosyltransferase (Makagama et al., 9. Viospet. 104: 165-168 (1988)), as well as Nez. 38: 234-242 (1994)). Other suitable galactosyltransferases are, for example, α1,2-galactosyltransferases (isolated, for example, from 5spigosasspagotusez Rotbe, Sparei!ei a!., Moi. Vioi. Seii. 5: 519-528 (1994)).

Sialyltransferases

Sialyltransferases are glycosyltransferases of another type, which can be used in recombinant cells and in reaction mixtures according to the invention. Cells producing recombinant sialyltransferases also produce CIMF-sialic acid, which is the donor sialic acid for sialyltransferases. Examples of sialyltransferases that can be used in the present invention are 5TZ3Sani (e.g., rat or mouse 5tTZ3saii), tZzaaiM, TZ(Saui, teza, eT6bsaii!, 5TZSam, 5t6saiI, ztb(za!mAsi, z tTb(zatmAsi! and 5tTb (zatMmAsI! (the nomenclature used to denote sialyltransferases is in Tvtsiya ei ai., Siusobriodu 6b:u-chem (1996)). A representative da(2,3)sialyltransferase, called d(2,3)sialyltransferase (E. S.2.4.99.6), transfers sialic acid to the non-reducing terminal side of the disaccharide or glycoside Ca!B1-»Z3ais. See Map Dep. a!., U. Bioi. Spet. 257: 13845 (1985) and Mep ei ai., U. Vioi. Spet. 267: 21011 (1992). Another representative a2,3- sialyltransferase (ES.2.4.99.4 ) also transfers sialic acid to the non-reducing terminal C of a disaccharide or glycoside, see Neapsk et al., U. Viol. Spect. 254: 4444 (1979) and SiPezrie e al!., 9. Viol. Snet. 267: 21004 ( 1992) Another representative enzyme is Ca!1 -8-1,4-cISMAs-α-2,6-sialyltransferase (see

Kygozama ei ai., Yeshig. U. Viospet. 219: 375-381 (1994)).

For the glycosylation of carbohydrates of glycopeptides, it is preferable that the sialyltransferase has the ability to transfer sialic acid to the sequence Са/!В1,4 СисСМАс-, that is, to the most common sequence, which is the penultimate sequence before the terminal sialic acid on fully sialylated carbohydrate structures (see Table 2 ).

Table 2

Sialyltransferases that use the sequence of саIр1,4с1ИсМас as a substrate-acceptor

MetzpAsag, 3 Sa! 1.3 СсИSMAs

MetspAsag,3 SaIV1,3SsIsMAS

M. dopoppoyeae 1) Soospeveeega)., Vio/Tesppoiodu 9: 1347-1355 (1991) 2) Matatatou vy ai., U. Viospet. 120: 104-110 (1996)

C) Siret ei ai., 9. Vioi. Spent 271: 28271-28276 (1996)

An example of a sialyltransferase used in the claimed methods is 5t13Sanshi, which is also called d(2,3)sialyltransferase (E.S.2.4.99.6). This enzyme catalyzes the transfer of sialic acid to

Sai! of the glycoside saIr1,CsIsMAs or Sa!Ir1,4S1IsMas (see, for example, Uuep eii ai., y. Vioi. Speth. 267: 21011 (1992);

Map dep Ei|pdep ei ai., 9). Vioi Spent 256: 3159 (1991)) and is responsible for sialylation of asparagine-linked oligosaccharides in glycopeptides. This sialic acid binds to Sai, as a result of which an α bond is formed between the two saccharides. Linking (attachment) of saccharides occurs between the 2-position of MepAs and the 3-position of CyY. This specific enzyme can be isolated from the liver of rats (M/eipvivip ei ai., .). Axes

Spent 257: 13845 (1982)); and kKDNK (Zavzaki ei ai., (1993) 9. Vioi. Speth. 268: 22782-22787; Kiadama is

Raptzisop (1994) 3. Vioi. Spent 269: 1394-1401) and the genomic DNA sequence of the human enzyme (Kigadama et al. (1996) 9. Vioi. Speth. 271: 931-938) are known to facilitate the production of this enzyme by recombinant expression. In a preferred embodiment of the invention, the claimed methods of sialylation involve the use of rat 5TZSsanpi.

Other representative sialyltransferases used in the present invention are sialyltransferases isolated from Satruiobrasieg |je)ipi, including C5T-I and C51-0I, and sialyltransferases that create a(2,3)-bonds. See for example, UMO 99/49051.

Other sialyltransferases not listed in Table 2 may also be used for large-scale, inexpensive and efficient glycopeptide sialylation procedures of commercial importance. The simplest test to determine the effectiveness of these other enzymes is to react different amounts of each of these enzymes (1-100 mU/mg protein) from asialo-α-AGF (at 1-10mg/ml) to compare the ability of the sialyltransferase of interest to sialylate glycopeptides with the ability to sialylate cow 5T6sai, 57Z3Sanshi, or both sialyltransferases.

Alternatively, for such an assessment, other glycopeptides or M-linked oligosaccharides, which are enzymatically released from the peptide backbone, can be used instead of asialo-d1-AGF. Sialyltransferases, which have the ability to more effectively sialylate M-linked oligosaccharides of glycopeptides than 576aa!, can be used for large-scale peptide sialylation procedures.

These and other sialyltransferases are shown in Figure 11, which provides a table of sialyltransferases used to transfer to the acceptor the modified sialic acid molecules described in this application and unmodified sialic acid.

Sa!Mas-transferases

M-acetylgalactosaminyltransferases are used for the implementation of this invention, and in particular, for the binding of a molecule of CaIMas with the amino acid of the O-linked glycosylation site of the peptide. Suitable M-acetylgalactosaminyltransferases include, but are not limited to, F(1,33(M-acetylgalactosaminyltransferase), B(1,4)(M-acetylgalactosaminyltransferase) (Madaya et al., 9. Vioi. Speth. 267: 12082-12089 (1992) ) and tn ei a)". 9. Viol. Spec. 269: 15162 (1994)) and polypeptide /M- acetylgalactosaminyl transferase (Nota ei al!., 9. Viol. Spec. 268: 12609 (1993)).

Obtaining proteins, such as the enzyme CaIIMAs Ti-xx from cloned genes by means of genetic engineering, is well known to specialists. See, for example, US Pat. No. 4,761,371. One method involves collecting a sufficient number of samples and then determining the amino acid sequence of the enzyme by M-end sequencing. Such information can then be used to isolate a cDNA clone encoding a full-length (membrane-bound) transferase, which, after its expression in the insect cell line 519, allows the synthesis of a fully active enzyme. The specificity of this enzyme to the acceptor is then determined by semiquantitative analysis of amino acids surrounding known glycosylation sites in 16 different proteins, followed by a study of the glycosylation of synthetic peptides. In this work, it was demonstrated that some amino acid residues are present in glycosylated peptide segments in excessive amounts, and that residues in specific positions surrounding glycosylated serine and threonine residues can affect acceptor efficiency more markedly than other amino acid residues.

Cell-bound glycosyltransferases

In another variant of the invention, the enzymes used in the method according to the invention are cell-bound glycosyltransferases. Although many soluble glycosyltransferases are known (see, for example, patent

USA Me5032519), however, these glycosyltransferases, when they bind to cells, are mainly present in a membrane-bound form. Many of the membrane-bound enzymes studied in this way are mainly considered to be internal proteins, that is, they are not released from the membranes during ultrasound treatment, and detergents are required for their solubilization. Surface glycosyltransferases have been identified on the surfaces of vertebrate and invertebrate cells, and it has also been established that these surface transferases retain catalytic activity under physiological conditions. However, the more well-known function of such cell surface glycosyltransferases is intracellular recognition (Roy, MoiIesshag Arrgoasnez o ZirgaseishShiag Rnepotepa, 1990).

Methods of modifying glycosyltransferases expressed by cells were developed. So, for example, in the work I ageep ei ai., Rgos. Maya). Asad 5si., OBA, 86: 8227-8231 (1989) described a genetic method for isolating cloned cDNA sequences that determine the expression level of oligosaccharide structures of the cell surface and their cognate glycosyltransferases. A cDNA library generated from mRNA isolated from a mouse cell line known to express UDF-galactose: B-O-galactosyl-1,4-M-acetyl-O-glucosaminid-α-1,3-galactosyltransferase, was transfected into CO5-1 cells. Then the transfected cells were cultured and analyzed for α-1,3-galactosyltransferase activity.

In the work of Egapsizso ei a!., Rhos. Mai). Asad 5si., OBA, 89: 2713-2717 (1992) described the method of anchoring D-lactamase on the outer surface of Euspegispia soybean. A three-component hybrid consisting of () the outer membrane protein signal sequence, (ii) the transmembrane portion of the outer membrane protein, and (ii) the full-length mature VD-lactamase sequence was produced and creates an active β-lactamase molecule bound to the cell surface. However, Francisco's method is limited to prokaryotic cell systems and, as the authors know, a complete three-component hybrid is required for this lactamase to function properly.

Sulfotransferases

The present invention also relates to methods of obtaining peptides that contain sulfated molecules, including, for example, sulfated polysaccharides such as heparin, heparan sulfate, carrageenan, and related compounds. Suitable sulfotransferases are, for example, chondroitin-6-sulfotransferase (chicken cDNA, described by Rickitsia et al., 9). Vioi Spent 270: 18575-18580 (1995); SepVapk, reg.Me049915), glycosaminoglycan-M-acetylglucosamine-M-deacetylase/M-sulfotransferase 1 (Oihop eii aiYi., Sepotisv 26: 239-241 (1995);

Sh118918) and glycosaminoglycan-M-acetylglucosamine-M-deacetylase/M-sulfotransferase 2 (mouse cDNA,

described OgeiIapa ei ai., U. Vioi. Spent 269: 2270-2276 (1994) and Epkwzop ei ai., 9). Vioi Snet 269: 10438-10443 (1994); and human cDNA, which is in Seprapk Assayop Mo.OO2304).

Glycosidases

The present invention also relates to the use of wild-type glycosidases and mutant glycosidases. Mutant B-galactosidases have been shown to catalyze the formation of disaccharides by binding ss-glycosyl fluoride to a galactosyl acceptor molecule (M/yNegv5, US patent Mob284494, issued September 4, 2001). Other glycosidases used in the present invention are, for example, p-glucosidases, B-galactosidases, B-mannosidases, B-acetylglucosaminidase, p-M-acetylgalactosaminidase, B-xylosidases, B-fucosidases, cellulases, xylanases, galactanases, mannanases , hemicellulases, amylases, glucoamylases, α-glucosidases, α-galactosidases, α-mannosidases, α-M-acetylglucosaminidase, α-M-acetylgalactosaminidase, α-xylosidases, α-fucosidases and neuraminidase/sialidase.

Immobilized enzymes

The present invention also relates to the use of enzymes immobilized on a solid and/or soluble carrier. In its representative version, the present invention relates to a glycosyltransferase, which is conjugated to PEG using an intact glycosyl linker by methods according to the invention. The "PEG-linker-enzyme" conjugate is attached, but not necessarily, to a solid carrier. The use of enzymes on solid supports in the methods according to the invention simplifies the processing of the reaction mixture and the purification of the reaction product, and also facilitates the recovery of the enzyme. In the methods according to the invention, a glycosyltransferase conjugate is used. Other combinations of enzymes and carriers are known to those skilled in the art.

Hybrid proteins

In other representative variants, hybrid proteins having more than one enzymatic activity involved in the synthesis of the desired glycopeptide conjugate are used in the methods according to the invention. Hybrid polypeptides can consist, for example, of a catalytically active domain of a glycosyltransferase, which is attached to a catalytically active domain of an auxiliary enzyme. The catalytic domain of an auxiliary enzyme can, for example, catalyze the stage of formation of a nucleotide sugar, which is a donor for a glycosyltransferase, or catalyze a reaction involved in the glycosyltransferase cycle.

So, for example, a polynucleotide encoding a glycosyltransferase can be joined, while preserving the reading frame, to a polynucleotide encoding an enzyme involved in the synthesis of a nucleotide sugar. Then the obtained hybrid protein can catalyze not only the synthesis of the sugar nucleotide, but also transfer the sugar group to the acceptor molecule. A hybrid protein can consist of two or more enzymes participating in this cycle and linked to one expressed nucleotide sequence. In other embodiments of the invention, this hybrid protein includes the catalytically active domains of two or more glycosyltransferases. See, for example, patent 5641668. Modified glycopeptides according to the invention can be easily designed and produced using various suitable hybrid proteins (see, for example, patent application PCT/СА98/01180, published on June 24, 1999 under number UMO 99/31224) .

Obtaining modified sugar

In general terms, the sugar moiety or the "sugar moiety - linker" cluster and the PEG group or groups of the "PEG-linker" cluster are linked to each other by means of reactive groups, which, in the course of the addition reaction, are usually transformed into a new organic functional group or in a non-reactive group.

The reactive functional group(s) of the sugar is located in any position of the sugar part.

Of course, for the implementation of this invention, reactive groups and class of reactions are used, which are well known to specialists in the field of bioconjugate chemistry. At this time, the most preferred classes of reactions with reactive sugar groups are reactions that proceed under relatively mild conditions. Such reactions include, but are not limited to, nucleophilic substitutions (for example, reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (for example, reactions with enamines), and addition reactions with the formation of carbon-carbon and carbon-heteroatom multiple bonds connections (for example, reaction

Michael, Diels-Alder addition reaction). These and other relevant reactions are discussed, for example, in the publications of Magsp, Admapsei Ogdapis Spitivigu, Za Ed., dopp UMieeu and ope, Mem MogKk, 1985; Neptaphzopus,

Viosopitsdase Tespidnev5, Asadetis Rgev5, Zap Oiedo, 1996 and in Geepeu vy ai., Modiysayop oi Rgoieipv; Admapsez ip

Spetivigu Zepez, MoiI.198, Ategisap Spetisa! bosieiu, UMazPpipdiyup, O.S., 1982.

