MXPA06007725A

MXPA06007725A - O-linked glycosylation of peptides

Info

Publication number: MXPA06007725A
Application number: MXPA/A/2006/007725A
Authority: MX
Inventors: A Zopf David; Clausen Henrik; Defrees Shawn; Wang Zhiguang
Original assignee: Clausen Henrik; Defrees Shawn; Neose Technologies Inc; Wang Zhiguang; A Zopf David
Priority date: 2004-01-08
Filing date: 2006-07-05
Publication date: 2006-12-13

Abstract

The present invention provides polypeptides that include an O-linked glycosylation site that is not present in the wild-type peptide. The polypeptides of the invention include glycoconjugates in which a species such as a water-soluble polymer, a therapeutic agent of a biomolecule is covalently linked through an intactO-linked glycosyl residue to the polypeptide. Also provided are methods of making the peptides of the invention and methods, pharmaceutical compositions containing the peptides and methods of treating, ameliorating or preventing diseased in mammals by administering an amount of a peptide of the invention sufficient to achieve the desired response.

Description

GLUCOSILATION OF PEPTIDES LINKED IN O Field of the Invention The present invention relates to glycosylated glycosides linked at 0 particularly therapeutic peptides and peptide mutants including sites of "glycosylation bound at 0 not present in the wild-type peptide The administration of glycosylated and non-glycosylated peptides to generate a particular physiological response is well known in the arts of medicine For example, both purified and recombinant hGH are used for the treatment of conditions and diseases due to hGH deficiency, for example, dwarfism in children, interferon has a known antiviral activity and the stimulating factor of the granulocyte colony stimulates the production of white blood cells. limited the use of therapeutic peptides, is the inherent difficulty in engineering an expression system to express a peptide having the glycosylation pattern of the wild-type peptide.As known in the art, the glycosylated peptides inappropriately or incompletely can be immunogenic, which leads to the neutralization of the ptido and / or leads to the development of an allergic response. Other Ref .: 174048 Deficiencies of glycopeptides that are produced recombinantly include suboptimal potency and rapid clearance rates. One method for solving problems inherent in the production of therapeutics for glycosylated peptides has been to modify the peptides in vitro after they are expressed. Modification of subsequent expression in vitro has been used both to modify glucan structures and to introduce glucans into novel sites. A detailed tool of recombinant eukaryotic glycosyltransferases has been available, making the in vitro enzymatic synthesis of mammalian glycoconjugates with custom designed glycosylation patterns and possible glycosyl structures. See for example U.S. Patent No. 5,876,980; 6,030,815; 5,728,554; 5,922,577; and WO / 9831826; US2003180835; and WO 03/031464. In addition to manipulating the structure of the glucosyl groups in the polypeptides, the glycopeptides can be prepared with one or more non-saccharide modifying groups, such as water-soluble polymers. An exemplary polymer that has been conjugated to the peptides is poly (ethylene glycol) ("PEG"). The use of PEG to derive therapeutic peptides has been shown to reduce the immunogenicity of the peptides. For example, U.S. Patent No. 4,179,337 (Davis et al.) Discloses non-immunogenic polypeptides such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. In addition to the reduced immogeneity, the depuration time in circulation is prolonged due to the increasing size of the PEG conjugate of the polypeptides in question. The main mode of placement of the PEG and its derivatives, to the peptides is a non-specific binding through a peptide amino acid residue (see for example, US Patent No. 4,088,538, US Patent No. 4,496,689, US Pat. No. 4,414,147, U.S. Patent No. 4,055,635 and PCT WO 87/00056). Another way of binding PEG to the peptides is through a non-specific oxidation of the glycosyl residues on a glycopeptide (see for example WO 94/05332). In these non-specific methods, random poly (ethylene glycol) does not specify the reactive residues in a peptide column. Of course, the random addition of the PEG molecules has its drawbacks, including a lack of homogeneity of the final product and the possibility of reduction in the biological or enzymatic activity of the peptide. Therefore, for the production of therapeutic peptides, a derivation strategy that results in the formation of a specifically labeled, easily characterized, essentially homogeneous product is superior.

Therapeutically specifically labeled, homogeneous peptides can be produced in vitro through the action of enzymes. Unlike typical non-specific methods for attaching a synthetic polymer or other label to a peptide, enzyme-based syntheses have the advantages of a regioselectivity and stereoselectivity. Two major classes of enzymes for use in the synthesis of labeled peptides are glycosyltransferases (eg, sialyltransferase, oligosaccharyltransferase, N-acetylglucosaminyltransferase) and glycosidases. These enzymes can be used for the specific binding of sugars which can subsequently be modified to comprise a therapeutic moiety. Alternatively, glycosyltransferases and modified glycosidases can be used to directly transfer modified sugars to a peptide column (see for example U.S. Patent No. 6,399,336 and U.S. Patent Application Publications 20030040037, 20040132640, 20040137557, 20040126838 and 20040142856, each of which is incorporated herein by reference). Methods of combining both chemical and synthetic enzymatic elements are also known (see for example Yamamoto et al., Carbohydr Res. 305: 415-422 (1998) and US Patent Application Publication 20040137557 which is incorporated in the present as a reference).

I Carbohydrates bind to glycopeptides in various forms, from which N-linked to asparagine and linked at 0 of mucin type to serine and threonine are the most relevant for recombinant glycoprotein therapeutics. Unfortunately, not all polypeptides comprise an N-linked glycosylation site or linked at 0 as part of their primary amino acid sequence. In other cases, an existing glycosylation site may be inconvenient for the binding of a modifying group (eg water-soluble or water-insoluble polymers, therapeutic moieties and / or biomolecules) to the polypeptide, or the binding of such portions in that site may cause an undesirable decrease in the biological activity of the polypeptide. Thus, there is a need in the art for methods that allow both the precise creation of glycosylation sites and the ability to precisely direct the modification of those sites.

Brief Description of the Invention It is a discovery of the present invention that enzymatic glycoconjugation reactions can be directed specifically to O-linked glycosylation sites and to glucosyl residues that bind to the glycosylation sites linked at 0. The linked glycosylation sites at 0 targets may be native sites to a wild type peptide or alternatively, they may be introduced into a peptide by mutation. In this manner, the present invention provides polypeptides comprising mutated sites suitable for 0-linked glycosylation and pharmaceutical compositions thereof. In addition, the present invention provides methods of making such polypeptides and the use of polypeptides and / or pharmaceutical compositions thereof for therapeutic treatments. Thus, in a first aspect, the invention provides an ated polypeptide comprising a sequence of a mutant peptide wherein the mutant peptide sequence encodes an O-linked glycosylation site that does not exist in the corresponding wild-type polypeptide. In one embodiment, the ated polypeptide is a G-CSF polypeptide. In one embodiment, the G-CSF polypeptide comprises a sequence of mutant peptides with the formula of 1XnTPLGP or M1B0PZmXnTPLGP. In this embodiment, the supra index 1 denotes the first position of the amino acid sequence of the wild-type G-CSF sequence (SEQ ID NO: 3), the subscripts n and m are integers selected from 0 to 3, and at least one of X and B is threonine and serine, and when more than one of X and B is threonine or serine, the identity of these portions is independently selected. Also in this modality, Z is selected from glutamate, any non-charged amino acid or dipeptide combination including MQ, GQ and MV. In another embodiment, the G-CSF polypeptide comprises a mutant peptide sequence selected from the sequences consisting of MVTPLGP, MQTPLGP, MIATPLGP, MATPLGP, MPTWGAMPLGP, MVQTPLGP, MQSTPLGP, MGQTPLGP, MAPTSSSPLGP and MAPTPLGPA. In another embodiment, the G-CSF polypeptide comprises a mutant peptide sequence with the formula of M1TPXBOrP. In this embodiment the superscript, 1, denotes the first position of the amino acid sequence of the wild-type G-CSF sequence (SEQ ID NO: 3), and the subscript r is an integer selected from 0 to 3, and at least one of X, B and O is threonine or serine, and when more than one of X, B and O is threonine or serine, the identity of these portions is independently selected. In another embodiment, the G-CSF polypeptide comprises a mutant peptide sequence selected from the sequences consisting of: MTPTLGP, MTPTQLGP, MTPTSLGP, MTPTQGP, MTPTSSP, M ^ PQTP, M ^ PTGP, MXTPLTP, MXTPNTGP, MTPLGP, M ^ PVTP , M ^ PMVTP and MT ^ TQGL ^ P ^ S7. In another embodiment, the G-CSF polypeptide comprises a mutant peptide sequence with the formula of LGX53BoLGI, wherein the superscript denotes the position of the amino acid in the wild type G-CSF amino acid sequence, and X is histidine, serine, arginine , glutamic acid or tyrosine, and B is either threonine or serine, I is an integer from 0 to 3. In another embodiment, the G-CSF polypeptide comprises a mutant peptide sequence selected from sequences consisting of: LGHTLGI, LGSSLGI, LGYSLGI, LGESLGI, and LGSTLGI. In another embodiment, the G-CSF polypeptide comprises a mutant peptide sequence with the formula P129ZmJq0rXnPT wherein the superscript denotes the position of the amino acid in the wild type G-CSF amino acid sequence, and Z, J, 0 and X are independently selected from threonine or serine, and m, q, r, and n are independently selected from 0 to 3. In another embodiment, the G-CSF polypeptide comprises a mutant peptide sequence selected from the sequences consisting of: P129ATQPT, P129TLGPT, P129TQGPT, P129TSSPT, P129TQGAPT, P129NTGPT, PALQPTQT, P129ALTPT, P129MVTPT, P129ASSTPT, P129TTQP, P129NTLP, P129TLQP, MAP129ATQPTQGAM, and MP129ATTQPTQGAM. In another embodiment, the G-CSF polypeptide comprises a mutant peptide sequence with the formula of PZmU? JqP61OrXnBoC wherein the superscript denotes the position of the amino acid in the wild-type G-CSF amino acid sequence, and at least one of Z, J, O, and U is selected from threonine or serine, and when more than one of Z, J, 0 and D is threonine or serine, each is independently selected, X and B are any uncharged amino acids or glutamates, and m, s, q, r, n, I are independently selected from 0 to 3. In another embodiment the G-CSF polypeptide comprises a mutant peptide sequence selected from the sequences consisting of: P61TSSC, P61TSSAC, LGIPTAP61LSSC, LGIPTQP61LSSC, LGIPTQGP61LSSC, LGIPQTP61LSSC, LGIPTSP61LSSC, LGIPTSP61LSSC, LGIPTQP61LSSC, LGTPWAP61LSSC, LGTPFAP61LSSC, P61FTP, and SLGAP58TAP € 1LSS. In another embodiment the G-CSF polypeptide comprises a mutant peptide sequence with the formula 0aGpJqOrP175XnBoZmUs? T wherein the superscript denotes the position of the amino acid in SEQ ID NO: 3, and at least one of Z, U, O, J, Gr. 0, B and X are threonine or serine and when more than one of Z, U, O, J, G, 0, B and X are threonine or serine, which are independently selected. 0 is optionally R, and G is optionally H. the symbol? represents any non-loaded amino acid residue or glutamate, ya, p, q, r, n, o, m, s, and t are integers independently selected from 0 to 3. In another embodiment the G-CSF polypeptide comprises a mutant peptide sequence selected from the sequences consisting of: RHLAQTP175, RHLAGQTP175, QP175TQGAMP, RHLAQTP175AM, QP175TSSAP, QP175TSSAP, QP175TQGAMP, QP175TQGAM, QP175TQGA, QP175TVM, QP175NTGP, and QP175QTLP. In another embodiment the G-CSF polypeptide comprises a mutant peptide sequence selected from the sequences P133TQTAMP139, P133TQGTMP, P133TQGTNP, P133TQGTLP, and PALQP133TQTAMPA. In another embodiment, the "isolated polypeptide" is an hGH polypeptide In one embodiment, the hGH polypeptide comprises a mutant peptide sequence with the formula of P133JXBOZUK140QTYS, wherein the superscript denotes the position of the amino acid in (SEQ ID NO: 20); and J is selected from threonine and arginine; X is selected from alanine, glutamine, isoleucine, and threonine; B is selected from glycine, alanine, leucine, valine, asparagine, glutamine, and threonine; Or it is selected from tyrosine, serine, alanine, and -threonine; and Z is selected from isoleucine and methionine; and U is selected from phenylalanine and proline. In another embodiment, the hGH polypeptide comprises a mutant peptide sequence selected from the sequences consisting of: PTTGQIFK, PTTAQIFK, PTTLQIFK, PTTLYVFK, PTTVQIFK, PTTVSIFK, PTTNQIFK, PTTQQIFK, PTATQIFK, PTQGQIFK, PTQGAIFK, PTQGAMFK, PTIGQIFK, PTINQIFK, PTINTIFK , PTILQIFK, PTIVQIFK, PTIQQIFK, PTIAQIFK, P133TTTQIFK140QTYS, and P133TQGAMPK140QTYS. In another embodiment, the hGH polypeptide comprises a mutant peptide sequence, with the formula of P133RTGQIPTQBYS wherein the superscript denotes the position of the amino acid in SEQ ID NO: 20; and B is selected from alanine and threonine. In another embodiment, the hGH polypeptide comprises a mutant peptide sequence selected from the sequences consisting of: PRTGQIPTQTYS and PRTGQIPTQAYS. In another embodiment, the hGH polypeptide comprises a mutant peptide sequence with the formula L128XTBOP133UTG wherein the superscript denotes the position of the amino acid in SEQ ID NO: 20; and X is selected from glutamic acid, valine and alanine; B is selected from glutamine, glutamic acid, and glycine; 0 is selected from serine and threonine; and U is selected from arginine, serine, alanine and leucine. In another embodiment, the hGH polypeptide comprises a mutant peptide sequence selected from the sequences consisting of: LETQSP133RTG, LETQSP133STG, LETQSP133ATG, - LETQSP133LTG, LETETP1 3R, LETETP133A, LVTQSP133RTG, LVTETP133RTG, LVTETP133ATG, and LATGSP133RTG. In another embodiment, the hGH polypeptide comprises a mutant peptide sequence with the formula M1BPTXnZmOPLSRL wherein the superscript 1 denotes the position of the amino acid in SEQ ID NO: 19; and B is selected from phenylalanine, valine and alanine or a combination thereof; X is selected from glutamate, valine and proline Z is threonine; 0 is selected from leucine and isoleucine; and when X is proline, Z is threonine; and wherein n-y m are selected integers of 0 and 2. In another embodiment, the hGH polypeptide comprises a mutant peptide sequence selected from the sequences consisting of: M ^ -FPTE IPLSRL, MXFPTV LPLSRL, and M ^ PTPTIPLSRL. In yet another embodiment the hGH polypeptide comprises the following mutant peptide sequence: M ^ TPTIPLSRL. In yet another embodiment the hGH polypeptide comprises a mutant peptide sequence selected from M1APTSSPTIPL7SR9 and DGSP133NTGQIFK140. In another embodiment, the isolated polypeptide is an IFN alpha polypeptide. In one embodiment, the INF alpha polypeptide has a peptide sequence comprising a mutant amino acid sequence, and the peptide sequence corresponds to the region of INF alpha 2 having a sequence as shown in SEQ NO: 22, and wherein the mutant amino acid sequence contains a mutation at a position corresponding to T106 of INF alpha 2. In another embodiment the IFN alpha polypeptide is selected from the group consisting of IFN alpha, IFN alpha 4, IFN alpha 5, IFN alpha 6, IFN alpha 7, IFN alpha 8, IFN alpha 10, IFN alpha 14, IFN alpha 16, IFN alpha 17, and IFN alpha 21. Still in another embodiment, the IFN alpha polypeptide is an IFN alpha polypeptide comprising a mutant amino acid sequence selected from the group consisting of: 99CVMQEERVTETPLMNADSIL118, 99CVMQEEGVTETPLMNADSIL118, and "CVMQGVGVTETPLMNADSIL118. Still in another embodiment, the IFN alpha polypeptide is an IFN alpha 4 polypeptide comprising a mutant amino acid sequence selected from the group. It consists of: 99CVIQEVGVTETPLMNVDSIL118, and "CVIQGVGVTETPLMKEDSIL118. In another embodiment, the IFN alpha polypeptide is an IFN alpha 5 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: 99CMMQEVGVTDTPLMNVDSIL118, "CMMQEVGVTETPLMNVDSIL118- and" CMMQGVGVTDTPLMNVDSIL118. In another embodiment, the IFN alpha polypeptide is an IFN alpha 6 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVMQEVWVTGTPLMNEDSIL118, 99CVMQEVGVTGTPLMNEDSIL118, and" CVMQGVGVTETPLMNEDSIL118. In yet another embodiment, the IFN alpha polypeptide is an IFN alpha 7 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: 99CVIQEVGVTETPLMNEDFIL118, and "CVIQGVGVTETPLMNEDFIL118. Still in another embodiment, the IFN alpha polypeptide is an IFN alpha 8 polypeptide. comprising a mutant amino acid sequence selected from the group consisting of: "CVMQEVGVTESPLMYEDSIL118," and "CVMQGVGVTESPLMYEDSIL118." In another embodiment, the IFN alpha polypeptide is an IFN alpha 10 polypeptide comprising an amino acid sequence selected from the group consisting of: 99CVIQEVGVTETPLMNEDSIL118, and 99CVIQGVGVTETPLMNEDSIL118 In another embodiment, the IFN alpha polypeptide is an IFN alpha 14 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVIQEVGVTETPLMNEDSIL118, and 99CVIQGVGVTETPLMNEDSIL118. In another embodiment, the IFN alpha polypeptide is an IFN alpha 16 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVTQEVGVTÉIPLMNEDSIL118, -" CVTQEVGVTETPLMNEDSIL118, and "CVTQGVGVTETPLMNEDSIL118. Still in another embodiment, the IFN alpha polypeptide is a IFN alpha 17 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: 99CVIQEVGMTETPLMNEDSIL118, "CVIQEVGVTETPLMNEDSIL118, and 99CVIQGVGMTETPLMNEDSIL118. In a further embodiment, the IFN alpha polypeptide is an IFN alpha 21 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: 99CVIQEVGVTETPLMNVDSIL118, and 99CVIQGVGVTETPLMNVDSIL118. In a second aspect, the invention provides an isolated nucleic acid encoding a polypeptide comprising a mutant peptide sequence, wherein the mutant peptide sequence encodes a 0-linked glycosylation site that does not exist in the corresponding wild-type polypeptide. In one embodiment the nucleic acid encodes a polypeptide corresponding to a mutant peptide sequence is comprised within an expression cassette. In a relative embodiment, the present invention provides a cell comprising the nucleic acid of the present invention. In a third aspect, the isolated polypeptide comprises a mutant peptide sequence, which encodes an O-linked glycosylation site that does not exist in the wild-type polypeptide, has a formula selected from: O- GaíNAc- X wherein AA is an amino acid side chain comprising a hydroxyl portion that is within the sequence; and X is a modification group or a saccharyl moiety. In an X-modality it comprises a selected group of sialyl, galactosyl and Gal-Sia portions, wherein at least one of sialyl, galactosyl and Gal-Sia comprise a modified group. In another embodiment X comprises the portion: wherein D is a member selected from -OH and RX-L-HN-; G is a member selected from R1-L- and -C (0) alkyl (C6C6); R1 is a portion comprising a member selected from a portion comprising a straight chain or branched poly (ethylene 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 R1-L-, and when G is -C ( O) alkyl (C? -C6), D is R ^ L-NH-. In another modality X comprises the structure: wherein, L is a substituted or unsubstituted alkyl group or substituted or unsubstituted heteroalkyl; and n is selected from the integers from 0 to about 500.

In another embodiment, X comprises the structure: where s is selected from the integers from 0 to 20.

In a fourth aspect, the invention provides a method for making a glycoconjugate of an isolated polypeptide comprising a mutant peptide sequence encoding a 0-linked glycosylation site that does not exist in the corresponding wild-type polypeptide comprising the steps of: ) producing recombinantly the mutant polypeptide, and (b) enzymatically glycosylating the mutant polypeptide with a modified sugar at the 0-linked glycosylation site. In a fifth aspect, the invention provides a pharmaceutical composition of an isolated polypeptide comprising a sequence of a mutant peptide, wherein the mutant peptide sequence encodes a 0-linked glycosylation site that does not exist in the corresponding wild-type polypeptide. In one embodiment, the pharmaceutical composition comprises an effective amount of a G-CSF polypeptide of the invention glycoconjugated with a modified sugar. In a related embodiment, the modified sugar is modified with a member selected from poly (ethylene glycol) and methoxy-poly (ethylene glycol) (m-PEG). In another embodiment, the pharmaceutical composition comprises an effective amount of a hGH polypeptide of the invention glycoconjugate with a modified sugar. In a related embodiment, the modified sugar is modified with a member selected from poly (ethylene glycol) and methoxy-poly (ethylene glycol) (m-PEG). In another embodiment, the pharmaceutical composition comprises an effective amount of a polypeptide - of a granulocyte macrophage colony stimulating factor of the invention glycoconjugated with a modified sugar. In a related embodiment, the modified sugar is modified with a member selected from poly (ethylene glycol) and methoxy-poly (ethylene glycol) (m-PEG). In another embodiment, the pharmaceutical composition comprises an effective amount of an IFN alpha polypeptide of the invention glycoconjugated with a modified sugar. In a related embodiment, the modified sugar is modified with a member selected from poly (ethylene glycol) and methoxy-poly (ethylene glycol) (m-PEG). In a sixth aspect the invention provides a method of delivering therapy to a subject in need of therapy, wherein the method comprises administering to the subject an effective amount of a pharmaceutical composition of the invention. In one embodiment, the therapy delivered is G-CSF therapy. In another embodiment, the therapy provided is a granulocyte macrophage colony stimulating factor therapy. In another embodiment, the therapy provided is alpha interferon therapy. In still another modality the therapy provided is growth hormone therapy. Aspects, advantages and additional objectives of the present invention will be apparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph of absorbance versus GCSF concentration for unmodified G-CSF and glyco-PEG-like analogs in an NSF-60 cell proliferation assay. Figure 2 is a graph of counts per minute (CPM) versus time for a pharmacokinetic study in rats using radioiodinated G- ^ CSF and glycol-PEG derivatives thereof. Figure 3 is a plot of μg / mL G-CSF in blood versus time (h) for a pharmacokinetic study in rats using radioiodinated G-CSF and glycol-PEG-side derivatives thereof. Figure 4 is a graph showing the induction of white blood cell cells in mice using unmodified G-CSF and chemical and glycopegylated G-CSF. Figure 5 is a graph of the results of an aggregation assay that follows the radioiodination with the Bolton-Hunter reagent. Figure 6 is a graph of the results of an accelerated stability study of glycopegylated G-CSF.

Figure 7 is an expanded view of Figure 6. Figure 8 is a graph of the results of the IV pK study of rats using the radiolabelling process Bolton Hunter (protein precipitated from plasma). Figure 9 is a graph of the results of the rat IV pK study using unlabeled G-CSF, chemical and glycopegylated G-CSF detected by ELISA. Figures 10A-10N show representative sialyltransferases for use in the present invention.

Detailed Description of the Invention Abbreviations PEG, poly (ethylene glycol); m-PEG, methoxy-poly (ethylene glycol); PPG, poly (propylene glycol); m-PPG, methoxy-poly (propylene glycol); It was, fucosilo; Gal, galactosyl; GalNAc, - N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc, N-acetylglucosaminyl; Man, mannosyl; ManAc, manosaminyl acetate; Sia, sialic acid; and NeuAc, N-acetylneuraminyl.

Definitions Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly employed in the art. Standard techniques are used for synthesis of peptides and nucleic acids. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al.) MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor; NY, which is incorporated herein by reference), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry and organic synthesis described below are those well known and commonly employed in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis. All the oligosaccharides described herein are described by the name or abbreviation for the non-reducing saccharide (ie, Gal), followed by the configuration of the glycosidic bond (a or β), the ring bond (1 or 2), the position in the reducing saccharide ring involved in the bond (2, 3, 4, 6 or 8) and then the name or abbreviation of the reducing saccharide (ie, GlcNAc). Each saccharide is preferably a pyranose. For a review of the standard glycobiology nomenclature see, Essentials of Glycobiology Varki et al., Eds. CSHL, Press (1999). The oligosaccharides are considered to have a reducing end and a non-reducing end, whether the saccharide at the reducing end is in fact a reducing sugar. In accordance with the accepted nomenclature, oligosaccharides are detailed in the present with the non-reducing end in the left and the reducing end in the right. The term"nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single or double stranded form, unless specifically limited, the term encompasses nucleic acids which contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides, unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as the explicitly indicated sequence .. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected codons (or all) were substi tuye with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). The term "nucleic acid" is used interchangeably with the gene, cDNA and mRNA encoded by a gene. The term "gene" means the segment of DNA involved in the production of a polypeptide chain. It can include regions that precede and follow the coding region (leader and trailer) as well as intervention sequences (introns) between individual coding segments (exons). The term "isolated" when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it associates in a natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined by using analytical chemistry techniques such as electrophoresis or polyacrylamide gel or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from the open reading frames that flank the gene and encode a protein different from the gene of interest. The term "purified" denotes that a nucleic acid or protein essentially gives rise to a band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure and even more preferably at least 99% pure. The term "amino acid" refers to synthetic and naturally occurring amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. The naturally occurring amino acids are those encoded by the genetic code as well as those amino acids that are subsequently modified for example hydroxyproline, β-carboxyglutamate and 0-phosphoserine. "Amino acid analogs" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, ie a carbon a that binds to hydrogen, a carboxyl group, an amino group, and an R group, eg, homoserine , norleucine, methionine sulfoxide, methionine methyl sulfonyl. Such analogs have modified R groups (for example norleucine) or columns of modified peptides, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimics" refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid but that functions in a manner similar to a naturally occurring amino acid. There are several methods known in the art that allow the incorporation of an unnatural amino acid derivative or analogue within a polypeptide chain into a specific form of the site, see for example WO 02/086075. The amino acids can be referred to herein either by the commonly known 3 letter symbols or by the letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides can be referred to by their commonly accepted simple letter codes. "Variants conservatively modified" applies to both amino acid sequences and nucleic acids. With respect to particular sequences of nucleic acids, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acids in their sequences or wherein the nucleic acid does not encode an amino acid sequence for essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the GCA, GCC, GCG - and GCU codons all code for the amino acid alanine. Thus, in each position where. an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such variations of nucleic acids are silent variations which are a kind of conservatively modified variations. Each nucleic acid sequence herein that encodes a polypeptide also describes each possible silent variation of the nucleic acid. One of experience will recognize that each codon in a nucleic acid (except AUG which is ordinarily the only codon for methionine, and TGG which is ordinarily the only codon for tryptophan) can be modified to produce a functionally identical molecule. In this manner, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence. As for amino acid sequences, someone of experience will recognize that individual substitutions, deletions or additions for a nucleic acid, peptide, polypeptide or protein sequence which alters, adds or eliminates a single amino acid or a small percentage of amino acids in the encoded sequence, is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, homologues between species and alleles of the invention. The following 8 groups each contain amino acids that are conservative substitutions of one another: 1) Alanine (A), glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) Cysteine (c), methionine (M) (see, e.g., Creighton, Proteins (1984)). The amino acids can be referred to herein either by their commonly known 3 letter symbols or by the letter symbols recommended by the chemical nomenclature commission of IUPAC-IUB. Nucleotides can similarly be referred to by their commonly accepted plain-letter codes. In the current application, the amino acid residues are listed according to their relative positions from the residue plus in Terminal N which is listed as 1 in a unmodified wild-type polypeptide sequence. "Peptide" refers to a polymer in which the monomers are amino acids and are joined together via amide bonds. The peptides of the present invention can vary in size for example from two amino acids to hundreds or thousands of amino acids which are referred to alternatively as a polypeptide. Additionally, non-natural amino acids for example β-alamin, phenylglycine and homoarginine are also included. Amino acids that are not encoded by genes can also be used in the present invention. Additionally, amino acids that have been modified to include reactive groups, glycosylation cycles, polymers, therapeutic, bio-olecular portions and the like can also be used in the invention. All amino acids used in the present invention can be either D- or L-isomers. The L-isomer is generally preferred. In addition, other mimetic peptides are also useful in the present invention. As used herein, "peptide" refers to glycosylated and non-glycosylated peptides. Also included are peptides that are incompletely glycosylated by a system that expresses the peptide. For a general review see, AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weintein, eds, Marcel Dekker, New York, p. 267 (1983). In the current application, amino acid residues are listed according to their relative positions from Teal N for example, the leftmost residue which is listed 1, is a sequence of peptides. The term "mutant polypeptide" or "mutein" refers to a form of a peptide that differs from its corresponding wild type or naturally occurring form. A mutant peptide can contain one or more mutations for example replacement insertion elimination etc. which results in the mutant peptide. The term "peptide conjugate" refers to a species of the invention in which a peptide is glycoconjugated with a modified sugar as set forth herein. In a representative example, the peptide is a mutant peptide having a 0-linked glycosylation site that is not present in the wild-type peptide. "Near to a proline residue" as used herein, refers to an amino acid having less than about 10 amino acids removed from a proline residue preferably less than about 9, 8, 7, 6 or 5 amino acids withdrawn from a proline residue more preferably less than about 4, 3, 2 or 1 residues removed from a proline residue. The amino acid close to a proline residue may be on either the N-teal or the C-teal side of the proline residue. The term "sialic acid" refers to any member - of a family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is the acid N-acety-neuraminic acid (2-keto-5- acetamido-3,5-dideoxy-D-galactononulopyran-l-onic often abbreviated as Neu5Ac, NeuAc, or NANA). A second member of the family is N-glycolyl-neurominic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. A third member of the sialic acid family is 2-keto-3-deoxy-nonulosomal acid (KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J Biol. Chem. 265: 21811-21819 (1990)). Also included are 9-substituted sialic acids such as 9-0-C1-C6 acyl-Neu5Ac type 9-0-lactyl-Neu5Ac or 9-0-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-acid- 9-deoxy-Neu5Ac. For a review of the sialic acid family see, VGaki, Glycobiology 2: 25-40 (1992); sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New York (1992)). The synthesis and use of the sialic acid compounds in a sialylation process is described in international application WO 92/16640, published on October 1, 1992. As used herein, the term "modified sugar" refers to a carbohydrate which it occurs naturally or not naturally that is added enzymatically on an amino acid or a glucosyl residue of a peptide in a process of the invention. The modified sugar is selected from various enzyme substrates including but not limited to modified sugar nucleotides selected from various enzyme substrates including but not limited to sugar nucleotides (mono-, di-, and triphosphates), sugars activated (for example, glucosyl halide, glucosyl mesylates) and sugars that are neither activated nor nucleotides. The "modifier sugar" is functionalized covalently with a modifying group. Useful modifying groups include but are not limited to water soluble polymers, therapeutic portions, diagnostic portions of biomolecules and the like. The modifying group is preferably the non-carbohydrate occurring naturally or an unmodified carbohydrate. The site of functionalization with the modifier group is selected such that it does not prevent the modified sugar from being added enzymatically to a peptide. The term "water-soluble" refers to portions that have some detectable degree of solubility in water. Methods for detecting and / or quantifying solubility in water are well known in the art. Exemplary water-soluble polymers include peptide poly (ethers) poly (amines), polycarboxylic acids and the like. The peptides may have mixed sequences or be composed of a single amino acid, for example, poly (lysine). An exemplary polysaccharide is polysialic acid. An exemplary poly (ether) is polyethylene glycol, for example, m-PEG poly (ethylene imine) is an exemplary polyamine and the poly (acrylic acid) is a representative poly (carboxylic acid).

The polymer column of the water-soluble polymer can be poly (ethylene glycol) (ie, PEG). However, it should be understood that other related polymers are also suitable for use in the practice of this invention and that use of the term PEG or polyethylene glycol is intended to be inclusive and not exclusive in this respect. The term PEG includes polyethylene glycol in any of its forms including PEG alkoxy, difunctional PEG, PEG with multirabilities, PEG in the form of a fork, branched PEG, pendant PEG (ie, PEG or related polymers having one or more pendant functional groups of the column of polymers), or PEG with degradable bonds therein. The polymer column can be linear or branched. Branched polymer columns are generally known in the art. Typically, a branched polymer has a portion of a central branching core and a plurality of linear polymer chains linked to the central branching core. PEG is commonly used in branched forms which can be prepared by the addition of ethylene oxide to various polyols such as glycerol, pentaerythritol and sorbitol. The central branching portion can also be derived from various amino acids such as power plants. The branched polyethylene glycol can be represented in general form as R (-PEG-OH) m in which R represents the core portion such as glycerol or pentaerythritol and m represents the number of arms. The multiple arm PEG molecules such as those described in US Patent E.U.A. No. 5,932,462, which is incorporated herein by reference in its entirety can be used as the polymer column. Many other polymers are also suitable for the invention. Polymer columns that are non-peptide and water soluble with from 2 to about 300 terminations are particularly useful in the invention. Examples of suitable polymers include but are not limited to other polyalkylene glycols, such as polypropylene glycol ("PPG"), copolymers of ethylene glycol and propylene glycol and the like, polyoxyethylated polyol, - polyolefin alcohol, polyvinylpyrrolidone, polyhydrocipropylmethacrylamide, polya-hydroxy acid, alcohol polyvinyl, polyphospazene, polixazoline, polyN-acryloylmorpholine, such as is described in U.S. Patent No. 5,629,384 which is incorporated herein by reference in its entirety and copolymers, terpolymers, and mixtures thereof. Although the molecular weight of each chain of the polymer column must vary, it is typically in the range from about 100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da. The term "glycoconjugation" as used herein, refers to the enzymatically mediated conjugation of a modified sugar species to an amino acid or glucosyl residue of a polypeptide, for example, a mutant human growth hormone of the present invention. A subgenus of "glycoconjugation" is "glycol-PEGylation", in which the modified sugar modifier group is poly (ethylene glycol), and the alkyl derivative (eg, m-PEG) or reactive derivative (e.g. , H2N-PEG, HOOC-PEG) thereof. The terms "large scale" and "industrial scale" are used interchangeably and refer to a reaction cycle that produces at least about 250 mg, preferably about 500 mg, and more preferably about 1 gram of a glycoconjugate at end of a simple reaction cycle. The term "glucosyl binding group" as used herein, refers to a glycosyl residue wherein a modifying group (eg, PEG portion, therapeutic biomolecule portion) is covalently linked, the glucosyl binding group is attached to a group modifier to the rest of the conjugate. In the methods of the invention, the glucosyl ligation group becomes covalently linked to a glycosylated or non-glycosylated peptide, whereby the binding of the agent to an amino acid and / or glycosyl residue on the peptide. A glucosyl ligation group is generally derived from a sugar modified by the enzymatic binding of the modified sugar to an amino acid and / or glucosyl residue of the peptide. The glucosyl ligation group can be a structure derived from saccharides that are degraded during the formation of a cassette of a modified sugar with a modifying group (eg, oxidation- »Schiff-base-reduction), or the Group of the glucosyl ligation may be intact. An intact group of a glucosyl ligature refers to a binding group that is derived from a portion of glucosyl in which the saccharide monomer that binds the modifying group and the remainder of the conjugate is not degraded, for example, is oxidized, example, by 'sodium metaperiodate. The intact glucosyl ligation groups of the invention can be derived from an oligosaccharide that occurs naturally by the addition of glucosyl units or the removal of one or more glucosyl units from a saccharide precursor structure. The term "targeting portion", as used herein, refers to a species that will selectively localize to a particular tissue or region of the body. The location is mediated by the specific knowledge of the molecular determinants, the molecular size of the targeting or conjugate agent, ionic interactions, hydrophobic interactions and the like. Other targeting mechanisms of an agent to a particular region or tissue are known to those skilled in the art. Exemplary targeting moieties include antibodies, antibody fragments, transferrin, H-glycoprotein, coagulation factors, β-glycoprotein serum proteins, G-CSF, GM-CSF, M-CSF, EPO and the like. As used herein, "therapeutic moiety" means any agent useful for therapy including, but not limited to, antibiotics, agents, anti-inflammatories, anti-tumor drugs, cytotoxins, and radioactive agents. "Therapeutic portion" includes prodrugs of bioactive agents, constructs in which more than one therapeutic moiety is linked to a carrier, eg, multivalent agents. The therapeutic portion also includes proteins and constructs that include proteins. Exemplary proteins include but are not limited to erythropoietin (EPO), Granulocyte Colony Stimulating Factor (GCSF), Granulocyte Macrophage Colony Stimulating Factor (GMCSF), Inferred (e.g., Interferon-a, -ß, ~ ?), interleukin (e.g., Interleukin II), serum proteins (e.g., Factors VII, Vlla, VIII, IX and X), Human Chorionic Gonadotropin (HCG), Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) ) and antibody fusion proteins (e.g., Tumor Necrosis Factor Receptor ((TNFR) / Fc domain fusion protein.) As used herein, an "antitumor drug" means any agent useful in combating the cancer, which includes but is not limited to, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procabazine, hydroxyurea, asparaginase, corticosteroids, interferons and radioactive agents. conjugates of peptides with anti-tumor activity, for example, TNF-a, are encompassed within the scope of the term "anti-tumor drug". The conjugates include, but are not limited to, those formed within a therapeutic protein and a glycoprotein of the invention. A representative conjugate is that formed between PSGL-1 and TNF-a. As used herein, "a cytotoxic agent or cytotoxin" means any agent that is detrimental to the cell. Examples include, taxol, cytochalasin B, gramicidin D, etidinium bormuro, emethine, mitomycin, etoposide, tenoposide, vincristine, vinbastine, colchicine, doxorubicin, daunorubicin, dihydroxy, anthracinedione, metixantrone, mitramycin, antinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Other toxins include, for example, ricin, CC-1065 and the like, duocarmycins. Still other toxins include diphtheria toxin and viper venom (eg, cobra venom). As used herein, a radioactive agent includes any radioisotope that is effective in the diagnosis or destruction of a tumor. Examples include but are not limited to indium-111, cobalt-60. Additionally, naturally occurring radioactive elements such as uranium, radium, and thorium, which typically represent radioisotope mixtures, are suitable examples of a radioactive agent. Metal ions are typically chelated with an organic chelation moiety. Many useful chelating groups, crown ethers, cryptands "and the like are known in the art and can be incorporated into the compounds of the invention (e.g., EDTA, DTPA, DOTA, NTA, HDTA, etc. and their analogs). of phosphonate such as DTPP, EDTP, HDTP, NTP, etc.) See for example, Pitt et al., "The Design of Chelating Agents for the Treatment of Iron Overload," In, INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE; Martell, Ed., American Chemical Society, Washington, D.C., 1980, pp. 279-312; Lindoy, THE CHEMISTRY OF -MACROCYCLIC LIGAND COMPLEXES; Cambridge University Press, Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989 and references contained therein. Additionally, a multitude of pathways are available that allow the linking of chelating agents, crown ethers and cyclodextrins to other molecules for those skilled in the art. See for example Meares et al., "Properties of In Vivo Chelate-Tagged Proteins and Polypeptides". In, MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICAL ASPECTS, "Feeney, et al., Eds., American Chemical Society, Washington, D.C., 1982, pp. 370-387; Kasina et al., Bioconjugate Chem., 9: 108-117 (1998); Song et al., Bioconjugate Chem., 8: 249-255 (1997). As used herein, "pharmaceutically acceptable carrier" includes any material which when combined with the conjugate retains the activity of the conjugates and does not react with the immune systems of the subject. Examples include but are not limited to any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as an oil in water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets including coated tablets and capsules. Typically such carriers may contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, or salts thereof, calcium stearate, or magnesium, talc, fats or vegetable oils, gums, glycols, other known excipients. Such carriers may also include flavor and color additives or other ingredients. The compositions comprising such carriers are formulated by conventional well-known methods. As used herein, "administration" means oral administration, administration as a suppository, topical, intravenous, intraperitoneal, intramuscular, intralesional, or subcutaneous administration, administration by inhalation, or implantation of a slow release device, eg, a miniosmotic pump at subject. Administration by any means including parenteral and transmucosal (eg, oral, nasal, vaginal, rectal, or transdermal), particularly by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Furthermore, where the injection is for treating a tumor for example, inducing apoptosis, the administration can be directly to the tumor and / or within the tissues surrounding the tumor. Other modes of administration include but are not limited to the use of liposome formulations, intravenous infusion, cross patches, etc. The term "improve" or "ameliorate" refers to any indication of success in the treatment of a pathology or condition including any objective or subjective parameter, such as abatement, remission or reduction of symptoms, or an improvement in the physical or mental well-being of a patient. patient. The reduction of symptoms can be based on objective or subjective parameters including the results of a physical examination and / or a psychiatric evaluation. The term "therapy" refers to the treatment or treatment of a disease or condition including the prevention of the disease or condition that occurs in an animal, which may be predisposed to the disease but which. still does not experience or show symptoms of the disease (prophylactic treatment), inhibit the disease (slow down or stop its development), by providing relief of symptoms or side effects of the disease (including palliative treatment) and relief of disease (causing a regression of the disease). The term "effective amount" or "an effective amount" or "a therapeutically effective amount" or any grammatically equivalent term means the amount that when administered to an animal for the treatment of the disease, is sufficient to effect the treatment for that disease . The term "isolated" refers to a material that is substantially or essentially free of components which are used to produce the material. For the peptide conjugates of the invention, the term "isolated" refers to a material that is substantially or essentially free of components which normally accompany the material in the mixture used to prepare the peptide conjugate. Isolated and pure are used interchangeably. Typically, the conjugates of the isolated peptide of the invention have a level of purity that is preferably expressed as a range. The lower end of the purity range for the peptide conjugates is about 60% about 70% or about 80% and the upper end of the purity range is about70% around 80% around 90% or more around 90%.

When the peptide conjugates are more than about 90% pure, their purities are also preferably expressed as a range. The lower end of the purity range is around 90% around 92% around 94% or about 98%. The upper end of the purity range is around 92%, around 94%, around 98% or about 100% pure. Purity is determined by any assay method recognized in the art (e.g., band intensity on a silver stained gel, polyacrylamide gel electrophoresis, HPLC, or a similar means). "Essentially each member of the population", as used herein, describes a characteristic of a population of peptide conjugate of the invention in which a selected percentage of the modified sugars added to a peptide are added to multiple identical acceptor sites in the peptide. Essentially each member of the population, speaks of the homogeneity of the sites in the peptide conjugated with a modified sugar and refers to the conjugates of the invention which are at least about 80% preferably at least about 90% and more preferably at least about 95% homogeneous. "Homogeneity" refers to the structural consistency through a population of acceptor portions to which the modified sugars are conjugated. Thus, a peptide conjugate of the invention in which each modified sugar portion is conjugated to an acceptor site having the same structure as the acceptor site to which each other modified sugar is conjugated, the peptide conjugate is said to be which is about 100% homogeneous. Homogeneity is typically expressed as an interval. The inner end of the homogeneity range for the peptide conjugates is about 60% about 70% or about 80% and the upper end of the purity range is about 70% about 80% about 90% or more around 90%. When the peptide conjugates are more than or equal to about 90% homogeneous, their homogeneity is also preferably expressed as a range. The lower end of the homogeneity range is about 90% about 92% about 94% about 96% or about 98%. The upper end of the purity range is about 92% about 94% about 96% about 98% or about 100% homogeneity. The purity of the peptide conjugates is typically determined by one or more methods known to those skilled in the art for example mass spectroscopy, liquid chromatography (LC-MS), laser-assisted desorption mass time by flight spectrometry matrix. (MALDITOF), capillary electrophoresis and the like. "Substantially uniform glycoforms" or a substantially uniform glycosylation pattern when referring to a glycopeptide species, refers to the percentage of acceptor portions that are glycosylated by the glycosyltransferase of interest (eg, fucosyltransferase). For example, in the case of an al, 2 fucosyltransferase, a substantially uniform fucosylation pattern exists if substantially all (as defined below) of the Galßl, 4-GlcNAc-R and sialylated analogues thereof "are fu-cosylated. In a peptide conjugate of the invention, it will be understood by one skilled in the art that the starting materials may contain glycosylated acceptor portions (eg, Galßl, fucosylated 4-GlcNAc-R portions). The calculated glycosylation will include acceptor moieties that are glycosylated by the methods of the invention, as well as those acceptor moieties that are already glycosylated in the starting material.The term "substantially" in the above definitions of "substantially uniform" generally means at least around 40%, at least about 70%, at least about 80%, or more preferably at least about 90%, and still more preferably at least about 95% of the acceptor portions for a particular glycosyltransferase are glycosylated. Where the substituent groups are specific by their conventional chemical formulas, written from left to right, these also cover the identical chemical substituents, which will result from the structure written from right to left, for example -CH20- is also intended to recite - OCH2-. The term "alkyl", by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono or polyunsaturated and may include di and multivalent radicals, having the designated carbon atom number (ie, C1-C10 means one to ten carbons). Examples of saturated hydrocarbons include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, -n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, and 3-propynyl, 3-butynyl and homologues e-higher isomers. The term "alkyl", unless otherwise noted, also means that it includes those alkyl derivatives defined in more detail below, such as "heteroalkyl". Alkyl groups that are limited to hydrocarbon groups are named "homoalkyl". The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by -CH2CH2CH2CH2- and further includes those groups described below as "heteroalkylene". Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or some carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a short chain alkyl or alkylene group, generally having 8 or some carbon atoms. The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) were used in their conventional sense, and refer to those alkyl groups linked to the remainder 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 stated, a straight or branched chain, or cyclic, hydrocarbon radical, or combinations thereof, consisting of the number carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N and S and Si can be placed at any internal position of the heteroalkyl group or at the position at which the alkyl group is bonded to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-0-CH3, -CH2-CH2-NH-CH3, -CH2-CH2- N (CH3) -CH3, -CH2-S-CH2-CH3, -CH2 -CH2, -S (0) -CH3, -CH3-CH2-S (O) 2 -CH3, CH = CH-0-CH3, Si (CH3) 3, -CH2-CH = N-OCH3 and -CH = N (CH3) -CH3. Up to 2 heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-0-Si (CH3) 3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a radical divalent heteroalkyl derivative, as exemplified, but not limited to, -CH2-CH2-S-CH2-CH2 and -CH2-S-CH2-CH2-NH-CH2. For heteroalkylene groups, the heteroatoms can also occupy any or both chain terminals (for example, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino and the like). Still further, for the alkylene and heteroalkylene linked groups, without orientation of the linking group, it is implied by the direction in which the formula of the linking group is written. For example, the formula -C (0) 2R'- represents both -C (0) 2R 'and -R'C (0) 2-. The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle binds to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1, 2, 5, 5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2 ilo, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl and the like. The terms "halo" or "halogen", by themselves or as part of another substituent, means, unless stated otherwise, a fluorine, chlorine, bromine or iodine atom. Additionally, terms such as "haloalkyl", means that they include monohaloalkyl and polyhaloalkyl. For example, the term "(C 1 -C 4) haloalkyl" means including, but not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. The term "aryl" means, unless otherwise stated, an aromatic, polyunsaturated substituent which may be a single ring or multiple rings(preferably from 1 to 3 rings), which are fused together or covalently linked. The term "heteroaryl" refers to aryl groups (or rings) containing from 1 to 4 heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atoms are optionally Quaternized A heteroaryl group can be linked to the rest of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, α-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, 3-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5- isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazilyl, benzo [b] furanyl, benzo [b] thienyl, 2,3-dihydrobenzo [1,4] dioxin-6-yl, benzo [1, 3] dioxol-5-yl and 6-quinolyl. The substituents for each of the ring-heteroaryl and aryl systems noted above are selected from the group of acceptable substituents described below. For brevity, the term "aryl" when used in combination with other terms (eg, aryloxy, arylthioxy, arylalkyl) include both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" means that it includes those radicals in which an aryl group is linked to an alkyl group (for example, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom ( for example, a methyl group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl and the like). Each of the foregoing terms (eg, "alkyl", "heteroalkyl", "aryl" and "heteroaryl") means that it includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are given below. Substituents for the alkyl and heteroalkyl radicals (include those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are generically referred to as "alkyl group substituents" and can be one or more of a variety of groups selected from, but not limited to: -OR '', = 0, = NR ', = N-0R', -NR'R '', -SR ', halogen, -SiR 'R "R'", -OC (0) R ', -C (0) R', -C02R ', -CONR'R ", -0C (0) R'R", -NR "C (0) R ', -NR' -C (0) NR "R '", -NR "C (0) 2R', -NR-C (NR'R" R "') - = NR" ", -NR-C (NR 'R ") = NR'", -S (0) R ', S (0) 2R', -S (0) 2NR'R ", -NRS02R ', -CN and -N02 is a number in the interval wants zero to (2m '+ 1), where m' is the total number of carbon atoms in such a radical, R ', R' ', R' '' and R '' '' each preferably independently referred to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, per axis mplo, aryl groups substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or arylalkyl groups. When a compound of the invention includes more than one group R, for example, each of the groups R is independently selected as are each of the groups R ', R ", R' '' and R" "when more of one of these groups are present When R 'and R "are linked to the same nitrogen atom, which can be combined with the nitrogen atom to form a 5-6-7 membered ring. For example, -NR'R "means that it includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the foregoing discussion of substituents, a person skilled in the art will understand that the term "alkyl" means that it includes groups that include carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., CF3 and -CH2CF3) and acyl (e.g., -C (0) CH3, -C (0) CF3, -C (0) CH20CH3 and the like). Similar to the substituents described by the alkyl radical, the substituents for the aryl and heteroaryl groups are generically referred to as "aryl group substituents". The substituents are selected from, for example: halogen, -OR ', = 0, = NR', = N-0R ', -NR'R ", -SR', halogen, -SiR'R" R '", - 0C (0) R ', -C (0) R', -C02R ', -CONR'R ", -OC (0) NR'R", -NR "C (0) R', -NR '-C (0) NR "R '", -NR "C (0) 2R', -NR-C (NR'R" R "') = NR" ", -NR-C (NR' R") = NR " ', -S (O) R', S (0) 2R ', -S (0) 2NR'R ", -NRS02R', -CN and -N02, -R ', -N3, -CH (Ph) 2 , fluoroalkoxy (C1-C4) and "fluoroalkyl (Ci-C4), in a number in the range from zero to the total number of open valencies in the aromatic ring system; and wherein R ', R ", R'" and R "" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one group R, for example, each of the groups R is independently selected as are each of the groups R ', R ", R'" and R "" when more than one of these groups are present In the schemes that follow, the symbol X represents "R" as described above Two of the substituents on adjacent atoms of the aryl or heteroaryl ring can optionally be replaced with a substituent of the formula -TC (0) - (CRR ') qU-, where T and U are independently -NR-, -0-, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, 2 of the substituents on adjacent atoms of the aryl or heteroaryl ring can optionally be replaced with a substituents of the formula -A- (CH2) rB-, wherein A and B are independently -CRR ', -0-, -NR-, -S-, -S (0 ) -, -S (0) 2-, -S (0) 2NR'- or a single bond, and r is an integer from 1 to 4. One of the single bonds of the newly formed ring It can optionally be replaced with a double link. Alternatively, 2 of the substituents of adjacent atoms of the aryl or heteroaryl ring can optionally be replaced with a substituent of the formula - (CRR ') SX- (CR "R"') d-, where syd are independently integers from 0 to 3, and X is -0-, -NR '-, -S-, -S (0) -, -S (0) 2- or -S (0) 2 -NR'-. The substituents R, R ', R "and R"' are preferably independently selected from hydrogen or substituted (C? -C6) alkyl or unsubstituted. As used herein, the term "heteroatom" means that it includes oxygen (0), nitrogen (N), sulfur (S) and silicon (Si).

Introduction The present invention provides conjugates of glycopeptides that modify a portion of sugar that is linked either directly or indirectly (eg, through and by intervening the glucosyl residue) to a glycosylation site linked to 0 in the peptide. Methods for producing the conjugates of the invention are also provided. The glycosylation site bound to 0 is generally the hydroxy side chain of a natural amino acid (eg, serine, threonine) or unnatural amino acid (eg, 5-hydroxyproline or 5-hydroxylysine). The. Saccharyl residues linked to 0 examples include N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose or xylose. The methods of the invention can be practiced on any peptide having a glycosylation site linked to 0. For example, the methods are of use to produce glucoconjugates linked to 0 in which the glucosyl portion binds to a glycosylation site linked to 0. which is present in the wild-type peptide. In this manner, the present invention provides glucoconjugates of wild-type peptides that include a glycosylation site linked to 0. Exemplary peptides according to this disclosure include G-CSF, GM-CSF, IL-2 and interferon. In exemplary embodiments the invention also provides novel mutant peptides that include one or more glycosylation sites linked to 0 that are not present in the corresponding wild-type peptide. In one embodiment the mutant polypeptide is a G-CSF polypeptide. In other exemplary embodiments the mutant polypeptide is a hGH polypeptide, an IFN alpha polypeptide or a GM-CSF polypeptide. Glycosylated versions linked to 0 of the mutant peptides are also provided, and methods to prepare glycosylated mutant peptides linked at 0. Additional methods include the elaboration, trimming and / or modification of the glycosyl residue bound to 0 and glucosyl residues that are bound to N, instead of 0. In an exemplary aspect, the invention provides a mutant peptide having the formula: O- GalNAc- X wherein AA is an amino acid with a side chain that includes a hydroxyl portion. Exemplary hydroxyamino acids are threonine and serine. The GalNAc portion is "bound to AA through the oxygen atom of the hydroxyl portion, AA may be present in the wild type peptide or, alternatively, be added or relocated by the mutation of the wild-type peptide sequence. a modified group, a saccharyl portion, for example, sialyl, galactosyl and Gal-Sia groups, or a saccharyl portion and a modified group In an exemplary embodiment, wherein X is a saccharyl portion, which includes a modified group, described herein The glycosylated amino acid may be on the peptide at the C or N terminus or internal to the peptide sequence In an exemplary embodiment, X comprises a group selected from sialyl, galactosyl and Gal-Sia portions, wherein at least one of sialyl, galactosyl and Gal-Sia comprises a modified group In an exemplary embodiment X comprises the portion: wherein D is a member selected from -OH and R -L-HN-; G is a member selected from R ^ L- and -C (0) alkyl (C? -C6); R1 is a portion comprising a member selected from a portion comprising a straight or branched chain poly (ethylene glycol) residue; and L is a bond that is a member selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, such that when D is OH, G is R1-L- and when G is -C (O) alkyl (C? -C6), D is RX-L-NH. In another exemplary embodiment X comprises the structure: wherein L is a substituted or unsubstituted alkyl group or substituted or unsubstituted heteroalkyl, and n is selected from the integers from 0 to about 2500, Still in another exemplary embodiment X comprises the structure: in which s is selected from the integers from 0 to 20. In another exemplary mode, AA is located within a proline-rich segment of the mutant peptide and / or approaches a proline residue. Appropriate sequences that form O-linked glycosylation sites are readily determined by cross-pollination of short peptide-linked enzymatic O-linked glycosylation containing one or more putative 0-linked glycosylation sites. The conjugates of the invention are formed between peptides and various species such as water-soluble polymers, portions. therapeutic, diagnostic portions, target portions and the like. Conjugates are also provided which include 2 or more peptides linked together through a binding arm, that is, multifunctional conjugates; at least one peptide that is O-glycosylated or includes an O-linked glycosylation site. The multiple functional conjugates of the invention may include 2 or more copies of the same peptide or a collection of various peptides with different structures and / or properties. In conjugated specimens according to this embodiment, the binding between the 2 peptides is linked to at least one of the peptides through a glucosyl residue bound to 0, such as an intact glycosyl-linked glucosyl group bonded to 0. The Conjugates of the invention are formed by the enzymatic binding of a sugar modified to the glycosylated or non-glycosylated peptide. " The modified sugar is added directly to a glycosylation site bound at 0 or to a glucosyl residue bound either directly or indirectly (eg, through one or more glycosyl residues) to a glycosylation site linked at 0. invention also provides a conjugate of a glycosylated peptide bonded at 0 in which a modified sugar is directly linked to an N-linked site, or to a glycosyl residue linked either directly or indirectly to an N-linked glycosylation site. modified, when it is interposed between the peptide (or .glucosyl residue) and the modifying group in the sugar becomes what is referred to herein as "intact glucosyl ligation group". By using the exquisite selectivity of enzymes, such as glycosyltransferase, the current method provides peptides that support a desired group in one or more specific locations. Thus, according to the present invention, a modified sugar is directly linked to a selected site on the peptide chain, or alternatively, the modified sugar is attached to a carbohydrate moiety of a glycopeptide. Peptides in which the modified sugars bind to both a glycopeptide carbohydrate and directly to an amino acid residue of the peptide column are also within the scope of the present invention. In contrast to the known strategies for the preparation of chemical and enzymatic peptides, the methods of the invention make it possible to assemble peptides and glycopeptides having a substantially homogeneous derivation pattern; the enzymes used in the invention are generally selective for a particular amino acid residue or combination of amino acid residues of the peptide. The methods are also practical for the large-scale production of modified peptides and glycopeptides. Thus, the methods of the invention provide a practical means for the large scale preparation of glycopeptides having preselected uniform derivation patterns. The methods are particularly well suited for the modification of therapeutic peptides, including but not limited to glycopeptides that are incompletely glycosylated during production in cultured cells (eg, mammalian cells, insect cells, plant cells, fungal cells, yeast cells or prokaryotic cells) or transgenic plants or animals. The methods of the invention also provide conjugates of glycosylated and non-glycosylated peptides with an increased therapeutic half-life due, for example, to a reduced rate of clearance or a reduced rate of absorption by the immune and / or reticuloendothelial system (RES). Moreover, the methods of the invention provide a means to hide antigenic determinants in the peptides, thereby reducing or eliminating a host immune response against the peptide. Selective binding of the targeting agent to a peptide by using a suitable modified sugar can also be used to direct a peptide to a particular cell surface or tissue receptor that is specific to the particular targeting agent. . Furthermore, a class of peptides that are specifically modified with a modified therapeutic moiety through a glucosyl binding group is provided.