Reactive functional groups branching from the sugar nucleus or modifying groups used include, but are not limited to: (a) carboxyl groups and their various derivatives including, but not limited to, /-M-hydroxysuccinimido esters, M-hydroxybenzotriazole ester, acid halides , acylimidazoles of acids, thioesters, p-nitrophenyl ethers, alkyl, alkenyl, alkynyl and aromatic esters; (5) hydroxyl groups, which can be transformed, for example, into esters, into simple esters, into aldehydes, etc.; (c) haloalkyl groups where the halide may then be replaced by a nucleophilic group such as, for example, an amine, carboxylate anion, thiol anion, carbanion or alkoxide ion, with covalent attachment of the new group to a functional group such as a halogen atom; (4) dienophilic groups that can participate in Diels-Alder reactions, such as, for example, maleimido groups; (c) aldehyde or ketone groups which may then be derivatized by the formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or by mechanisms such as the Grignard addition reaction or the alkyllithium addition reaction; () sulfonyl halide groups for further reaction with amines, for example, with the formation of sulfonamides; (9) thiol groups, which can, for example, be converted into disulfides or react with acyl halides; (n) amino groups or sulfhydryl groups, which can be, for example, acylated, alkylated or oxidized;

() alkenes that can undergo, for example, cycloaddition reactions, acylation, addition reactions

Michael, etc.; and (I) epoxides that can react, for example, with amines and hydroxyl compounds.

Reactive functional groups can be chosen so that they do not participate in the reactions, or do not interfere with the reactions that are necessary to assemble the reactive sugar nucleus or modifying group. Alternatively, a reactive functional group may be protected from participating in the reaction by the presence of a protecting group. A method of protecting a specific functional group is well known to those skilled in the art to prevent its participation in a selected series of reactions carried out under certain conditions. Examples of appropriate protection groups can be found, for example, in the guidelines of Stgeeepe ei ai., Rgoyesiime Stoyrz ip Ogdapis

Zupipeviv, Chuopp UMieu 5 5op5, Mem Moi, 1991.

A number of specific examples of modified sugars that may be used in the practice of this invention are discussed below. In representative embodiments of the invention, a sialylic acid derivative is used as a sugar core to which a modifying group is attached. The discussion of sialic acid derivatives is provided for illustration purposes only and should not be construed as limiting the scope of the invention. Those skilled in the art will appreciate that various other sugar groups can be activated and derivatized by methods similar to the sialic acid methods described by way of example. Thus, for example, there are many methods suitable for the modification of galactose, glucose, M-acetylgalactosamine and fucose, not to mention other sugar substrates, and such modification can be easily carried out by known methods. See example,

Einaiparbi eyi a, Ssht. Honey. Spent 6: 93 (1999) and Snageg Yei a!., U. Oga. Spent 65: 24 (2000)).

In a representative embodiment of the invention, the 2-C5E peptide modified by the method according to the invention is a glycopeptide that is produced in mammalian cells (for example, in CHO cells) or in a transgenic animal, and therefore it contains M- and/or O-chains of oligosaccharides that are incompletely sialylated.

Glycopeptide oligosaccharide chains that do not contain sialic acid but contain a terminal galactose residue may be PEGylated, PPGylated, or otherwise modified with a modified sialic acid.

As shown in scheme 4, aminoglycoside 1 is treated with an active complex ester of a protected amino acid derivative (for example, glycine), as a result of which the amine residue of the sugar is transformed into the corresponding protected amide product of amino acid addition. This addition product is treated with aldolase and α-hydroxycarboxylate 2 is obtained. Compound 2 is converted into the corresponding CMF derivative under the action of CMF-SK synthetase with subsequent catalytic hydrogenation of the CMF derivative and compound 3 is obtained. The amine introduced as a result of the formation of the addition product of glycine is used as a site addition of PEG by reaction of compound 3 with an activated PEG or PPG derivative (eg, PEG-C(O)MH5, PEG-OC(O)O-p-nitrophenyl), resulting in compounds such as 4 or 5, respectively.

Scheme 4

And he 1. TsMF-SK-synthetase, CTF "VD Kenlsallv., pyruvate "Z deuet z nougas 7777 er N shi ; sho le oche -y still no Te toDnne no he u S o on that ---- M. g g"Mma BUT he

RESy vv ore mo on vet zer n i -schl z

SMR-FOR-UMNSOSNINA-REV theo Logos SMR-FOR-ZNNSOSNOMNO

SMR. ZA-5YANSOSNIM NA--S(OYU-REO

Table C shows representative examples of molecularly derivatized sugar monophosphates

PEG. Some of these compounds of Table C are prepared by the method illustrated in Scheme 4. Other derivatives are prepared by well known methods. See e.g., Kerrieg et al., Susorioiyodo 11: 118 (2001) and Spapeg et al.,

Sspusobrioiodu 10: 1049 (2000)). Other amine-reactive analogs of PEG and PPG are commercially available or can be prepared by methods readily available to those skilled in the art.

Table 3

Mnchne n " 5th SA, 5-o ts "Zara el po-de oiue s, jann-- do knn-yd o

СМе.5А-5-МН-К nn СМЕ-Mevas-9-0-Я. nn sia po nya oto o-R-o ch'o zi Zyaeyet no o "Ma no On vno svin no on pa-on 9 Asnn. 9

SMR. KetsAo MN dn

SMRZHOM-50-k mne ka 9 S, yang gom v y to ego SA nyu TYA ebuet n domn-nidnO donn-dno o SMR-Mezle-d MY, chna

SMRAMemAs-V OK shk for WHY o-y-0 no no Mna u no 9-y de cen eh n no rotn n dochn--"dn

AsmN-"on mele che СМР-МечАс-7-МН-В nu with -Y oyu dschn p lomne i ok

SMR-Mends-4-0.A SMR Keshkeina V

The modified sugar phosphates used in the practice of this invention may be substituted in other positions as well as in the positions specified above. According to the present invention, preferred substitutions for sialic acid are those illustrated in the formula below:

Mn; es sh mo bot z ud dia ona

КК и--0 Oma no On 4 o

E-A 7-ni where X is a linker group, preferably selected from -O-, -M(H)-, -5, SNeo- and M(A)2, where each of A is a member independently selected with B"-H5. Each of the symbols M, 7, A, and B represents a group selected from the groups specified above for X. Each of X, Y, 2, A, and B is independently selected, and therefore these groups may be the same or different. The symbols B', B2, VU, B?, and BZ denote H, a PEG moiety, a therapeutic moiety, a biomolecule, or another moiety. Alternatively, these symbols denote a linker linked to a PEG moiety, a therapeutic moiety, a biomolecule, or another moiety .

Representative moieties linked to the conjugates described herein include, but are not limited to, PEG derivatives (e.g., acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG, carbamoyl-PEG, aryl-PEG), PPG derivatives (eg, acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPG, carbamoyl-PPG, aryl-PPG), therapeutic fragments, diagnostic fragments, mannose-β6-phosphate, heparin, heparan, 5 ex, mannose , mannose-6-phosphate, sialyl-I eummi5-X,

ESE, MES, proteins, chondrotin, keratan, dermatan, albumin, integrins, antennal oligosaccharides, peptides, etc.

Methods of conjugation of various modifying groups with a saccharide group are easily available to specialists in this field (Roiuu(EShuiepe Spuso! Spetivigu: Vioyesppisa! apa Vioteadisa! Arriisayiopv, U). Miyup Naigtiz, Ed., Riepyt Ryb.

Sogr., 1992; Rowe (EiShuiepe Spousoi) Spetis! apa Viioodisa! Arriisayopv, U. Miyop Naittiz, Ed., AS5 Zutroviyt

Zepeb5 Mob80, Ategpsap SPpetisa! Bosieiu, 1997; Negptapzop, Viosopiidaie Tesppidoez, Asadetis Rgez5, Zap

Oivedo, 1996 8 Oipp eii ai., Eaz. Roiutegis YuOgydz apa Ogpd Oeiimegu Zuzietvs, AS5 Zutrovisht Zepev Mo!1.469,

Ategisap Spetis! bosieiu, Mazpipdiyup, O.S. 1991).

Linker groups (crosslinking groups)

Obtaining the modified sugar used in the methods according to the invention includes the attachment of a PEG fragment to the sugar residue, and preferably, the formation of a stable addition product that is a substrate for glycosyltransferase. So, for example, to obtain a conjugate of PEG and sugar, it is often preferable to use a linker, for example, a linker formed as a result of the reaction of PEG and a sugar group with a cross-linking agent. Representative bifunctional compounds that can be used to attach modifying groups to carbohydrate moieties include, but are not limited to, bifunctional polyethylene glycols, polyamides, polyesters, polyesters, and the like. General methods of joining carbohydrates to other molecules are known from the literature. See, for example, And! ее ей ай., Viospetizigu 28: 1856 (1989); Vnaya ey a!., Apa!. Viospet. 178: 408 (1989); Udapaa eyi ai., U. At. Spent boss. 112: 8886 (1990) and VedpagekKi ei ai., UMO 92/18135. Discussed below are reactive groups treated to favor the growth of the sugar chain of the modified sugar. This discussion is provided for illustrative purposes only.

Those skilled in the art will appreciate that the discussion regarding reactive groups also applies to modifying groups.

Various reagents are used to modify the components of the modified sugar with intramolecular chemical cross-links (a description of cross-linking reagents and cross-linking procedures can be found in the works of Uoya R., May Epgutoi. 25: 623-651, 1972; Meeiai! NN, Soopeu 5 0.A., Ip: Eplutev az YuOgade5 (Noisepregud 5 Nobreigiv5, ed5) pp. 395-442, MMPeu, Mem MogKk, 1981; Shchi T.N., Meiy. Epgutoi. 91: 580-609, 1983;

Maiwop ei ai., Moi. Axes Aer. 17: 167-183, 1993, which are incorporated herein by reference).

Preferred cross-linking reagents are derived from various zero-order, homo-bifunctional and hetero-bifunctional cross-linking reagents. Cross-linking reagents of zero order are reagents for direct conjugation of two internal chemical groups without the introduction of external reagents. This category includes agents that catalyze the formation of a disulfide bond. Other examples are reagents that induce the condensation reaction of the carboxyl and primary amino groups to form an amide bond, and such reagents are carbodiimides, ethyl chloroformate, reagent

Woodward K (2-ethyl-5-phenylisoxazolium-3i--sulfonate) and carbonyldiimidazole. In addition to these chemical reagents, the enzyme transglutaminase (glutamic peptide-y-glutamyltransferase; OS 2.3.2.13) can be used as a zero-order cross-linking reagent. This enzyme catalyzes acyl transfer reactions to carboxamide groups of protein-bound glutaminyl residues, of course, using the primary amino group as a substrate. Preferred homo- and heterobifunctional reagents contain two identical or two different sites, respectively, which can react with an amino group, with a sulfhydryl group, with a guanidino group, with an indole group, or with a non-specific group.

Purification of conjugates -C5E

Refolding of insoluble 1-C5E

Many recombinant proteins expressed in bacteria are expressed as insoluble aggregates in bacterial inclusion bodies. Inclusion bodies are protein deposits found in the cytoplasmic and periplasmic space of a bacterial cell (see, for example, Siagk,

Whitefish. Or. Vioyesp. 12: 202-207 (2001)). Recombinant proteins 2-C5E are expressed in bacterial inclusion bodies, and this application describes methods of refolding these proteins in order to produce active proteins -SOBE.

A. Conditions for refolding of active -C5E

To produce active proteins (14-C5E from bacterial cells, proteins (3-C5E) are expressed in bacterial inclusion bodies, after which the bacteria are collected, subjected to disruption, and the inclusion bodies are isolated and washed. In one of the variants of the invention, three washings are carried out: the first washing is carried out in a buffer at pH 6.0-9.0, containing a monovalent salt, for example, sodium chloride, a nonionic detergent, for example, Triton X-100, an ionic detergent, for example, sodium deoxycholate, and EOTA; the second washing is carried out in a buffer , which does not contain detergent; and the third wash is carried out in Ngo. The proteins in the inclusion bodies are then solubilized. Solubilization can be carried out using denaturing agents, guanidinium chloride or urea, at extreme pH values; or detergents, or any combination thereof. In one according to the variants of the invention, for solubilization (14-C5E use 5-6M guanidine-HCI or urea. In another variant of the invention, DTT is added.

After solubilization, denaturing agents are removed from the 1-C5E protein mixture. Removal of the denaturing agent can be accomplished by various methods, including dilution in refolding buffer or buffer exchange methods. Buffer exchange methods include dialysis, diafiltration, gel filtration, and immobilization of protein on a solid support (see, for example, Siagk, Sig. Or. Bioesp. 12: 202-207 (2001)). A combination of any of the above methods may be used to remove denaturing agents.

The formation of a disulfide bond in 4-C5E proteins is stimulated by the addition of a refolding buffer containing a redox couple. Redox pairs are reduced and oxidized glutathione (9U5B-ya550), cysteine/cystine, cysteamine/cystamine, ОТТ/355(12 and ОТЕ/25502 (see, for example, Siaik, Sig. Or.

Vioyesn. 12: 202-207 (2001)). In one of the variants of the invention, the redox couple is 45H/55OI in a ratio of 10:1.

Refolding can be carried out in a buffer with a pH in the range, for example, from 6.0 to 10.0. Buffers for refolding can include other additives to enhance refolding, for example, I -arginine (0.4-1M); PEG; denaturing agents in low concentrations, such as urea (1-2M) and guanidinium chloride (0.5-1.5M), and detergents (for example, Sparz, DSN, STAV, laurylmaltoside, Tween-80, and Triton X-100).

After refolding, the -C5E protein can be dialyzed to remove the redox pair or other unwanted buffer components. In one of the variants of the invention, dialysis is carried out using a buffer that includes sodium acetate, glycerin and a nonionic detergent, for example, Tween-80. After dialysis, protein

C2-C5E can be further purified and/or concentrated using ion exchange chromatography. In one of the variants of the invention, cation exchange resin 5P-sepharose is used.

Those skilled in the art know that "correct" refolding of a protein occurs if such a refoldable protein has detectable biological activity. Biological activity of protein (-

C5E can be measured by various methods. So, for example, biologically active C-C5E proteins are substrates for

O-linked glycosylation described in US patent application 60/535284, filed January 8, 2004; Application No. 60/544411, filed February 12, 2004, and Application No. Assigned by Patent Attorney, 019957-01882005, filed February 20, 2004, each of which is incorporated in its entirety and for all purposes into this description of the invention using a link. 3-C5E protein activity can be measured using cell proliferation assays or rat leukocyte formula assays (such assays are also described in US patent application 60/535284, filed Jan. 8, 2004; in application 60/544411, filed Feb. 12, 2004 and in the application bearing registration number assigned by the patent attorney, 019957 -01882005, filed February 20, 2004, each of which is incorporated in its entirety and for all purposes of the invention into this specification by reference Proliferation Assays and Leukocyte Formula Assays can be carried out before or after O-linked glycosylation of refolded proteins (3-C5.