O-Glucosylation The present invention provides 0-linked glycosylated peptides, conjugates of these species and methods for the formation of 0-linked glycosylated peptides that include a sequence of selected amino acids ("a 0-linked glycosylation site"). Of particular interest are mutant peptides that include a 0-linked glycosylation site that is not present in the corresponding wild-type peptide. The glycosylation site bound at 0 is a site for the binding of a glycosyl residue carrying a modifying group. Mucin-linked glycosylation in 0, one of the most abundant forms of protein glycosylation, is found on glycoproteins associated with the cell surface and secreted from all eukaryotic cells. There is great diversity in the structures created by glycosylation linked at 0 (hundreds of potential structures), which are produced by the catalytic activity of hundreds of glycosyltransferase enzymes that are resident in the Golgi complex. There is diversity at the level of the glucan structure and in binding positions of the 0-glucans to the protein columns. Despite a high degree of potential diversity, it is clear that glycosylation bound at 0 is a highly regulated process that shows a high degree of conservation among multicellular organisms. The first step in a mucin-type 0-linked glycosylation is catalyzed by one or more members of a large family of UDP-GalNAc: N-acetylgalactosaminyltransferase polypeptide (GalNAc-transferases) (EC 2.4.1.41), which transfers GalNAc to the acceptor of serine and threonine in the sites (Asan et al., J. Niol, Chem. 275: 38197-38205 (2000)). To date, twelve members of the mammalian GalNAc-transferase family have been identified and characterized (Schwientek et al., J. Biol. Chem. 277: 22623-22638 (2002)) and various additional putative members of the family have been predicted. this family of genes from the analysis of the genome databases. The isoforms of GalNAc-transferase have different kinetic properties and show temporal and especially differential expression patterns, suggesting that they have different biological functions (Asan et al., J. Biol. Chem. 275: 38197-38205 (2000)). Sequence analysis of GalNAc-transferase has led to the hypothesis that these enzymes contain two different subunits; a central catalytic unit and a terminal C unit with a sequence similarity to the plant lactin resin designated the lectin domain (Hagen et al., J. Biol. Chem. 274: 6797-6803 (1999); Hazes, Protein Eng. 10: 1353-1356 (1997); Breton et al., Curr. 'Opin. Struct. Biol. 9: 563-571 (1999)). Previous experiments involving site-specific mutagenesis of the selected conserved residues confirmed that mutations in the catalytic domain eliminated the catalytic activity. In contrast, mutations in the "lectin domain" had no significant effects on the catalytic activity of the GalNAc-transferase isomer, GalNAc-Tl (Tenno et al., J. Biol. Chem. 277 (49): 47088- 96 (2002)). Thus, the C-terminal lectin domain is thought to be non-functional and does not play roles in the enzymatic functions of GalNAc-transferases (Hagen et al., J. Biol. Chem. 274: 6797-6803 (1999)). ). However, recent evidence shows that some GalNAc-transferase show particular activities with partially glycosylated glycopeptides with GalNAc. The catalytic actions of at least three isoforms of GalNAc-transferases, GaINAc-T4, -T7 and -Tio, act selectively on the glycopeptides corresponding to mucin tandem repeat domains where only some of the potential glycosylation sites in group have been glycosylated with GalNAc by other GalNAc-transferases (Bennett et al., FEBS Letters 460: 226-230 (1999); Ten Hagen et al., J. Biol. Chem. 276: 17395-17404 (2001); Bennett et al., J. Biol. Chem. 273: 30472-30481 (1998); Ten Hagen et al., J. Biol. Chem. 274: 27867-27874 (1999)). GaINAc-T4 and -T7 recognize different glycosylates in GalNAc and catalyze the transfer of GalNAc to acceptor substrate sites in addition to those previously used. One of the functions of such activities of GalNAc-transferase is predicted to represent a stage of controlling the density of O-glucan occupancy in mucins and mucin-like glycoproteins with high density of a glycosylation linked at 0. An example of these is the glycosylation of mucin associated with MUCl cancer. MUC1 contains a glycosylated region bound in 0 tandem repeat of 20 residues (HGVTSAPDTRPAPGSTAPPA) with five glycosylation sites bound in 0 potentials. GalNAc-Tl, -T2 and -T3 can initiate glycosylation of the tandem repeat MUC1 and incorporate only three sites (HGVTSAPDTRPAPGSTAPPA, GalNAc underlined placement sites). GaINAc-T4 is unique in that it is the only isoform of GalNAc-transferase identified so far that it can complete the binding of glucans bound at 0 to all five acceptor sites in the 20 amino acid tandem repeat sequence of the mucin associated with breast cancer MUCl. GaINAc-T4 transfers GalNAc to at least two sites unused by other isoforms of GalNAc-transferase in the GaINAc4TAP24 glycopeptide (TAPPAHGVTSAPDTRPAPGSTAPP, unique placement sites of GaINAc-T4 are in bold) (Bennett et al., J. Biol. Chem. 273: 30472-30481 (1998). An activity such as that shown by GaINAc-T4 appears to be required for the production of the glycoform of MUC1 expressed by cancer cells where all potential sites are glycosylated (Muller et al. , J. Biol. Chem. 274: 18165-18172 (1999).) Normal MUC1 from lactating mammary glands has approximately 2.6 glucans linked at 0 per repetition (Muller et al., J. Biol. Chem. 272 : 24780-24793 (1997) and MUC1 derived from cancer line T47D has 4.8 glucans bound at 0 per repetition (Muller et al., J. Biol. Chem. 274: 18165-18172 (1999)). to MUC1 cancer is therefore associated by a higher density of occupation of O-linked glycans and this is achieved by an activity of GalNAc-transferase identical to or similar to that of GaINAc-T4. GalNAc-transferases of polypeptides, which have not displayed apparent specificities of GalNAc-glycopeptide, also appear to be modulated by their presumed lectin domains (PCT WO 01/85215 A2). Recently, it was found that mutations in the assumed lectin domain of GalNAc-Tl, similarly to those previously analyzed in GaINAc-T4 (Asan et al., J. Biol. Chem. 275: 38197-38205 (2000)), modified the activity of the enzyme in a similar way as GaINAc-T4. Thus, although the wild type GalNAc-Tl added consecutive multiple residues of GalNAc to a peptide substrate with multiple acceptor sites, the mutated GalNAc-Tl failed to aggregate more than one GalNAc residue to the same substrate (Tenno et al., J .

Biol. Chem ~. 277 (49): 47088-96 (2002)). Since it has been demonstrated that GalNAc-transferase mutations can be used to produce glycosylation patterns that are different from those produced by wild-type enzymes, it is within the scope of the present invention to use one or more mutant GalNAc-transferase in the preparation of O-linked glycosylated peptides of the invention.

Mutant peptides with O-linked glycosylation sites The peptides provided by the present invention include an amino acid sequence that is recognized as a GalNAc acceptor by one or more mutant or wild-type GalNAc-transferases. The amino acid sequence of the peptide is either wild-type, for those peptides that include a 0-linked glycosylation site, a mutant sequence in which an O-linked glycosylation site that does not occur naturally is introduced, or a polypeptide which comprises both glycosylation sites linked to or occurring naturally and which do not occur naturally. Exemplary peptides with which the present invention is practiced include granulocyte colony stimulating factor (G-CSF), eg, 175 and 178 wild-type amino acids (with or without methionine residues at the N-terminus), interferon (e.g., interferon alpha, e.g., interferon alpha 2b, or interferon alpha 2a), granulocyte macrophage colony stimulating factor (GM-CSF), human growth hormone, and interleukin (e.g., interleukin 2). The emphasis of the following discussion on G-CSF, GM-CSF and INFa 2ß is for clarity of illustration. Any number in the superscript of an amino acid indicates the amino acid position relative to methionine at the N-terminus of the polypeptide. These numbers can be easily adjusted to reflect the absence of methionine at the N-terminus if the N-terminus of the polypeptide starts without a methionine. It is understood that the N terminals of the exemplary peptides can be initiated with or without a methionine. further, those skilled in the art will understand that the strategy set forth herein for the preparation of O-linked glycoconjugates of mutant and wild-type peptides is applicable to any peptide. In an exemplary embodiment, the peptide is a biologically active G-CSF mutant that includes one or more mutations at a site selected from the N-terminus, adjuvant to or encompassing H53, P61, P129, P133 and P175. The biologically active G-CSF mutants of the present invention include any G-CSF polypeptide in part or in its entirety, with one or more mutations that do not result in a substantial or complete loss of its biological activity when measured by any of the assays suitable functionalities known to one skilled in the art. In one embodiment, mutations within the biologically active G-CSF mutants of the present invention are located within one or more 0-linked glycosylation sites that do not naturally occur in wild-type G-CSF. In another embodiment, mutations within the biologically active G-CSF mutants of the present invention reside within as well as outside one or more glycosylation sites linked in 0 of the G-CSF mutants. Polypeptides G-CSF wild type and representative having sequences selected from: SEQ ID NO: 1 (178 amino acids of wild type) mtplgpasslp qsfllkcleq vrkiqgdgaa Iqeklvseca tyklchpeel vllghslgip waplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ ID NO: 2 (178 wild-type amino acids without methionine at the N-terminus) tplgpasslp qsfllkcleq vrkiqgdgaa Iqeklvseca tyklchpeel vllghslgip waplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ ID NO: 3 (175 wild-type amino acids) mtplgpasslp qsfllkcleq vrkiqgdgaa Iqeklca tyklchpeel vllghslgip waplsscpsq alqlagclsq Ihsglflyqg llqalegisp elgptldtlq Idvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ ID NO: 4 (175 amino acids of wild type without methionine in the N-terminus) mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklca tyklchpeel vllghslgip waplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ ID NO: 5 mvtplgpasslp qsfllkcleq vrkiqgdgaa Iqeklca tyklchpeel vllghslgip waplsscpsq alqlagclsq Ihsglflyqg llqalegisp elgptldtlq Idvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashi qsflevsyrv lrhlaqp; SEQ ID NO: 6 mvtplgpasslp qsfllkcleq vrkiqgdgaa lqeklca tyklchpeel vllghtlgip waplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv Irhlaqp; SEQ ID NO: 7 mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklca tyklchpeel vllghtlgip waplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlq Idvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ ID NO: 8 mvtplgpasslp qsfllkcleq vrkiqgdgaa lqeklca tyklchpeel vllgsslgip waplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ ID NO: 9 mqtplgpasslp qsfllkcleq vrkiqgdgaa Iqeklca tyklchpeel vllghslgip waplsscpsq alqlagclsq Ihsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ ID NO: 10 mtplgpasslp qsfllkcleq vrkiqgdgaa Iqeklca tyklchpeel vllghslgip waplsscpsq alqlagclsq Ihsglflyqg llqalegisp elgptldtlq Idvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqptqgamp; and SEQ ID NO: 11 mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklca tyklchpeel vllgsslgip waplsscpsq alqlagclsq Ihsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp SEQ ID NO: 12 maitplgpasslp qsfllkcleq vrkiqgdgaa Iqeklcatyk lchpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq alegispelg ptidtlqldv adfatdwqq meelgmapal qptqgampaf asafqrragg vivashlqsflevsyrvlrh laqp SEQ ID NO: 13 mgvtetplgpasslp qsfllkcleq vrkiqgdgaa lqeklcatyk lchpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp SEQ ID NO: 14 maptplgpasslp qsfllkcleq vrkiqgdgaa Iqeklcatyk Ichpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq alegispelg ptidtlqldv adfattiwqq meelgmapal qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp SEQ ID NO: 15 Mtptgglgpasslp qsfllkcleq vrkiqgdgaa lqeklcatyk Ichpeelvll ghslgipwap Isscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmap to qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp SEQ ID NO: 16 mtplgpasslp qsfllkcleq vrkiqgdgaa Iqeklcatyk Ichpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmapatqptqgampaf asafqrragg vivashlqsf levsyrvlrh laqp SEQ ID NO: 17 Mtplgpasslp qsfllkcleq vrkiqgdgaa Iqeklcatyk Ichpeelvll ghslgipftp Isscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmapaL qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp SEQ ID NO: 18 mtplgpasslpqsfllkcleqvrkiqgdgaalqeklcatyklchpeelvllghslgi pwaplsscpsqalqlagclsqlhsglflyqgllqalegispelgptldtlqldvadfa ttiwqqmeelgmapalqptqtampafasafqrraggvlvashlqsflevsyrvlr hlaqp. In another exemplary embodiment, the peptide is a biologically active hGH mutant that includes one or more mutations at a site selected from the N terminus or adjacent to or encompassing P133. The biologically active hGH mutants of. The present invention includes any hGH polypeptide in part, or in its entirety, with one or more mutations that do not result in a substantial or complete loss of their biological activity when measured by any of the suitable functional assays known to one skilled in the art. . In one embodiment, mutations within the biologically active hGH mutants of the present invention are located within one or more O-linked glycosylation sites that do not naturally occur in wild-type hGH. In another modality, the Mutations within the biologically active hGH mutants of the present invention reside within as well as outside of one or more O-linked glycosylation sites of the hGH mutants. Representative mutant and wild-type hGH polypeptides have the sequences that are selected from: SEQ. ID NO: 19 (192 amino acids of wild type hGH pituitary-derived wild type comprising a methionine at the N terminal) mfptiplsrlfdnamlrahrlhqlafdtyqefeeayipkeqkysflqnpqtslcfse siptpsnreetqqksnlellrislliqswlepvqflrsvfanslvygasdsnvydllk dleegiqtlmgrledgsprtgqifkqtyskfdtnshnddallknygllycfrkdm dkvetflrivqcrsvegscgf - SEQ ID NO: 20 (191 amino acids hGH pituitary-derived wild type lacking a the N terminal methionine) fptiplsrlfdnamlrahrlhqlafdtyqefeeayipkeqkysflqnpqtslcfsesi ptpsnreetqqksniellrisllliqswlepvqflrsvfanslvygasdsnvydllkd leegiqtlmgrledgsprtgqifkqtyskfdtnshnddallknygllycfrkdmd kvetflrivqcrsvegscgf SEQ ID NO: 21 (wild type) MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYI PKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLE LLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVY DLLKDLEEGIQTLMGRLEDGSPRTGOIFKOTYSKFDT NSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCR SVEGSCGF following are representative sequences of mutant peptides corresponding to the region underlined wild type SEQ ID NO: 21; LEDGSPTTGQIFKQTYS, LEDGSPTTAQIFKQTYS, LEDGSPTATQIFKQTYS, LEDGSPTQGAMFKQTYS, LEDGSPTQGAIFKQTYS, LEDGSPTQGQIFKQTYS, LEDGSPTTLYVPKQTYS, LEDGSPTINTIFKQTYS, LEDGSPT VSIPKQTYS, LEDGSPRTGQIPTQTYS, LEDGSPRTGQIPTQAYS, LEDGSPTTLQIFKQTYS, LETETPRTGQIFKQTYS, LVTETPRTGQIFKQTYS, LETQSPRTGQIFKQTYS, LVTQSPRTGQIFKQTYS, LVTETPATGQIFKQTYS, LEDGSPTQGA PKQTYS and LEDGSPTTTQIFKQTYS. In another exemplary embodiment, the peptide is an IFN alpha mutant that includes one or more mutations at a site corresponding to T106 of INP alpha 2, for example, adjacent to or spanning an amino acid position in wild-type IFN alpha, which corresponds to or is aligned with T106 of INF alpha 2. The biologically active IFN alpha mutants of the present invention include any IFN alpha polypeptide, in part or as a whole with one or more mutations that do not result in a complete or substantial loss of their biological activity when measured by any of the suitable functional assays known to one skilled in the art. In one embodiment, mutations within the biologically active IFN alpha mutants of the present invention are located within one or more O-linked glycosylation sites that do not naturally occur in wild-type IFN alpha. In another embodiment, mutations within the biologically active IFN alpha mutants of the present invention reside within as well as outside one or more crosslinking sites linked in 0 of the IFN alpha mutants. A mutant and wild-type IFN alpha polypeptide is shown below: SEQ ID NO: 22 (from wild-type IFN 2b) 98CVIQGVGVTETPLMKEDSIL117 Other appropriate glycosylation sequences linked in O for G-CSF and peptides other than G-CSF are it can be determined by preparing a polypeptide that incorporates an assumed glycosylation site bound at 0 and proposes that the polypeptide at the appropriate glycosylation conditions bound at 0, thereby confirming its ability to serve as an acceptor for a GalNac transferase. Moreover, as will be apparent to one skilled in the art, peptides that include one or more mutations are within the scope of the present invention. The mutations are designed to allow adjustment of desirable properties of the peptides, for example, activity and number and position of the glycosylation sites linked in 0 and / or N on the peptide.

Acquisition of peptide coding sequences Recombinant technology in general This invention is based on routine techniques in the field of recombinant genetics. Basic texts describing the general methods of use in this invention include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology (1994). For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from the electrophoresis with agarose gel or acylamide, from. of nucleic acids formed in sequences or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or in the number of amino acid residues. Protein sizes are estimated from gel electrophoresis, from proteins formed in sequences, from derived amino acid sequences, or from published protein sequences. Oligonucleotides that are not commercially available can be chemically synthesized, for example according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Lett. 22: - 1859-1862 (1981), by using an automated synthesizer as described in Van Devanter et al., Nucleic Acids Res. 12: 6159-6168 (1984). Complete genes can also be synthesized chemically. The oligonucleotide purification is carried out using any strategy recognized in the art, for example native acrylamide gel electrophoresis or anion exchange HPLC as described in Pearson & Reanier, J. Chrom. 255: 137-149 (1983). The sequence of cloned wild type peptide genes, mutant peptides encoding polynucleotides and synthetic oligonucleotides can be verified after cloning by using for example the chain termination method for the double stranded sequence forming templates of Wallace et al. ., Gene 16: 21-26 (1981). Cloning and Subcloning of a Wild-type Peptide Encoding Sequence Various polynucleotide sequences encoding wild-type peptides have been determined and are available from a commercial supplier for example human growth hormone eg Access Nos. GanBank NM 000515 , NM 002059, NM 022556, NM 022557, NM 022558, NM 022559, NM 0022560, NM 022561 and NM 022562. Rapid progress in human genome studies, has made possible a cloning methodology where a database of the Human DNA sequence can be searched for any segment of genes having a certain percentage of sequence homology with a known nucleotide sequence, such as one encoding a previously identified peptide. Any DNA sequence thus identified can be obtained later by chemical synthesis and / or by a polymerase chain reaction (PCR) technique such as the overlap extension method. For a short sequence, a completely de novo synthesis may be sufficient; while additional isolation of the full-length coding sequence from a human cDNA or a genomic library using a synthetic probe may be necessary to obtain a larger gene. Alternatively, a nucleic acid sequence encoding a peptide can be isolated from a human cDNA or a collection of genomic DNA by using standard cloning techniques such as the polymerase chain reaction (PCR) where the primers based on the homology can often be derived from that known sequence of "nucleic acids encoding a peptide." The techniques most commonly used for this purpose are described in standard texts for example Sambrook and Russell, supra. obtaining a coding sequence for a wild-type peptide can be commercially available or can be constructed The general methods of isolating the mRNA by making the cDNA by reverse transcription, by ligating the cDNA into a recombinant vector, transfecting into a recombinant host for propagation, exclusion by exclusion and cloning are well known (see for example Emplo Gubler and Hoffman, Gene 25: 263-269 (1983); Ausubel et al., Supra). By obtaining an amplified segment of the nucleotide sequence by PCR, the segment can further be used as a probe to isolate the full-length polynucleotide sequence encoding the wild-type peptide from the cDNA library. A general description of the appropriate procedures can be found in Sambrook and Russell, supra. A similar procedure can be followed to obtain a full-length sequence encoding a wild-type peptide for example, any of the aforementioned Genbank accession numbers from a human genomic library. Human genomic libraries are commercially available or can be constructed according to various methods recognized in the art In general, to construct a genomic library, DNA is first extracted from a tissue where a peptide is likely to be found. DNA is then either mechanically disrupted or enzymatically digested to produce fragments of about 12-20 kb in length.The fragments are subsequently separated by gradient centrifugation from polynucleotide fragments of undesirable sizes and inserted into the bacteriophage vectors. These vectors and phages are packaged in vitro.The recombinant phages are analyzed by plaque hybridization as described in Benton and Davis, Science, 196: 180-182 (1977) .Colonial hybridization is carried out as described by Grunstein et al., Proc. Nati, Acad. Sci. USA, 72: 3691-3965 (1975). Based on sequence homology, oligonucleotides degenerates can be designed as primer sets and PCR can be performed under suitable conditions (see for example, White et al., PCR Protocols: Current Methods and Application, 1993; Griffin and Griffin, PCR Technology, CRC Press Inc. 1994) to amplify a segment of the nucleotide sequence from a cDNA or a genomic library. By using the amplified segment as a probe, the full-length nucleic acid encoding a wild-type peptide is obtained. By acquiring a nucleic acid sequence encoding a wild-type peptide, the coding sequence can be subcloned into a vector for example, an expression vector so that a wild-type recombinant peptide can be produced from the resulting construct. Further modifications to the coding sequence of the wild-type peptide for example, nucleotide substitutions, can be done subsequently to alter the characteristics of the molecule.

Introduction of mutations in a peptide sequence From a polynucleotide coding sequence, the amino acid sequence of a wild-type peptide can be determined. Subsequently, this amino acid sequence can be modified to alter the protein glycosylation pattern by introducing additional glycosylation sites at various locations in the amino acid sequence. Various types of protein glycosylation sites are well known in the art. For example, in eukaryotes, N-linked glycosylation occurs in the asparagine of the consensus sequence Asn-Xaa-Ser / Thr in which Xaa is any amino acid except proline (Kornfeld et al., An RevBiochem 54: 631-664 (1985), Kukuruzinska et al., Proc. Nati, Acad. Sci. USA 84: 2145-2149 (1987), Herscovics et al., FASEB J 7: 540-550 (1993), and Orlean, Saccharomyces Vol. (nineteen ninety six) ) . O-linked glycosylation occurs in the serine or threonine residues (Tanner et al., Biochim Biophys., Acta 906: 81-91 (1987), and Hounsell et al., Glycoconj. J. 13: 19- 26 (1996)). Other glycosylation patterns are formed by ligating the glycosylphosphatidylinositol with the carboxyl group at the carboxyl terminus of the protein (Takeda et al., Trends Biochem, Sci. 20: 367-371 (1995); and Udenfriend et al., Ann. Rev. Biochem. 64: 593-591 (1995) Based on this knowledge, it is thus possible to introduce suitable mutations into a wild-type peptide sequence to form new glycosylation sites, although the direct modification of an amino acid residue within A sequence of polypeptides and peptides may be suitable for introducing a new O-linked or N-linked glycosylation site, the introduction of a new glycosylation site is more often accomplished by assembling the polynucleotide sequence encoding a peptide. achieved by using any of the known methods of mutagenesis, some of which are discussed below. Exemplary modifications to a G-CSF peptide include those used in SEQ ID NO: 5-18. A variety of mutation-generating protocols are established and described in the art see, for example, Zhang et al., Proc. Nati Acad. Sci. USA, 94: 4504-4509 (1997); and Stemmer, Nature, 370: 389-391 (1994). The methods can be used separately or in combination to produce variants of a set of nucleic acids and thus variants of encoded polypeptides. Kits for mutagenesis, building collections and other diversity-generating methods are commercially available. Mutation methods for generating diversity include, for example, site-directed mutagenesis (Botstein and Shortle, Science, 229: 1193-1201 (1985)), mutagenesis using templates containing uracil (Kunkel, Proc. Nati, Acad. Sci. USA, 82: 488-492 (1985)), oligonucleotide-directed mutagenesis (Zoller and Smith, Nucí Acids Res., 10: 6487-6500 (1982)), mutagenesis of phosphorothioate-modified DNA (Taylor et al., Nucí Acids Res., 13: 8749-8764 and 8765-8787)), and mutagenesis using a spaced duplex DNA (Kramer et al., Nucí Acids Res., 12: 9441-9456 (1984)). Other methods for generating the mutations include spot-free repair (Kramer et al., Cell, 38: 879-887 (1984)), mutagenesis using host strains deficient in repair (Cárter et al., Nucí. Acids. Res., 13: 4431-4443 (1985)), elimination mutagenesis (Eghtedarzadeh and Henikoff, Nucí Acids Res., 14: 5115 (1986)), selection-restriction and purification-restriction (Wells et al., Phil. Trans R. Soc. Lond. A., 317: 415-423 (1986)), mutagenesis by total gene synthesis (Nambiar et al., Science, 223: 1299-1301 (1984)), repair of strand break double (Mandecki, Proc. Nati, Acad. Sci. USA, 83: 7177-7181 (1986)), mutagenesis by polynucleotide chain termination methods (U.S. Patent No. 5,965,408), and error-prone PCR (Leung et al. al., Biotechniques, 1: 11-15 (1989)).

Modifying Nucleic Acids for a Preferred Use of the Codon in a Host Organism The polynucleotide sequence encoding a mutant peptide can be further altered to coincide with the preferred use of the codon of a particular host. For example, the preferred use of the codon of a strand of bacterial cells can be used to derive a polynucleotide that encodes a mutant peptide of the invention and includes the codons favored by this strain. The frequency of preferred use of the codon shown by a host cell can be calculated by averaging the frequency of the codon's preferred use in a large number of genes expressed by the host cell (for example, calculation service is available from the host site). Kazusa DNA Research Institute, Japan). This analysis is preferably limited to genes that are highly expressed by the host cell. U.S. Patent No. 5,824,864, for example, provides the frequency of use of the codon by highly expressed genes shown by dicotyledonous plants and monocotyledonous plants. At the end of the modification, the mutant peptide coding sequences are verified by the formation of sequences and then subcloned into an expression vector suitable for recombinant production in the same manner as the wild-type peptides.

Expression and purification of the killing peptide After sequence verification, the mutant peptide of the present invention can be produced by using routine techniques in the field of recombinant genetics, by relying on the polynucleotide sequences encoding the polypeptide described herein.

System of. Expression To obtain a high level expression of a nucleic acid encoding a mutant peptide of the present invention, a polynucleotide encoding the mutant peptide is typically subcloned into an expression vector containing a strong promoter to direct transcription, a terminator transcription / translation and a ribosome binding site for the start of translation. Suitable bacterial promoters are well known in the art and described for example in Sambrook and Russell, supra, and Ausubel et al., Supra. Bacterial expression systems for expressing the mutant or wild-type peptide are available in, for example, E. coli, Bacillus sp., Salmonella, and Caulobacter. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In one embodiment, the eukaryotic expression vector in an adenoviral vector, a vector associated with adeno, or a retroviral vector. The promoter used to direct the expression of a heterologous nucleic acid depends on the particular application. The promoter is optionally placed about the same distance from the starting site of the heterologous transcription since it is from the start site of the 7 transcription in its natural parameter. As is known in the art however, some variation in this distance can be adjusted without loss of the function of the promoter. In addition to the promoter, the expression vector typically includes a transcription unit or an expression cassette that contains all of the additional elements required for the expression of the mutant peptide in host cells. A typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding the mutant peptide and the signals required for a polyadenylation. Efficient transcript, ribosome binding sites and translation termination. The nucleic acid sequence encoding the peptide is typically linked to a sequence of unfolded signal peptides to promote secretion of the peptide by the transformed cell. Such signal peptides include among others, the signal peptides from the tissue plasminogen activator, insulin and neuron growth factor and juvenile hormone esterase from Heliothis virescens. Additional elements of the cassette may include enhancers and if genomic DNA is used as the structural gene, introns with functional splice acceptor and donor sites. In addition to a promoter sequence, the expression cassette must also contain a transcription termination region in the downstream direction of the structural gene to provide efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes. The expression vector - in particular used to transport the genetic information within a cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells can be used. Standard vectors of bacterial expression include plasmids such as plasmids based on pBR322, pSKF, pET23D, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to the recombinant proteins to provide convenient isolation methods for example, c-myc. Expression vectors containing regulatory elements from eukaryotic viruses are typically used eukaryotic expression vectors for example, SV40 vectors, papilloma virus vectors and vectors derived from the Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009 / A +, pMTO10 / A +, pMAMneo-5, baculovirus pDSVE, and any other vector that allows the expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, promoter of the murine mammary tumor virus, Rous del-saccharoma virus promoter, polyhedrin promoter, or other promoters that are effective for expression in eukaryotic cells. In some exemplary embodiments, the expression vector is chosen from pCWinl, pCWin2, pCWin2 / MBP, pCWin2-MBP-SBD (pMS39) and pCWin2-MBP-MCS-SBD (pMXS39) as described in the US Pat. co-ownership filed on April 9, 2004, which is incorporated herein by reference. Some expression systems have markers that provide an amplification of genes such as thymidine kinase, hygromycin B phosphotransferase and dihydrofolate reductase. Alternatively, high throughput expression systems that do not involve gene amplification are also suitable such as a baculovirus vector in insect cells, with a polynucleotide sequence encoding the mutant peptide under the direction of the polyhedrin promoter or other promoters. of the baculovirus. The elements that are typically included in the expression vectors also include a replicon that functions in E. coli, a gene that encodes the resistance to 'antibiotics to allow the selection of bacteria harboring the recombinant plasmids, and unique restriction sites in non-essential regions of the plasmid to allow the insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. The prokaryotic sequences are optionally chosen such that they do not interfere with DNA replication in eukaryotic cells if necessary. When periplasmic expression of a recombinant prn (eg, a high mutant of the present invention) is desired, the expression vector further comprises a sequence encoding a secretion signal such as E. coli OppA (periplasmic oligopeptide binding prn). secretion signal or a modified version thereof which is directly connected to 5 'of the coding sequence of the prn to be expressed. This signal sequence directs the recombinant prn produced in the cytoplasm through the cell membrane in the periplasmic space. The expression vector may further comprise a coding sequence for the signal peptidase 1 which can enzymatically unbind the signal sequence when the recombinant prn is introduced into the periplasmic space. A more detailed description for the periplasmic production of a recombinant prn can be found in for example, Gray et al., Gene 39: 247-254 - (1985), U.S. Patent Nos. 6,160,089 and 6,436,674. As discussed above, a person skilled in the art will recognize that various conservative substitutions can be made for any mutant or wild-type peptide or their coding sequence while still preserving the biological activity of the peptide. Moreover, modifications of a polynucleotide coding sequence can also be made to fit a preferred use of the codon in a particular expression host without altering the resulting amino acid sequence.

Transfection Methods Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large amounts of the mutant peptide, which are then purified using standard techniques (see, for example, Colley et al., Biol. Chem. 264: 17619-17622 (1989), Guide to Prn Purification, in Methods in Enzymology, vol.182 (Deutscher, ed., 1990)). The transformation of eukaryotic and prokaryotic cells is carried out according to standard techniques (see for example, Morrison, J. Bact., 132: 349-351 (1977); Clark-Curtiss &Curtiss, Methods in Enzyomology 101: 347-362. (Wu et al., Eds., 1983).

Any of the well-known procedures for introducing external nucleotide sequences into host cells can be used, these include the use of a transfection with calcium phosphate, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, vectors viruses, and any of the other well-known methods for the introduction of cloned genomic DNA, cDNA, synthetic DNA, or other external genetic material into a host cell (see, for example, Sambrook and Russell, supra). It is only necessary that the particular genetic engineering method used can successfully introduce at least one gene into a host cell capable of expressing the mutant peptide.

Detection of the expression of the mutant peptide in host cells After the expression vector is introduced into suitable host cells, the transfected cells are cultured under conditions that favor the expression of the mutant peptide. The cells are then removed by exclusion for the expression of the recombinant polypeptide which is subsequently recovered from the culture using standard techniques (see, for example, Scopes, Prn Purification: Principles and Practice (1982); US Patent No. 4,673,641; Ausubel et al. ., supra; and Sambrook and Russell, supra).