Other methods of isolation of conjugates according to the invention

Alternatively, the products obtained by the above methods can be used without purification.

However, of course, it is preferable that this product be selected. For this, standard well-known methods for the isolation of glycosylated saccharides, such as thin-layer and thick-layer chromatography, column chromatography, ion exchange chromatography or membrane filtration, can be used. At the same time, it is preferable to carry out membrane filtration for isolation, and more preferably to use a reverse osmosis membrane or one or more methods of column chromatography, as discussed earlier and in the literature cited here. Thus, for example, membrane filtration can be used to remove proteins such as glycosyltransferases, where the membranes have a molecular weight cutoff of approximately 3,000 to 10,000.

Then, nanofiltration or reverse osmosis can be used to remove salts and/or to purify saccharide products (see, for example, UUO98/15581). Nanofiltration membranes are a class of reverse osmosis membranes that pass monovalent salts but retain polyvalent salts and uncharged solutes having a molecular weight greater than approximately 1000-2000 Daltons, depending on the membrane used. Thus, in typical applications, the saccharides produced by the methods of the invention will be retained in the membrane, and contaminating salts will pass through the membrane.

If the modified glycoprotein is produced inside the cell, as in the first step, then the debris of large particles, or host cells, or lysed fragments is removed, for example, by centrifugation or ultrafiltration, and this protein can, but not necessarily, be concentrated using commercial an available filter for protein concentration with subsequent separation of polypeptide variants from other impurities in one or more stages selected from the stages of immunoaffinity chromatography, fractionation on an ion exchange column (for example, on diethylaminoethyl (OEAE) or on matrices containing carboxymethyl or sulfopropyl groups), Chromatography on Vipe-Sepharose, SM-Vipe-Sepharose, chromatography on MOMO-O, MOMO-5, chromatography on lectin-Sepharose, chromatography on M/CA-Sepharose, chromatography on Sop A-Sepharose, chromatography on Tourea ether, on butyl-Toureai!, phenyl-

Towreap! or on protein A-Sepharose, SDS-PAGE chromatography, silica chromatography, chromatofocusing, reversed-phase HPLC (for example, on silica gel to which aliphatic groups are attached), gel filtration, for example, on Sephadex and molecular sieves, or exclusion chromatography, chromatography on columns selectively bound to the polypeptide, and precipitation with ethanol or ammonium sulfate.

Modified glycopeptides produced in culture are usually first isolated by extraction from cells, enzymes, etc., followed by one or more steps of concentration, salting out, aqueous ion exchange chromatography, or size exclusion chromatography. In addition, the modified glycoprotein can be purified by affinity chromatography. And finally, HPLC can be used to carry out the final stages of purification.

A protease inhibitor such as methylsulfonyl fluoride (PM5BE) may be included in any of the preceding steps to inhibit proteolysis, and antibiotics may be included to prevent accidental contamination.

In another embodiment of the invention, supernatants from systems that produce a modified glycopeptide according to the invention are first concentrated using a commercially available filter for protein concentration, for example, an Artichoke ultrafiltration device or MiSchrohe Reisop. After the concentration stage, the concentrate can be applied to a suitable matrix for cleaning. For example, a suitable affinity matrix may contain a ligand for a peptide, lectin or antibody molecule bound to a suitable carrier. Alternatively, an anion exchange resin, such as a matrix or substrate having OEAE side groups, can be used. Suitable matrices are acrylamide, agarose, dextran, cellulose and other types of substances commonly used for protein purification. Alternatively, a cation exchange chromatography step can be performed. Suitable cation exchangers are various insoluble matrices containing sulfopropyl or carboxymethyl groups. Particularly preferred are sulfopropyl groups.

And, finally, for additional purification of the composition of the polypeptide variant, one or more ZF-HPLC stages can be carried out, in which hydrophobic ZF-HPLC media are used, for example, silica gel with side methyl or other aliphatic groups. To obtain a homogeneous modified glycoprotein, some or all of the above-mentioned stages of purification can also be carried out in various combinations.

The modified glycopeptide according to the invention, obtained as a result of carrying out large-scale fermentation, can be purified by methods similar to those described by Ogaai!ei a!., U. Spgotaload. 296: 171 (1984). In this work, two consecutive ZF-HPLC are described -purification steps of recombinant human II-2 on a preparative HPLC column Alternatively, methods such as affinity chromatography can be used to purify the modified glycoprotein.

Pharmaceutical compositions

In another aspect, the present invention relates to a pharmaceutical composition. This pharmaceutical composition includes a pharmaceutically acceptable diluent and a covalent conjugate containing an unnatural PEG molecule, a therapeutic molecule or biomolecule and a glycosylated or non-glycosylated peptide.

The specified polymer, therapeutic molecule or biomolecule is conjugated to the C2-C5E peptide using an intact glycosyl linker group located between them and covalently bound to the C-C5BE peptide and the polymer, therapeutic molecule or biomolecule.

Pharmaceutical compositions according to the invention are suitable for their use in various drug delivery systems. Compositions suitable for use in the present invention can be found in the manual of Vetipdiopye Rpaptaseshis! bsiepse5, Mase Rybrizpipd Sotrapu, Rpiaade!rnia, RA, 171y ej (1985).

A brief review of drug delivery methods can be found in I apdeg, bsiepse 249: 1527-1533 (1990).

Pharmaceutical compositions can be prepared for any suitable method of administration, including, for example, topical, oral. intranasal, intravenous, intracranial,

intra-abdominal, subcutaneous or intramuscular administration. The carrier for parenteral administration, such as subcutaneous injection, preferably contains water, saline, alcohol, fat, wax or buffer. For oral administration, any of the aforementioned carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium-containing saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate can be used. Biodegradable microspheres (for example, polylactate-polyglycolate) can also be used as carriers for pharmaceutical compositions according to the invention. Suitable biodegradable microspheres are described, for example, in US patents MoMe 4,897,268 and 5,075,109.

Of course, pharmaceutical compositions are administered parenterally, for example, intravenously. Thus, the present invention relates to compositions for parenteral administration containing a compound dissolved or suspended in an acceptable carrier, preferably in an aqueous carrier, for example, in water, in buffered water, in physiological solution, РВ5, etc. Such compositions may contain pharmaceutically acceptable additives necessary for the correction of physiological conditions, such as pH-adjusting agents and buffering agents, as well as agents that add tonicity, wetting agents, detergents, etc.

These compositions can be sterilized by standard methods, or they can be subjected to sterile filtration. The obtained aqueous solutions can be packaged for direct use in the form in which they were obtained or lyophilized; moreover, the lyophilized drug, before its introduction, can be diluted with a sterile aqueous carrier. The pH of this drug is usually from 3 to 11, more preferably from 5 to 9, and most preferably from 7 to 8.

In some embodiments of the invention, glycopeptides according to the invention can be introduced into liposomes derived from standard vesicle-forming lipids. There are a number of methods for obtaining liposomes, described, for example, in the work of 520Ka vy a!., App. German Viorpuz. Vioepod. 9:467 (1980), in US Patents MoMe 4,235,871, 4,501,728, and 4,837,028.

Delivery of liposomes using various targeting agents (for example, sialyl galactosides according to the invention) is well known in the art (see, for example, US patents Me4957773 and 4604044).

Standard methods of attaching targeting agents to liposomes can be used. These methods typically include the introduction into liposomes of lipid components such as phosphatidylethanolamine, which can be activated to attach targeting agents or derivatized lipophilic compounds, such as the lipid derivatized glycopeptides of the invention.

Delivery mechanisms usually require that agents for directed delivery are on the surface of the liposome in such a position that such molecules will be available for interaction with the target, for example, with a cell surface receptor. Carbohydrates according to the invention can be attached to a lipid molecule before the formation of liposomes by methods known to those skilled in the art (for example, by alkylating or acylating the hydroxyl group present on the carbohydrate, with a long-chain alkyl halide or fatty acid, respectively). Alternatively, the liposome can be obtained in such a way that the binding part was first introduced into the membrane during its formation. Such a binding part should have a lipophilic part, which is firmly loaded into the membrane and anchored on it. This binding part must have a reactive part that is chemically accessible on the aqueous surface of the liposome. This reactive part is chosen so that, according to its chemical structure, it can form a stable chemical bond with the targeting agent or with the carbohydrate that is added later. In some cases, the targeting agent can be attached directly to the linking molecule, but in most cases, it is more preferable to use a third molecule that acts as a chemical bridge that allows the binding molecule located on the membrane to be attached to the targeting agent or to the carbohydrate , which protrudes beyond the surface of the vesicle into the surrounding space.

Compounds obtained by methods according to the invention can also be used as diagnostic reagents.

Thus, for example, labeled compounds can be used to determine the localization of areas of inflammation or tumor metastases in a patient with suspected inflammation. For such applications, the compounds may be labeled with 125|, 140, or tritium.

The active ingredient used in the pharmaceutical compositions according to the invention is glycopegylated (-C5E) or its derivatives, which have the biological properties of follicle-stimulating hormone, which stimulate, for example, ovulation. The composition (3-C5E according to the invention is preferably administered parenterally ( (e.g., IM, IV, IV, or IV) Obviously, effective doses can vary considerably depending on the condition being treated and the route of administration, but it is believed that these doses will range from about 0.1 (u7U) to 100 (-7000U) mcg of active substance/kg body weight.For the treatment of anemia, the preferred doses are about 50 to 300 units/kg and administered three times a week.

Since the present invention relates to 2-C5E with an increased time of residence of ip mimo, when introducing the composition according to the invention, the specified doses may be, but not necessarily, reduced.

The following examples are given to illustrate the conjugates and methods of their preparation according to the invention, however, these examples should not be considered as limitations of the claimed invention.

Examples

Example 1

Glycopegylated C-C5E produced in CHO cells a. Obtaining asialo-granulocyte colony-stimulating factor (2-C51E) c2-C5B, produced in CHO cells, was dissolved at a density of 2.5 mg/ml in 50 mM Tris-HCI, pH 7.4, 015 M mass, 5 mM Sas" and concentrated to 500 μl on a Sepiasop Rii 20 filter centrifuge. The resulting solution was incubated with ZObmOd/ml of neuraminidase I! (Mirgpo spoiegaye) for 16 hours at 32"C. To monitor this reaction, a small aliquot of the reaction mixture was diluted with the appropriate buffer and performed isoelectric focusing (EF) in the gel. Then the reaction mixture was added to the previously washed conjugate "M-(n- aminophenyl)oxamic acid-agarose" (800 μl/ml of the volume of the reaction mixture), and the washed spheres were slightly mixed by rotation for 24 hours at 4"C. The mixture was centrifuged at 10,000 rpm, and the supernatant was collected. The spheres were washed 3 times with Tris-EOTA buffer, once with 0.4 ml of Tris-EOTA buffer and once with 0.2 ml of Tris-EOTA buffer, and all supernatants were combined. The supernatant was dialyzed at 4"C against 50 mM Tris-HCII, pH

7.4, 1M mass, 0.0595 MamMz, and then two more times against 50mM Tris-HCl, pH 7.4, 1M mass, 0.0595 MaMz. After that, the dialyzed solution was concentrated on a Sepiasop Riv 20 filter centrifuge and stored at -20"C. IEF in the gel was performed under conditions corresponding to the procedures and reagents from Ipmygodep. Samples of native and desialylated 2-C5BE were dialyzed against water and analyzed using MA! 0I -TOE-M5. Preparation of C-C5E-(alphag,3)-sialyl-PEG

Desialylated 2-C5E was dissolved at a density of 2.5 mg/ml in 50 mM Tris-HCl, 0.15 M mass, 0.0595 mM, pH 7.2. The resulting solution was incubated with 1 mM CMFf-sialic acid-PEG and 0.1 mU/ml 5TZ3(a!!) at 32"C for 2 days. To monitor the incorporation of sialic acid-PEG, a fluorescent dye was added to a small aliquot of the reaction mixture of CMF-SC-PEG ligand, and the label incorporated into the peptide was separated from the free label by gel filtration on an analytical column Tozho Naaz 230005 using PB5 buffer (pH 7.1).Incorporation of the fluorescent label into the peptide was quantified using a connected fluorescent detector.

After 2 days, the reaction mixture was purified on a Tozo Naaz SZ0O005UM preparative column using a buffer

РВ5 (pH 7.1), and fractions were collected based on UV absorption. The reaction product was analyzed using electrophoresis in SDS-PAGE and IEF analysis was performed using procedures and reagents from Ipmigodep.

Samples of native and PEGylated C-C5E were dialyzed against water and analyzed using MA! 0BI-TOB-M5. with. Preparation of C-C5E-(alphag,8)-sialyl-PEG a-C5E, produced in CHO cells and containing alphag,3-sialylated O-linked glycan, was dissolved at a density of 2.5 mg/ml in 50 mM Tris-HCI , 0.15M Mass, 0.0595 MamMz, pH 7.2. The resulting solution was incubated with 1 mM of the conjugate CMF-sialic acid-PEG and 01 U/ml C51-III at 32"C for 2 days. To monitor the incorporation of sialic acid-PEG, a fluorescent ligand was added to a small aliquot of the reaction mixture of CMF-SC-PEG , and the label included in the peptide was separated from the free label using gel filtration on an analytical column

Tozo Naaz C2Z30005UU using buffer РВ5 (pH 7.1). Incorporation of the fluorescent label into the peptide was quantified using a connected fluorescent detector. After 2 days, the reaction mixture was purified on a preparative Tozho Naaz SZ0005UU column using buffer РВ5 (pH 7.1), and fractions were collected based on UV absorption. The reaction product was analyzed using electrophoresis in SDS-PAGE and

IEF analysis was performed using procedures and reagents from Ipmigodep. Samples of native and PEGylated 2-C5E were dialyzed against water and analyzed using MAI 0BI-TOE-M5. and. Preparation of 2-C5E-(alphag,6)-sialyl-PEG 2-C5BE, containing only O-linked CaIMAs, was dissolved at a density of 2.5 mg/ml in 50 mM Tris-HCl, 0.15 M mass, 0.0595 MamMz, pH 7.2. The resulting solution was incubated with 1 mM CMF-sialic acid-PEG conjugate and 01 U/ml

ZztbaaiMAS I or II at 32"C for 2 days. To monitor the incorporation of sialic acid-PEG, a fluorescent ligand was added to a small aliquot of the CMF-SK-PEG reaction mixture, and the label incorporated into the peptide was separated from the free label using gel filtration on a Toho Naaz SZO00O5UU analytical column using PB5 buffer (pH 7.1). Incorporation of the fluorescent label into the peptide was quantified using a connected fluorescent detector. After 2 days, the reaction mixture was purified on a Toho Naaz SZO0005UM preparative column using PB5 buffer (pH 7 ,1), and fractions were collected based on UV absorption. The reaction product was analyzed by electrophoresis in SDS-PAGE and IEF analysis was performed using procedures and reagents from Ipmigodep. Samples of native and PEGylated 2-C5E were dialyzed against water and analyzed using MAG! 0BI-TOE-M5.