Various general methods for the separation by exclusion of gene expression are well known among those skilled in the art. First, gene expression can be detected at the level of the nucleic acid. A variety of specific measurement methods of DNA and RNA using nucleic acid hybridization techniques are commonly used (eg, Sambrook and Russell, supra). Some methods involve electrophoretic separation (eg, Southern immunoblotting to detect DNA and Northern blotting to detect RNA), but detection of DNA or RNA can be carried out without electrophoresis as well (such as spot immunoblotting). The presence of the nucleic acid encoding a mutant peptide in the transfected cells can also be detected by PCR or RT-PCR by using sequence specific primers. Second, gene expression can be detected at the level of the polypeptide. Various immunological assays are routinely used by those skilled in the art to measure the level of a gene product, particularly by using polyclonal or monoclonal antibodies that specifically react with a mutant peptide of the present invention such as a polypeptide having the amino acid sequence of SEQ ID NO: 1-7 (for example, Harlow and Lane, Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988; Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniques require the preparation of antibodies by selecting antibodies with high specificity against the mutant peptide or an antigenic portion thereof. Methods of polyclonal and monoclonal antibody preparation are well established and their descriptions can be found in the literature, see for example, Harlow and Lane, supra; Kohler and Milstein, Eur. J. Immunol. , 6: 511-519 (1976). More detailed descriptions of antibody preparation against the mutant peptide of the present invention and performing immunological assays upon detection of the mutant peptide are provided in a later section.

Purification of a mutant peptide produced recombinantly Once the expression of a recombinant mutant peptide in transfected host cells is confirmed, the host cells are then cultured on a scale suitable for the purpose of purifying the recombinant polypeptide.

Purification of a mutant peptide produced recombinantly from bacteria When the mutant peptides of the present invention are produced recombinantly by bacteria transformed in large amounts, typically after induction of the promoter, although the expression may be constitutive, the proteins they can form insoluble aggregates. There are several protocols that are suitable for the purification of protein inclusion bodies. For example, the purification of aggregate proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and / or purification of inclusion bodies by the disruption of bacterial cells for example, by incubation in a buffer solution of about 100-150 μg / ml lysozyme and 0.1% Nonidet P40, non-ionic detergent. The cell suspension can be ground using a Polytron mill (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternative methods for lysing bacteria are described in Ausubel et al., And Sambrook and Russell, both supra, and will be apparent to those skilled in the art. The cell suspension is generally centrifuged and the pelletizing containing the inclusion bodies is resuspended in buffer solution which does not dissolve but washes the inclusion bodies for example 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 NaCl mM and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the washing step to remove as much cellular debris as possible. The remaining pelletizing of the inclusion bodies can be resuspended in a suitable buffer solution (psr example, 20 mM sodium phosphate, pH 6.8, 150 mM NaCl).

Other suitable buffer solutions will be apparent to those skilled in the art. After the washing step, the inclusion bodies are solubilized by the addition of a solvent which is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties). The proteins that formed the inclusion bodies can then be renatured by dilution or dialysis with a compatible buffer solution. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80% on a volume to volume basis), and guanidine hydrochloride (from about 4 M) up to around 8 M). Some solvents that can solubilize aggregate-forming proteins such as SDS (sodium dodecyl sulfate) and 70% formic acid, may be unsuitable for use in this procedure due to the possibility of irreversible denaturation of the proteins accompanied by a lack of immunogenicity and / or activity. Although guanidine chlorohydrate and similar agents are denaturing, this denaturation is not irreversible and renatrualization can happen with removal (by dialysis, for example) or dilution of the denaturant, allowing the reformation of an immunologically and / or biologically active protein of interest. . After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques. For a further description of the purification of a recombinant peptide from a bacterial inclusion body see for example PATRA et al., Protein Expression and Purification 18: 182-190 (2000). Alternatively, it is possible to purify recombinant polypeptides for example a mutant peptide from the bacterial periplasm. Where the recombinant protein is exported within the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those skilled in the art, (see for example, Ausubel et al., Supra. ). To isolate the recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer solution containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and pelleted again in cold ice MgSO4 5 mM, and kept in an ice bath for about 10 minutes. The cell suspension is centrifuged and the supernatant is decanted and stored. The recombinant proteins present in the supernatant can be separated from host proteins by standard separation techniques well known to those skilled in the art. 2. Standard protein separation techniques for purification When a recombinant polypeptide, for example the mutant peptide of the present invention, is expressed in host cells in a soluble form, its purification may follow the standard procedure of protein purification described below. i. Dissolution of solubility Often as an initial step, and if the protein mixture is complex, an initial fractionation of salt can remove many of the undesirable proteins from host cells (or proteins derived from the cell culture media) from the Recombinant protein of interest, for example, a mutant peptide of the present invention, the preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Then the proteins are precipitated on the basis of their solubility. The more hydrophobic a protein is, the more likely it is that it will precipitate at lower concentrations of ammonium sulfate. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resulting concentration of ammonium sulfate is between 20-30%. This will precipitate more hydrophobic proteins. The precipitate is discharged (unless the protein of interest is hydrophobic) and the ammonium sulfate is added to the supernatant at a known concentration to precipitate the protein of interest. The precipitate is then solubilized in buffer solution and if necessary excess salt is removed through either dialysis or diafiltration. Other methods that are based on protein solubility, such as precipitation with cold ethanol, are well known to those skilled in the art and can be used to fractionate complex mixtures of proteins. ii Differential filtration by size Based on the calculated molecular weight, a larger and smaller protein can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size having a lower molecular weight cutoff than the molecular weight of the protein of interest eg a mutant peptide. The retention product of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane in the filtrate. Then the filtrate could be processed by chromatography as described below. iii Column chromatography The proteins of interest (such as mutant peptide of the present invention) can also be separated from other proteins based on their size, net surface charge, hydrophobic capacity or affinity for the ligands. In addition, antibodies formulated against the peptide can be conjugated to column matrices and can be immunopurified from peptides. All these methods are well known in the art.

It will be apparent to someone skilled in the art that chromatographic techniques can be carried out at any scale and using equipment from many different manufacturers (for example, Pharmacia Biotech).

Immunoassays for the Detection of Methyl Peptide Expression To confirm the production of a recombinant mutant peptide, immunological assays can be useful for detecting polypeptide expression in a sample. Immunogenic assays are also useful for quantifying the level of expression of the recombinant hormone. Antibodies against a mutant peptide are necessary to carry out these immunological assays.

Production of Mutant Peptide Antibodies Methods for the production of polyclonal and monoclonal antibodies that specifically react with an immunogen of interest are known to those skilled in the art (see for example, Coligan, Current Protocols in Immunology Wiley / Greene, NY, 1991; Harlow and Lane, Antibodies: A Laboratory Manual Colds Spring Harbor Press, NY, 1989; Suites et al. (Eds) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA and references cited therein Goding, Monoclonal Antibodies: Principles and Practice (2d ed.) Academia Press, New York, NY 1986; and Kohler and Milstein Nature 256: 495-497, 1995). Such techniques include the preparation of antibodies by selection of antibodies from collections of recombinant antibodies in phage or similar vectors (see, Huse et al., Science 246: 1275-1281, 1989; and Ward et al., Nature 341: 544-546 , 1989). In order to produce antibodies containing antisera with desired specificity, the polypeptide of interest (eg, a mutant peptide of the present invention) or an antigenic fragment thereof can be used to immunize suitable animals for example mice, rabbits or primates. A standard adjuvant such as Freund's adjuvant can be used according to a standard immunization protocol. Alternatively, a synthetic antigenic peptide derived from that particular polypeptide can be conjugated to a carrier protein and subsequently used as an immunogen. The immune response of the animal to the preparation of the immunogen is observed by taking test bleeds and finishing the concentration of the reactivity with the antigen of interest. When suitably high concentrations of the antibody for the antigen are obtained, the blood of the animals is collected and anti-sera are prepared. The additional fractionation of the anti-sera to enrich antibodies specifically reactive for the antigen and the purification of the antibodies can be done later see, Harlow and Lane, supra, and the general descriptions of protein purification given above. Monoclonal antibodies are obtained by using various techniques familiar to those skilled in the art. Typically, spleen cells from an animal immunized with a desired antigen are commonly immortalized by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol 6: 511-519, 1976). Alternative methods of immortalization include for example transformation with Epstein Barr virus, oncogenes, or retroviruses or other methods well known in the art. Colonies resulting from single immortalized cells are removed by exclusion for the production of antibodies of the desired specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by such cells can be enhanced by various techniques including injection into the peritoneal cavity. of a vertebrate host. Additionally, monoclonal antibodies can be produced recombinantly by identifying nucleotide acid sequences encoding an antibody with desired specificity or a binding fragment of such an antibody by screening out a collection of cDNAs from C h manas cells according to the invention. with the general protocol detailed by Huse et al., supra. The general principles and methods of production of recombinant polypeptides discussed above apply to the production of antibodies by recombinant methods. When desired, antibodies that can specifically recognize a mutant peptide of the present invention can be tested for their cross-reactivity against the wild-type peptide and thus differentiated from the antibodies against the wild-type protein. For example, antisera obtained from an animal immunized with a mutant peptide can run through a column on which a wild-type peptide is immobilized. The portion of antibodies passing through the column recognizes only mutant peptide and not the wild-type peptide. Similarly, monoclonal antibodies against a mutant peptide can also be excluded by exclusion because of their uniqueness in recognizing only the mutant in recognizing only the mutant.e but not the wild-type peptide. Polyclonal or monoclonal antibodies that specifically recognize only the mutant peptide of the present invention but not the wild-type peptide, are useful in isolating the mutant protein from the wild-type protein for example, by incubating a sample with a monoclonal or polyclonal antibody. specific for an immobilized mutant peptide or on a solid support.

Immunoassays for Detecting the Expression of Mutant Peptides Once antibodies specific for a mutant peptide of the present invention are available, the amount of the polypeptide in a sample pro, a cell lysate can be measured by a variety of immunoassay methods. that provide qualitative and quantitative results to an experimental technician. For a review of the immunological and immunoassay procedures in general see for example Suites, supra; U.S. Patent 4,366,241; 4,376,110; 4,517,288; and 4,837,168.

Labeling in immunoassays Immunoassays often use a labeled agent to bind specifically and label the binding complex formed by the antibody and the target protein. The labeling agent was in itself to be one of the portions that comprise the antibody / target protein complex or may be a third portion such as another antibody that specifically binds to the antibody / target protein complex. A label - can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optimal or chemical means. Examples include but are not limited to magnetic beads (eg, Dynabeads ™), fluorescent dyes (eg, fluorescein isothiocyanate, Texas red, rhodamine and the like), radiolabels (eg, 3H, 125I, 35S, 14C, or 32P), enzymes (eg, horseradish peroxidase, alkaline phosphatase, and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or glass or colored plastic (eg, polystyrene, polypropylene, latex, etc.) .) in pearls. In some cases, the labeling agent is a secondary antibody that carries a detectable label. Alternatively, the second antibody may lack a label but may in turn be linked by a third labeled antibody specific to the antibodies of the species from which the second antibody is derived. The second antibody can be modified with a detectable portion such as biotin to which a third labeled molecule such as streptavidin labeled with enzymes can specifically bind. Other proteins that can bind specifically, the immunoglobulin constant regions such as protein A or protein G can also be used as the labeling agents. These proteins are normal constituents of the cell walls of streptococcal bacteria. They show a strong non-immunogenic reactivity with the immunoglobulin constant regions from a variety of species (see generally, Kronval, et al., J. Immunol., 111: 1401-1406 (1973); and Akerstrom, et al., J. Immunol., 135: 2589-2542 (1985)).

Immunoassay Formats Immunoassays for detecting a target protein of interest (eg, a mutant human growth hormone) from samples can be competitive or non-competitive. Non-competitive immunoassays are assays in which the amount of the target protein captured is measured directly. In a preferred sandwich assay for example, the antibody specific for the target protein can be directly linked to a solid substrate where the antibody is immobilized. Then it captures the target protein in the test samples. The thus immobilized antibody / target protein complex is then linked by a labeling agent such as a second or third antibody that carries a label as described above. In competitive assays, the amount of the target protein in a sample is measured indirectly by measuring the amount of an aggregated target protein (exogenous) displaced (or competed) from an antibody specific for the target protein by the target protein present in the sample. In a typical example of such an assay, the antibody is immobilized and labeled to the exogenous target protein. Since the amount of exogenous target protein bound to the antibody is inversely proportional to the concentration of the target protein present in the sample, the level of target protein in the sample can thus be determined based on the amount of the exogenous target protein bound to the antibody and thus immobilized. In some cases, western blot analysis (immunoblotting) is used to detect and quantify the presence of a mutant peptide in the samples. The technique generally comprises separating the sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as a nitrocellulose filter, a nylon filter, or a derivative nylon filter). in incubating the samples with antibodies that bind specifically to the target protein. These antibodies can be directly labeled or can be subsequently detected alternatively using labeled antibodies. (e.g., labeled sheep anti-mouse antibodies) that specifically bind to antibodies against a mutant peptide. Other assay formats include liposome immunoassays (LIA) which utilize liposomes designed to bind to specific molecules (e.g., antibodies) and release reagents or encapsulated labels. The released chemicals are then detected according to standard techniques (see, Monroe et al., Clin. Prod. Rev., 5: 34-41 (1986)).

The Conjugates In a representative aspect, the present invention provides a glycoconjugate between a peptide and a selected modifier group, in which the modifier group is conjugated to a peptide through a glucosyl binding group for example, a binding group of intact glucosyl. The glucosyl ligation group binds directly to a glycosylation site linked at 0 on the peptide or alternatively common is linked to a glycosylation site linked at 0 through one or more additional glycosyl residues. Iso conjugate preparation methods are set forth herein and in U.S. Patent Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; WO 98/31826; US2003180835; and WO 03/031464. Exemplary peptides include an O-linked GalINAc residue that binds to the O-linked glycosylation site through the action of GalINAc transferase. GalINAc itself can be an intact glucosyl ligature group. The GalINAc can also be further elaborated by for example, a residue of Gal or Sia any of which can act as the intact glucosyl ligation group. In representative embodiments, the saccharil residue bound at 0 is GalINAc-X, GalINAc-Gal-Sia-X, or GalINAc-Gal-Gal-Sia-X, wherein X is a modifying group. In an exemplary embodiment, the peptide is a mutant peptide that includes a 0-linked glycosylation site that is not present in the wild-type peptide. The peptide is preferably glycosylated at 0 at the site mutated with a GalINAc residue. The immediately preceding discussion concerning the structure of the saccharin portion is also relevant here. The link between the peptide and the selected portion includes an intact glucosyl ligation group interposed between the peptide and the modifier portion. As discussed herein, the portion selected is essentially any species that can bind to a unit of saccharides, resulting in a modified sugar that is recognized by an appropriate transferase enzyme which attaches the modified sugar to the peptide. The saccharide component of the modified sugar when tripeptide is interposed and a selected portion becomes an intact glucosyl ligation group. The glycosyl ligation group is formed from any mono or oligosaccharide which after modification with a selected portion is a substrate for a suitable transferase. The conjugates of the invention will typically correspond to the general structure: in which the symbols a, b, c, d and s represent a positive integer that is not 0 and T is C to zero or a positive integer. The agent is a therapeutic agent, a bioactive agent, a detectable label, soluble portion, in water, or the like. The agent can be a peptide for example, above, antibody, antigen, etc. The ligation can be any of a broad configuration of infra-binding groups. Alternatively, the ligation can be a single bond or a zero order ligation. The identity of the peptide is without limitation. In an exemplary embodiment, the selected portion is a water-soluble polymer, for example, PEG, m-PEG, PPG, m-PPG, etc. The water soluble polymer is covalently bound to the peptide by means of a glucosyl ligation groups. The glycosyl ligation group is covalently linked to either an amino acid residue or a glycosyl residue of the peptide. Alternatively, the glucosyl ligation group is linked to one or more glycosyl ligands of a glycopeptide. The invention also provides conjugates in which the glucosyl ligation group (e.g., GalINAc) binds to an amino acid residue (e.g., Thr or Ser). In an exemplary embodiment, the protein is an interferon. Interferons are antiviral glycoproteins that are secreted in humans by human primary fibroblasts after induction with a virus or double-stranded RNA. Interferons are of interest as therapeutics for example, antiviral agents (e.g., hepatitis B and C) antitumor agents (e.g., hepatocellular carcinoma) and in the treatment of multiple sclerosis. For relevant reference to interferon-a see, Asano, et al., Eur. J. Cancer, 27 (Suppl 4): S21-S25 (1991); Negy, et al., Anticancer Research, 8 (3): 467-470 (1988); Dron, et al., J. Biol. Regul. Homeost. Agents, 3 (1): 13-19 (1989); Habib, et al., Am. Sirg., 67 (3): 257-260 (3/2001); and Sugyiama, et al., Eur. J. Biochem., 217: 921-927 (1993). For references that discuss interferon-β see for example, Yu et al., J. Neuroimmunol. , 64 (1): 91-00 (1996); J., J. Neurosci. Res., 65 (l): 59-67 (2001); Wender, et al., Folia Neuropathol, 39 (2): 91-93 (2001); Martin, et al., Springer Semen. Immunopathol. , 18 (l): l-24 (1996); Takane, et al., J. Pharmacol. Exp. Ther., 294 (2): 746-752 (2000); Sburlati, et al., Biotechnol. Prog., 14: 189-192 (1998); Dodd, et al., Biochimica et Biophysica Acta, 787: 183-187 (1984); Edelbaum, et al., J. Inferieron Res., 12: 449-453 (1992); Conrado, et al., J. Biol. Chem, 262 (30): 14600-14605 (1987); Covas, et al., Eur. J. Biochem., 173: 311-316 (1988); Demolder, et al., J. Biotechnol, 32: 179-189 (1994); Sedmak, et al., J. Inferieron Res., 9 (Suppl 1): S61-S65 (1989); Kagawa, et al., J. Biol. Chem, 263 (33): 17508-17515 (1988); Hershenson, et al., U.S. Patent No. 4,894,330; Jayaram, et al., J. Inferieron Res., 3 (2): 177-180 (1983); Menge, et al., Develop- Biol. Standard, 66: 391-401 (1987); Vonk, et al., J. Infer Res., 3 (2): 169-175 (1983); and Adoli, et al., J. Inferieron Res., 10: 255-267 (1990). In an exemplary conjugate of inferred, the inferred alias, for example, inferred alias 2b and 2a, is conjugated to a water soluble polymer through an intact glucosyl ligation. In a further exemplary embodiment, the invention provides a conjugate of a human granulocyte colony stimulating factor (G-CSF). G-CSF is a glycoprotein that stimulates proliferation differentiation and activation of neutropoietic progenitor cells in functionally mature neutrophils. Injected G-CSF is rapidly cleared from the body. See for example, Nohynek, et al., Cancer Chemother. Pharmacol., 39: 259-266 (1997); Lord, et al., Clinical Cancer Research, 7 (7): 2085-2090 (07/2001); Rotondaro, et., Molecular Biotechnology, 11 (2): 117-128 (1999); and Bónig et al., Bone Marrow Transplantation, 28: 259-264 (2001). The present invention encompasses a method for the modification of GM-CSF. GM-CSF is well known in the art as a cytokine produced by activated T cells, macrophages, endothelial cells and stromal fibroblasts. GM-CSF acts mainly in the bone marrow to increase the production of inflammatory leukocytes, and also functions as an endocrine hormone to initiate the replenishment of neutrophils consumed during the inflammatory function. In addition, GM-CSF is a macrophage activating factor and promotes the differentiation of Lagerhans cells into dendritic cells. Like G-CSF, GM-CSF also has "clinical applications in bone marrow replacement following chemotherapy." In addition to supplying conjugates that are formed through an aggregated intact glucosyl ligature group, the present invention provides conjugates which are highly homogeneous in their substitution patterns.When using the methods of the invention, it is possible to form peptide conjugates in which essentially all portions of sugars modified through a population of conjugates of the invention bind to an amino acid structurally identical or a glycosyl residue Thus, in a second aspect, the invention provides a peptide conjugate having a population of water soluble polymer portions, which are covalently linked to the peptide through a group of a glucosyl ligature intact In another conjugate of the invention, essentially each member of the population is linked by means of The glucosyl ligation group to a glycosyl residue of the peptide, and each glucosyl residue of the peptide to which the glucosyl ligation group is attached has the same structure. A conjugate of peptides having a population of water soluble polymer portions is also provided, linked covalently to it through a glucosyl ligation group. In another embodiment, essentially each member of the population of water-soluble polymer portions is linked to an amino acid residue of the peptide by means of an intact glucosyl ligation group and each amino acid residue having an intact glucosyl ligation group. attached to it has the same structure. The present invention also provides conjugates analogous to those described above in which the peptide is conjugated to a therapeutic moiety, diagnostic moiety, targeting moiety, toxin moiety or the like by means of a glucosyl ligation group. Each of the aforementioned portions may be a small molecule, natural polymer (e.g., polypeptide) or synthetic polymer. Still in a further embodiment, the invention provides conjugates that are selectively localized to a particular tissue due to the presence of a targeting agent as a component of the conjugate. In an exemplary embodiment, the targeting agent is a protein. Exemplary proteins include transfer (brain, accumulated blood), glycoprotein-HS (bone, brain, accumulated blood), antibodies (brain, tissue with antigens, specific antibodies, accumulated blood), coagulation factors V-XII ( damaged tissue, coagulation, accumulated blood cancer), whey proteins, for example, α-acid glycoprotein, fetuin, α-fetal protein (accumulated blood brain) β 21-glycoprotein (liver, atherosclerotic plaques, accumulated blood brain) G-CSF, GM-CSF, M-CSF, and EPO (immune stimulation, cancers, accumulated blood, overproduction of red blood cell neuroprotection), albumin (increase in half-life), IL-2 and IFN-a. In a direct exemplary conjugate, interferon alpha 2β (IFN-a 2ß) is conjugated to transferrin by means of a bifunctional ligation that includes a group and an intact glucosyl ligature at each termination of the PEG portion. (reaction scheme 1). For example, one termination of the PEG ligature is functionalized with an intact slurry of sialic acid that binds to transferrin and the other is functionalized with an intact ligature of O-linked GalNAc that binds to IFN-α 2β. The conjugates of the invention may include glucosyl ligation groups that are monovalent or multivalent (e.g., antennal structures). Thus, the conjugates of the invention include both species in which a selected portion is linked to a peptide by means of a group and a monovalent glucosyl ligature. Also included within the invention are conjugates in which more than one selected portion is linked to a peptide by means of a group of a multivalent ligature.

The Methods In addition to the conjugates described above, the present invention provides methods for the preparation of these and other conjugates. In addition, the invention provides methods of preventing cure or improvement of a disease state by the administration of a conjugate of the invention to a subject at risk of developing the disease or to a subject having the disease. Additionally, the invention provides methods for targeting the conjugates of the invention to a particular tissue or region of the body. Thus, the invention provides a method of forming a covalent conjugate between a selected portion and a peptide. In exemplary embodiments, the conjugate is formed between a water-soluble polymer a therapeutic portion, a targeting moiety or a bio-molecule, and a glycosylated or non-glycosylated peptide. The polymer, therapeutic moiety or bio-molecule is conjugated to the peptide by means of a group of a glucosyl ligation that is interposed and covalently linked to both the peptide and the modifying group (e.g., the water-soluble polymer). The method includes contacting the peptide with a mixture containing a modified sugar and a glycosyltransferase for which the modified sugar is a substrate. The reaction is carried out under suitable conditions to form a covalent bond between the modified sugar and the peptide. The sugar portion of the modified sugar is preferably selected from sugars of nucleotides, activated sugars and sugars which are neither nucleotides nor activated. The acceptor peptide (glycosylated-0 or non-glycosylated) is synthesized 'typically de novo, or is expressed recombinantly in a prokaryotic cell (eg, bacterial cell, such as E. coli) or in a eukaryotic cell such as a cell of mammal, yeast, insect, fungus or plant. The peptide can be either a full-length protein or a fragment. In addition, the peptide can be a mutated or wild-type peptide. In an exemplary embodiment, the peptide includes a mutation that adds one or more glycosylation sites N-linked or O-linked to the peptide sequence. In an exemplary embodiment, the peptide is O-glycosylated and functionalized with a water-soluble polymer in the following manner. The peptide is produced with a glycosylation site of available amino acids or if glycosylated, the glucosyl portion is trimmed to expose the amino acid. For example, GallNAc is added to a serine or threonine and the galacto silated peptide is sialylated with a cassette of a sialic acid modifying group using ST6Gal-1. Alternatively, the galactosylated peptide is galactosylated using Core-1-GalT-1 and the product is sialylated with a cassette of a sialic acid modifying group using ST3GalTl. An exemplary conjugate according to this method has the following ligations: Thr-a-1-GalNAc-β-1, 3-Gal-a2, 3-Sia *, in which Sia * is the cassette of the sialic acid modifying group . In the methods of the invention, such as those established, by using multiple enzymes and saccharyl donors, the individual glycosylation steps may be carried out separately or combined in a single vessel reaction. For example, in the reaction of three enzymes previously established, the GalNAc transferase, GalT and SiaT and their donors can be combined in a single container. Alternatively, the reaction with GalNAc can be performed alone and both of GalT and SiaT and the appropriate saccharyl donors added as a single step. Another way of running the reactions involves adding each enzyme and a suitable donor sequentially and carrying out the reaction in a single container portion. The combinations of each of the methods set forth above are of use in the preparation of the compounds of the invention. In the conjugates of the invention, the cassette of the Sia modifier group can be ligated to the Gal in a bond a-2.6, or a-2.3. For example, in one embodiment, G-CSF is expressed in a mammalian system and is modified by sialidase treatment to report residues in the sialic acid terminus, followed by PEGylation using ST3Gal3 and a PEG-acid donor. sialic. The method of the invention also provides for the modification of incompletely glycosylated peptides that are produced recombinantly. Many recombinantly produced glycoproteins are incompletely glycosylated by exposing the carbohydrate residues that may have undesirable properties, for example, immunogenicity, recognition by the RES. By employing a sugar modified in the method of the invention, the peptide can be simultaneously glycosylated and further derivatized with for example, a water soluble polymer, therapeutic agent or the like. The sugar portion of the modified sugar may be the residue that would be suitably conjugated to the acceptor in a fully glycosylated peptide, or other sugar portion with desirable properties. Peptides modified by the methods of the invention may be synthetic or wild-type peptides or they may be peptides mutated or produced by methods known in the art, such as site-directed mutagenesis. The glycosylation of the peptides is typically either N-linked or O-linked. An exemplary N-ligation is the placement of the modified sugar to the side chain of an asparagine residue. The sequence of tripeptides asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline, are the recognition sequences for the enzymatic binding of the carbohydrate moiety to the side chain of asparagine. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of a sugar (eg, N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose or xylose) to the hydroxy side chain of a hydroxyamino acid, preferably serine or threonine, although amino acids are not natural or unusual, for example, 5-hydroxyproline or 5-hydroxylysine can also be used. Additionally, in addition to the peptides, the methods of the present invention can be practiced with subsequent biological structures (eg, glycolipids, lipids, sphingoids, ceramides, whole cells and the like containing a 0-linked glycosylation site). The addition of the glycosylation sites to a peptide or other structure is conveniently achieved by altering the amino acid sequence that it contains one or more glycosylation sites. Addition can also be made by the incorporation of one or more species having an OH group, preferably serine or threonine residues, within the peptide sequence (for the 0-linked glycosylation sites). The addition can be made by mutation or by complete chemical synthesis of the peptide. The amino acid sequence of the peptide is preferably altered through changes in the level of the DNA particularly by mutating the DNA encoding the peptide into preselected bases such as codons that are generated that will result in the desired amino acids. DNA mutations are preferably made by using methods known in the art. In an exemplary mode, the glycosylation site is added by mixing polynucleotides. Polynucleotides encoding a candidate peptide can be modulated with DNA mixing protocols. DNA blending is a process of recursive recombination and mutation effected by random fragmentation of an accumulation of related genes, followed by the reassembly of the fragments by a process of the polymerase chain reaction type. See, for example, Stemmer, Proc. Nati Acad. Sci. USA 91: 10747-10751 (1994); Stemmer, Nature 370: 389-391 (1994); and Patent of E.U.A. Nos. 5,605,793, 5,837,458, 5,830,721 and 5,811,238. The present invention also provides methods for adding (or removing) one or more selected glycosyl residues to a peptide, after which a modified sugar is conjugated to at least one of the selected glucosyl residues of the peptide. The present embodiment is useful, for example, when it is desired to conjugate the modified sugar to a selected glycosyl residue that is either not present in a peptide or is not present in a desired amount. Thus, prior to coupling a sugar-modified peptide, the selected glycosyl residue is conjugated to the peptide by enzymatic or chemical coupling. In another embodiment, the glycosylation pattern of a glycopeptide is altered prior to the conjugation of the modified sugar by the removal of the glycopeptide carbohydrate residue. See, for example WO 98/31826. The addition or removal of some carbohydrate moieties present in the glycopeptide are achieved either chemically or enzymatically. Chemical deglycosylation is preferably presented by exposure of the polypeptide variant to trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most of all sugars except the ligation sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the peptide intact. Chemical deglycosylation is described by Hakimuddin et al., Arch. Biochem. Biophys. 259: 52 '(1987) and by Edge et al., Anal. Biochem. 118: 131 (1981). Enzymatic cleavage of the carbohydrate moieties in the polypeptide variants can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138: 350 (1987). The chemical addition of glucosyl portions is carried out by any method known in the art. The enzymatic addition of sugar portions is preferably achieved by using a modification of the methods established herein. By replacing native glucosyl units with the modified sugars used in the invention. Other methods of adding portions of sugar are described in U.S. Pat. No. 5,876,980, 6,030,815, 5,728,554 and 5,922,577. Exemplary binding sites for the selected glycosyl residue include but are not limited to: (a) consensus sites for N-linked glycosylation and site for O-linked glycosylation; (b) terminal glucosyl moieties that are acceptors for a glycosyltransferase; (c) arginine, asparagine and histidine; (d) free carboxyl groups; (e) free sulfhydryl groups such as those of cysteine; (f) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (g) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (h) the glutamine amide group. Exemplary methods of use in the present invention are described in WO 87/05330 published September 11, 1987, and in Aplin and Wriston, CRC CRIT. REV. BIOCHEM., Pp. 259-306 (1981).

In one embodiment, the invention provides a method for ligating two or more peptides through a binding group. The ligature group is of any useful structure and can be selected from straight and branched chain structures. Preferably, each termination of the ligation, which binds to a peptide, includes a modified sugar (i.e., a nascent intact glucosyl ligation group). In an exemplary method of the invention, two peptides are ligated together by means of a ligation portion that includes a PEG ligation. The construct conforms to the general structure established in the previous sheet. As described herein, the construct of the invention includes two intact glucosyl ligation groups (ie, s + 1 = 1). The focus on a PEG ligature that includes two glucosyl groups for purposes of clarity and should not be construed as limiting the identity of the ends of ligatures for use in this embodiment of the invention. Thus, a portion of PEG is functionalized in a first term with a first glucosyl unit and in a second term with a second glucosyl unit. The first and second glucosyl units are preferably substrates for different transferases, which allows the orthogonal linkage of the first and second peptides to the first and second glucosyl units, respectively. In practice, the ligation (glycosyl) 1-PEG- (glycosyl) 2 makes contact with the first peptide and a first transferase to which in the first unit of glycosyl is a substrate, whereby it is formed (peptide) 1 - (glycosyl) 1-PEG- (glycosyl) 2. The transferase peptide and / or unreacted is then optionally removed from the reaction mixture. The second peptide and a second transferase for which the second glucosyl unit is a substrate are added to the conjugate (peptide) 1- (glycosyl) 1-PEG- (glycosyl) 2, forming, (peptide) 1- (glycosyl) 1 - PEG- (glycosyl) 2- (peptide) 2; at least one of the glucosyl residues is directly or indirectly bound in O. Those skilled in the art will appreciate that the above-detailed method is also applicable to the formation of conjugates between more than two peptides by, for example, the use of branched PEG, dendrimer, poly (amino acid), polsaccharide or the like. In an exemplary embodiment, interferon alpha 2β (IFN-α 2β) is conjugated to transferrin by means of a bifunctional ligation that includes an intact glucosyl ligation group in each portion of the PEG terminus (Reaction Scheme 1). The IFN conjugate has an in vivo half-life that increases over that of IFN alone by virtue of a higher molecular dimensioning of the conjugate. Moreover, the conjugation of IFN to transferrin serves to selectively direct the conjugate to the brain. For example, one termination of the PEG ligature is functionalized with a CMP sialic acid and the other is functionalized with a UDP GalNAc. The ligation is combined with IFN in the presence of a GalNAc transferase, in the binding of the GalNAc of the linker arm to a serine and / or threonine residue in the IFN.

Reaction scheme 1 The processes described above can be carried out through as many cycles as desired and is not limited to the formation of a conjugate between two peptides with a single ligation. Moreover, those skilled in the art will appreciate that reactions that functionalize the intact glucosyl ligation groups at the ends of the PEG (or other) ligation with the peptide can be presented simultaneously in the same reaction vessel, or can be carried performed in a phased manner. When the reactions are carried out in a stepwise fashion, the product conjugate in each step is optionally purified from one or more reaction components (eg, enzymes, peptides). An exemplary yet additional embodiment is set forth in reaction scheme 2. Reaction scheme 2 shows a method of preparing a conjugate that directs a selected protein, eg, GM-CSF, to the bones and increases the circulatory half-life of the selected protein.

Reaction scheme 2 GM-CSF transferase wherein G is a glycosyl residue on an activated sugar portion (eg, nucleotide sugar), which is converted to an intact glucosyl ligation group in the conjugate. When s is greater than 0, L is a group of saccharyl ligatures such as GalNAc, or GalNAc-Gal. The use of reactive derivatives of PEG (or other ligatures) to bind one or more portions of peptides to the ligation is within the scope of the present invention. The invention is not limited by the identity of the PEG reactive analogue. Many activated derivatives of poly (ethylene glycol) are commercially available and in the literature. It is well within the capabilities of one skilled in the art to choose and synthesize, if necessary, a suitable activated PEG derivative with which to prepare a substrate useful in the present invention. See, Abuchowski et al. Cancer Biochem. Biophys., 7: 175-186 (1984); Abuchowski et al, J. Biol. Chem., 252: 3582-3586 (1977); Jackson et al., Anal. Biochem., 165: '114-127 (1987); Koide et al., Biochem Biophys. Res. Commun., 111: 659-667 (1983)), tresylated (Nilsson et al.-, Methods Enzymol., 104: 56-69 (1984); Delgado et al., Biotechnol. Appl. Biochem., 12: 119-128 (1990)); active ester derived from N-hydroxysuccinimide (Buckmann et al., Makromol. Chem., 182: 1379-1384 (1981); Joppich et al., Makromol. Chem., 180: 1381-1384 (1979); Abuchowski et al. Cancer Biochem. Biophys., 1: 175-186 (1984), Katreetal, Proc. Nati, Acad. Sci. USA, 84: 1487-1491 (1987), Kitamura et al., Cancer Res., 51: 4310- 4315 (1991), Boccu et al., Z. Naturforsch., 38C: 94-99 (1983), carbonates (Zaiipsky 'et al., POLY (ETHYLENE GLYCOL) CHEMISTRY: BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, Harris, Ed., Plenum Press, New York, 1992, pp. 347-370, Zalipsky et al., Biotechnol.Appl. Biochem., 15: 100-114 (1992), Veronese et al., Appl. Biochem. Biotech., 11: 141. -152 (1985)), imidazolyl formates (Beauchamp et al., Anal. Biochem., 131: 25-33 (1983), Berger et al., Blood, 71; 1641-1647 (1988)), 4-dithiopyridines (Woghiren et al., Bioconjugate Chem., 4: 314-318 (1993)), isocyanates (Byun et al., ASAIO Journal, M649-M-653 (1992)) and epoxides (U.S. Patent No. 4,806,595, grant to Noishiki et al., (1989). Other groups of ligatures include urethane ligation between the amino groups and the activated PEG. See, Veronese, etal., Appl. Biochem. Biotechnol., 11: 141-152 (1985). In another exemplary embodiment in which a reactive PEG derivative utilizes it, it provides a method for extending the average circulation life in the blood of a selected peptide, essentially targeting the peptide accumulated in the blood by conjugating a peptide to a natural polymer. or synthetic of a size sufficient to retard filtration by glomerulus (e.g., albumin). See reaction scheme 3. This embodiment of the invention is illustrated in the reaction scheme in which G-CSF is conjugated to albumin by means of a PEG ligation using a combination of chemical and enzymatic modification. Reaction scheme 3 T X- PEG-SA-CMP * - (albumin) - PEG-SA-CMP Thus, as shown in reaction scheme 3, a residue (eg, an amino acid side chain) of albumin is modified with a reactive PEG derivative such as X-PEG- (CMP-sialic acid), in which X is an activating group (for example, reactive ester, isothiocyanate, etc.). The PEG derivative and G-CSF are combined and contacted by a transferase for which CMP-sialic acid is a substrate. In an illustrative further embodiment, a lysine e-amine is reacted with the N-hydroxysuccinimide ester of the PEG ligation to form the albumin conjugate. The CMP-sialic acid is enzymatically conjugated to a suitable residue on GCSF, for example, Gal or GalNAc whereupon the conjugate is formed. Those skilled in the art will appreciate that the method described above is not limited to established reaction partners. In addition, the method can be practiced to form conjugates that include more than two protein portions, for example, the use of a branched ligature that has more than two terminations.

Modified sugars Modified glucosyl donor species ("modified sugars") are preferably selected from modified sugar nucleotides, activated modified sugars and modified sugars that are simple saccharides that are neither nucleotides nor activated. Any desired structure of carbohydrates can be added to a peptide by using the methods of the invention. Typically, the structure will be a monosaccharide, but the present invention is not limited to the use of modified monosaccharide sugars, oligosaccharides and polysaccharides are also useful. The modifying group is attached to a portion of sugar by enzymatic means, chemical means or a combination thereof by which a modified sugar is produced. The sugars are substituted in any portion that allows the binding of the modifying portion, although which still allows the sugar to function as a substrate for the enzyme used to bind the modified sugar to the peptide. In another embodiment, when the sialic acid is sugar, the sialic acid is replaced with the modifying group either at position 9 on the pyruvane side chain or at position 5 on the amine portion which is normally acetylated at sialic acid . In certain embodiments of the present invention, a modified sugar nucleotide is used to add the modified sugar to the peptide. Exemplary sugar nucleotides which are used in the present invention in their modified form include mono, di or triphosphate of nucleotides or analogs thereof. In another embodiment, the modified sugar nucleotide is selected from UDP-glycoside, CMP-glycoside or a GDP-glycoside. Even more preferably, the modified sugar nucleotide is selected from a UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid or CMP-NeuAc. N-acetylamine derivatives of the sugar nucleotides are also to be used in the method of the invention. The invention also provides methods for synthesizing a modified peptide by using a modified sugar for example modified galactorsa, modified fucose, modified GalNAc and modified sialic acid. When a modified sialic acid is used, either either sialyltransferase or a trans-sialidase (for only a sialic acid bound at a2.3) can be used in these methods. In other embodiments, the modified sugar is an activated sugar. The activated modified sugars which are useful in the present invention are typically glycosides which have been synthetically altered to include an activated starting group. As used herein, the term "activated starting group" refers to those portions which readily move in nucleophilic substitution reactions regulated by enzyme. Many activated sugars are known in the art. See, for example, Vocadlo et al., In CARBOHYDRATE CHEMISTRY AND BIOLOGY, vol. 2, Ernst et al. Ed., Wiley-VCH Verlag: Weinheim, Germany, 2000; Kodama et al., Tetrahedron Lett. 34: 6419 (1993); Lougheed, et al. J. Biol. Chem. 274: 37717 (1999)). Examples of the activating groups (starting groups) include fluoro, chloro, bromo, tosylated ester, mesylated ester, triflate ester and the like. The preferred activated starting groups for use in the present invention are those which do not significantly sterically obstruct the enzymatic transfer of the glycoside to the acceptor. Thus, preferred embodiments of the activated glycoside derivatives include glucosyl fluoride and glucosyl mesylate, with the glucosyl fluorides being particularly preferred. Among the glucosyl fluorides, α-galactosyl fluoride, α-mannosyl fluoride, α-glucosyl fluoride, α-fucosyl fluoride, α-xylosyl fluoride, α-sialyl fluoride, αN-acetylglucosaminyl fluoride, fluoride aN-acetylgalactosaminyl, ß-galactosyl fluoride, ß-mannosyl fluoride, β-glucosyl fluoride, β-fucosyl fluoride, β-xylosyl fluoride, β-sialyl fluoride, β-N-acetylglucosaminyl fluoride and β-N-acetylgalactosaminyl fluoride are most preferred. By way of illustration, glucosyl fluorides can be prepared from free sugar by first acetylating the sugar and then treating it with HF / pyridine. This generates the thermodynamically more stable anomer of the protected (acetylated) glucosyl fluoride (ie, α-glucosyl fluoride). If the less stable anomer (ie, ß-glucosyl fluoride) is desired, it can be prepared by converting the peracetylated sugar with HBr / Hoc or with HCl to generate the anomeric bromide or chloride. This intermediate reacts with a fluoride salt such as silver fluoride to generate the glucosyl fluoride. The acetylated glucosyl fluorides can be deprotected by reaction with a moderate (catalytic) base in methanol (for example NaOMe / MeOH). In addition, many glucosyl fluorides are commercially available. Other activated glucosyl derivatives can be prepared by using conventional methods known to those skilled in the art. For example, glucosyl mesylates can be prepared by the treatment of a fully benzylated hemiacetal form of the sugar with benzyl chloride, followed by catalytic hydrogenation to remove the benzyl groups. In a further exemplary embodiment, the modified sugar is an oligosaccharide having an antennal structure. In another embodiment, one or more of the terminations of the antennas carries the modifier portion. When more than one modifier portion is linked to an oligosaccharide having an antennal structure, the oligosaccharide is useful for amplifying the modifier portion, each oligosaccharide unit is conjugated to the peptide and binds multiple copies of the modifier group to the peptide. The general structure of a typical conjugate of the invention as set out in the previous drawing, encompasses multivalent species resulting from the preparation of a conjugate of the invention when using an antennal structure. Many antennal saccharide structures are known in the art and the current method can be practiced with them without limitation.

Exemplary modifying groups are discussed below. Modifying groups can be selected for their ability to impart to a peptide one or more desirable properties. Exemplary properties include, but are not limited to, potentiated pharmacokinetics, enhanced pharmacodynamics, improved biodistribution, providing a polyvalent species, improved water solubility, decreased or enhanced lipophilicity, and tissue targeting.

Water soluble polymers Many water soluble polymers are known to those skilled in the art and are useful in the practice of the present invention. The term "water-soluble polymer" encompasses species such as saccharides (eg, dedxtran, amylose, hyaluronic acid, poly (sialic acid), heparans, heparins, .etc.); poly (amino acids), for example, poly (aspartic acid) and poly (glutamic acid); nucleic acids; synthetic polymers (eg, poly (acrylic acid), poly (ethers), for example poly (ethylene glycol); peptides, proteins and the like The present invention can be practiced with any water-soluble polymer with the sole limitation that the The polymer should include in which the remainder of the conjugate can be bound, Methods for the activation of polymers can also be found in WO 94/17039, US Patent No. 5,324,844, WO 94/18247, WO 94/04193, US Patent. No. 5,219,564, U.S. Patent No. 5,122,614, WO 90/13540; U.S. Patent No. ,281,698 and more WO 93/15189, and for the conjugation between activated polymers and peptides for example, coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen transporting molecule (US Patent No. 4,412,989 ), ribonuclease and superoxide dismutase (Veronese et al., App. Biochem, Biotech, 11: 141-45 (1985)). Preferred water soluble polymers are those in which a substantial proportion of the polymer molecules in a sample of the polymer of the same molecular weight, such polymers are "odispersed". The present invention is further illustrated with reference to a polyethylene glycol conjugate. Several reviews and monographs on the functionalization and conjugation of PEG are available. See for example, Harris, Macronol. Chem Phys, C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie, 57: 5-29 (2002). The routes for the preparation of reactive PEG molecules and the formation of conjugates using the reactive molecules are known in the art. For example, U.S. Patent No. 5,672,662 discloses a water-soluble, isolable conjugate of an active ester of an acid polymer selected from poly (alkylene oxides), poly (oxyethylated polyols), poly (olefinic alcohols) and linear poly (acrylomorpholine) or branched. U.S. Patent No. 6,376,604 establishes a method for the preparation of a water-soluble 1-benzotriazolylcarbonate ester or a non-peptidic, water-soluble polymer by reacting a terminal hydroxyl of the polymer with di (1-benzotriazoyl) carbonate in a solvent organic. The active ester was used to form conjugates with a biologically active agent such as a protein or peptide. WO 99/45964 describes a conjugate comprising a biologically active agent and an activated water soluble polymer comprising a polymer column having at least one ligation of terms to the polymer column through a stable ligation, wherein at least one term comprises a branched portion having nearby reactive groups that are linked to the branched portion, in which the biologically active agent is bound to at least one of the next reactive groups. Other branched poly (ethylene glycols) are described in WO 96/21469, Patent E.U.A. No. 5,932,462 which describes a conjugate formed with a branched PEG molecule that includes a branched term including reactive functional groups.

Free reae groups are available to be reacted with a biologically ae species, such as proteins or peptides, which form conjugates between poly (ethylene glycol) and the biologically ae species. The patent of E.U.A. No. 5,446,090 discloses a bifunnal PEG ligation and this is used in the formation of conjugate having a peptide for each of the termini of the PEG linker. Conjugates that include degradable PEG linkers are described in WO 99/34833; and WO 99/14259, as well as in the patent of E.U.A. No. 6,348,558. Such degradable linkers are applied in the present invention. The methods recognized in the art of aating polymers set out above are for use in the context of the present invention in the formation of branched polymers set forth herein and also for the conjugation of these branched polymers to other species, for example , sugars, sugar nucleotides and the like. Exemplary poly (ethylene glycol) molecules of use in the invention include, but are not limited to, those of the formula: (CH2) b-X (CH2CH20) e (CH2) d-A1-R8 In which R8 is H, OH, NH2, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted heterocycloalkyl or unsubstituted, substituted or unsubstituted heteroalkyl, for example, acetal, OHC, H2N- (CH2) q-, HS- (CH2) q, or - (CH2) qC (Y) Z1. The index "e" represents an integer from 1 to "2500. The indices b, dyq independently represent integers from 0 to 20. The symbols Z and Z1 independently represent OH, NH2, starting groups, for example, imidazole, p-nitrophenyl , HOBT, tetrazole, halide, S-R9, the alcohol portion of aated esters; - (CH2) PC (Y1) V, or - { CH2) p? {Cñ2) sC {Y1) v The symbol Y represents H (2), = 0, = S, = N-R 10. The symbols X, Y, Y1, Aa, and U independently represent the portions O, S, N-R11, the symbol V represents OH , NH2, halogen, S-R12, the alcohol component of aated esters, the amine component of aated amides, sugar nucleotides and proteins The indices p, q, syv are members independently selected from the integers from 0 to 20. The symbols R9, R10, R11 and R12 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 exemplary embodiments, the poly (ethylene glycol) molecule is selected from the following: M? - (OCH2CH2) T-0 ^ / ^ Me- (OCH2CH2) e- O ^^ Z O Y O The poly (ethylene glycol) useful in forming the conjugate of the invention is either linear or branched. Branched poly (ethylene glycol) molecules suitable for use in the invention include, but are not limited to, those described by the general formula: In which selected from the groups defined by R8 above. A1 and A2 are members independently selected from the groups defined by A1 above. The indices e, f o and q are as described above. Z and Y are as described above. X1 and X1 'are members independently selected from S, SC (0) NH, HNC (0) S, SC (0) 0, O, NH, NHC (O), (O) CNH and NHC (O) O, OC (O) H. In other exemplary embodiments, the branched PEG is based on a core of cysteine, serine or di-lysine. Thus, the exemplary branched PEG also includes: In yet another embodiment, the branched PEG portion is based on a tri-lysine peptide. The tri-lysine can be mono, di or tetra-PEG-ilada. The species according to this modality have the formula: in which e, f and f are independently selected from integers from 1 to 2500; and q, q 'and q "are independently selected from integers from 1 to 20. In exemplary embodiments of the invention, the PEG in m-PEG (5 kd, 10 kD, or 20 kD). An exemplary branched PEG species is a serine or cysteine- (m-PEG) 2 in which the m-PEG is a 20 kD m-PEG, as will be apparent to those of skill in art, branched polymers for use in the invention include variations in the terms set forth above. For example, the conjugated di-lysine-PEG shown above may include three polymer subunits, the third linkage to the a-amine shown is not modified. in the structure above. Similarly, the use of a tri-lysine functionalized with three or four polymer subunits is within the scope of the invention. Specific embodiments according to the invention include: and activated carbonates and esters of these species, such as: Other activation groups, or starting groups, suitable for the activation of linear PEG for use in the preparation of the compounds set forth herein include, but are not limited to, the species: PEG molecules that are activated with this and other species and methods for making PEG activity are set forth in WO 04/083259. Those of skill in the art will appreciate that one or more of the m-PEG arms of the branched polymer can be replaced by a PEG portion with a different term, for example, OH, COOH, NH2, C2-C6alkyl, etc. However, the structures above are easily modified by inserting alkyl linkers (or removing carbon atoms) between the α-carbon atoms and the functional groups of the side chain. Thus, the "homo" derivative and higher homologs, as well as lower homologs are within the scope of the branched PEG nuclei for use in the present invention. The branched PEG species set forth herein are readily prepared by methods such as those set forth in the reaction scheme below: in which Xa is O u S and r is an integer from 1 to 5. The indices e and f are independently selected from integers from 1 to 2500. Thus, according to this reaction scheme, a natural or unnatural amino acid is contacted with a m-PEG derivative activated, in this case the tosylate, forms 1 by renting the heteroatom Xa side chain. The mono-functionalized m-PEG amino acid was subjected to N-acylation conditions with a m-PEG derivative reagent, thereby assembling the branched m-PEG2. As someone of skill will appreciate, the tosylate starting group can be replaced with any suitable starting group, for example, -halogen, mesylate, triflate, etc. Similarly, the reactive carbonate is used to acylate the amine can be replaced with an active ester, for example, N-hydroxysuccinimide, etc., or the acid can be activated in situ using a dehydrating agent such as dicyclohexylcarbodiimide, carbonyldiimidazole, etc. In an exemplary embodiment, the modified group is a PEG portion, however, any modified group, eg, water soluble polymer, water insoluble polymer, therapeutic moiety, etc., may be incorporated in a portion of glycosyl through of an appropriate ligature. The modified sugar is formed by enzymatic means, chemical means or in combination thereof, thereby producing a modified sugar. In an exemplary embodiment, the sugars are substituted with an active amine in any position that is allowed to bind from the modified portion, yet still the sugar allows the function as a substrate for an enzyme capable of coupling the modified sugar to the G peptide. -CSF. In an exemplary embodiment, when the galacotosamine is the modified sugar, the amine moiety is bonded to the carbon atoms in the 6-position.

Modified species of the water-soluble polymer Water-soluble polymer modified nucleotide sugar species in which the sugar portion is modified with a water-soluble polymer are for use in the present invention. An exemplary modified sugar nucleotide carries a sugar group that is modified through an amine moiety in the sugar. Modified sugar nucleotides, for example, saccharylamine derivatives of a sugar nucleotide, are also of use in the methods of the invention. For example, a saccharyl amine (without the modified group) can be enzymatically conjugated to a peptide (or other species) and the free saccharyl amine portion is subsequently conjugated to a desired modified group. Alternatively, the modified sugar nucleotide can function as a substrate for an enzyme that transfers the modified sugar to a saccharyl receptor on a substrate, for example, a peptide, glycopeptide, liquid, aglycone, glycolipid, etc. In a modality in which the saccharide nucleus is galactose or glucose; R5 is NHC (O) Y. In an exemplary embodiment, the modified sugar is based on a 6-amino-N-acetyl-glucosyl moiety. As shown below by N-acetylgalactosamine ,. The 6-amino sugar portion is easily prepared by standard methods. to. g In the reaction scheme above, the index n represents an integer from 1 to 2500, preferably from 10 to 1500, and more preferably from 10 to 1200. The symbol "A" represents an activated group, for example, a halo, an component of an activated ester (e.g., an N-hydroxysuccinimide ester), a carbonate component (e.g., p-nitrophenyl carbonate), and the like. Those of skill in the art will appreciate that other PEG-amide nucleotide sugars are readily prepared by this and analogous methods.

In other exemplary embodiments, the amide moiety is replaced by a group such as a urethane or a urea. In still other embodiments, R1 is a branched PEG, for example, one of those species set forth above. Illustrative compounds according to this embodiment include: In which X is a bond u 0, and J is S u 0.

In addition, as discussed above, the present invention provides conjugated peptides that are formed using nucleotide sugars that are modified with a water soluble polymer, which are either a straight or branched chain. For example, compounds having the formula shown below are within the scope of the present invention: wherein X is O or a bond, and J is S or O. Similarly, the invention provides peptide conjugates that are formed using nucleotide sugars from those modified sugar species in which the carbon in the 6-position is modified: wherein X4 is a bond or O, J is S or O and Y is O or 1. Conjugates of peptides and glycopeptides, liquids and glycolipids including the compositions of the invention are also provided. For example, the invention provides conjugates having the following formulas: Where J is S or O.

Water insoluble polymers In another embodiment, analogous to those described above, the modified sugars include a water insoluble polymer, rather than a water soluble polymer. The conjugates of the invention may also include one or more water-insoluble polymers. This embodiment of the invention is illustrated by the use of the conjugate as a vehicle to deliver a therapeutic peptide in a controlled manner. Polymeric drug delivery systems are known in the art. See, for example, Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C. 1991. Those skilled in the art will appreciate that substantially any known drug delivery system is applied to the conjugates of the present invention. Representative water-insoluble polymers include, but are not limited to, polyphosphazines, poly (vinyl) alcohols, polyamides, polycarbonates, polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkylene oxide, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidone, polyglycolides, polysloxanes, polyurethanes, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isoecyl methacrylate), poly ( lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate) polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), poly (vinyl acetate), polyvinyl chloride, polystyrene, polyvinyl pyrrolidone, pluronic and polyvinylphenol and copolymers thereof. Synthetically modified natural polymers for use in conjugates of the invention include, but are not limited to, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters and nitrocelluloses. Preferred members particularly of the broad classes of the synthetic polymers modified include, but are not limited to, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacete, sodium sulfate salt of cellulose, and polymers of acrylic and methacrylic esters and alginic acid. These and other polymers described herein can be readily obtained from commercial sources such as Sigma Chemical Co. (St. Louis, MO.), Polisciences (Warrenton, PA.), Aldrich (Milwaukke, Wis.), Fluka (Ronkonkoma, NY. ) and BioRad (Richmond, CA) or are also synthesized from monomers obtained from these suppliers using standard techniques. Representative biodegradable polymers for use in the conjugates of the invention include, but are not limited to, polylactides, polyglycolides and copolymers thereof, poly (ethylene terephthalate), poly (butyric acid), poly (valeric) acid, poly (lactide) -co-caprolactone), poly (lactide-co-glycolide), polyanhydrides, polyorthoesters, mixtures and copolymers thereof. Of particular use are compositions that form gels, such as those that include collagen, pluronic and the like. Polymers for use in the invention include "hydride" polymers that include water insoluble materials that have within it at least a portion of its structure, a bioabsorbable molecule. An example of such a polymer is one that includes a water insoluble copolymer, which has a bioabsorbable region, a hydrophilic region and a plurality of crosslinking functional groups per polymer chain.

For purposes of the present invention, "water-insoluble materials" include materials that are substantially insoluble in water or a water-containing environment. Thus, through certain regions or segments of the copolymers can be hydrophilic or even soluble in water, the polymer molecule, as a complete, is not any substantial measure dissolved in water.

For purposes of the present invention, the term "bioabsorbable molecule" includes a region that is capable of being metabolized or broken and reabsorbed and / or eliminated through normal excretory pathways by the body. Such metabolites or fragmentation products are preferably substantially non-toxic to the body. The bioabsorption region can be either hydrophobic or hydrophilic, as long as the copolymer composition as a whole is not soluble in water. Thus, the bioabsorption region is selected based on the preference that the polymer as a whole remains insoluble in water. In this way, the relative properties, that is, the functional type groups contained and the relative proportions of the bioabsorption region and the hydrophilic region are selected to ensure that the useful bioabsorption compositions remain insoluble in water. Exemplary resorption polymers include, for example, the resorbable block copolymers produced synthetically from poly (α-hydroxy carboxylic acid) / poly (oxyalkylene) (see, Cohn et al., US Patent No. 4,826,945). they cross-link and are soluble in water so that the body can excrete degraded block copolymer compositions See, Younes et al., J. Biomed, Mater. Res. 21: 1301-1316 (1987); and Cohn et al. , J Biomed, Mater. Res. 22: 993-1009 (1988).

Presently preferred bioresorption polymers include one or more selected components of poly (esters), poly (hydroxy acid), poly (lactones), poly (amides), poly (ester-amides), poly (amino acids), poly (anhydrides) ), poly (orthoesters), poly (carbonates), poly (phosphazines), poly (phosphoesters), poly (thioesters), polysaccharides, and mixtures thereof. Still more preferably, the bioresorption polymer includes a component of a poly (hydroxy) acid. of poly (hydroxy) acids, polylactic acid, polyglycolic acid, polycaproic acid, polybutyric acid, polyvaleric acid and copolymers and mixtures thereof are preferred. In addition to forming fragments that are absorbed in vivo ("bioresorption") preferred polymer coatings for use in the methods of the invention may also form a metabolizable and / or excretable fragment. Higher order copolymers can also be used in the present invention, for example, Casey et al., US Patent NO. No. 4,438,253, which issued on March 20, 1984, discloses triblock copolymers produced from the transesterification of poly (glycolic acid) and a hydroxylated poly (alkylene glycol). Such compositions are described for use as resorbable monofilament sutures. The flexibility of such compositions is controlled by the incorporation of an aromatic orthocarbonate such as tetra-p-tolyl orthocarbonate into the co-polymer structure.