C2-C5E, produced in CHO cells, was treated with sialidase of bacteria of the genus Agpipgobrassieg, after which it was purified by size-exclusion chromatography on Zyregaech 75 and treated with 573sa or 5TZSai2, and then with the conjugate

TsMF-SK-PEG, 20 kDa. The resulting molecule was purified by ion-exchange chromatography and gel filtration, and analysis performed using SDS-PAGE electrophoresis indicated the completion of PEGylation. This is the first evidence of glycoPEGylation of an O-linked glycan.

Example 2

Expression, Refolding and Purification of Recombinant -C5E - Cells are collected by centrifugation and the supernatant is discarded. The results of cultivation on different media are shown in Fig.9. - The cell pellet is resuspended in 1O0 ml/g of lysis buffer containing 10 mM Tris pH 7.4, 75 mM mass, 5 mM EOTA. - Cells are microfluidized (also using a French press) - Cells are centrifuged for 30 minutes, at 4" and at 500006 rpm, and the supernatant is discarded. - The pellet is resuspended in lysis buffer and centrifuged as described above. - The IVs are washed in 25mM Tris, RNAe, 100mM Masi, 195 TXx-100, 195 MarosS, 5mM EOTA. The sediments are resuspended by pipetting and shaking. Then it is centrifuged for 15 minutes at 4"C and at 5000 rpm. This stage is repeated one more time (two washes in total). - The sediments are washed twice in 25mM Tris, rnHn8e, 100mM mass, 5mM EOTA to remove detergents and centrifuged as described above. - Precipitates are resuspended in 4H2O for separation into aliquots and centrifuged as described above. Precipitates are frozen at -2070. - IV is resuspended at 20mg/ml in 6M guanidine-HCI, 5mM EOTA, 100mM Masi, 100mM Tris pH 8, 10mM DTT, using a pipettor with subsequent mixing for 2-4 hours at room temperature. - The solubilized IV is centrifuged during their at room temperature at 14000o06./min.

Save the supernatant. - The supernatant is diluted 1:20 with refolding buffer containing 10 mM ME5, pH 6, 240 mM Masi, 10 mM. - Then a buffer containing KSI, 0.3 mM laurylmaltoside, 0.05595 PEGZ3Z350, iM a5H, 0 1M 555, 0O.5M arginine is added, and refolding is carried out on a rotary shaker overnight at 470. - The refolding mixture is transferred to a Riegse coil with a molecular weight cut-off (MU/SO) of 7 kDa for dialysis. Dialysis is carried out in a buffer containing 20 mM MabOAc, pH 4, 50 mM Masi, 0.00595 Tween-80, 0.1 mM EOTA.

Dialysis is carried out only 3 times against at least a 200-fold excess, at 47s.

- After dialysis, the material is passed through a 0.45 μM filter. - The 5P-sepharose column is equilibrated with dialysis buffer and the sample is applied. The column is washed with dialysis buffer and eluted with dialysis buffer containing a salt gradient up to 1M Massey. Protein is usually eluted at 300-400 mM mass. - The material is analyzed using electrophoresis in DSN-PAGE (see, for example, Fig. 10).

Example C

Method using two enzymes in two vessels In the following example, the preparation of 1-C5E-CaiMmAs-5A-REY in two consecutive stages, where each intermediate product was purified before its use in the next stage, is illustrated. and. Preparation of 1-C5E-CaIMAs (pH 6.2) from -C5E and OOR-CaIMAs using cCaIMAs-T2 a-C5E (960 μg) in 3.2 ml of packing buffer was concentrated by ultrafiltration using

UV filter (MUSO 5K), and then diluted with 25 mM MEZ5 buffer (pH 6.2, 0.00595 MaMz). Then added

OOR-SiaIMAs (bmg, 9.24mM), SaiMmAs-T2 (40μl, 0.04 units) and 100mM Mpsig (40μl, 4mM), and the resulting solution was incubated at room temperature.

After 24 hours, MY OI analysis indicated the completion of the reaction. The reaction mixture was subjected to direct

HPLC-purification using 5ES (Zyregaeh 75 and Zuyregaeh 200) and elution buffer containing РВ5 (phosphate-buffered saline, pH 4.9, and 0.00595 Tween 80). The collected 2-C5BE-(zaiMAS) peak was concentrated using a Sepigysop filter with MM/CO 5 kDa to a concentration of 15 Ωl, and the volume was adjusted to 1Tml using РВ5 (phosphate-buffered saline, pH 4.9 and 0.00595 Tween 80) The final protein concentration was 1 mg/ml (A280), the yield was 10095. The sample was stored at 47 °C. and mouse 5T6SaiIMAs-TtT1 (rn 6.2)

In the C-C5E-CaIMAs solution containing 1 mg of protein, the buffer was replaced with 25 mM MEZ buffer (pH 6.2, 0.00590

Mamz) and TsMF-SC-PEG (20 KDa) (5 mg, 0.25 μmol) were added. After dissolution, Mpsig (100 μl, 100 mM solution) and 5TZ(zaiMmAs-1 (100 μl, mouse enzyme) were added, and the reaction mixture was slowly stirred at 327 for three days. The reaction mixture was then concentrated by ultrafiltration (MUUSO 5K), and one buffer exchange was performed on 25 mM MaobAs (pH 4.9), after which it was concentrated to a volume of only ml. Then the product was purified using 5P-Sepharose (A: 25 mM MaOAsnO, 00595 Tween-80. pH 4.5; B: 25 mM MabAs-- 0.0059 o

Tween-80, pH 4.5-2M Mass) with a retention time of 13-18 minutes and 5ES (Zyregeh 75; РВ5-РН 7.2, 0.00595 tween-80) with a retention time of 8.6 minutes (Zyregeh 75; flow rate I1ml/min.) The required fractions were collected, concentrated to 0.5ml and stored at 47C.

Example 4

The method of obtaining 2-C5E-CaIMAs-SK-PEG in one vessel with the simultaneous addition of enzymes

The following example illustrates the preparation of (c-C5BE-(zaiMmAs-SK-PEG) in one vessel with the simultaneous addition of enzymes. 1. Reaction in one vessel using mouse 5T6b(amAs-1 (pH 6.0) 2-C5E (960 μg of protein , dissolved in 3.2 ml of product preparation buffer) was concentrated by ultrafiltration (MUU/CO 5K) to 0.5 ml, and then diluted with 25 mM MEZ buffer (pH 6.0, 0.00595 mM) to a volume of only approximately 1 ml or to a protein concentration of 1 mg/ml. Then OOR-CaiMAs (6 mg, 9.21 μmol), CaIMAs-T2 (8 Ωcl, vbmOd), CMF-

SK-PEG (20kDa) (bmg, 0.µmol), after which mouse enzyme 5T6SzaIMAs-1 (120µl) and 100mM Mpsig (5µl) were added. The resulting solution was stirred at 327"C for 48 hours and purified under standard conditions of chromatography on 5P-Sepharose. Only 0.5 mg of protein (Agvo) was obtained, or the total yield was approximately 5095. The structure of the product was confirmed by MAI I analysis and electrophoresis in SDS -

PAAG. 2. The reaction in one vessel using chicken 5T6CaIMmAs-i (pH 6.0) 14 4mg of 2-C5E was concentrated to a final volume of 3ml, and then the buffer was replaced with 25mM MEZ5 buffer (pH 6.0, 0.0595 mM, 0 ,0049o5 Tween-80) and the volume was adjusted to 1Zml. Then OOR-CaIMAs (9 Ωg, 150 μmol) was added,

SaMmAs-T2 (0.590 units), TsMF-SC-PEG (20kDa)(9Omg), chicken 5Tb(zaiMmAs-1 (0.44 units) and 100mM Mpsi" (60Omcl).

The resulting mixture was kept at room temperature for 60 hours. Then the reaction mixture was concentrated by ultrafiltration (MUU/CO 5K) and centrifuged. The residue (approximately 2 ml) was dissolved in 25 mM NaOH buffer (pH 4.5) and concentrated again to a final volume of only 5 ml. This sample was purified using 5P-Sepharose for approximately 10-23 minutes, 5ES (Zyregadeh 75; 17 minutes, flow rate 0.5ml/min.) and again 5ES (Zyregadeh 200; 23 minutes, flow rate 0.5ml/min. ), as a result of which 3.6 mg (total yield 2595) of 1-C5E-CaiMAs-SK-PEG (20kDa)x(A280 and BSA-method) were obtained.

Example 5

The method of obtaining 1-C5E-CaIMAs-Ca!I-SK-PEG in one vessel with sequential addition of enzymes

The following example illustrates the preparation of 2-C5BE-CaIMAs-cCaI-SK-PEG in one vessel with sequential addition of enzymes. 1. Obtaining based on SaiMAs-a-S5E a. Preparation of 1-C5E-CaIMAs (pH 6.2) from -C5E and OOR-CaAIMAs using cCaIMAs-T2 a-C5E (960 μg) in 3.2 ml of packing buffer was concentrated on a filter for ultrafiltration (MUU/CO 5K), and then they were diluted with 25 mM buffer ME5 (rnb,2, 0.00595 MaMz). Then OOR-CaiMAS (bmg, 9.24 mM) was added,

CyMAS-T2 (40 μl, 0.04 units) and 100 mM Mys" (40 μl, 4 mM), and the resulting solution was incubated at room temperature. r. Preparation of 1-C5E-CaIMAs-CaI-SC-PEG from 2-C5E-CaIMAs, UDP-galactose, SC-PEG (20KDa) and relevant enzymes

UDf-galactose (4mg, 6.5μmol), "skeleton-1-CaI-T" conjugate (320μl, 160 med), CMF-SC-PEG (20kDa) (mg,

0.4 μmol), 51Z3(3αi2 (8 Ωl, 0.07 med) and 100 mM Mpsi" (8 Ωl) were directly added to the crude reaction mixture of 2-C5E-CaiIMAs (1.5 mg) in 1.5 ml of 25 mM MEZ buffer (pH 6, 0) of the above-mentioned stage (a). The resulting mixture was incubated at 32"C for 60 hours. The reaction mixture was centrifuged and the solution was concentrated using ultrafiltration (MM CO 5K) to 0.2 ml, and then dissolved again in 25 mM MaOAc (pH 4.5) to the final volume of 1 ml. The product was purified on 5P-Sepharose (retention time 10-15 minutes), and then the peak fraction was concentrated on a filter centrifuge (MMUSO 5K) and the residue was again purified using 5ES (Biregaekh 75, retention time 10 2 min. ).After concentration on a filter centrifuge (MU/CO 5K), the protein was diluted to 1 ml using a buffer to obtain a composition containing РВ5, 2.595 mannitol, 0.00590 polysorbate, pH 6.5, and the composition was obtained at a concentration of 850 μg of protein per ml of product (Agvo) The total yield was 5595.

Example 6

The method of obtaining 2-C5E-SaIMAs-Sa!I-SK-PEG in one vessel with the simultaneous addition of enzymes a. Obtained from 1-C5E a-C5E (960 kg, 3.2 ml) was concentrated by ultrafiltration (MUUSO 5K), and then diluted with 25 mM ME5 buffer (pH 6.0, 0.00595 MaMz). The total volume of the 2-C5E solution was adjusted to approximately mg/ml. Then added SHOR-(zaIMAs (bmg), saIMmAs-T2 (8µl, (ZVOµOd), OOR-Cai (bmg), "skeleton 1-Sa!t" conjugate (160µl, 8µOd), CMF-SC-PEG ( 2OK) (bmg), 2,3-(0)-sialyltransferase (16bO0µl, 120µU) and 100mM Mpsig (4µl).

The resulting mixture was incubated at 32"C for 48 hours. Purification was performed as described below using IEF and 5ES. The resulting fraction containing the product was concentrated by ultrafiltration (MU/CO 5K), and the volume was brought to approximately 1 ml by adding buffer .The protein concentration, as determined by Agvo, was 0.392 mg/ml, and the total yield from the 2-C5E calculation was 40905.

It is obvious that the examples and variants described here are given for illustrative purposes only, and based on the description of this invention, a specialist in this field can make various modifications or changes that do not go beyond the scope and essence of this invention and the appended claims. All publications, patents and patent applications cited herein in their entirety and for all purposes of the invention are incorporated herein by reference. now: z zho She i i A aiva x a-Ssche

Her (her l. Zh

And lem and i

Glycan | FA, Shchy Y, Y x m Y; o, KK Ai o sy ShK) dk "educational center

Sho rt and v. In I de yo - preschi and I S and 4 ОЖ. s B xx zl K. x oi » y ( ke fig. dk s oh

O-baiMAS T i | Kk laliptraneferase ek foonn - In a pig

OR -th SMR-ZA-RES b-SBE

Produced in

B. dream

Fig. 2 im-injection booster dose | CD-tagged protein in rats: 10,000 - sn a in Meshavia Shin 0OODMYOU not stonn d "Meprodeip

That's it! s-i- shko 0 5 10 5 2 95 30 35 40 45 BO

Time (hours)

Fig. WITH

Lenkocytic response in mice with variant O-C5E (350 μg/ml) iu-Introduction at 0 h. in a picnic ho - y ni o t h LY ШЙ С-С5Е-РЕО.