Other polymers based on lactic and / or glycolic acids can also be used, for example, Spinu, US Patent No. 5,202, 413 which was issued on April 13, 1993, discloses biodegradable multiblock copolymers having blocks sequentially. ordered from a polylactide and / or polyglycolide produced by an aperture polymerization of lactide and / or glycolide rings within either an oligomeric diol or a diamine residue followed by a chain extension with a difunctional compound such as diisocyanate, diacylchloride or Dichlorosilane The bioabsorption regions of the coatings useful in the present invention can be designed to be hydrolytically and / or enzymatically unfoldable For the purposes of the present invention"Hydrolytically unfoldable" refers to the susceptibility of the copolymer especially the bioabsorption region for hydrolysis in water or in a water-containing environment. Similarly, "enzymatically unfoldable" as used herein, refers to the susceptibility of the copolymer especially the bioresorption region, to cleavage by endogenous or exogenous enzymes. When placed inside the body, the hydrophilic region can be processed into fragments that are excreted and / or metabolized. Thus, the hydrophilic region can include for example polyethers, polyalkylene oxides, polyols, poly (vinyl pyrrolidine), poly (vinyl alcohol), poly (alkyl oxazolines), polysaccharides, carbides, peptides, proteins and copolymers and mixtures thereof. Additionally, the hydrophilic region may also be, for example, a polyalkylene oxide, such poly (alkylene) oxides may include, for example, poly (ethylene oxide), poly (propylene oxide), and mixtures and copolymers of Polymers that are components of hydrogels are also useful in the present invention Hydrogels are polymeric materials that can absorb relatively large amounts of water Examples of hydrogel-forming compounds include but are not limited to polyacrylic acids, sodium carboxymethylcellulose. , polyvinyl alcohol, polyvinyl pyrrolidone, gelatin, carrageenan and other polysaccharides, hydroxyethylene methacrylate acid (HEMA) as well as derivatives thereof and the like .. Hydrogels can be produced which are stable, biodegradable and bioabsorbable. subunits that show one or more of these properties. They are biocompatible whose integrity can be controlled through the crosslinking are known and are currently preferred for use in the present invention. For example, Hubbell et al., U.S. Patent No. 5,410,016 which issued April 25, 1995 and 5,529,914 which issued on June 25, 1996, describe water-soluble systems, which are cross-linked block copolymers which they have a water-soluble central block segment sandwiched between 2 hydrolytically related extensions. Such copolymers are also closed at one end with photopolymerizable acrylate-functionalities. When reticulated, these systems become hydrogels. The water-soluble core block of such copolymers can include poly (ethylene glycol); while, hydrolytically extensions can be a poly (a-hydroxy acids), such as polyglycolic acid or polylactic acid. See, Sawhney et al., Macromolecules 26: 581-587 (1993). In another embodiment, the gel is a thermoreversible gel. Term-reversible gels include such components as pluronic, collagen, gelatin, hyaluronic acid, polysaccharides, polyurethane hydrogel, urea polyurethane hydrogel and combinations thereof are currently preferred. In yet another exemplary embodiment, the conjugate of the invention includes a component of a liposome. Liposomes can be prepared according to methods known to those skilled in the art for example as described in Eppstein et al., U.S. Patent No. 4,522,811, which was conceived on June 11, 1985. For example, the formulations of liposomes can be prepared by dissolving suitable lipids (such as phosphatidyl-stearoyl ethanolamine, phosphatidyl stearolyl choline, phosphatidyl aracadoyl choline, and cholesterol) in an organic solvent which then evaporates leaving behind a thin film of a dry lipid in the surface of the container. An aqueous solution of the active compound or its pharmaceutically acceptable salt is then introduced into the container. The container is then stirred by hand to release the lipid from the sides of the container and disperse the lipid aggregates thereby forming the liposomal suspension. The aforementioned microparticles and methods of preparing the microparticles are offered by way of example and are not intended to define the scope of the microparticles for use in the present invention. it will be apparent to those skilled in the art that a configuration of microparticles made by different methods are for use in the present invention. The structural formats discussed above in the context of water soluble polymers, both straight chain and branched are generally applicable with respect to the water insoluble polymers as well. Thus, for example, the branching nuclei of cysteine, serine, dilisin and trilisin can be functionalized with 2 portions of water insoluble polymers. The methods used to produce these species are generally closely analogous to those for producing the water soluble polymers. The in vivo half life of the therapeutic glycopeptides can also be enhanced with portions of PEG such as polyethylene glycol (PEG). For example, the chemical modification of proteins with "- PEG (PEGylation) increases their molecular size and decreases their access to a functional and surface group, each of which depends on the size of the PEG bound to the protein. of plasma half-lives and proteolytic stability and a decrease in hepatic absorption and immunogenicity (Chaffee et al., J. Clin, Invest, 89: 1643-1651 (1992); Pyatak et al., Res. Common, Chem. Pathol Pharmacol., 29: 113-127 (1980).) It has been reported that PEGylation of interleukin-2 increases its antitumor potency in vivo (Catre et al., Proc. Nati. Acad. Sci. USA, 84: 1487-1491 (1987)) and PEGylation of F (ab ') 2 derived from monoclonal antibody A7 has improved tumor localization (Kitamura et al., Biochem. Biophys., Res. Commun. 28: 1387-1394 (1990)). in another embodiment, the average in vivo life of a peptide derived from a PEG portion by an invertebrate method. tion is highly elevated for the average in vivo life of the non-derived peptide. The increase in the peptide of the average life in vivo is best expressed as a range of increase in the percentage of that amount. The lower end of the percentage increase range is around 40% up to around 60%, around 80%, around 100%, around 150% or about 200%. The upper end of the range is around 60%, around 80%, around 100%, around 150%, or more than about 250%.

Biomolecules In another embodiment, the modified sugar transports a biomolecule. Even in the additional embodiments, the biomolecule is a functional protein, enzyme, antigen, antibody, peptide, nucleic acid (e.g., single nucleotide or nucleoside oligonucleotides, polynucleotides and nucleic acids of single and higher strands), lectin, receptors or a combination thereof. Preferred biomolecules are essentially non-fluorescent or emit such minimal amounts of fluorescence that are unsuitable for use as a fluorescent label in an assay. On the other hand, it is generally preferred to use molecules other than sugars. An exception to this preference is the use of a sugar that otherwise occurs naturally, which is modified by a covalent bond of another entity (eg, PEG, biomolecule, therapeutic portion, diagnostic portion, etc.). In an exemplary embodiment, a sugar portion, which is a biomolecule, is conjugated to a linker arm and the cassette of the sugar linker arm is subsequently conjugated to a peptide via a method of the invention. The biomolecules useful in the practice of the present invention can be derived from any source. Biomolecules can be isolated from natural sources or can be produced by synthetic methods. The peptides can be natural peptides or mutant peptides. Mutations can be effected by chemical mutagenesis, site-directed mutagenesis, or other induced mutation media known to those skilled in the art. Peptides useful in practicing the present invention include, for example, enzymes, antigens, antibodies and receptors. The antibodies can be both polyclonal and monoclonal; either intact or fragmented. Peptides are optionally the product of a direct evolution program. Both nucleic acids, synthetic peptides and derivatives are naturally used in conjunction with the present invention; these molecules can be linked to a sugar residue component or a crosslinking agent by any available reactive group. For example, the peptides can be linked through a reactive amine, carboxyl, sulfhydryl, or a hydrophilic group. The reactive group can reside in a peptide term or in a site internal to the peptide chain. The nucleic acids can be linked through a reactive group on a base (eg, exocyclic amine) or a hydroxyl group available on a portion of the sugar (eg, 3 'or 5' hydrophilic). The peptide and the chains of the nucleic acid can be derived, in addition at one or more sites to allow the binding of appropriate reactive groups on the chain. See, Chrisey et al. Nucleic Acids Res. 24: 3031-3039 (1996). In a further embodiment, the biomolecule is selected to direct the modified peptide by methods of the invention to a specific tissue, thereby improving the delivery of the peptide to the relative tissue with the amount of non-derived peptide that is delivered to the tissue. In a further embodiment the amount of derived peptide delivered to a specific tissue within a selected time period is improved by derivatizing at least about 20%, more preferably at least about 40%, and even more preferably at less than around 100%. Currently the preferred biomolecules for directing applications include antibodies, hormones and ligands for cell surface receptors. In a still more exemplified embodiment, it is provided as a conjugate with biotin. Although, for example, a biotinylated peptide is selectively made by linking a portion of streptavidin or avidin carrying one or more modified groups.

Therapeutic portions In another embodiment the modified sugar includes a therapeutic portion. Those skilled in the art will appreciate that there is an overlap between the category of therapeutic portions and biomolecules; many biomolecules have properties or therapeutic potential. The therapeutic portions can be agents already accepted for clinical use or they can be drugs whose use is experimental or whose activity or mechanism of action is under investigation. The therapeutic portions may have a proven action in a given disease state or may be only a hypothesis that shows a desirable action for a given disease state. In another embodiment, the therapeutic portions are compounds that are separated by exclusion for their ability to interact with a tissue of choice. Therapeutic portions, which are useful in the practice of the present invention include drugs that form a broad range of classes of drugs that have a variety of pharmacological activities. Preferred therapeutic portions are essentially non-fluorescent or emit such as a minimum amount of fluorescence than that which is unsuitable for use as a fluorescent label in an assay. However, this is generally preferred for use of therapeutic portions that are not sugars. An exception to this preference is the use of a sugar that is modified by a covalent linkage of another entity, such as a PEG, biomolecule, therapeutic portion, diagnostic portion, and the like. In another exemplary embodiment, a portion of therapeutic sugar was conjugated to a cassette of high sugar linker and ligated end which is subsequently conjugated to a peptide by means of a method of the invention. - Methods to conjugate therapeutic agents and diagnostics for several other species are concentrated by those of skill in the art. See, for example, Hermanson, BIOCONJUGATE TECHNIQUES, 'Academic Press, San Diego, 1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C. 1991. In an exemplary embodiment, the therapeutic portion is linked to the modified sugar by means of a ligation that unfolds under selected conditions. Exemplary conditions include, but are not limited to, a selected pH (e.g., stomach, intestine, endocytotic vacuole), the presence of an active enzyme (e.g., esterase, reductase, oxidase), light, heat and the like. Many splitting groups are known in the art. See, for example, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147 (1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning et al., J. Immunol., 143: 1859-1867 (1989).

The classes of useful therapeutic portions include, for example, nonsteroidal anti-inflammatory drugs (NSAIDS). The NSAIDS can, for example, be selected from the following categories: (eg, propionic acid derivatives, acetic acid derivatives, fenamic acid derivative, biphenylcarboxylic acid derivatives and oxicams); Steroidal anti-inflammatory drugs include hydrocortisone and the like; antihistamine drugs (for example, chlorpheniramine, triprolidine); antitussive drugs (for example, dextromethorphan, codeine, caramiphen and carbetapentane); antipruritic drugs (for example, metdilazine and trimeprazine); anticholinergic drugs (for example, scopolamine, atropine, homatropine, levodopa); anti-emetic and anti-nausea drugs (e.g., cyclizine, meclizine, chlorpromazine, buclizine), anorexic drugs (e.g., benzfetamine, phentermine, chlorphentermine, fenfluramine); central stimulant drugs (eg, amphetamine, metamfetamine, dextroamphetamine and methylphenidate); antiarrhythmic drugs (e.g., propanolol, procainamide, disopyramide, quinidine, encainide); ß-adrenergic blocking drugs (eg, metoprolol, acebutolol, betaxolol, labetalol and timolol); cardiotonic drugs (for example, milrinone, amrinone and dobutamine); antihypertensive drugs (for example, enalapril, clonidine, hydralazine, minoxidil, guanadrel, guanethidine); diuretic drugs (for example, amilodira and thiazide hydrochloride); vasodilator drugs (for example, diltiazem, amiodarone, isoxsuprine, nilidrine, tolazoline and verapamil); vasoconstrictor drugs (e.g., dihydroergotamine, ergotamine, and methylmerged); anti-ulcer drugs (for example, ranitidine and cimetidine); anesthetic drugs (for example, imipramine, desipramine, amitriptyline, nortriptyline); tranquilizing and sedative drugs (for example, chlordiazepoxide, benacitizine, benzquinamide, flurazepam, hydroxyzine, loxapine and promazine); anti-psychotic drugs (for example, chlorprothixene, fluphenazine, haloperidol, molindone, thioridazine and trifluoroperazine); anti-microbial drugs (anti-bacterial, antifungal, antiprotozoal and antiviral drugs). Antimicrobial drugs which are preferred for incorporation into the present composition include, for example, pharmaceutically acceptable salts of β-lactam drugs, quinoline drugs, ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin, triclosan, doxycycline, capreomycin, chlorhexidine, chlortetracycline, oxytetracycline, clindamycin, ethambutol, hexamidine isothionate, metronidazole, pentamidine, gentamicin, kanamycin, lineomycin, methacycline, methenamine, minocycline, neomycin, netilmicin, paromomycin, streptomycin, tobramycin, miconazole and amantadine. Other drug portions of use in the practice of the present invention include anti-neoplastic drugs (e.g., leuprolide or flutamide); cytocidal agents (e.g., adriamycin, doxorubicin, taxol, cyclophosphamide, buslfan, cisplatin, β-2-interferon), anti-estrogens (e.g., tamoxifen), anti-metabolites (e.g., fluorouracil, methotrexate, mercaptopurine, thioguanine) . Also included within this class are radioisotope-based agents for both diagnosis and therapy and conjugated toxins, such as racin, geldanamycin, mitansin, CC-1065, duocarmycins, cliqueamicin and related structures and analogues thereof. The therapeutic portion is also a hormone (eg, medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide, or somatostatin); muscle relaxant drugs (eg, cinnamedrine, cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine, idaverine, ritodrine, diphenoxylate, dantrolene and azumolene); anti-spasmodic drugs; active drugs for bones (for example, diphosphonate and phosphonoalkylphosphinate drug compounds); endocrine modulating drugs (e.g., contraceptives (e.g., ethinoldiol, ethinyl estradiol, norethindrone, mestranol, desogestrel, medroxyprogesterone), modulators of diabetes (e.g., glyburides or chlorpropamide), anabolics, such as testoiactone or stanozolol, androgens (e.g. , methyltestosterone, testosterone or fluoxymesterone), antidiuretics (eg, desmopressin and calcitonins) Also of use in the present invention are estrogens (eg, diethylstilbesterol), glucocorticoids (eg, triamcinolone, betamethasone, etc.) and progestogens, such such as norethindrone, ethinodiol, norethindrone, levonorgestrel, thyroid agents (eg, liothyronine or levothyroxine) or antithyroid agents (eg, methimazole), antihyperprolactinemic drugs (eg, cabergoline), hormone suppressants (eg, danazol or goserelin) ), oxytocics - (for example, methylergonovine or oxytocin) and prostaglandins, such as Myoprostol, alprostadil or dinoprostane are also used. Other useful modification groups include immunomodulatory drugs (eg, antihistamines, mast cell stabilizers, such as yodoxamide and / or cromolyn, steroids (eg, triamcinolone, beclomethazone, cortisone, dexamethasone, prednisolone, methylprednisolone, beclomethasone or clobetasol), antagonists of H2 histamine (for example, famotidine, cimetidine, ranitidine), immunosuppressants (eg, azathioprine, cyclosporine), etc. Groups with anti-inflammatory activity, such as sulindac, etodolac, ketoprofen, and ketorolac were also used. with the present invention they will be apparent to those of skill in the art.

Preparation of modified sugars In general, the sugar portion and the modified group is linked together through the use of reactive groups, which are typically transformed by the binding process into a new organic functional group or non-reactive species. Reactive sugar functional groups are located at any position in the sugar portion. The reactive groups and classes of reactions useful in the practice of the present invention are generally those that are known in the art of bioconjugate chemistry. The currently favored classes of reactions available with reactive sugar portions are those, which are produced under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (eg, reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and carbon-carbon additions and multiple heteroatom-carbon bonds ( for example, Michael reaction, Diles-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, ORGANIC CHEMISTRY, 3rd ED., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATIÓN OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982. Useful reactive functional groups pendant from a sugar core or modified group include, but are not limited to: (a) carboxyl groups and various derivatives thereof include, but are not limited to, N-hydroxysuccinimide ester, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkyl and aromatic esters; (b) hydroxyl groups, which can be converted to, for example, esters, ethers, aldehydes, etc. (c) haloalkyl groups, wherein the halide can be further displaced with a γ-nucleophilic group such as, for example, an amine, a carboxylate anion, anion of lime, carbanion, or an alkoxide ion, thereby resulting in the covalent bonding of a new group to the functional group of the halogen atom; (d) dienophile groups, which are capable of participating in Dil-Alder reactions such as, for example, maleimido group; (e) aldehyde or ketone groups, such to that subsequent derivation is possible by means of formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or by means of such mechanisms as Grignard addition or addition of alkylthio; (f) sulfonyl halide groups for the subsequent reaction with amines, for example, to form sulfonamides; (g) thiol groups, which may be, for example, converters for disulfides or react with acyl halides; (h) amine or sulfhydryl groups, which may be, for example, acylated, alkylated or oxidized; (i) alkanes, which can under, for example, cycloadition, is acylation, Michael addition, etc; and (j) epoxides, which can be reacted with, for example, amines and hydroxyl compounds. The reactive functional groups can be chosen such that they do not participate in, or interfere with, the reactions necessary to join the reactive sugar nuclei or modification group. Alternatively, a reactive functional group can be protected from participating in the reaction by the presence of a protecting group. Those of skill in the art understand how to protect a particular functional group such as that which does not interfere with a set of selected reaction conditions. For examples of useful protection groups, see, for example, Greene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991. In the discussion that follows, a number of specific examples of modified sugars that are useful in the practice of the present invention are established. In the exemplary modalities, a sialic acid derivative is used as the sugar core to which the modified group is linked. The focus of the discussion on sialic acid derivatives is for illustration clarity only and should not be constructed to limit the scope of the invention. Those of skill in the art will appreciate that a variety of. other sugar portions can be activated and derived in a manner analogous to that established using sialic acid with an example. For example, numerous methods are available to modify galactose, glucose, N-acetylgalactosamine and fucose to name a few sugar substrates, which are easily modified by art recognized methods. See, for example, Elhalibi et al., Curr. Med. Chem. 6: 93 (1999); and Schafer et al., J. Org. Chem. 65:24 (2000)). In an exemplary embodiment, the peptide that is modified by a method of the invention is a glycopeptide that is produced in prokaryotic cells (e.g., E. Coli), eukaryotic cells include yeast and mammalian cells (e.g., CHO cells) , or in a transgenic animal and thus contain chains of oligosaccharides linked to N and / or 0, which were incompletely sialylated. The oligosaccharide chains of the glycopeptide lack a sialic acid and contain a terminal galactose residue which may be glyco-PEG-ylated, glyco-PPG-ylated or other modification with a modified sialic acid. In reaction scheme 4, amino glucoside 1 was treated with the active ester of a protected amino acid derivative (eg, glycine), converting the sugar amine residue to the amino acid amide adduct protected accordingly. The adduct was treated with an -aldolase to form α-hydroxy carboxylate 2. Compound 2 was converted to the corresponding CMP derivative by the action of CMP-SA synthetase, followed by catalytic hydrogenation of the CMP derivative to produce compound 3. The amine introduced by the formation of the glycine adduct was used as a locus of PEG or PPG bound by reacting compound 3 with an activated (m-) PEG or derivative of (m-) PPG (e.g., PEG-C) (0) NHS, PPG-C (0) NHS), yielding 4 or 5, respectively.

Reaction scheme 4 CMP-SA-5-NHCOCH2NH-PPG (m-PPG) 5 Table 2 sets forth the representative examples of sugar monophosphates that are derived with a portion of PEG or PPG. Some of the compounds of Table 2 were prepared by the method of reaction scheme 4. Other derivatives were prepared by methods recognized in the art. See, for example, Keppler et al, Glycobiology 11: 11R (2001); and Charte et al. , Glycobiology 10: 1049 (2000)). Other PEG reactive amines and PPG analogues are commercially available or these can be prepared by methods readily accessible by those of ordinary skill in the art.

Table 2 CMP-SA-5-NH-R CMP-NeuAc-9-O-R C P-NeuAc-8-NH-R CMP-NeuAc-4-OR CMP-NeuAc-4-NH-R The modified sugar phosphates for use in the practice of the present invention can be substituted in other positions as well as those stated above . The preferred present substitutions of sialic acid are set forth in formula I: wherein X is a linking group, which is preferably selected from -O-, -N (H) -, -S-, CH2-, and -N (R) 2, in which each R is a. member independently selected from R1-R5. The symbols Y, Z, A and B each represent a group that is selected from the group set up above for the identity of X. X, Y, Z, A and B are each independently selected and therefore, these can be be the same or different. The symbols R1, R2, R3, R4 and R5 represent H, a water soluble polymer, therapeutic portion, biomolecule or other portion. Alternatively, these symbols represent a ligature that binds to a water soluble polymer, therapeutic moiety, biomolecule or other portion. Exemplary portions linked to the conjugates described herein include, but are not limited to, PEG derivative (eg, alkyl-PEG, acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-PEG, aryl- PEG), PPG derivatives (for example-, alkyl-PPG, acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPG carbamoyl-PPG, aryl-PPG), therapeutic portions, diagnostic portions, mannose-6-phosphate , heparin, heparan, SLex, mannose, mannose-6-phosphate, Sialyl Lewis X, FGF, VFGF, proteins, chondroitin, ceratan, dermatan, albumin, integrins, antennary oligosaccharides, peptides and the like. The methods to conjugate the various modification groups for a portion of saccharides are readily accessible to those of skill in the art (POLY (ETHYLENE GLYCOL CHEMISTRY: BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; POLY (ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS, J. Milton Harris, Ed., ACS Symposium Series No. 680, American Chemical Society, 1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C. 1991).

Cross-linking groups The preparation of the sugar modification for use in the methods of the present invention include linking a modified group to a sugar residue and forming a stable adduct, which is a substrate for a glycosyltransferase. The sugar and the modified group can be coupled by a crosslinking agent of zero or higher order. Exemplary bifunctional compounds which can be used to link modified groups for carbohydrate moieties include, but are not limited to, bifunctional poly (ethylene glycols), polyamides, polyethers, polyesters, and the like. The general procedures for carbohydrates linked to other molecules are known in the literature. See, for example, Lee et al., Biochemistry 28: 1856 '(1989); Bhatia et al., Anal. Biochem. 178: 408 (1989); Janda et al., J. Am. Chem. Soc. 112: 8886 (1990) and Bednarski et al., WO 92/18135. In the discussion that follows, the reactive groups were treated as they started in the sugar portion of the modified sugar growing. The focus of the discussion is for clarity of illustration. Those of skill in the art will appreciate that the discussion is relevant to reactive groups in the modified group. An exemplary strategy involves the incorporation of a protected sulfhydryl in the sugar using the heterobifunctional crosslinked (n-succinimidyl-3- (2-pyridylthio) propionate SPDP and then deprotects the sulfhydryl for the formation of a disulfide bond with another sulfhydryl in the modified group .

If SPDO detrimentally affects the ability of the modified sugar to act as a glycosyltransferase substrate, one of a formation of other crosslinks such as 2-iminothiolane or N-succinimidyl S-acetylthioacetate (SATA) is bound to form a disulfide bond . The 2-iminothiolane is reacted with primary amines, instantaneously incorporating an unprotected sulfhydryl in the amine-containing molecule. SATA is also reacted with primary amines, but incorporates a protected sulfhydryl, which is later deacetylated using hydroxylamine to produce a free sulfhydryl. In each case, the incorporated sulfhydryl is free to be reacted with other protected, similar sulfhydryls or sulfhydryls, SPDP, forming the required disulfide bond. The strategy described above is exemplary, and is not limited to ligatures for use in the invention. Other cross-links are available to those that can be used in different strategies to cross-link the peptide modifying group. For example, TPCH hydrazide (S- (2-thiopyridyl) -L-cysteine and TPMPH ((S- (2-thiopyridyl) mercapto-propionohydrazide) are reacted with carbohydrate moieties that are previously oxidized by mild periodate treatment, thus forming a hydrazone linkage between the hydrazide portion of the crosslinking and the aldehydes generated from periodate TPCH and TPMPH introduce a sulfhydryl protected group 2-pyridylthione in the sugar, which can be deprotected with DTT and then subsequently used by conjugation, such as disulfide bonds formed between the components If the disulphide bond is unsuitable for producing stable modified sugars, other crosslinks can be used as those that incorporate more stable bonds between the compounds The heterobifunctional crosslinks (N-gamma-malimidobutyryloxy) succinimide) GMBS and (succinimidyl 4- (N-maleimido-methyl) cyclohexane) SMCC are reacted with primary amines, thus introducing a g rupo maleimide in the component. The maleimide group can be subsequently reacted with sulfhydryls in the other component, which can be introduced by previously mentioned crosslinks, thus forming a stable thioether bond between the components. If the steric hindrance between the components interferes with either the activity of the components or the ability of the modified sugar to act as a glycosyltransferase substrate, the crosslinkers can be used, which introduce long spacer arms between the components and include derivatives of some of the aforementioned crosslinks (that is, SPDP). Thus, there is an abundance of suitable crosslinks, which are useful; each of which is selected dependent on the effects this is an optimal peptide conjugate and modified sugar production.

A variety of reagents were used to modify the components of the modified sugar with intramolecular chemical crosslinkers (for the review of crosslinking agents and cross-linking procedures, See: Wold, F., Meth. Enzymol., 25: 623-651, 1972; Weetall, HH, and Cooney, DA, In: ENCIMES AS DRUGS (Holcenberg, and Roberts, eds.) Pp. 395-442, Wiley, New York, 1981; Ji, TH, Meth. Enzymol. 91: 580- 609, 1983; Mattson et al., Mol. Biol. Rep. 17: 167-183, 1993, all of which are incorporated herein by reference). Preferred crosslinking reagents are derived from various crosslinking reagents of zero length, homobifunctional and heterobifunctional. Zero-length crosslinking reagents include direct conjugation of two intrinsic chemistry groups without introduction of extrinsic material. The agents that catalyze the formation of a disulfide bond belong to this category. Another example are reagents that induce the condensation of a carboxyl and a primary amino group to form an amide bond such as carbodiimides, ethyl chloroformate, K Woodward's reagent (2-ethyl-5-phenylisoxazolium-3'-sulfonate), and carbonyldiimidazole . In addition to these chemical reagents, the enzyme transglutaminase (glutamyl-peptide? -glutamyltransferase; EC 2.3.2.13) can be used as a zero-length crosslinking reagent. This enzyme catalyzes acyl transferase reactions in the carboxamide groups of the glutaminyl residues bound to the protein, usually with an amino-pyramidal group as a substrate. The preferred homo- and bifunctional reactants contain two identical sites or two different sites, respectively, which may be reactive for the amino, sulfhydryl, guanidino, indole, or non-specific groups. i. Preferred specific sites in crosslinking reagents 1. Reagent amino groups In one embodiment, the sites in the reticulazens are reactive amino groups. Useful non-limiting examples of the reactive amino groups include N-hydroxysuccinimide (NHS) esters, imidoesters, isocyanates, acyl halides, arylazides, p-nitrophenyl esters, aldehydes and sulfonyl chlorides. The NHS esters are preferentially reacted with the primary amino groups (including aromatics) of a modified sugar component. The imidazole groups of histidines are known to complete the reaction with primary amines, but the reaction products are unstable and easily hydrolyzed. The reaction involves the nucleophilic attack of an amine on the carboxyl acid of an NHS ester to form an amide, releasing the N-hydroxysuccinimide. Thus, the positive charge of the original amino group was lost. The imidoesters are the specific acylating reagents for the reaction with the amine groups of the modified sugar components. At a pH between 7 and 10, the imidoesters are reacted only with primary amines. The primary amines attack the imidates nucleophilically to produce an intermediate that cleaves to an amidine at a high pH or to a new imidate at a low pH. The new imidate can be reacted with another primary amine, thus the two amino groups of crosslinking, a case of supposedly monofunctional imidate that reacts with bifunctionality. The main product of the reaction with primary amines is an amidine which is a strong base than the original amine. The positive charge of the original amino group is retained later. The isocyanates (and isothiocyanates) are reacted with the primary amines of the modified sugar components to form stable bonds. Their reactions with sulfhydryl, imidazole and tyrosyl groups give relatively unstable products. The acylazides can also be used as specific amino reagents in which the nucleophilic amines of the affinity component attack the carboxyl acidic groups under mild alkaline conditions, for example pH 8.5. Aryl halides such as 1,5-difluoro-2,4-dinitrobenzene are preferentially reacted with the amino groups and phenolic tyrosine groups of modified sugar components, but also with sulfhydryl and imidazole groups. The p-nitrophenyl esters of mono and dicarboxylic acids are also useful reactive amino groups. Although the specific reagent is not very high, the a and e-amino groups appear to react faster. Aldehydes such as glutaraldehyde are reacted with primary amines of modified sugar. Although the non-stable Schiff bases are formed during the reaction of the amino groups with the aldehydes of the aldehydes, the glutaraldehyde is able to modify the modified sugar with stable cross-links. A pH 6-8, the pH of the typical crosslinking conditions, the cyclic polymers under a dehydration to form α-β unsaturated aldehyde polymers. The Schiff bases, however, are stable, when joined to another double bond. The resonant interaction of both double bonds prevent the hydrolysis of the Schiff binding. In addition, amines at high local concentrations can attack the ethylenic double bond to form a stable Michael addition product.

The aromatic sulfonyl chlorides are reacted with a variety of sites of the modified sugar compounds, but the reaction with the amino groups is the most important, resulting in a stable sulfonamide ligation. '2. Reactive sulfhydryl groups In another embodiment, the sites are reactive sulfhydryl groups. Non-limiting examples are useful from reactive sulfhydryl groups including maleimides, alkyl halides, pyridyl disulfides, and thiophthalamides. The maleimides are preferentially reacted with the sulfhydryl group of the modified sugar components to form the stable thioether bonds. These are also reacted at a slower ratio with primary amino groups and the imidazole groups of histidines. Nevertheless, at a pH7 the maleimide group can be considered a specific sulfhydryl group, since at this pH the reaction range of simple thiols is 1000 times greater than that of the corresponding amine. The alkyl halides are reacted with sulfhydryl group, sulfides, imidazoles, and amino groups. At a neutral to slightly alkaline pH, the alkyl halides are reacted primarily with sulfhydryl groups to form thioether linkages. At a high pH, the reaction with amino groups is favorable. The pyridyl disulfides are reacted with free sulfhydryls by the exchange of disulfide to give a mixture of disulfides. As a result, pyridyl disulfides are the most specific reactive sulfhydryl groups. The thiophthalimides are reacted with the free sulfhydryl groups to form disulfides. 3. Reactive carboxyl residue In another embodiment, the carbodiimides soluble in both water and organic solvents were used as reactive carboxyl reactants. These compounds are reacted with the free carboxyl groups which form a pseudourea which can then be coupled to available amines by providing an amide linkage which teaches how to modify a carboxyl group with carbodiimide (Yamada et al., Biochemistry 20: 4836-4842, 1981) . ii. Preferred non-specific sites in cross-linking reagents In addition to the use of the site-specific reactive portions, the present invention contemplates the use of non-specific reactive groups to link the sugar to the modified group. Exemplary nonspecific crosslinks include fully inert darkness-active groups, which are converted to reactive species during absorption of a photon of appropriate energy. In one embodiment, the photoactivatable groups are selected from nitrene precursors generated during the heating or azide photolysis. The deficient electron nitrenes are extremely reactive and can be reacted with a variety of chemical bonds including N-H, 0-H, C-H and C = C. Although three types of azides (aryl, alkyl and acyl derivatives) can be employed, arylazides are present. The activity of arylazides during photolysis is better with N-H and O-H than the C-H bonds. The deficient electron arylnitrenes rapidly extend the ring to form dehydroazepines, which are taken care of to be reacted with nucleophiles, before the C-H insertion products are formed. The reactivity of the arylazides can be increased by the presence of substituents that remove electrons such as nitro or hydroxyl groups in the ring. Such substituents push the maximum absorption of arylazides along their wavelength. Unsubstituted arylazides have a maximum absorption in the range of 260-280 nm, while hydroxy and nitroaryl azides significantly absorb light beyond 305 nm. Therefore, hydroxy and nitroarilazides are more preferable as they are allowed to employ fewer photolysis conditions harmful to the affinity component than the unsubstituted arylazides.