With KE I | | and Meuvodey 5 A pos e B : ; : - ! "Mo -B0 o for 100 150

Withdrawal time (hours)

Fig. 4

G" o in vmos Yich- og" nor exact no. in ON iy - i . o uchi ns diny nos so tk EMOs doing ovov on Mernovum o on Aldolase (В В 5 no ssh vlpe CTP, (PM F-solic acid) and synthetase we have

I think it's summer! memes What he o7frtog no ele) shen)

Sosna -y i at natr v Y EMOS: fl no by

X takes oyu, chn 2 is 7, it is true that Vi Y eye HC is the same, not SO Ma 7 ato t 2 Z no on

CMF-sialic acid THIS

Fig. 5

A variant consisting of 175 amino acids

MTRIORAZBEROZELKSTVOUKKIUPSOSAASOVKOSA

TUKESIREVEI AND ONVSOTRZHARI APPEAL ASSY

SNIV HO OA JAVIZRESORT OT OBBUAREAT

TUVYMEVSOMARASORTOSAMRRARAHAKSKKASOTI,

MABNEOBKIKUVUKUYUANYAOR (5EO TV MO: 3).

Variant consisting of: 174 amino acids "TREORABVIROBNLZHSIVOUVKYUOOISALATOVKISATU

KISNREBILL LYONU RULREZVSRVO AOS ZOY,

BUT OSIOABEBRESYUV TOM OY OUAOKATTI

MOOMEVOMARATORTOSAMRAVAVATOVKASOUEU

AZNSOSEEMBUKUTANIAOR (BE LO MO: 2),

Fig. 6

Testing growth conditions - washing IV

З7С, 1 mm (final IRTE Output ЯВ (- 05751 g/l) « i.

Sere- IRN, What Time | Raw osakh TV With the same Shk dovi- and correct- | imi) "KK Zhe Exre sya buffer. |. ; ! 15 UK 2 i te |ta tk) 9yu mo aK i z) tv | emEvu zol t " dk I psyMe et chic m and tako oyu sh 1234158149

Cell samples loaded in equal quantities (μg (expression was best observed overnight) - m E 7,

IK Elizat, supernvtant NOYA Lzizau, supernatane she Zk o I 2 Y 1 lyzat. bewl GI and lysate, sediment i 5. 1 final IV 12. On final IV » g u. Ah

Ya. Ya o lysate, supernatant 15. 5 5 lysate, supernatant their nya 5. lysate, osvl. 14, 8 5 lyzat, all and he: - , - I and yah t o. final IV 15:Ж5 final IV shk 7. and U.lizat, superniitant shchi her 8. and Z nizit, vsad "vy 42 43 44 35 95 final IV

Purified IP is shown in lanes 3, 6,912 15.

Note: The IV tracks are loaded with approximately 1/2 of what WAS IN the previous track (based on predicted sediment mass)

FCyg. 7

Western blot analysis of C-SBE subjected to refolding by additional electrophoresis in native polyacrylamide gel 1 23.

Track I, Meyrorop and Track 2, C"

SBRN2OYA (atiany

Track 3, 0- ""ShShchyOsegozyum

Hpedistic)

Track 4.0"

OBROZIVOKH (yaknii pOznyse be proznalyzopannya)

SeSeEO20604 kO-SYAKO21004 are obtained from the same series of refolding reactions, with the only difference that Ch-S5KO21004 is isolated from the flow

SO SBROOBOYA by means of pH correction and re-loading on 5P-sepharose. - SUKO2I VO was the product of a separate series of refoliation reactions

After XR-sepharose, only one band was observed, as determined by Coomassie-assisted staining. Western blotting.

Fig. 8 okonostsknisnaniya o zhav ; - B inn He certain MIO sleep I puvognatsestsno asn uyaN!

In M "en nos o v i / sht ih, in "yaneoyum, shodo | VYSH v - onkhtknohny okisno sn s ONA Nya v; he Us their vindictiveness to me oknsnoM

B. not about She! ra o s I | sya y " 4 Y Chovooonoosu m, osnonyunyumsnverisnanni o yuyu y,. A What; b i oyu, - he NO b di SNONSVKOniNNNISNu IN! 9 yu ki D sewed in: ; - all | foot

I'm sorry. b a Chonokoptsnytsonsn ad OsnenikyunymNA zho

Y H kosh to their i, o no t New sNIIUMSNIYUMISNNNohKen osn amer

Codes nno(dream - that would be nekoo! I didn't dream. 14th: -

H no Sho Os ek sonya b v rt, b Kk dosh -- U still he

Ah, oh

Fig. 9 in | » ovyutteonaeetemosyute o on le y fih F cho noos. yo UsnyunyunyunyutON - S te chnoihsnannoyuyuirnanyustsnonsnduchnya!

And he

In pose anya

BUT m сщОНИСНЮОНЮЧОН

Ufi pp and

NOOS.IB sSNIRNEENOYUSNyuYUM Y

Backgrounds " svoe K and -3 ve, v. - no: snu sn sna oetynni noOs. and AschONEtsonenvom and se Still about nnegoyu (snomoyetsy lyut!

In Sho e

BUT. oh, m otoyusnuyuston shk - "S 7 tr tnnooyuyesnyusnyusnvyusnannu o ve Sho

Fig. 10

Protein Organs EC bevvanvtoschtei War

AidoVovo o Atvshchyurzy pe ikhnachapy AS MMAZVAARTIVIA ooo fayvpya OO MACHALY S VTOOdBaZAAOTVIA 0095602

SHY NI MS SOZOTOMR, etc

Aipovbaka2O1334 AgeOKurVyU mevyznachali ASYUZOYATAAROVITVYA OM file ATO VAZY SON KO

AH124Ba? AAMTOVIV me OO? 1723481 mm 1806509 MR VBObaVA

AZOV In AgviibOrziv DUPYAOBYAZAAI Ob pecks were not determined

Tayvla DUTZZVISAAMOYAI0 o5mzoi

AGLZ29O3SANOTYO

KM 14741? 1964541

And Vozhivyh undefined dova savyavivya

IZ3bayem) and e-2b--silileranoferniai Vovivshie did not determine AB SAROBVEOYA (Biitb) and vnsvaliltraneferase; F2ALOV ANVOVVSASETVOO (WAT) | god ivshtv

VE. the world of ransferase Voziavopv did not define Ame cASII VZA (more than I ! eV -sitattransayuriza pkovtseiya UTA 98

VTEoK-NI (5iviv) Vakhivutya NOT Determined | and

CM ee in Vo ivitye 454 UTB119 SAVA 0 O18974 sialiatransferose (668 MM STVI ME, vozav31

In sialyltransferase Z Vezizitiya MeVvyNachali dra AASITOYUVA OBUNIZ ibraementi smiltrinsferio vTZbayu Vobiantya Not determined dav saSAYAYAVOYIA

He gave half of the pizhtyatranepheriza ZTZOvB Vozivshcha unspecified

Bean sivnintranepherase ЗТзбаю боеиаютв Not specified Dutmavazs Asalyaa4

UNvano) And what

BtaSan Vokhizstpih Nevnznachaln duzovovosASoYAVOV d9VTe

BIBOVINAET Vobishnyv not specified AyulyausSAROVSYAV SOBYA ZVvysliovyuv not specified AEZO1289AAMIVVISA ZY ve

Setoriyesikh 2458, AROMOTOBAARITUTOVA OGO

Retnrsgment ZT zaibre u polychalyltransferase Sepornyosikh 28 DE OziaAARTIOKA OTTO (VT fragmvnt) VTVviv eviniora ee esivdiltransferase Syupa Ivan s determined duvuvvI in SAK26173 vt3va (im) her, Z-sialyltransveraze They could not determine the phenomenon of Av ASAVYAVTM

Va (Zigm) sho pe In nlisialiltransterim Stseyyuya dpznoe AV, o "ALERVVZK 0 SfiEVo

ZVU gav SANY ba ya va sumaAshe Spseyiv dpeetsa undefined arc ADNOOAIYA OO O gd-snopytraneferase "HIV in vyya Wilzy-Oismaye gi blog dpzeiv did not determine Ugo ARIA OKO aonkivnltransferase vyv Y fragment). p 2, With eidnltransferase Fepizteno che determined duv SANTA

Zrzsa(vak | Y vam sialpiltraneferase Yuelutetyu Do not define duv сАНОНВ

Btzbanni (from Zinv

Kay. sialyltransferases were not determined in any case.

Fe, ve sialyltranepheriza Ovpiuteno VE determined dutayava sao

ZziZzawnm (zi)

Fig. PAS

Protein Organam EK bevvanvtoschtei Viyny as oz--sialyltransferase ener a AV SANOOV

MEK did not determine in Besiyliltrvaneferkiz OSapiotenia NeVvyZnAliY dUv SAS

Tv vi i sn vch ssno b-sialyltransferase with Ovpiuteu You have not started dvzyBasAOzevtO

Tabata pe B-sialyltvansferase Oepiyusvno did not determine divavatasSAVIVTOVY vTesvimAye U (Viai?E) "2 b-sivliltraneferacha Tatoteniv Mevnachyli duvavossAvovMoYa

BTOSVIMAYE M (VIVTE) fvalnent) What sog.Ve sivliyatransferiza Oanyufeyo OMEVZNAChchaLy duzi sAvgozTA

VZTYABiv (ZIVA; (fragments! under Vesivnltraneferase Vevpioteyo nezhzhzchanali duti sAsKVIVI aa sha: 80) (fragment) o whether V-sivliltraveferase Fapivlyu was not determined ANT sSAOVOVIVA

BIV M (ZI vo) "fragments of fe, vesalyl transdurase Batotelo did not determine the thought of ssgozava tazi U (Big VE) ifrayments that vyg, b-sialyltransferase: Oalibtnio nevnznakali 0 div sSAstezYa0

VTB M (Viv VE) (fragment) Y o

Y alactozamil bab" Yuepivtepyo was not determined /AYUIVOT SAKE A sivlviranererase and

M-glycan o B-EV" Ovliotayo did not determine VsoBosazAANEOYUVIU OT2C silyltranepheriza AOUOBOYAO2AAKITIVIII UN . n MM 153885 MR. 7О594VY fraimeit, rolenny TZI YuOvpioteno chevyznachali vBSO531оААНЕЗИТОИ 73 days2b820САКЯВВЛ

SHY MM 200355 59648. yaabeu Paioigenyu nezhizhinachalty didivavsakovosil vyuvsamAsm Rapyutepo not determined VO080932 AANVOVIgI 2 b sidnleraisferase resgorihiv gav ABOOZVBAARYATIVV tsevOoi pe KAN SE tevpodayev DE2IV2Z7AASIVOYA OA.

DEZV75Z2 AKOT

AEVOZIVBAAMTN A nm ote12a me, 523953 ! MMm.1vvba4ne, hab41 vv zesialivtransferase Sayikh bayene determined AZI SAVA (tov) What AIVOTOKSABINNYA with aa sivliltransferase beitsa dah bedom 0 KhVOBOZSAAVOVEVA 1300 and shi | nm o c2o621zme vol pe Zsialyltraneferata | Sayye deya 2AB. AROZBUBOAASiYAI 973724 stages

Fairy Sivliptransferase Saivanizes did not define Duabuvuvuvisaeniizao (Ztzva-N fragment and "aa bsovlltravsferase, the site Rao did not mark two saras (a. ADEBOOZVL svo taz 5TvsachAye I "AAKvVOgV ktavaasSAAbIVOgYa m 205240 MR. poOBU

Fa Vsyltovnefeoraz sashe selyu 5 8akh ХТТТТБААЕЯВОВОЙ 092184

ZTBSaMAYE AND MM ZHOB2ZZSAANVYAA

MR VBOBVCHI koe, v-acylyltransferase by sons: unspecified dumavosAvoVIA

BTvOviMAye IN(VIAT?S)

Fig IV

. Protein Organism ESE peobank take BV and 2 b-sialyltransferase did not determine Ame SAT

SOMETIMES (YET) lin is HIGHER

KseVesivlyuttraneverazy fighter gave z495. PTB ASIVVVN 279753 (POC synthase) VTEVIv | howled I

VE sialiyatransfereza baychaceya Chevyznachali dove sAohtvv (TATOVI ooesivniyatravoferiza Sayie dai: Me determined duvovagosAsyaTVVEYA (ATS | !

To all allyltrenoferase biozovyche MeVYZZDEDOVAZAOZTVAV (BIATVKH 0 bsidvlyltransferiza baizediaz They did not define the dog saSIVVYAT

BIB ps my Y and

In galactosaminia b2.v-Saileoviyua, ABITv2vSsARTYAYAOT sialiytoanofveral (zt) was not determined.

YaMasiktase (BIT bayse deyov 24599 АТВИБОБААеВВЙ shi polysilyliatransferase bayiz data 2409. АРООВ1ИЯАЕАААНОЯАЙОЯЯЯЯ 042399 ziva yad Zesialintraneferase Noto zavivlya 24594 KeZBBBAAAZOST. o zZzva! ARKSII ASV OT

ISOTOAASIII I 00 OZ

AR1Z5233 AAYUZNEZV

AEMOTV AA OTIT

Ov AANTYAZOI

MM. split; 0030241

MM 1334 MR 7154794 o-2, Z sevlyltransferase Noto zvarnaya 54 PIaZO9sAdVOZVO I bib842 etzsani BSOZBTTTTAANIETTT.: 000854 o KON SAAVNYAYAT

And | mm, oao27 MR. COLLAPSE

In ksialiatraneferniaa Nato will appear on the 24th of Goa A IIT soot

LLC VSOBOZVODANOYUZVOY OvByNE

AREA Sen

ARASZVOIDAZOV SOBUKE

AROOVUZAAOV shvbOka

AESV5AAKOVEH and

DEYAEIZOO5ZAAOIZKZY t

DEaOBaHeADOIZVAYAYA 0 OB

AraBavtААОИюея 00 ХО

AEYOBOZVAAST AGAIN aa

AKAOVVBVAAOI REMOVED avihao

ARYASIVODATSI ZVOV 1 Onikha

AKYAVOV1AAS NVO sVIhA?

AEDOIZVO2ААОИИВІІ ovha

DEFIZVOZAATZVI ah

DRaBOBAAAOTITO I'm here

AERO AASITSI 00 OVIH

AgabNOVADOIZVTAt 00 OH

ARAESTAAOT VV with Xu

AUB AvOZVVI SvihoYa

AUbTZaZAAOZANY OkhB5

ATTBTVOAAAZEVOVY OV'HVK

AHIVBTYAZBAAOZVNOYA I SVIKH5I and DUET OOVADOZYAVIO VIKO

KU 1B79Я7ААОЗВОТ mm OhetoNe bu . and topic. 006270, nm conspiracy MR tt

MM 1l4ove Me Tttvovy and MIS YOU

Mnmobyavvt me gg

MM 1740695 177925

MM 178970, 777830 nm opalym tal

Fig. 11C

Booky Organism! BO spepryavv o borror vymyrtoi TV 4 v-sialyltraneferase Rito varu 24 ah KEZ/VTAAAIVYAVM 00006 YATZAVNU DROBOM DASIYAYUVIA 00097

CULTIVATION OF SUVOYUO

ATOZOVOVAAKOZTVOYA VM

ARB1ve02AAMOvVEZI OMA

ARBNOZAAMOVIZO AND OVNKOZ LEVYAZmMAADMOVYAZAL OVMEO?