In another preferred embodiment, the photoactivatable groups are selected from arylazides treated with fluorine. The photolysis products of the arylazides treated with fluorine are arinitrenes, all of which under the reactions characteristic of this group, including insertion of the CH bond, with high efficiency (Keana et al., J. Org. Chem. 55: 3640-3647 , 1990) . In another embodiment, the photoactivatable groups are selected from benzophenone residues. Benzophenone reagents generally give high crosslinking performance than arylazide reagents. In another embodiment, the photoactivatable groups are selected from the diazo compounds, which form a deficient electron carbene during photolysis. These carbenes under a variety of reactions include C-H bond insertion, addition to double bonds (including aromatic systems), hydrogen attraction and coordination for nucleophilic centers of carbon ions. In still another embodiment, the photoactivatable groups are selected from diazopyruvates. For example, the p-nitrophenyl ester of p-nitrophenyl diazopyruvate is reacted with aliphatic amines to give diazopyruvic acid amides that under ultraviolet photolysis to form aldehydes. The affinity component of modified photolyzed diazopyruvate is reacted with cross-links formed by -formaldehyde or glutaraldehyde. iii. Uniform homobifiable reagents 1. Homobi functional reactive crosslinkers with primary amines synthesis, properties and applications of reactive amine crosslinks are commercially described in the literature (to review the crosslinking and reagent procedures, see above). Some reagents are available (e.g., Pierce Chemical Company, Rockford, Ill .: Sigma Chemical Company, St. Louis, Mo .; 'Molecular Probes, Inc., Eugene, OR.). Preferred, non-limiting examples of homobifunctional NHS esters include disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis-2- (succinimidoxycarbonyloxy) ethylsufone (BSOCOES), bis-2- (sulfosuccinimidooxycarbonyloxy) ethylsufone (sulfo-BSOCOES), ethylene glycolbis (succinimidylsuccinate) (EGS) , ethylene glycosol (sulfosuccinimidylsuccinate) (sulfo-EGS), dithiobis (succinimidyl-propionate (DSP), and dithiobis (sulfosuccinimidylpropionate (sulfo-DSP).) Preferred, non-limiting examples of homobifunctional imidoesters include dimethyl malonimidate (DMM), succini idato of dimethyl (DMSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethylimperimidate (DMS), dimethyl-3, 3'-oxydipropionimidate (DODP), dimethyl-3, 3 '- (methylenedioxy) dipropionimidate (DMDP), dimethyl-3 '- (dimethylenedioxy) dipropionimidate (DDDP), dimethyl-3, 3' - (tetramethylenedioxy) -dipropionimidate (DTDP), and dimethyl-3,3 '-dithiobispropionimidate (DTBP). Non-limiting examples of homobifunctional isothiocyanate .include: p-phenylenedisothiocyanate (DITC), and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). Preferred are non-limiting examples of homobifunctional isocyanates including xylene diisocyanate, toluene-2,4-diisocyanate, toluene-2-isocyanato-4-isothiocyanate, 3-methoxydiphenylmethane-4,4'-diisocyanate, 2, 2 '- dicarboxy-4, 4 '-azophenyldiisocyanate and hexamethylene diisocyanate. Preferred are non-limiting examples of homobifunctional arylhalides including 1,5-difluoro-2,4-dinitrobenzene (DFDNB) and 4,4'-difluoro-3'3-dinitrophenyl-sulfone. HE. prefer, non-limiting examples of homobifunctional aliphatic aldehyde reagents including, glyoxal, malondialdehyde and glutaraldehyde. Preferred are non-limiting examples of homobifunctional acyl reagents including nitrophenyl esters of dicarboxylic acids. Preferred are non-limiting examples of homobifunctional aromatic sulfonyl chloride including phenol-2,4-disulfonyl chloride and α-naphthol-2,4-disulfonyl chloride. Preferred are non-limiting examples of homobifunctional amino reactive reagents including erythritolbiscarbonate which is reacted with amines to give biscarbamates. 2. Homobifunctional reactive crosslinkers with free sulfhydryl groups The synthesis, properties and applications of such reagents are described in the literature (for review of crosslinking and reagent procedures, see above). Some of the reagents are commercially available (eg, Pierce Chemical Company, Rockford, Ill .; Sigma Chemical Company, St. Louis, Mo .; Molecular Probes, Inc., Eugene, OR). Preferred are non-limiting examples of homobifunctional maleimides including bismaleimidohexanes (BMH), N, N '- (1,3-phenylene) bismaleimide, N, N'- (1, 2-phenylene) bismaleimide, azofenyldimaleimide and bis (N) -maleimidomethyl) ether.

Preferred are non-limiting examples of homobifunctional pyridyl disulfides including 1,4-di-3 '- (2'-pyridyldithio) propionamidobutane (DPD.PB). Preferred are non-limiting examples of homobifunctional alkyl halides including 2,2 '-dicarboxy-4,4'-diiodoacetamidoazobenzene, a, a'-diiodo-p-xylene sulfonic acid, a, a'-dibromo-p- xylene sulfonic acid, N, N'-bis (b-bromoethyl) benzylamine, N, N '-di (bromoacetyl) phenylhydrazine and 1,2-di (bromoacetyl) amino-3-phenylpropane. 3. Photoactivable homobifunctional crosslinkers- The synthesis, property and applications of such reagents are described in the literature (for review of the crosslinking process and reagents, see above).

Some of the reagents are commercially available (for example, 'Pierce Chemical Company, Rockford, III .; Sigma Chemical Company, St. Louis, Mo.; Molecular Probes, Inc., Eugene, OR). Preferred, non-limiting examples of homobifunctional photoactivatable crosslinkers include bis-β- (4-azidosalicylamido) ethyldisulfide (BASED), di-N- (2-nitro-4-azidophenyl) -cystamine-S, S-dioxide (DNCO) , and 4,4'-dithiobisphenylazide. iv. Heterobifunctional reagents 1. Heterobifunctional reagents of amino reactive with a portion of pyridyl disulfide ka synthesis, properties and applications of such reagents are described in the literature (for review of the crosslinking and reagent procedures, see above). Some of the reagents are commercially available (e.g., Pierce Chemical Company, Rockford, III; Sigma Chemical Company, St Louis, Mo .; Molecular Probes, Inc., Eugene, OR). Preferred are non-limiting examples of hetero-bifunctional reagents with a pyridyl disulfide moiety and an NHS reactive amino ester including N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), 6-3- (2 -pyridyldithio) propionamidohexanoate succinimidyl (LC-SPDP), 6-3- (2-pyridyldithio) ropionamidohexanoate sulfosuccinimidyl (sulfo-LCSPDP), 4-succinimidyloxycarbonyl-a-methyl-a- (2-pyridyldithio) toluene (SMPT), and sulfosuccinimidyl 6-a-methyl-a- (2-pyridyldithio) toluamidohexanoate (sulfo-LC-SMPT). 2. Heterobifunctional amino reactants reactive with a portion of maleimide ka synthesis, properties and applications of such reagents are described in the literature. Non-limiting examples of the hetero-bifunctional reactants are preferred with a portion of maleimide and an NHS reactive amino ester including succinimidyl maleimidylacetate (AMAS), succinimidyl 3-maleimidylpropionate (BMPS), N-α-maleimidobutyryloxysuccinimide ester __ (GMBS), N -? - maleimidobutyryloxy sulfo succinimide ester (sulfo-GMBS), succinimidyl 6-maleimidylhexanoate (EMCS), succinimidyl 3-maleimidylbenzoate (SMB), m-maleimidibenzoyl-N-hydroxysuccinimide ester (MBS), ester of m-maleimidobenzoyl-N-hydroxysulfosuccinimide (sulfo-MBS), 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC), 4- (N-maleimidomethyl) cyclohexane-1-carboxylate of sulfosuccinimidyl (sulfo-SMCC) ), 4- (p-maleimidophenyl) succinimidyl utirate (SMPB) and sulfosuccinimidyl 4- (p-maleimidophenyl) butyrate sulfo-SMPB). 3. Reactive heterobifunctional amino reactive with a portion of alkyl halide ka synthesis, portions and applications of such reagents are described in the preferred literature, non-limiting examples of heterobifunctional reagents with an alkyl halide portion and an NHS reactive amino ester include N -succinimidyl- (4-iodoacetyl) aminobenzoate (SIAB), sulfosuccinimidyl- (4-iodoacetyl) aminobenzoate (sulfo-SIAB), succinimidyl-6- (iodoacetyl) aminohexanoate (SIAX), succinimidyl-6- (6- ((iodoacetyl) -amino) hexanoylamino) hexanoate (SIAXX), succinimidyl-6- (((4-iodoacetyl) -amino) -methyl) -cyclohexane-1-carbonyl) aminohexanoate (SIACX), and succinimidyl-4 ((iodoacetyl) -amino) methylcyclohexane-1-carboxylate (SIAC). An example of a hetero-bifunctional reagent with a reactive NHS-amino ester and an alkyl halide portion is 2,3-hydroxysuccinimidyl dibromopropionate (SDBP). SDBP introduces intramolecular crosslinks to the affinity component by conjugating these amino groups. The reactivity of the dibromopropionyl moiety with respect to the primary amine groups is controlled by the reaction temperature (McKenzie et al., Protein Chem. 7: 581-592 (1988)). Preferred, non-limiting examples of hetero-bifunctional reactants with an alkyl halide portion and a reactive p-nitrophenyl amino ester moiety include p-nitrophenyl iodoacetate (NPIA). Other crosslinking agents are known to those skilled in the art. See, for example, Pamato et al., Patent of E.U.A. No. 5,965,106. This is within the abilities of someone of skill in the art to choose an appropriate crosslinking agent for a particular application. v. Locking groups of ligatures Still in a further embodiment, the linking group is provided with a group that can be split to release the modified group of the reduced sugar. Some splitting groups are known in the art. See, for example, Jung et al., Biochem. Biophys. Acta 761: 152-162 (1983); Joshi et al., J. Buiol. Chem. 265: 14518-14525 (1990); Zarling et al., J. Immunol. 124: 913-920 (1980); Bouizar et al., Eur. J. Biochem. 155: 141-147 (1986); Park et al., J. Biol. Chem. 261: 205-210 (1986); Browning et al., J. Immunol. 143: 1859-1867 (1989). However, a wide range of bifunctional linker groups (both homo and hetero-bifunctional), with cleavage are commercially available from suppliers such as Pierce. Exemplary cleavage portions can be split up using light, heat or reagents such as thiols, hydroxylamine, bases, periodate and the like. However, certain preferred groups are desdiblated in vivo in response to endocytosis (eg, Cis-aconityl; see, Shen et al., Biochem. Biophys., Res. Commun. 102: 1048 (1991)). Preferred cleavage groups comprise a cleavage portion which is a member selected from the group consisting of disulfide, ester, imide, carbonate, nitrobenzyl, phenacyl and benzoin groups.

Conjugation of modified sugars to peptides Modified sugars are conjugated to a glycosylated or unglycosylated peptide using an appropriate enzyme to measure conjugation. Preferably, the concentrations of the modified donor sugars, enzymes and acceptor peptides are selected such as those glycosylation procedures until the accept was consumed. The considerations discussed below, while being established in the context of a sialyltransferase, are generally applicable to other glycosyltransferase reactions. A number of methods of using glycosyltransferase to synthesize desired oligosaccharide structures are known and are generally applicable to the present invention. Exemplary methods are described, for example, WO 96/32491, Ito et al., Puré Appl. Chem. 65: 753 (1993) and Patent of E.U.A. No. 5,352,670, 5374,541 or 5,545,553. The present invention is practiced using a single glycosyltransferase or a combination of glucosyltransferase. For example, one can be used in combination of a sialyltransferase and a galactosyltransferase. In those embodiments using more than one enzyme, the enzymes and substrates are preferably combined in an initial reaction mixture, or the enzymes and reagents for a second enzymatic reaction are added to the reaction medium once the first enzymatic reaction is complete or full approximately. To conduct two enzymatic reactions in the sequence in a single container, the total yield is improved during the procedures in which a spice of intermediate was isolated. However, the cleaning and removal of extra solvents and by-products is reduced. In another embodiment, each of the primary and secondary enzymes is a glycosyltransferase. In another embodiment, an enzyme is an endoglycosidase. In a further embodiment, more than two enzymes were used to assemble the modified glycoprotein of the invention. The enzymes were used to alter a structure sacharide in the peptide at any point either before or after the addition of the modified sugar to the peptide. The O-linked glycosyl moieties of the conjugates of the invention generally originate with a GalNAc moiety that binds to the peptide. Any number of the family of the GalNAc transferases can be used to bind a GalNAc portion to the peptide (Asan H, Bennett EP, Mandel U, Hollingsworth MA and Clausen H (2000) Control of Mucin-Type O-Glycosylation: 0-glycan Occupancy is Directed by Substrate Specificities of Polypeptide GalNAc-Transferases (Eds. Ernst, Hart, and Sinay), Wiley-VCH chapter "Carbohydrates in Chemistry and Biology - a Comprehension Handbook," 273-292).

The GalNAc portion by itself may be the intact glucosyl ligation. Alternatively, the saccharyl residue is constructed using an "enzyme plus and one or more glycosyl substrates appropriate for the enzyme, the modified sugar was added to construct the glucosyl portion." In another embodiment, the method makes use of one or more exo -or endoglycosidase.Glycosidase is typically a mutant that is designed to form glycosyl bonds rather than unfolding them.The mutant glycanase typically includes a substitution of an amino acid residue for an active site of acidic amino acid residues.For example, when the endoglycanase is endo-H, the residues of the substituted active site will typically be Asp at position 130, Glu at position 132 or a combination thereof.Amino acids are generally replaced with serine alanine asparagine or glutamine.The mutant enzyme catalyzes the reaction, usually by a synthesis step that is analogous to the reverse reaction of the endoglycan hydrolysis step In these embodiments, the glycosyl donor molecule (eg, an oligo or mono saccharide structure) containing a starting group and the reaction proceeds with the addition of the donor molecule to a GlcNAc residue in the protein. For example, the starting group can be a halogen such as a fluoride. In other embodiments, the starting group is an Asn, or a portion of the Asn peptide. Even in the additional embodiments, the GlcNAc residue is modified in the glycosyl donor molecule. For example, the GlcNAc residue may comprise a 1,2-oxazolino moiety. In another modality, each one of the. Enzymes used to produce a conjugate of the invention are presented in a catalytic amount. The catalytic amount of a particular enzyme varies according to the substrate concentration of that enzyme as well as the reaction conditions such as temperature, time and pH value. The means for determining the catalytic amount for a given enzyme under preselected substrate concentrations and reaction conditions are well known to those skilled in the art.

The temperature at which the above process is carried out can be in the range from above the freezing point to the temperature at which most of the sensitive enzymes are denatured. Preferred temperature ranges are from about 0 ° C, up to about 55 ° C, and more preferably around 20 ° C, up to about 30 ° C. In another exemplary embodiment, one or more components of the present method are conducted at an elevated temperature using a thermophilic enzyme. The reaction mixture is maintained for a sufficient period of time for the acceptor to be glycosylated, thus forming the desired conjugate. Some of the conjugates can be detected frequently after a few hours, with recoverable amounts usually obtained within 24 hours or less. Those skilled in the art will understand that the reaction rate depends on a variety of variable factors (e.g., enzyme concentration, donor concentration, acceptor concentration, temperature, solvent volume), which is optimized for a selected system. The present invention also provides the production on an industrial scale of modified peptides. As used herein, an industrial scale generally produces by at least about 250 mg, preferably at least about 500 mg, and more preferably at least about 1 gram of purified purified conjugate, preferably after a simple reaction cycle. , that is, the conjugate is not a combination of products of the reaction of successively identical iteration cycles. In the discussion that follows, the invention is exemplified by the conjugation of the sialic acid portions for a glycosylated peptide. Exemplary modified sialic acid is labeled with (m-) PEG. The point of view of the following discussion about the use of glycosylated peptides and sialic acid modified with PEG is to clarify the illustration and is not intended to assume that the invention is limited to the conjugation of those two patterns. One skilled in the art will understand that the discussion is generally applicable to additions of the modified glucosyl portions contrary to those of sialic acid. On the other hand, the discussion is equally applicable to the modification of a glucosyl unit with agents other than PEG that include other water-soluble polymers, therapeutic portions and biomolecules. An enzymatic methodology can be used for the selective introduction of carbohydrates (m-) PEG-ilates or (m-) PPG-ilates on a peptide or glycopeptide. The method uses modified sugars containing PEG, PPG, or a masked reactive functional group, and is combined with an appropriate glycosyltransferase or glycosylate. By selecting the glycosyltransferase that will bind the desired carbohydrate and using the modified sugar as the donor substrate, the PEG or PPG can be introduced directly into the structure of the peptide, over the existing sugar residues of a glycopeptide or into the residues of sugar that have been added to a peptide. An acceptor for the sialyltransferase is presented on the peptide to be modified by the methods of the present invention either as a naturally occurring structure or one that is replaced either recombinantly, enzymatically or chemically. Suitable acceptors include for example, galactosyl acceptors such as GalNAc, Galßl, 4GlcNAc, Galßl, 4GalINAc, Galßl, 3GalNAc, lacto-N-tetraose, Galßl, 3GlcNAc, Galßl, 3 Ara, Galßl, dGlcNAc, Galßl, 4 Glc (lactose ) and other acceptors known as those skilled in the art (see, for example, Paulson et al., Biol. Chem. 253: 5617-5624 (1978)). In one embodiment, an acceptor for the sialyltransferase is present in the glycopeptide to be modified after the in vivo synthesis of the glycopeptide. Such glycopeptides can be sialylated using the claimed methods without prior modification of the glycoylation pattern of the glycopeptide. Alternatively, the methods of the invention can be used to sialylate a peptide that does not include a convenient acceptor; a first modifies the peptide to include an acceptor by methods known to those skilled in the art. In an exemplary embodiment, a GalNAC residue is added to an O-linked glycosylation site by the action of a GalNAc transferase. Hassan H, Bennett EP, Mandel U, Hollingsworth MA, and Clausen H (2000). Control of Mucin-Type O Glycosilation: O-Glycan Occupancy is Directed by Substrate Specificities (Eds. Ernst, Hart, and Sinay). Wiley-VCH in Chemistry and Biology-a Comprehension Handbook, 273-292 In an exemplary embodiment, the galactosyl acceptor is assembled by binding a galactose residue to an appropriate acceptor linked to the peptide, eg, a GalNAc The method includes incubating the peptide to be modified with a reaction mixture containing an appropriate amount of galactosyltransferase (eg, Galβ1.3 or Galβ1, 4) and a suitable galactosyl donor (eg, galactose UDP). The reaction is allowed to proceed substantially to completion or, alternatively, the reaction is terminated when a preselected amount of the galactose residue is added Other methods of assembling a selected saccharide acceptor will be apparent to those skilled in the art. In this embodiment, the oligosaccharides linked to the glycopeptide are first "fragmented" either completely or in part, to expose either an acceptor to the sialyltrasferase or a portion in which one or more appropriate residues can be added to obtain an adequate acceptor. Enzymes such as glycosyltransferase and endoglycosidases (see for example, Patent No. 5,716, 812) are useful for binding and fragmentation reactions. In the following discussion, the method of the invention is exemplified by the use of modified sugars having water-soluble polymers attached to them. The focus of the discussion is to clarify the illustration. Those skilled in the art will appreciate that the discussion is equally relevant to those embodiments in which the modified sugar produces a therapeutic moiety, biomolecule or the like. In an exemplary embodiment, a residue of the O-linked carbohydrate is "fragmented" prior to the addition of the modified sugar. For example, a GalNAc-Gal residue is fragmented again with GalNAc. A modified sugar carrying a water-soluble polymer is conjugated to one or more of the sugar residues exposed to "fragmentation". In one example, a glycopeptide is "fragmented" and added to the resulting O-side chain amino acids or glycol glycopeptide via a saccharyl moiety, for example, Sia, GalNAc portions conjugated to the water soluble polymer. The modified saccharyl moiety binds to an acceptor site in the "fragmented" glycopeptide. Alternatively, an unmodified saccharin portion, for example, Gal may be added at the end of the glycan bound to O. In another exemplary embodiment, a water soluble polymer is added to a GalNAc residue via a modified sugar having a residue of galactose. Alternatively, an unmodified Gal can be added to the terminal GalNAc residue. In a still more extensive example, a water soluble polymer is added to the Gal residue using a modified sialic acid.

In another exemplary embodiment, an O-linked glycosyl residue is "re-fragmented" to the GalNAc bound to the amino acid. In one example, a water soluble polymer is added via a Gal modified with the polymer. Alternatively, an unmodified Gal is added to the GalNAc preceded by a Gal with a water soluble polymer adhered. In yet another embodiment, one or more unmodified Gal residues are added to the GalNAc preceded by a portion of sialic acid modified with a water-soluble polymer. The exemplary modalities discussed above provide an illustration of the power of the methods set forth herein. By using the methods of the invention, it is possible to "re-fragment" and accumulate a carbohydrate residue of substantially any desired structure. The modified sugar can be added to. term of the carbohydrate moiety set forth above, or they may be intermediates between the peptide core and the carbohydrate terminus. In an exemplary embodiment, the water soluble polymer is added to a Gal terminal residue using a modified sialic acid polymer. An appropriate sialyltransferase is used to add a modified sialic acid. The methodology is summarized in Scheme 5.

Reaction scheme 5 Sialyltransferase a'ÍP-SA-S-HHCOCH2NH - PEG (PPG Glycoprotein Still in an additional method, summarized in reaction scheme 6, hidden reactive functionality is present sialic acid. The hidden reactive group is preferably not affected by the conditions used to bind the modified sialic acid to the peptide. After covalent attachment of modified sialic acid to the peptide, the coat is removed and the peptide is conjugated with an agent such as PEG, PPG, a therapeutic, biomolecular, or other agent. The agent is conjugated to the peptide in a specific manner by its reaction with the reactive group without hiding on the residue of the modified sugar.

Reaction scheme 6 Any modified sugar can be used with its suitable glycosyltransferase depending on the terminal sugars of the oligosaccharide side chains of the glycopeptide (Table 3). As discussed above, the terminal sugar of the glycopeptide required for the introduction of the PEG-ylated or PPG-ylated structure may be introduced naturally during expression or may occur post-expression using the appropriate glycosidases, glycosyltransferases or a mixture of glycosidases and glycosyltranspheres.

Table 3 Derivatives of UDP-galactose (where A = NH, E can be acetyl) Derivatives of UDP-glucosamine Derivatives of UDP-glucose (when A ^ MH, ^ can be acetyl) Derivatives of GDP-anosa X-03NH3 StCH2 > N "(Rrs) 2. Y-Jfc Z = X; A = X; B" X Ligand of interest = acyl-PEG, acyl-PPG, alkyl-PEG, acyl-alkyl-PEG, acyl-alkyl-PEG, carbamoyl -PEG, carbamoyl-PPG, PEG, PPG, acyl-aryl-PEG, acyl-aryl- PPG, aryl-PEG, aryl-PPG, mannose-6-phosphate, heparin, heparan, Slex, mannose, FGF, VFGF, protein , R, Rl-4 = B, linker? M. chondroitin, ceratan, dermatan, albumin, integrins, peptides, etc. M33 Ligand of interest In an alternative embodiment, the modified sugar is added directly to the structure of the peptide using a glycosyltransferase known to transfer the sugar residues to the glycosylation site linked to O in the structure of the peptide. This exemplary embodiment is set forth in Reaction Scheme 7. Exemplary glucosyltransferases useful in the practice of the present invention include but are not limited to, GalNAc (GalNAc Tl-20) transferases, GalNAc transferases, fucosyltransferases, glucosyltransferases, xylosyltransferases, mannosyltransferases and similar. The use of this methodology allows the direct addition of the modified sugars on the peptides lacking any carbohydrate or, alternatively, on the existing glycopeptides. In both cases, - the addition of the modified sugar occurs at specific positions in the structure of the peptide as defined by the substrate specificity of the glycosyltransferase and not in a random fashion as occurs during the modification of the peptide structure of the protein using chemical methods. A series of agents can be introduced into proteins or glycopeptides lacking the peptide sequence of the glycosyltransferase substrate by genetically engineering the appropriate amino acid sequence in the polypeptide chain.

Scheme of reaction 7 Protein or glycoprotein ?? X GalNH-CO (CH2) 4NH-PEG GalNAc Transferase (GalNAc 13) GalNH-CO (CH2) 4NH-PEG In each of the exemplary embodiments set forth above, one or more additional enzymatic or chemical modification steps can be used following conjugation of the sugar modified for the peptide. In an exemplary embodiment, an enzyme (e.g., fucosyltransferase) is used to add a glucosyl unit (e.g., fucose) to the terminal modified sugar linked to the peptide. In another example, an enzymatic reaction is used to "capsulate" sites (e.g., sialylate) in . which the modified sugar was not conjugated. Alternatively, a chemical reaction is used to alter the structure of the conjugated modified sugar. For example, the conjugated modified sugar is reacted with agents that stabilize or destabilize its bond with the component of the peptide to which the modified sugar is bound. In another example, a component of the modified sugar is deprotected following its conjugation to the peptide. An expert will appreciate that there are a number of enzymatic and chemical procedures that are useful in the methods of. the invention in a step subsequent to the conjugation of the modified sugar to the peptide. Further elaboration of the modified sugar peptide conjugate is within the scope of the invention. In another exemplary embodiment, the glycopeptide is conjugated with a targeting agent, e.g., transferrin (to deliver the peptide through the cerebral blood barrier and endosomes), carnitine (to deliver the peptide to the muscle cells; see for example, LeBorgne et al Biochem, Pharmacol 59: 1357-63 (2000), and phosphonates, for example, bisphosphonate (to direct the peptide to bones and other calciferous tissues, see for example, Modern Drug Discovery, August 2002 , page 10) Other useful agents for directing are apparent to those skilled in the art.For example, glucose, glutamine and IGF are also useful for the target muscle.The targeting portion and the therapeutic peptide are conjugated by any method. discussed herein or otherwise known in the art Those skilled in the art will appreciate that the additional peptides set forth above may also be derived as is The exemplary peptides are set forth in the appended appendix to the co-pending, usually provisional patent application No. 60/328, 523 owned by the US. filed on October 10, 2001. In an exemplary embodiment, the subject agent and the therapeutic peptide are coupled via a binding moiety. In this embodiment, at least one of the therapeutic peptides or the target agent is coupled to the linking portion via an intact glucosyl linkage group according to a method of the invention. In an exemplary embodiment, the linking portion includes a poly (ether) such as poly (ethylene glycol). In another exemplary embodiment, the binding portion includes at least one linkage that degrades in vivo, releasing the therapeutic peptide from the target agent, following delivery of the conjugate to the targeted tissue or region of the body. In yet another exemplary embodiment, in the in vivo distribution of the therapeutic portion via alteration of a glycoform in the therapeutic portion without conjugating the therapeutic peptide to an object moiety. For example, the therapeutic peptide can be deviated out of the uptake by the endothelial network system by capsulating a terminal galactose portion of a glucosyl group with the sialic acid (or a derivative thereof). i. Enzymes 1. Glucosyltransferase Glucosyltransferases catalyze the addition of activated sugars (NDP sugar donors), in a prudent stage form, to a protein, glycoprotein, lipid or glycolipid or at the end without increasing oligosaccharide reduction. N-linked glycopeptides are synthesized via a transferase and a Dol-PP-NAG2Glc3Mang donor of the oligosaccharide linked to the lipid in a block transfer followed by core fragmentation. In this case, the nature of the saccharide "nucleus" is in some way - different from the subsequent unions. A large number of glucosyltransferases is known in the art. The glycosyltransferase to be used in the present invention can be so extensive that the modified sugar can be used as a sugar donor. Examples of such enzymes include glycosyltransferase with Leloir path, such as galactosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, fucosyltransferase, sialyltransferase, mannosyltransferase, xylosyltransferase and the like. For the synthesis of enzymatic saccharides involving glucosyltransferase reactions, the glucosyltransferases can be cloned, or isolated from any source. Many cloned glycosyltransferases are known as their polynucleotide sequences. See for example, "Te WWW Guide To Cloned Glycosyltransferases," (http: // www. Vei.co.uk/TGN/gt_guide.htm) The amino acid sequences of glycosyltransferases and nucleotide sequences that encode glycosyltransferases from which amino acid sequences can be deduced are also found in several publicly available databases, including GenBank, Swiss-prot, EMBL, and others. The glycosyltransferases that can be employed in the methods of the invention include, but are not limited to, galactosyltransferases, fucosyltransferases, glucosyltransferases, N-acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases, - glucuronyltransferases, sialyltransferases, mannosyltransferases, glucuronic acid transferases, galacturonic acid transferases, and oligosaccharyltransferases. Suitable glycosyltransferases include those obtained from eukaryotes as well as from prokaryotes. The DNA encoding the glycosyltransferases can be obtained by chemical synthesis, by separation by exclusion of reverse transcripts of mRNA from appropriate cells or cultures of cell lines, by genomic collections by exclusion of appropriate cells, or by combinations of these procedures . Separation by exclusion of mRNA or genomic DNA can be carried out with oligonucleotide probes generated from a sequence of glycosyltransferase genes. The probes can be labeled with detectable groups such as the fluorescent group, or a radioactive atom or a chemiluminescent group according to methods known and used in conventional hybridization assays. In an alternative, the glycosyltransferase gene sequences can be obtained by using the polymerase chain reaction (PCR) method, with the PCR oligonucleotide primers that are produced from the glycosyltransferase gene sequence . See, for example, U.S. Patent No. 4,683,195 by Mullis et al and U.S. Pat. No. 4,683,202 by Mullís. The glycosyltransferase can be synthesized in host cells transformed with vectors containing DNA encoding the glycosyltransferase enzyme. The vectors are used either to amplify the DNA encoding the glycosyltransferase enzyme and / or to express the DNA encoding the enzyme of the glycosyltransferases. An expression vector is a replicable DNA construct in which the DNA sequence encoding the glycosyltransferase enzyme is operably linked to the appropriate control sequences capable of affecting the expression of the glycosyltransferase enzymes in a suitable host. The need for such control sequences will vary depending on the selected host and the choice of transformation method. Generally, the control sequences include a transcriptional promoter, an optional sequence of operation to control transcription, a sequence encoding the ribosomal binding sites of the appropriate mRNA, and the sequences that control translation termination and transcription. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication and a selection gene to facilitate the recognition of transformants. In an exemplary embodiment, the invention utilizes a prokaryotic enzyme. Such glycosyltransferases include enzymes involved in the synthesis of lipooligosaccharides (LOS), which are produced by many gram-negative bacteria (Preston et al., Critical Reviews in Microbiology 23 (3): 139-180 (1996)). Such enzymes include, but are not limited to, the proteins of the rfa-operons of species such as E. coli and Salmonella typhimurium, which include a galactosyltransferase β1, 6 and a galactosyltransferase β1 / -3 (see for example, EMBL Nos. Access M80599 and M86935 (E. coli); EMBL Accession No. S56361 (S. typhimurium)), a glycosyltransferase (Swiss-Prot Accession No. P25740 (E. coli), a βl, 2-glucosyltransferase (rfaJ) (Swiss-Prot Accession No. P27129 (E coli) and Swiss-Prot accession no. P19817 (S. typhimurium) and a ßl, 2-N-acetylglucosaminyltransferase (rfaK) (EMBL Accession No. U00039 (E. coli), other glycosyltransferases for which sequences amino acids are known to be those encoded by operons such as rfaB, which have been characterized in organisms such as Klebsiella pneumoniae, E. coli, Salmonella typhimurium, enteric Salmonella, Yersinia enterocolitica, Mycobacterium leprosum and the rhl operon of Pseudomonas aeruginosa. Also suitable for use in the present invention are glycosyltransferases which are involved in the production of structures containing lacto-N-neotetraose, D-galactosyl-β-1,4-N-acetyl-D-glucoosaminyl-β-1, 3 -D-galactosyl-β-1,4-D-glucose, and the sequence tr isacid of the blood group Pk, D-galactosyl-a-1,4-D-galactosyl-β-1, 4-D-glucose, which have been identified in the LOS of mucosal pathogens Neisseria gonnorhoeae and N. meningitidis (Scholten et al. ., J. Med. Microbiol. 41: 236-243 (1994)). The genes of N. 'meningitidis and N. gonorrhoeae coding for the glycosyltransferases involved in the biosynthesis of these structures have been identified from the immunotypes L3 and Ll of N. meningitidis (Jennings et al., Mol. Microbiol. 18: 729-740 (1995)) and the F62 mutant of N. gonorrhoeae (Gotshlich, J. Exp. Med. 180: 2181-2190 (1994)). In the ?. meningitidis, a site consisting of three genes lgtA, and IgtB and Ig E, which encodes the glycosyltransferase enzymes required for the addition of the last three sugars in the lacto -? - neotetraose chain (Wakarchuk et al., J. Biol. Chem. 271: 19166-73 (1996)).