AEVE MBA DAME1318.1

HTZ SAvVaVOg

SleBvava SAS 02 Zsivliltransf 24894 Shi tik shi oi ansipliltraneferase ! ! 2499. MA, to her Saula vtasai m But couple VSO2ZZIZAANIZN I

AOS IVVAATVOV

Akhvitivslaenovov I

AHVZBP2ZSAKOIMOI A

In Siililtranoferaza Noto, the comparison was determined by Vs AANVEvo ava (VTOOai and; KALIVT?) AvovlovANIVO ovsho

AITYASAYUZYAYUOVYA o long:

AHTOVOZ SABO and

AHTeVMOZSABKVVIVI A mm oz2BovnR 5 vav sivliltoa ke ferase Note zoriyut not determined / VSOBozbZAANYAVIVZ Om (STVOATSCHAS NI AUZVAMOALOVVOYUMA OVO

AKOZI2I5VASOZ1

AZNOVISAVAVNMT p KE i 1A HE ve steel raneferase Neto hariel NOT DETERMINED VSOOI20h NOR OTI sooVUNI (BTBOIMAs MI AKOZVIEVAVTIT

AGOZBAVO SAVA

: diBoteVa Sasha

MM OZ0OB5 MR 142227 still besivliltraneferachv Noshyu variv 2485 MAO AAABOV O9Nu3I i5Tamutodamasli VSOAOBAANYAYUMOV shi

AOOBTOBZSAVOYIAZ.

M. OBOV. 00

I osivliltransferase and Noto veriglh 24991 VSOZITOAANIZYAYATYAYA 0 RIBOOT

Ivsen vOOFO009 ANI

AITZV2SAAOYAZIT de SAVOOVA

XTT247 SAVA

KV SAAZVIYAV 1 hvzag2 sSAAYAV mm rososa Me 0o3093 1

MM'T732iaMe 7763034

KU sialileranoferoz Note variants gas VOMOIAANYAYAVoM otv

EtaseichAs APV AAMOZVOO domo

ATVZVOVAAOVYAIT M denHOi

AKOT ZVAAOYUVYUI

ATTIZZOSAATITV and ME IB An. 0609841 no in Notoe varivpe 2.4.09- BY BOYUAASYATITV O5MmiRa pelicidlyltranoferase | Sov AANoTVOVI 099181 tach NSOBZVBTAANEVET 92893.

MM. Fo5898 MR. 005859 ee b sivniltrayuferiza Noteo zvrilya 24.55.58 mze8v7 AADBOZVV shop (YUZsnnuaza) ZT | zay AASZTBOKYA 092185

VSOZOIVVAANYAVIVO 0 p93ob4 "AASZaAO

AHOVVZTaAAVTOTVL

OVO VALOVI I

HTTOde SALI nm oozosa Me 0oz025 more V-srliltraneveraza Noto harivnya 2.A88.- tzob5vАААЗВВЙЯА se

Fig. 110th Stupid Organism! вы кейнавк шеррире варе 59518 и POST ААВОИО42А 0 492670

PZ tAASIVIUVYA 002748 še | MM must not. ooo "o Vestalil transferna No vyriy 2ade. ARYMBAAVITVKYA at:

BTI ех АРИ ААСИИ И вжи

MM (BER. 0569531 7, ve sialintranepherase oto zvrivyh 2454- MOVA AASIT O15486 zastascha IN SNaBhOZ37SAVIZZIVA nm s133O5NR. 0374371 eEMeROOOSOSOGOY did not determine deozov- (fragment)

DLaktaznaneram hey and . U.S

ЖЗксівлілтранецераз Мото бориюпу 24998

VSOZBYAZAANeBOZV dutBevivlASivVOV

DUZ5BOTO5 LASIA

AVOT535O NAAZZOVOI street, phone number Me 003887.

M-nnetylgalactosaminidi be Noto varivpu 2458. VSOOBBAAANOVOK O989X2 "gbestalyltraneferase VSOOVozaAANOgVOg and SuNVA?

Yours in ВСО16290ААНИВго 4 secs va

AUB gAAOVOOV

AVOHLTZVAAVIMZA

AQUA

AUT 29ZSAPIVIUZ

AHVVOKOOSAEYA V

MM biz4a3mr. oZVagi 2 SNO VSAOZZ59

That MM OIZAvZMR. 0384712

Meacetiyagalactosaminiy b" Noto Varna" 7483. ARISHTI4aAAROVYU OeNagi "sidipiitransferase S VsoztoBAANIITOVAI dn (zTaZanaYet "VAREZYAAO1 SOBKO

Av vAket?ozya Obi Ve

AKOOObO0BADVTOVIYAI this

TITYAV1SAVlYAZVIYA Oh

JSC Ta SASOTYAOM

AHOVIV2O SAS

AHOVv265SASIT2501 dHOBVeBy SAK14301

STE

. . Whose! | eat

VTAVIA-MI (fragment) Notovars were not determined by AV SAKgT and neglentvfication of M o ZHM and EHR yah; the protein product Moto Varyuta was not determined AKOgiogoBAVIZYAYOM OVNAHLO; and AH SAYEVIZVI 4 se 1.3m Oismde p. Mezobysvyut 24595 AYUuVOoSAVIZMMYA SROHEYA

Z-sialiptransferase is higher (Tue! I)

Fig. PE

Protein Body! that SES Oeateri bio svo dan Za OkMAvve MmeBostevin. У7.4.95.6 give SANEIUUYEYA OK og Z sialyltraneferase vyg

BOM; AND

Oz-syntata (fragment): Mazostovie unspecified deia1651ААрИЙВТО ояшА

Pelvic weight what! | sho pev ria Mavospsevo ba ANYA SAVOZNMEI O9OHg4 : ashcheh : , 4-2 sivliltrapsferazv bo M KSO zada / ABRIMOVAAROOVTZI ROVI tu iltraneferaza sa 0 Myztivenov 4 AKOT MeVASTYAVSYA vyho

AKatVAVZNASZGOVO ayu

HTIZEZSAAVIYA VM

; pe M OB177NR. 03352031

FV, B-sialiitraneferase BIO Myvtovsye grove VSOVoveAaNeVOVMYA p204

Iza vOopobOvELANOSYUVA OBR

AkKOozIBbanASeVTvei OVNA

AK VASoVOIU ObB5E8

ACKOoBMegpaSZONIiY 0 dme.

HTVVVV SANYA mm. Os tane, 033205 that | mk stavav MR, 8251491 oo d-silyltranoferase bisral 0 Mus pvsia 2409 BOSOMRMOAANOVOYI ROTI

Tab AkKOoOBOBzaAVIZI 5 eh eKOiZOovBAVIVOOVA svo V o Hv sSAAZNOIZI 090885: what MMorOi?vMR OZ I uzksianltranoferase I will pass Mus vnyh g Ado4 VSOTMYATAANYAIAYAOYA ROSE BY

ROanUu VSOBORGZAANyaOTITYAI 061325 rova VAKOBOVVA 09144

AKObaBazVAV2YATI2i bo

AVOVIZOBBAYATVOVA O9SUEV

HUBBOV SAYABOTYA 1 va nm taMme 0332042 og de ssialyltransferase buzoviya Myvtiesyshe 94094 DEMOZORAAYUZVIZOI OK

ZO MI enemy AANOgIV OFVIMI

ANOFZZZgeVAVTYAAA OBUTVZ

AKOZZOVIVASOVUVOI SOMUSI

AKOT T7ZVASSOV runes that MM.O9754Ne, O61254 ee b-sivliachransferati dryovmasg Mine: Feb MM OOviVOovetov83 y

БТбОзиМас ВСО102О6ААНИОГOVA 0 090с54

AVOdTIovVANOYAZ7 0 O8LYM5 kori VAVIZaIO xKheazvovsadazvo1 1

HVO SAAVIVIgA! 5. MM. ooBivO Me 0332064 pub sypliltoanopherase Zhibrvi2 Movltvsnyukh 0 ZAVVY KAESVOZIi OVAvav vtvban BSO28ZZAANIIVII I SO8VMO? Опа Вайозввой 0 ОВК

AKOT VASIVOIVA

AKOT VAOZOT20. | between such VOBOYA8A

BF, B-sylyltranoferase BibreE and tyveiyane determined AKOVOVAYEVASZVIZYA OV 5tabaci AvovhozhzvASVT

AKIgO492BASVVETO and MM. 17202a Me, tob4171

Fe b--sidylyltransferase Zibovipasi Mo tiznia 2458:3 U 12u4sAAIIZI O80239

ETesaiMAei MM bat MROZAVOGI 095 sh-2b--sivliltranefsvaza bibovasi Mob pizeluene determined VSOZVZVTAANYAVIV? I OUAUg 5TVSVYNAE NO AkozavlavVASevVIVi 0 ObINe5

UNZazbAATOIVI u11343SAVZVON and av omMmobztne os

KE sevliltraneferase Bibavilya Muye tizenov 24997 VSOBVAEIAANOVYAVTi OVO

ZToZaimaAye M AKOVBIZOVASZOVEYA AND SO9INRe

AYUOIMOSAATSIYAVI Oo9K2v6

UT SAVIAZVOI O86t25

Fig. PE

Protein Organisms ESE about bepoaectEaiRere Vely vo.

ACCOUNTING 0 OZINRO

UTVOBBSAVOMVYA pesyura

MIVOB7SANOZVIVM o9nan what What MMOTRIZIZNR 0393 i f-sivliltransferase with ba? Miuvtshestnkh 24995 138077 AAABVOVI /OB4AVE cha» snmizai) Tab VSOZYAVe1AANYAEVOI I Ob ! AkoletvaBASZIVIV OBVIY

AKOBIYA4aVASZMOVMM AND DAVUYU

HA SAAOOT AND OVC

AMO2SASAYUTOVI. O9ERKO

MM bama. OZ I 2 B-sialyltransferase Biviaf We initially determined DVOSO5BAVASOI 265.1 O8niyaZ (5TV5ih M) AMOBBIO5VASZYUZET 00 OBKaTi and | MM ZABOZVNR. 6658371. What

VE In sraliltranepheraza ZNO Muye tibspche 2488» ХВЗБЕСААВаБЯВИ О35098

Btaziv and Kho9VAV SAAV? OV

HVO SYETOVL

Ha SAAVTOV Ya

BALL SAAVBTOV 1

HOBBY SAAVTYAV o HVO SAAVIOV. that MM bObivi me ot pgakstnyaltransferase schibviaye Bob ishieomye 24990 VSOBOM2AANeOOMI:L ObЯ692

ZTVve M AKOOZ6OVAVOTIET AND OVO

AKOgiteZVASZIVA la sAVAOVVA uovava SAATOVO Ya ry | mm bovivz KR 0332041 peF in -svyuvltrassferase vve lev pooshih 2.490. VSOZVO5AANZYAIVOOYA 0 RTOIV

TYAM AkOlevVASyuMA ORI?

HOVOTESAAEBEMOYA ROI havoMMsAAovyazA OA o KhevasSAAeYUUTA ObIOZ mm otZvev MR O38804. mm t5zggaMmR vat nm i7741v Me vo3i35 ok, B-sivaittransferase bib5aZ Movtevsyna 24shcha. VSOTOBBAANTYUOIYA OBOV.

BTEBIV NO ACCOZAT NAZK oesshshshcha

HVO SABO I

And mms o

SO sivtase BIibratasi Mykh tivsovya did not determine VSOBOTO?AANOVIUT. OATS? (INCLUDED in AvOZOvvVAAVYATATI OvSyH

ABOayaOVAAVOgOgI ovo

AKOZAMIVASOVVOHI OVKOKV decree of VASU 1

AKOCHIVEZVAZZIZZI I nm Ot2028me 0361582

OMmi-smintasm (23 bide Miyetezstyu padeyu ARZIVAAREVIYAGYAI obo -sivdiltranefervyza BT AAREMUZA OBSTO5 aapvoavAdaViYA 0 OBOMkO

ANOTZZO2VAATOCHVTY

AKO1BIVANDIUT

ZOH SAKII

NIK hundred WE

M-vcetilyuevlaktozamonikh 0 bibuvitasb Myv toasiviv: 2.avo. VSOZBeVBAANYAVAVI SOS i b-sivliluraneferazy ANOzBilaVAAVTOZVA OBIAMZ (stvovime u AVOZ5I23VALOEVOO! /097M55

AKOZOVAVVASITOVAL O9NOSe

MM.016073 MR. 0556801

I had vakhote ut NOT Determined 0 me 57VAAVOODVV A ! AKTTO72vAAEVIVIVA

Mota AAEVTZYAV

ARIBYUVA

MR. 051852 kg of salyltransferase Opeetbiyslvne was determined duets SAVVIZVA

Fig. 116

Back Organism! SHEB SheobeekoSvoyori Bamebnyu i (z3van" tuyuva | pk adb-sialintranepheraza Ovsolupeyov Me Vychanali d vo saROvVYANYA (Za tukizh oo v-poviszazviltransferacha: Opsotupstiya Ne VIZnachapi derzaponAstya loh raichyai (Tv M !

Olebtupelts' family did not determine AVOZIZ4VAST?VOV ono ve b-sialyleraneferase ppikkuvu (originating 0-2, Z-sialyltransferacha Opinogvye zada. ABIZ21eb?AAKIVYAT: ObMO57 ztasgu sieve

ONAnotOgu hieropis siyivge DROVAOVAVAVOTOTE mountain was not determined

OBIMVeaoo431 24.2 or Ogut aiyuya was not determined AKTZIvav SAVA oBINVOYU2Y 19 faropisa siyyeik DI.V829659 SAEOYA 74

ROVBZYU2 18 or Oguga wai Me determined by Drozovo VAVOZi VA

RAZVONOZL daropisa silynya drO)ZTva VANVOBVA fluor) "2.b-sivliltranoferazv. Oogive iabraene was determined by duvev576 CaO

STOBAmAye in (SHUT DOWN (frag Znanyaaisya ich zhe tav ore siyliltranepheriza Rep hodyudbuyuv not Defined childVOS SAFZYAVI8 tu ntiv | NOT DETERMINED via

Go! (with ; "They did not recognize ; : "and Zesialyleranoferase p boriyuduye and che BIB SAKIVTA

ЗИZ (Va I I I , ! : and m-sidznltransferase Rep odiyuduvane was determined by adding SAKovTIg 5tIZzban (bis)

And Yasialyltransferase vp godiyuduvine was determined by ATO SAM

Btasa m (vivNO) i -spliyatraisfera vp sodiyudutvane determined duteutosAavatv (BtA). yav sialyltranepherase Rap hyposioduvne determined Dutavta SAVA (vita) . that V" sizliltraneferase Rep vadiyoduvtne determined: АНвЗ454 SABO stosanA nasho! m iv4vato SAOIv098 be In saddlelyltransferase and pgodioduivnnye determined leva SAOTovoYu:

BTVOaIMAYE M (5SHYTOI (fragment) | ancient

From B sialiatranoferase Rai pofiodyuinely determined ALFIVET6 SAVIVTOv

Tobi m Vit | And pf in -sialyltraneferase Rep mountains was not determined AovbaVa SAVA

FTobamae YAN) (fragments

KA V-sialyltraneferzhiv a VA Raiyoobuevt 82996 lyuvolyuvsasivvovl (BivivA ! "eV sivliltranoferase 288 Rai iporiovyuth did not determine divata sasyvVog (Be.

Feb sevlyltransferase Ralp (odiodiezne was determined DUVYATOVISAOZISVIV I vo (BIS 2 B--cyaniztransferase Rep iodiodezene determined A97661cAO268991 0 (Zivao! y I vav sel uransferase VE Rapitodiyuvuehte determined: AUVVTVN» SAsobBOV (bag I ha

FV sivliliraisferaza VE Religorioduvy did not DETERMINE in deuvaZsASIVOS I cheek o, di, szamid vo» RBapigodiyuduvh 24904 AVIA SAKE siaviltrinerata thesis vm) I and I I ,

B. galactose ill. ey B- Bal uodyuvdoene determined ACTION 27825 SABOIVaYAI I eizlidtraneferase with ge une I I !

The use of ZT Rab was periodically determined twice

FIGLIN

Cart! that Body! Sho Be deppunk ideeler oUvny her (Zno pavvy of the poet was not determined by plaianim;

Viz sing Kazta MC OaBeMR. 052055 analysis of transferase Daniz pogodi 2496 MOo7BAdAAaoTaVi det vyk ne » MM. 031997 menvv dog d-sialyltraneferase z villa nevnznyachali DBeVVISAKOMVZ vTzbamivivms) p AZHIVT4ZSAROVVZ "g With sialyltranoferase Kaiiepoteristia Not IzNichalni a ; and

EM alizeraleferyata Kayiz half-spring: 24. КИВЖВСААБИВИИ

BTabatsi and | Sho Ma ozvo5nA Z13883 pz bsizliyatransferase I dana potmediyeih 24081 MyvTboAyaAOVY RYI vva. n М8Зт43ААВОГ2З33Й ee b-sivlyltransferase from Kaitsz pogevreiv was not determined! two

ZTbsaAde with BITAH | E st v-siylyltvansferase Kaiszpogedsiv indeterminate virgin cAOg5879.1

BTodviAe and pav GZBMADSADNO And le V. ssialiatraneferana nate poguvtsisnya 240- VS AANIOYUYA 0 OBivV6 yaTovals no MM me1azmR, ob'yaVEYA ve bsiyliltranepherosis Naiee pogmeritsya did not determine duvavaty savbzetovy vtezatmas M (WTO (fragment) ! ish AV -

Huh? bsialyltransferase 0 yetsepogmephisoya mevznachali det sACIVT

Stavamas m VishtEi and J 5 piznana dog, bsivlot ranoferaza Neba pogediyuva Me Vzynacheti dvivvv SAOIVT TOM

CALL ME (ZE) ifboatent) ee basaliltvaneferase Kene bvoguvsya 28 OBZYAOZASIAYA RUOBBA iZObUsyntazvi ZOBI babobBBadOVgI3A RYAUTI I ev bsaliatraneferase ЖKaerpoieriyeya undefined durRma SAMA

BE still esialyltranepheriza Vaisepoleieva nevniinasali! dovomezsdoezIVvn

SNATVEI. dovo ltraceferazi na popa 2488. ЗB AAAOIIAU 077 vtvvivii MM О5715e MR "7vlo? OBIVIZ i, B sialyltransferase 0 by pegterisov 2.Aav. SCHOVEVZVAANOOYUVI ROTU thesis I shk nm ovogaeni mya SS reg rsivviltranepherase Kaisya rogeztsieya 258.80 TUOO21BAANAONVOYAI OpveVa etaviya M

And balactozamii "and Dopierolerisye were not determined: DB SsAKVOM

SHK sho shi ge bOzsnntava ZTZOA U Leiuelomeritsya Not determined AVOIVO4OVAAZIYAOGY 098830

And And | and nm ban 1282 spkineraneferiosis vtZOn Nayszhlomediyev ze determined ADIAVVMOSAYELAV (called "y, a-sialyltransferasea Zymein yorisvye did not determine: duvavtoasAKVIZVT (webano !

Fairy b-sivliyatraneferazayu Zala orien did not define the dao SA cyB (atri I

Fo sialyltranioferase a 0 Zrpdyusevisv unspecified Diveviev sla (Z'Ezatasi ripanye kSANVIZAVYA pe Z laliluidanorevase Bie ve unspecified ALIBVA?OBSAEVTVV. (B13OASY «5 I-sioliyatraneferazna 0 biv ze. unspecified duvvivtya saEYAV295.1 (vT3vAAM | VY

Fo sim niraneferase according to ZM vOhoiV 24994 Me OZAAAZI25A 02745

BT and? Allyltransferase by sov 24991

And geuactosamid, Fah bik vola was not identified! OR SAECHNYAVYA I svaliptransferase (BtasaMmAEII I recognized sialyltransferase, which is not DEFINED, where AASVIZZYA both

Ifragment of ZTOO

Fig. 11

E Philoki Organism! SYA peiVank i Secheor obaivVyt eh

BTEOATSNASU ZHYFYASYMV 0 was not determined! AIHOVSANYAV in oferase tact I did not determine 4505 sAOZANY oh, sialiyatransferase so pibprev did not cause AdvovetvsAKEVI AND giving Y and y and AMS TSAKNIVA dkuy "kaleraneya ry Secrets and did not determine that? HC tue (ab) | translated you ar sialyltranoferase Takbods pibprez cheovyznapa doovnivsyaavovteya

WtZba beat! that about ts-2.v-silyltranofermza Takyroigivprez did not lower dota 4800 AZI vteba nevy play sialyltranepheriza SoNnots povprev it was determined VISA of the SAS

BTabaimAe Vita tovka V (VivOV- Such yanniiree did not determine the spon nab sidliltraneferase Z Takyiniishtrev pedefined desvasSAsIIVTV

From obynl IMS (fragment) | . You Vsyvliytryvsce times tTakyooy lispreg: 240 chitabasSaviazvi soyu eTobacnAE m (TO melon saSIVVOVA (fragment) that the zone of higher axis? bsialyltransferase Tachyodisutrye did not establish dovvttasslvteg

This is a MV fragment

VA? bsiachiptranepheriza Takiosoinirva unspecified AVM sSASOVIOVA

Ztabanaye MI (For?) I still ; and dutvzaskogo3134 avspiltranereraza TtTakiyvy pirprekh hviilyuachelyy VK yav dan zTaviya (Bin VA) Mf re x it was determined

The fragments of the Vsyaliltransfchnia Takyry chbprev o nevymchaly dugizzzvsASVT taza niv B) (frag) that I hi v-siviltraneferaa Takyosetyray did not vaznachali duli sAssIOVVo

BTOBIv (OS gfrragevnt that Veivlin and everaz Tekyoro piborev did not determine AItVasSlazazvt

STO's goat ace

BA B-sialytransferase Such pairs did not determine dBA SAT

TAB uma VE seg vsta ransferase in Takvtsye chryreya determined Dolma SAN dad U an VR (fralment that still ped uy sivliltransferachna chords swopree undefined 0 dDuadevosaoIv8

Table VN VN; 9 Zesialiltrapereraza Tvyzodov undefined 0 yutaAVOVSASZEVYA (Va | podomih that dov sialieeransfrevazh Tvcvodsp undefined DALA EVIVA btzbashvna o podkochekne What

Fe vivivtraneferazi and Teitodov did not define duvevagasSAKEVIVO vTaba (Zo podkone aga ba lintavneforaz Teikvodop did not underestimate dav odOevvyai

FATIGUE (iatvi podgo bej Allvliltovneferase tegeoi did not determine 0 dodevato bestatov

VTVbanas in (BE) podhomigknh sh-2, vsivliltraneferase Teiaoov pEOVYUNaChaE durivBZaSASVIV3v

Ltd.: (With VA subtovki; (regent) szyatsia: yes

BABI (Z VV) podhoripnya fragment)

AF Veizdiltovnereraza Tenvochop did not define dutlivvzaslag vi

Fig. PI a s Orga | mzi bizbavegogvlyuro svi i kov

OLEOOnraneferaa of Teigvoiog neyaninachali AlLIBMOSAVIOZTV

Tabia She VO prepared (frapnvnt) pe vesivliltraneferase Tenvodop did not determine AMS AOI VL zteviapmviayayu perkochikva (frag shchi

YAZ sedli feraz Heporyh events were not determined nava SAKVIZaOI om) oo av piatranepheraia Heporutidaeh were not determined dDOBOBTBVSAEU OY pal ranoferoz Heporizivechiv were not determined eq ov noi asana (wine) AMVVOZ SARA and Heporiz Vemiv dao. AVOOTeVVAAZOVI 083234 neolisivlizhtraneferase. and I ot Vsyalyltranoferaza x duv ; defines: AZOV ААСИОВаг vTVva (ZiniVA: veporikhye iz not to determine Aatadvt vla is 485 '

OZ enantase; Shchi ZOV 2 SAOTNEOV.

The unknown protein of lya Heporivivnyi was determined by 0 vsovetevAANEYATEVM

MOSS, и и ее rialiatransferase Heroriv yoriseiya Not determined DIVA SARIVOVI

In ettransferaya Heporikh na nevznakhali DvrzenasAKIgOvaya (Bivnoi) and pad sh-2 b-sialyltransferase Heporiz toriene neznachali /AoavVtasAsevtOg vtabaMmAS M (Zivitei

Ifragmekti and At and I oo Vgsiyliltranoferase Heporovivvemiya indeterminate pev SAVA

HAREEYA TO YOU 8)

Vekchaktosamiya. "two Heporich iorisvyav vne determined the left sSAKOVAVVA "bialittransfertza Z iIvtezai. And I am in hyalyltransferanzai Va Heporiz iorisyaye ze determined 0 UVBaTT5 ACTOM poly ee esivlosylsialyltraneferase keolelobea KI m malo dika soya (Me) o Bychko vet SAAE Ха en Z popisteutpransferase EvstepoinvoovKea zh; te shi s4taom eeybpolisial patra isferase Shi tepipoinv KA. MOBO53 AKASY, 1 Ana YIVO and KTVOVV SAAOCHOSV. OB1345.

BupE Nononi tevipoyye was not determined! OT5BBO AAVBZVYg I'm an owl

KAMIV nolysylyltransferase BIO Moivzena uvepiloikne unspecified AZI ogAAOVOOI (fragment) myste iy (fragments) Aivikhaetv peliprnkne unspecified HVO KARYITVVA

Ba (frasmeni) Mivizzan pepillonyuve ve determined / DUVIYUMAARZATET , but Mne that poured renefervza na) Language tepiuynye snevechanli A236191 ALOVBEVO I ia brragmeni Mevivantv lepipeniyeye: undefined AKA OLADRUKTO - 7 I owe amsiferase (88) Mvizopa telicnye nemzachali RUKh maga AALOVi yavyeva s pod (fragment) Ghivayeta petitioner was identified by AHOVTOVDARMTT; mibe2 happens ransferaa nenab) Mvizzola tepipudyuta was not determined KUA AAOVVYVI I

PMeNt, 5177 | ! sh

Bio (fragment) Meizzatov Tepiluyka. do not define AUZVMOYUZADRZATVVY io (frapyvnti mi teprotsyuya did not define AV MVBAARMIVaI

MET Mvivaot TeYOEE pe determined No opa me, 213134

Fig. THRESHING FLOOR

BIK Organ She bevbonetOvsireri Ziibvriei moya shchi denipopaya undefined de DAVOBEM oct Nestoryn: nNevnInachali moe AAAZAOTVI bila AYU S o tieves Navtorishyue undefined uzd ASM51 om yoepive KI Ne oMMOoTnR YAM 2 Z-siazyltranepherizy: Mvivzola 24834 Adpotovaya OBOBBIA RASIYATYAIV do 2

PV sivliltransferase KMeivvatv 24894 PVOBBgAASIAVO warm 1ZHENNOO yayu 2 Z sivliluvasferase Moigzniv 2aa9A vvi AAsYaVazA cho, MKS z03o bog. Mvizzon's sialyltranepherase 2404 iVOOVOVASYAYATSYA RT 0Yu? (kma8o922) pepipotsia OR BUT ZO mobe no ovane agvvg SHY

MMAZIV MZUTO did not determine: Bag BASAVaYAZVVI DMOUYE pelipoiki: mo voza me 285807 ha vMOBOv Rezishneye pedefined deFOOVAAKOZVOA ОЗСХСЯ pyosyuv MS O0VZ MR. back 1

RM

Ugan Baivopon was not identified, deBIvtVtAAMV2VOYAI OVO zaEVOV I recognized migan Yeainoleya S BIZNACHIALI DeBIduVadAMOOVOYAYA OBKVOg vygopsya BANI | mk man Boyniov was not identified AEBTVTVVOAAMVZOVYAA epvpsa.

BAKYAZE and ev Zaytyupena Me determined SOME AAMVONUV

Y BAK uva zatnlen onevnyznakhali AKVOCHIAAMNIVVYA I ZK vlgelsa

BANVB; zhizan Baiptopets was not identified 0 ARBINTOZAAMKOV (OK5VV anvney

BAKY DI uchvan Zaipoleiv did not define AR AAMVYABEKYA OBKEVO epneisa ZAKVYA

Zhavn Yalipnyshev was not belittled by ABe977ODAMeZ840, who was a victim

AND ZANSOTOM about what I van Zarlolene disagreed with DRU TVIAAMOVOO shi Bas shiya uuvan. krragaeni) bviluptela not insignificant dev dAMVaai OKO

Mtvvn (fragment) Baipopoysh 0 MevyZnYachalny AkEBouOZAAMOVVYAKYA OVKBOI epglsa yet FAVSTYAI shi

Uaen Zamyuopene did not define dev AASCHESVYAVYA OZKBYAE negise

ASV

Mavn Bvitopecha Me vyZnannyni DEBIYATYAZAAMOEVYAYA OKO vaivpsv.

BAWSTI

Fig. PI,

! ideohi Organism! I DepbayyutyuvyReeh Zenyk uva ifrapaenei Zvitopeiv Ye Determined: devIo a AAMNSHO i SKOAYA

Student's application

ZAKSY that shi Chuvan Faiyuvev determined it / AETOITYA AAMOVAZV I

VZAKSVV, what did the members of Хайпопей mean for the БИВВ77BAAMEVESI SVKBA?

ZAVOBNE in Zebpopeye Not determinable deEVIVTTTAAMOVEIVA OVKOAE ugaan ; of the National Academy of Sciences and and , .

Baiyupey's decrees undefined the debit of AAMINYMI OBKBAO

BANSFU. need to

UDF glucoho-k-12. Batovene 254 EV AAMaVIYEVA m natransferase venae BAKS

Bifenkytsianalna Satruevasivgiyshti pe vnznakhali dradgvovdATSOEOMA S OoVasI5 voza steel raneferiza ATOSyaYAY (see Svtruevasivtuvnoi nezchanali dDEZOBETI AATSOVOVA mhgh g; INN re Zesiiliykransfvevvza SB Satprusvasiveivdni ZD AHOVABv AKT.

These in ATS are 2-2, a-sialintranepherase (SV Setruyuvasiveiiyut 24.89". AKZOOOZT AKB its Atom What is sialiittranepherase (sy. Saprmovevivsivin 24.98. ACTIVE SYAZyV 0 ObKOMO about ZI sivliltransferaa E Satruyureasiv also determined AEZOMV DAK so9zZMII (point ATOM I of sivlileraneferikhy B Svprmodanyusivni 2488" DETO734 AEN and AOS yaznv | more. and

Fe Zesialyltranophorase (St. Satruyubastveluit 2498, drarivevAAoVIOM OZ pO5

Tsi Ateszhn eV V-siznitraneiyuraza Setruyuvasiegivin ba" kuOvavvAAMOVOSIA O938X6 (Sen AKsYANU still ory M simnatraneferyaza Satruyuvasivg vit o nezachanali ARe16847 LASYA

Sk ATOS 00297 Sho

ОВЕ Satruyuvayetvtivynya vetsychanali Ar 197ААНВОВТВ ot

YAZ sizlilernosferaza szyI Seapruyurasivye ii 248" AERTIBOBBAASOVA 6. MS and od a-silyliatransferase seni Zovgya AIlZvatsSAvVtIU5YA OR Mmea sao meto i468 24 MS Olya Mme ovoovy I a Zoben g raneferase Setruobaiennivili Not determined. "AAOZVVNYA

NeseCho by. | dhazaett Sava anode sialyltranscerazz betruyurasivgiannii unspecified AHeZA SAROIVK sa 019 sho shchi vani vav sialyltrapeferase Selrudesivg idiot pnezchanali Aa SAKVYT

Pp | in at. branches that TD ai-sialyltranoferase Oepruyurasiegishiti did not determine dhezaaz saKvyTOM osn | oh And what

OZ AB sivpiltraceferoz Sepruyuvasivgiviyti still determined -everywhere and (sya) ok "AATITOVT , nich AH gV SATIN es? With sialiitransferase and betruyuvasiyusivdii 2488. AETZOaOBAAEIiaI SVK OKi

Bifunctional Satruyuvysivhivrt! 2485- DE zzoove AVT 18076 ee ZOV sno ntranepherase neza AHOZAZVSAROkvv i 1NOVA (zm SHY What

Fig. 11M

B oki sho ori ani mishe osaVAyuO that ve vaivetkoya g.

НююзСвобиралывный! 5. and did not determine ZO AASTOY 0 ROI (fragile) Navtoriye fzpyovpuve KhBIZIBSALOVE?

Rmita Bazivolove picosuans determines deOdvivuAAKOZOVAYA pebiRI

The Ministry of Internal Affairs and Communications of the Russian Federation did not specify the AAOZVVIZ

This sequence 10, described by Unknown, was not attributed to Atize69

USA me 0503744 Addttovam

Succession of TO, enisatsa uva Unknown was not determined,

USA Me 6099705 AATTtol

Sequence 12, original in paten Unknown not determined -AATIZIZO

USA B 0699705

Peslidennost o", described in the pateny. It is unknown, it was determined

USA He o505744 and y

The sequence of Z. opisava in the PeVidomiy vonatei was not determined "AATYATOVO E

STA Mebo99705 om I E

The sequence 34, described in the patent, was not determined consciously, -VAOVOVOGA

USA M 6503744 eniavichni

Sequence 35, Described in the Bay of SOMY was not determined "Mar

SINA Me 65013744 ofpayementi o. - to her I ""AAeZHE

Sequence 45, described by the KChidemnI zatents, did not determine "AATATOVVA

SPAS va99705 Y y y Y

Sequence 5 described in B patent 0 New not identified "AATIT986 4

STA Me 6699705 shk | !

Sequence 9. is described in the note "Unknown Uncertainty"

SINA Me 603744

Fig. TI M

Claims (27)

1. Conjugate of the peptide of the granulocyte colony-stimulating factor (0-C5E), containing the following sugar fragment, which includes a PEG residue: on p o. /boon no 9th on where O is selected from -OH and KI ANM; C is selected from K.I, and C(OHCI-Cv)alkyl; K! represents a group selected from fragments that include a linear or branched residue of polyethylene glycol; 1 I. means a linker selected from a bond, substituted or unsubstituted alkyl 1 substituted or unsubstituted heteroalkyl, provided that if Y is OH, then C is K-I, and if C is C(O )X(Si-Se)alkyl, then Y represents K-I-MH-. 2. The peptide according to claim 1, where KI, has the formula: ев : о б where a is equal to a whole number from 0 to 20. 3. Peptide according to claim 1, where B! has a structure that is selected from: se) o; r pntta udo emnomkh Mne(0)sn,nuosn,sNUyusnN, Mnesyusn,snyOosn,sN osn, o s; 2 o--enenaech ro venoi" Mne(osn,snuosn,sNdusn, Mne(o)osn,snuosN,sNuyusN", where e 1 and are independently equal to integers selected from 1-2500, and 4 is equal to an integer from 0 to 20. 4. Peptide according to claim 1, where VE! has a structure selected from: o) what is Mneoryusnsnosnyosn;osn" h МН» " nm y ne(oyusnosnhosnsnoysn» Ge) ch ge! , mne(o)snsnuosNosno Osn» Z : ny ne(ohsnosnuosnsnyusn» o i (e) МNe(o)snsnhosnosno)v Osn» Z : 1 Mne(o) СнснОСносNo СН» nm Nekozhsнсноснсносно» (c) t o; chne(yusn,snuosnosNO Osn ; : чНе(ОюСснНsСНхоснисною СН» nm neсоснсноснасноснохосна o I where: e, T1T are equal to whole numbers, independently chosen from 1-2500, and d 14 mean integers numbers independently chosen from 1-20. 5. Peptide according to claim 1, where B! has a structure that is chosen from: o MNe(oyusnsnuosnsnovOSN from such AD tr niyuyusnisnosnsnayusn" Mn l nm Mn. I ne(o)osnisnhosnsnokosnz o Ф : o В Mne(oyesn,snuosnosnovOSN" with (c) ODA and r eyuisnisnaosnisnayusn" mn , НМ МН; I Ne(o)snosnIossnosnoosnya (c) h where e, T1 Y are independently equal to integers chosen from 1-2500, and d, 4 1 4" are equal to integers independently chosen from 1-20. 6. Peptide according to claim 1, where B! has a structure selected from: 8-Ф(0)СноснHOСНСно) ОС ;1 5-С(0)оСНаСН Osno СН jus where е 1 І are independently equal to integers selected from 1-2500. 7. Peptide О-С5Е according to claim 1, where the indicated fragment has the formula: о о о. oon no o-sa-3 v--nm on 8. Peptide О-С5Е according to claim 1, where the specified fragment has the formula: о о о. oon Pe) O-bai-SaimAs--2 5--nNyu on 9. Peptide O-C5E according to claim 1, where the specified fragment has the formula: it c) is) soon li no Oo--ba--batMmAs--AA v--nk li on where AA means the amino acid residue of the specified peptide. 10. The CO-C5E peptide according to claim 9, where the specified amino acid residue is selected from serine or threonine. 11. Peptide O-C5E according to clause I, where the indicated peptide has the amino acid sequence 5ZEO IJU MO.1. 12. Peptide O-C5E according to claim 10, where the indicated amino acid residue is threonine at position 134 of 5EO and MO 1. 13. Peptide O-SB5E according to claim 1, where the specified peptide has an amino acid sequence selected from BEO IU MO.1 1 5EO IU MO:2. 14. Peptide O-SB5E according to claim 1, where the indicated fragment has the formula: SismAs-(ba), |, (51a), - (V. (re r "Soky ko (i ik ! 5-AK SISMAs-SISMAs-Mai kli ! mak POiomAs-(bai) (bia);- (B) . e, I, 51 are independently equal to integers chosen from 0-6; MUK 11 t are independently equal to integers chosen from 0-100; у, м, х 1у are independently selected from 0 1 И, and at least one of у, ху, х 1 у is equal to 1; AA means the amino acid residue of the indicated C-C5E peptide; 51ia-(K) has the formula: no r nos o on 5-9 mn-s on where: I selected from -OH and VI ANM; C is selected from K-I.- 1-C(OXCi-Cv)alkyl; K! is a group containing a fragment selected from a linear or branched polyethylene glycol residue; 1 And, is a linker selected from 1 bond, substituted or unsubstituted alkyl, 1 substituted or unsubstituted heteroalkyl; provided that, if Y means OH, then C means KI, and if ОС means -С(ОЧСи-Св)alkyl, then Y means K/-I-AMN.-. 15. Peptide according to claim 14, where the indicated amino acid residue is an asparagine residue. 16. The peptide according to item I, where the specified peptide is a biologically active peptide of the granulocyte colony-stimulating factor. 17. The method of obtaining the O-C5E peptide conjugate containing the following sugar fragment, which includes a PEG residue: no or noos o on 5-9 Mmn-o on de O selected from -OH and KI ANM; C is selected from K.I, and C(OHCI-Cv)alkyl; K! represents a group containing a fragment selected from a linear or branched polyethylene glycol residue; 1 And, means a linker that is selected from 1 bond, substituted or unsubstituted alkyl 1 substituted or unsubstituted heteroalkyl, provided that, if Y is OH, then C is K-I, and if C is C (OX(Si-Ce)alkyl, then Y is K-I-MH-; which includes: (a) contacting the C-C5E peptide, which is a substrate, with the PEG-sialic acid donor fragment, which has the formula: on ( c) SoonN.. no o 7 no tra on 5--n Ge! (o, on o v chu Mne 1 with an enzyme that transfers the specified PEG-sialic acid to an amino acid residue or a glycosyl residue of the specified C-C5E peptide under conditions, that are suitable for such transfer. 18. The method according to claim 17, where B"-I, has the formula: veins : o 2 where a is equal to a whole number from 0 to 20. 19. The method according to claim 17, where B! has a structure selected from: o o r pnenayu udo emnomkh Mne()sn,nHosn,snosN, chnesyusn,snuiosn,sN osn, o (v; 2 o--enenaeh ro pnenoyuh Mne(osn,nosn,snuyusn, Mchne(o)osn,snuosn,sNDyusN , where е 1 І are independently equal to integers chosen from 1-2500, d is equal to an integer from 0 to 20. 20. The method according to claim 17, where E! has a structure that is selected from: Ch. MN" ' nm not (oyusn, snuosnosnosnya (in ch ). Mne(o)sNnuosNosNovO SN» Z : nyu Gu noryunenuosnenyos, o y o Mne()snsnuosnosnovOsN» Z : MNe(o)snsnuOosNosN yus nya ne(osnosn(osNsNayuSsn» i o i o Mne(oyusnosnosNosNo) OS E : MNesoyusnosnuosnIsnosnosnz nm U noryusnentosnendos about and where: e, T1T are equal to integers independently selected from 1-2500, and d 14 means integers independently selected from 1-20. 21. The method according to claim 17, where E! has a structure selected from: iv) MneyuiosnInhosnsnOIosNn" sum ODU tr ioyusnisnkosnisnoysn" Mn and nm Mn; С" ne(oyusnsnuosnosnohosn" o y : o Mne(osnisnhosnsnohosn s : m ODA and r eyuisnsnuosnosnohosn" Mn b NM Mne Ne(o)snisnhosnosnoosn" o h where e, T1 Y are independently equal to integers selected from 1-2500, and e, 4 1 4" are equal to integers independently chosen from 1-20. 22. The method according to claim 17, where E! has a structure selected from: 8-Ф(О)СНосно(ОСНоснодеО Сн» 1 8-б(ОЮСНснОСносНо Сн 2 where е 1 І are independently equal to integers selected from 1-2500. 23. The method according to claim 17, which, before carrying out stage (a), includes: (5) expression of the indicated granulocyte colony-stimulating factor peptide, used as a substrate, in a suitable host. 24. The method according to claim 17, where the specified host is selected from an insect cell and a mammalian cell. 25. A method of stimulating the production of inflammatory leukocytes in a mammal, which includes the administration of the peptide according to claim 1 to the indicated mammal. 26. A method of treating an infection in a patient in need thereof, which includes the step of administering to said patient an amount of the peptide according to item I, effective to relieve said condition in said patient. 27. Pharmaceutical composition containing the peptide of granulocyte colony-stimulating factor according to claim 1 1 pharmaceutically acceptable carrier.