Recently, the enzymatic activity of the product of the IgtB and IgtA gene was demonstrated, providing the first direct evidence for its proposed glycosyltransferase function (Wakarchuk et al., J. Biol. Chem. 271 (45): 28271-276 (1996 )). In N. gonorrhoeae, there are two additional genes, the IgtD that adds ß-D-Gal? Ac in position 3 of the terminal galactose of the lacto-? -neotetraose structure and IgtC that adds a terminal aD-Gal to the element of the lactose of a truncated LOS, thus creating the structure of the antigen of the blood group Pk (Gotshlich (1994), supra). In N. meningi tidis, a separate immunotype Ll also expresses the blood group antigen P and has been shown to contain an IgtC gene (Jennings et al., (1995), supra). Neisseria glycosyltransferases and associated genes are also described in USP? 5,545,553 (Gotschlich). The genes for a2-fucosyltransferase and al, 3-fucosyltransferase from Helicobacter pylori have also been characterized (Martin et al., J. Biol. Chem. 272: 21349-21356 (1997)). Also of use in the present invention are the glycosyltransferases of Campylobacter jejuni. { see for example, http: // afmb. cnrs-mrs. fr / ~ pedro / CAZY / gtf 42.html). a) Fucosyltransferase In some embodiments, a glycosyltransferase used in the method of the invention is a fucosyltransferase. Fucosyltransferases are known to those skilled in the art. Examples of fucosyltransferases include enzymes that transfer L-fucose from mucosa GDP to a hydroxy position of an acceptor sugar. Fucosyltransferases that transfer sugars without nucleotides to an acceptor are also used in the present invention. In some embodiments, the acceptor sugar is, for example, GlcNAc in a Galß (1? 3, 4) GlcNAcß group in an oligosaccharide glycoside. Suitable fucosyltransferases for this reaction include Galß (1? 3, 4) GlcNAc? L-a (l? 3, 4) fucosyltransferase (FTlI EC No. 2.4.1.65), which was first characterized from human milk (see, Palcic, et al, Carbohydrate Res. 190: 1-11 (1989), Prieels, et al., J. Biol. Chem. 256: 10456-10463 (1981), and Núñez, et al., Can. J. Chem. 59: 2086 -2059 (1981)) and the Galß (1? 4) GlcNAcß-afucosyltransferases (FTIV, FTV, FTVI) which are found in human serum. The FTVII (EC No. 2.4.1.65), a sialyl (2? 3) Gal ((l? 3GlcNAc? Fucosyltransferase, has also been characterized.) A recombinant form of the Gal? (1? 3, 4) GlcNAc? a (1? 3, 4) fucosyltransferase (see, Dumas, et al., Bioorg, Med. Letters 1: 425-428 (1991) and Kukowska-Latallo, et al., Genes anda Development 4: 1288-1303 (1990)) Other exemplary fuc siltransferases include, for example, 2-fucosyltransferase (EC No. 2.4.1.69) Enzymatic fucosylation can be carried out by the methods described in Mollicone et al., Eur. J. Biochem. 191: 169- 176 (1990) or US Patent No. ,374,655. The cells that are used to produce a fucosyltransferase will also include an enzymatic system to synthesize GDP-fucose. b) Galactosyltransferase In another group of modalities, the glucosyltransferase is a galactosyltransferase. Exemplary galactosyltransferases include (1, 3) galactosyltransferases (EC No. 2.4.1.151, see for example, Dabkowski et al., Transplant Proc. 25: 2921 (1993) and Yamamoto et al., Nature 345: 229-233 (1990). , Bovine (GenBank J04989, Joziasse et al., J. Biol. Chem. 264: 14290-14297 (1989)), murine GenBank m26925, Larsen et al., Proc. Nat'l. Acad. Sci. USA 86: 8227-8231 (1989)), swine (GenBank L36152; Strahan et al.

Immunogenetics 41: 101-105 (1995)). Another 1, 3 galactosyltransferase is one that is involved in the synthesis of the antigen of blood group B (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem. 265: 1146-1151 (1990) (human)). Still one more exemplary galactosyltransferase is the Gal-Tl nucleus. Β (1,4) galactosyltransferases are also suitable for use in the methods of the invention, which include, for example, EC 2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose synthetase) (bovine (D) 'Agostaro et al., Eur. J. Broche., 183: 211-217 (1989)), human (Masri et al., Biochem. Biophys., Res. Comm. 157: 657-663 (1988)), murine (Nakazawa et al. , J. Biochem, 104: 165-168 / 1988)), in addition to ED 2.4.1.38 and ceramide galactosyltransferase (EC 2.4.1.45, Stahl et al, J. Neurosci Ress. 38: 234-242 (1994) ). Other suitable galactosyltransferases include, for example, 2-galactosylatransferases (e.g., Schizosaccharomyces pombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)). Also soluble in the practice of the invention are soluble forms of al, 3-galactosyltransferase such as that reported by Cho, S.K. and Cummings, R. D. (1997) J. Biol. Chem., 272, 13622-13628. c) Sialiltransfrasas Sialitransferases are another type of glucosyltransferase that is useful in the recombinant cells and reaction mixtures of the invention. Cells that produce recombinant sialyltransferases will also produce CMP-sialic acid, which is a sialic acid donor for sialyltransferases. Examples of sialyltransferases that are useful for use in the present invention include ST3Gal III (eg, a mouse or human ST3Gal III), ST3Gal IV, ST3Gal I, ST6Gal I, ST3Gal V, STdGalII, ST6GalNAc I, ST6GalNAc II, and ST6GalNAc III (the nomenclature of the sialyltransferase used herein is as described in Tsuji et al., Glycobiology 6: - v-xiv (1996)). One (2,3) sialyltransferase referred to as (2, 3) sialyltransferase (EC 2.4.99.6) transfers sialic acid to the terminal without Gal reduction of a Galßl? 3Glc disaccharide or glycoside. See, Van den Eijnden et al., J. Biol. Chem. 256: 3159 (1981), Weinstein et al., J. Biol. Chem. 257: 13845 (1982) and Wen et al., J. Biol. Chem. 267: 21011 (1992). Another exemplary a2,3-sialylstransferase (EC 2.4.99.4) transfers sialic acid to the terminus without disaccharide or glycoside Gal reduction. See, Rearick et al., J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J. Biol. Chem. 267: 21004 (1992). More exemplary enzymes include Gal-ß-1, -GlcNAc α-2,6 sialyltransferase (See, Kurosawa et al., Kurosawa et al., Eur. J. Biochem. 219: 375-381 (1994)). Preferably, for glycopeptide carbohydrate glycosylation the sialyltransferase will be capable of transferring sialic acid to the Galßl, 4GlcNAc- sequence, the most common penultimate sequence that underlines the terminal sialic acid in sialylated carbohydrate structures (see, Table 5). Table 5. Sialyltransferase which uses the sequence Galßl, 4GlcNAc as an acceptor substrate. 1) Goochee et al., Bio / Technology 9: 1347-1355 (1991) 2) Yamamoto et al., J. Biochem. 120: 104-110 (1996) 3) Gilbert et al., J. Biol. Chem. 271: 28271-28276 (1996) An example of a sialyltransferase which is useful in the claimed methods is ST3Gal III, which is also referred to as a (2,3) sialyltransferase ( EC 2.4.99.6). This enzyme catalyzes the transfer of sialic acid to Gal from a glycoside Galßl, 3GlcNAc or Galßl, 4GlcNAc (see, for example, Wen et al., J. Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem. 256: 3159 (1991)) and is responsible for sialylation of oligosaccharides linked to asparagine in glycopeptides. Sialic acid binds to a Gal with the formation of a ligature between the two saccharides. The binding (ligation) between the saccharides is between position 2 of NeuAc and position 3 of Gal.

This particular enzyme can be isolated from rat liver (Weinstein et al., J. Biol. Chem. 257: 13845 (1982)); the human cDNA (Sasaki et al., (1993) J.

Biol. Chem. 268: 22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-1401) and genomic DNA sequences (Kitagawa et al., (1996) J. Biol. Chem. 271: 931-938) known, facilitating the production of this enzyme by recombinant expression. In another embodiment, the claimed sialylation methods use a rat ST3Gal III. Other exemplary sialyltransferases for use in the present invention include those isolated from Camppylobacter jejuni, including a (2,3). See, for example, WO99 / 49051. Other sialyltransferases other than those listed in Table 5 are also useful in an economical and efficient large-scale process for sialylation of commercially important glycopeptides. As a simple test to find the utility of these different enzymes, different amounts of each enzyme (1-100 mU / mg protein) are reacted with asialo-a? AGP (at 1-10 mg / ml) to compare the ability of the sialyltransferase of interest to sialylate glycopeptides relative to either or both of the ST6Gal I sialyltransferases, or bovine ST3Gal III. Alternatively, other N-linked glycopeptides or oligosaccharides enzymatically released from the peptide support can be used in place of asialo-ai AGP for this evaluation. Sialyltransferases with the ability to sialylate N-linked oligosaccharides of glycopeptides more efficiently than ST6Gal I are useful in a practical large-scale process for peptide sialylation (as illustrated for ST3Gal III in this description). Other exemplary sialyltransferases are shown in Figure 10. d) GalNAc Transferases N-acetylgalactosaminyltransferases are of use in the practice of the present invention, particularly for binding of a portion of GalNAc to an amino acid of the O-linked glycosylation site of the peptide. Suitable N-acetylgalactosaminyltransferases include, but are not limited to, (1, 3) N-acetylgalactosaminyltransferase, β (1,4) N-acetylgalactosaminyltransferase (Nagata et al., J. Biol. Chem. 267: 12082-12089 (1992 ) and Smith et al., J. Biol. Chem. 269: 15162 (1994) and polypeptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol. Chem. 268: 12609 (1993)). The production of proteins such as GalNAc T? -? enzyme from genes cloned by genetic engineering is well known See, for example, US Patent No. 4,761,371 One method involves the collection of sufficient samples, then the amino acid sequence of the enzyme is determined by sequencing This information is then used to isolate a cDNA clone that encodes a full-length transferase (bound membrane) which is over expressed in the Sf9 insect cell line resulting in the synthesis of an enzyme completely The specificity of the enzyme acceptor is then determined using a semiquantitative analysis of the amino acids surrounding the known glycosylation sites in 16 different proteins followed by in vitro glycosylation studies of synthetic peptides. This work has shown that certain amino acid residues are over-represented in segments of glycosylated peptides and those residues at specific positions that surround the glycosylated serine and threonine residues may have a more marked influence on the acceptor efficiency than other portions of amino acids. 2. Sulfur Transfers The invention also provides methods for the production of peptides including sulphated molecules, including, for example, sulfated polysaccharides such as heparin, heparan sulfate, carrageenan, and related compounds. Suitable sulfotransferase include, for example, chondroitin-6-sulfotransferase (chicken cDNA described by Fukuta et al., J. Biol. Chem. 270: 18575-18580 (1995); GenBank Accession No. D49915), glycosaminoglycan N-acetylglucosamine N deacetylase / N-deacetylase / N-sulfotransferase 1 (Dixon et al., Genomics 26: 239-241 (1995); UL18918), and glycosaminoglycan N-acetylglucosamine N-deacetylase / N-sulfotransferase 2 (murine cDNA described in Orellana and collaborators, J. Biol. Chem. 269: 2270-2276 (1994) and Eriksson et al., J. Biol. Chem. 269: 10438-10443 (1994); human cDNA described in GenBank Accession No.

U2304). 3. Glucosyltransferases Linked to Cells. In another embodiment, the enzymes used in the method of the invention are glycosyltransferases bound to cells. Although many soluble glucosyltransferases are known (see, for example, U.S. Patent No. 5,032,519), glycosyltransferases are generally in membrane-bound form when associated with cells. Many of the membrane-bound enzymes studied in this way are considered to be intrinsic proteins; that is, they are not released from the membranes by sonication and require detergents for solubilization. Surface glycosyltransferases have been identified on the surfaces of vertebrate and invertebrate cells, and it has also been recognized that these surface transfersase maintain catalytic activity under physiological conditions. However, the most recognized function of cell surface glycosyltransferases is for intercellular recognition (Roth, Molecular Approaches to Supracellular Phenomena, 1990). Methods have been developed to alter the glycosyltransferases expressed by cells. For example, Larsen et al., Proc. Nati Acad. Sci. USA 86: 8227-8231 (1989), reports a genetic approach for isolating cloned cDNA sequences that determines the expression of surface oligosaccharide structures in cell and their related glycosyltransferases. A cDNA library generated from mRNA isolated from a murine cell line known to express UDP-galactose:. H.H . -D-galactosyl-1, 4-N-acetyl-D-glucosaminide a-1,3-galactosyltransferase was transfected into COS-1 cells. The transfected cells were then cultured and analyzed for α-galactosyltransferase activity. Francisco et al., Proc. Nati Acad. Sci. USA 89: 2713-2717 (1992), describe a method of anchoring β-lactamase to the outer surface of Escherichia coli. A tripartite fusion consisting of (i) a signal sequence from an outer membrane protein, (ii) a membrane expansion section of an outer membrane protein, and (iii) a complete mature ß-lactamase sequence -se produces resulting in an active surface linked to ß-lactamase molecule. However, Francisco's method is limited only to systems of prokaryotic cells and as recognized by the authors, requires complete tripartite fusion for proper functioning. 4. Fusion proteins. In other exemplary embodiments, the methods of the invention utilize fusion proteins that have more than one enzymatic activity that is involved in the synthesis of a desired glycopeptide conjugate. The fusion polypeptides may be composed of, for example, a catalytically active domain of a glycosyltransferase that binds to a catalytically active domain of an accessory enzyme. The catalytic domain of accessory enzyme can, for example, catalyze a step in the formation of a nucleotide sugar that is a donor for the glycosyltransferase, or catalyze a reaction involved in a glycosyltransferase cycle. For example, a polynucleotide encoding a glycosyltransferase may be linked, in structure, to a polynucleotide that encodes an enzyme involved in nucleotide sugar synthesis. The resulting fusion protein can then catalyze not only the synthesis of the nucleotide sugar, but also the transfer of the sugar portion to the acceptor molecule. The fusion protein may be two or more cyclic enzymes linked to an expression nucleotide sequence. In other embodiments, the fusion protein includes the catalytic activity domains of two or more glycosyltransferases. See, for example, 5,641,668. The modified polypeptides of the present invention can be easily designed and manufactured using various appropriate fusion proteins (see, for example, PCT Patent Application PCT / CA98 / 01180, which was published as WO 99/31224 on June 24, 1999 ).

. Immobilized Enzymes. In addition to the enzymes linked to cells, the present invention is also provided for the use of enzymes that are immobilized on a solid and / or soluble support. In an exemplary embodiment, a glycosyltransferase is provided which is conjugated to a PEG via an intact glucosyl ligature in accordance with the methods of the invention. The PEG ligation enzyme conjugate optionally binds to the solid support. The use of solid supported enzymes in the methods of the invention simplifies the work of the reaction mixture and purification of the reaction product, and also makes possible the easy recovery of the enzyme. The glucosyltransferase conjugate is used in the methods of the invention. Other combinations of enzymes and supports will be palpable for those with experience in the technique.- ..

Purification of Peptide Conjugates. The products produced by the above processes can be used without purification. However, it is usually preferred to recover the product. Standard well known techniques for recovery of glycosylated saccharides such as thin or thick layer chromatography, column chromatography, ion exchange chromatography, or membrane filtration can be used. It is preferred to use membrane filtration, more preferably using a reverse osmotic membrane, or one or more column chromatographic techniques for recovery as described hereinafter and in the literature cited herein. For example, membrane filtration wherein the membranes have molecular weight cut-off from about 3000 to about 10,000 can be used to remove proteins such as glucosyl transferases. The nanofiltration or reverse osmosis can then be used to remove salts and / or purify the saccharides from the product (see, for example, WO 98/15581). Nanofilter membranes are a class of reverse osmosis membranes that pass monovalent salts but retain polyvalent salts and uncharged solutes greater than about 100 to about 2,000 Daltons, depending on the membrane used. Thus, in a common application, the saccharides prepared by the methods of the present invention will be retained in the membrane and the contaminating salts will pass through it. If the modified glycoprotein is produced intracellularly, as a first step, the particle is discarded, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration; optionally, the protein can be concentrated with a commercially available protein concentration filter, followed by separation of the variant polypeptide from other impurities by one or more steps selected from immunoaffinity chromatography, ion exchange column fractionation (e.g., in diethylaminoethyl) (DEAE) 'or matrices containing carboxymethyl or sulfopropyl groups), chromatography on Blue-sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, Gentle lecithin-Sepharose, WGA-Sepharose, With A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl, SP-Sepharose, or Protein A Sepharose, SDS-PAGE chromatography, silica chromatography, chromatofocusing, reverse phase CLAP (for example, silica gel with aliphatic groups attached), gel filtration using, for example, Sephadex molecular sieve or size exclusion chromatography, chromatography on columns that selectively bind the polypeptide, and precipitation of ethanol or sulfate of ammonium. Modified glycopeptides produced in culture are usually isolated by initial extraction of cells, enzymes, etc., followed by one or more concentrations, desalination, aqueous ion exchange, or size exclusion chromatography steps, for example, SP Sepharose. Additionally, the modified glycoprotein can be purified by affinity chromatography. CLAP can also be used for one or more purification steps. A protease inhibitor, for example, methylsulfonyl fluoride (PMSF), may be included in any of the preceding steps to inhibit proteolysis and antibiotics may be included to prevent the growth of future contaminants. Within another embodiment, the supernatants of systems that produce the modified glycopeptide of the invention are first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to an appropriate purification matrix. For example, an appropriate affinity matrix may comprise a ligand for the peptide, a lectin or antibody molecule bound to an appropriate support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant DEAE groups. Suitable matrices include acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include several insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are particularly preferred. Finally, one or more steps of CLAP RP employing hydrophobic CLAP RP media, for example, silica gel having pendant methyl or other aliphatic groups, can be used to further purify a variant polypeptide composition. - Some or all of the steps of purification, in various combinations, may also be employed to provide a homogeneous modified glycoprotein. The modified glycopeptide of the invention resulting from large-scale fermentation can be purified by methods analogous to those described by Urdal et al., J. Chromatog. 296: 171 (1984). This reference describes two sequential steps of CLAP RP for purification of recombinant human IL-2 in a preparative CLAP column. Alternatively, techniques such as affinity chromatography can be used to purify the modified glycoprotein. Pharmaceutical compositions. The modified O-linked glycosylation site-modified polypeptides according to the method of the present invention have a wide range of pharmaceutical applications. For example, modified erythropoietin (EPO) can be used to treat general anemia, aplastic anemia, chemoinduced injury (such as bone marrow injury), chronic renal failure, nephritis, and thalassemia. The modified EPO can also be used to treat neurological disorders such as brain / spine injury, multiple sclerosis, and Alzheimer's disease. A second example is interferon-a (IFN-a), which can be used to treat AIDS and hepatitis B or C, viral infections caused by a variety of viruses such as human papillomavirus (HBV), coronavirus, human immunodeficiency (HIV), simple herpes virus (HSV), and varicella zoster virus (VZV), cancers such as hairy cell leukemia, AIDS-related Kaposi's sarcoma, malignant melanoma, follicular Hodgkins's other lymphoma, chromosome Philadelphia (Ph) -positive, chronic phase myelogenous leukemia (CML), kidney cancer, myeloma, chronic myelogenous leukemia, cancers of the head and neck, bone cancer, in addition to cervical dysplasia and central nervous system disorders (CNS) such as multiple sclerosis. In addition the modified IFN-a according to the methods of the present invention is useful for the treatment of a variety of other diseases and conditions such as Sjogren's syndrome (an autoimmune disease), Behcet's disease (an autoimmune inflammatory disease), fibromyalgia (a musculoskeletal pain / fatigue disorder), aphthous ulcer (canker sore), chronic fatigue syndrome, and pulmonary fibrosis. Another example is interferon-ß, which is useful for the treatment of CNS disorders such as multiple sclerosis (either recurrent / chronic relapsing or progressive), AIDS and hepatitis B or C, viral infections caused by a variety of viruses such such as human papilloma virus (HBV), human immunodeficiency virus (HIV), simple herpes virus (HSV), and varicella-zoster virus (VZV), otological infections, musculoskeletal infections, as well as cancers that include cancer of the breast, brain cancer, colorectal cancer, non-small cell lung cancer, head and neck cancer, basal cell cancer, cervical dysplasia, melanoma, skin cancer, and liver cancer. INF-ß modified according to the methods of the present invention is also used in the treatment of other diseases and conditions such as transplant rejection (e.g., bone marrow transplantation), Huntington's disease, colitis, brain inflammation, pulmonary fibrosis, macular degeneration, liver cirrhosis, and keratoconj unctivitis. The granulocyte colony stimulation factor (G-CSF) is another example. The G-CSF modified according to the methods of the present invention can be used as an adjunct in chemotherapy for cancer treatment, and to prevent or alleviate conditions or complications associated with certain medical procedures, for example, bone marrow-induced injury. chemo leukopenia (general); febrile neutropenia induced by chemo; neutropenia associated with bone marrow transplants; and severe chronic neutropenia. The modified G-CSF can also be used for transplantation; mobilization of peripheral blood cell; mobilization of peripheral blood progenitor cells for collection in patients who will receive myeloablative or myelosuppressive chemotherapy; and reduction in duration of neutropenia, fever, antibiotic use, hospitalization followed by induction / consolidation treatment for acute myeloid leukemia (AML). Other conditions or disorders can be treated with modified G-CSF including asthma and allergic rhinitis. As a further example, human growth hormone (hGH) modified according to the methods of the present invention can be used to treat conditions related to growth such as infantilism, short stature in children and adults, cachexia / muscle wasting, General muscular atrophy, and sex chromosome abnormality (for example, Turner syndrome). Other conditions can be treated using modified hGH including: short bowel syndrome, lipodystrophy, osteoporosis, uremia, burns, female infertility, bone regeneration, general diabetes, type II diabetes, osteoarthritis, chronic obstructive pulmonary disease (COPD), and insomnia. Furthermore, modified hGH can also be used to promote various processes, e.g., general tissue regeneration, bone regeneration, and wound healing, or as a vaccine adjunct. Thus, in another aspect, the invention provides a pharmaceutical composition. The pharmaceutical composition includes a pharmaceutically acceptable diluent and a covalent conjugate between a non-naturally occurring water soluble polymer, therapeutic moiety or biomolecule, and a glycosylated or non-glycosylated peptide. The polymer, therapeutic moiety or biomolecule is conjugated to the peptide via a group that binds the intact glucosyl interposed in medium and covalently bound to both the peptide and the polymer, therapeutic moiety or biomolecule. The pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. Formulations suitable for use in the present invention are found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of the methods for drug delivery, see, Langer, Science 249: 1527-1533 (1990). The pharmaceutical compositions can be formulated for any suitable form of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer solution. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable matrices, such as microspheres (eg, polylactate polyglycolate), can also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are described, for example, in U.S. Patent No. 4,897,268 and 5,075,109. Commonly, the pharmaceutical compositions are administered subcutaneously or parenterally, for example, intravenously. Thus, the invention provides compositions for parenteral administration which comprises the compound dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, for example, water, buffer water, saline, PBS and the like. The compositions may also contain detergents such as Tween 20 and Tween 80; stabilizers such as mannitol, sorbitol, sucrose, and trehalose; and preservatives such as EDTA and m-cresol. The compositions may contain substantially pharmaceutically acceptable auxiliaries as required for approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. These compositions can be sterilized by conventional sterilization techniques, or they can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation that is combined with a sterile aqueous carrier prior to administration. The pH of the preparations will commonly be between 3 and 11, more preferably from 5 to 9 and more preferably from 7 to 8. In some embodiments the glycopeptides of the invention can be incorporated into the liposomes formed of lipids that form standard vesicles. A variety of methods are available for the preparation of liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S. Patent Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomes using a variety of targeting agents (eg, the sialyl galactosides of the invention) is well known in the art (see, for example, U.S. Patent Nos. 4,957,773 and 4,603,044). Standard methods for coupling targeting agents to liposomes can be used. These methods generally involve the incorporation into liposomes of lipid components, such as phosphatidylethanolamine, which can be activated for the binding of targeting agents, or derived lipophilic compounds, such as glycopeptides derived from lipid of the invention. The targeting mechanisms generally require that the targeting agents be positioned on the surface of the liposome in a manner such that the address portions are available for interaction with the target, eg, a cell surface receptor. The carbohydrates of the invention can be attached to a lipid molecule before the liposome is formed using methods known to those of skill in the art. (for example, alkylation or acylation of a hydroxyl group present in the carbohydrate with a long chain alkyl halide or with a fatty acid, respectively).

Alternatively, the liposome can be formed in such a way that a connector portion is first incorporated into the membrane while the membrane is formed. The connector portion must have a lipophilic portion, which is firmly embedded and anchored in the membrane. It must also have a reactive portion, which is chemically available in the aqueous surface of the liposome. The reactive portion is selected so that it will be chemically appropriate to form a stable chemical link with the targeting agent or carbohydrate, which is subsequently added. In some chaos it is possible to attach the directing agent to the linker molecule directly, but in most cases it is more appropriate to use a third molecule to act as a chemical bridge, thus linking the linker molecule which is in the membrane with the steering agent or carbohydrate which extends, three-dimensionally, out of the surface of the vesicle. The compounds prepared by the methods of the invention can also find use as diagnostic reagents. For example, labeled compounds can be used to localize areas of inflammation or tumor metastasis in a patient suspected of having inflammation. For this use, the compounds can be labeled with 1125I, 14C, or tritium. The following examples are provided to illustrate the conjugates, and methods of the present invention, but not to limit the claimed invention.

EXAMPLES EXAMPLE 1 1.1a Preparation of Interferon alfa-2β-GalNAc (pH 6.2) Interferon alfa-2β was reconstituted by addition of 200 μL of water to 4 mg of IFN alfa-2β. When the solid dissolved, 1.92 mL of reaction buffer (20 mM MES, pH 6.2, 150 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 0.05% polysorbate, and 0.05% NaN3), dissolve . Then the UDP-GalNAc (4.16 mg, 3 mM) and GalNAc T2 (80 μl, 80 μL) were added and the reaction mixture was incubated at 32 ° C with slow rotary motion. The reaction was monitored using MALDI analysis and was essentially complete after 72 h. Once complete, the reaction mixture was subjected to peptide mapping and site occupancy analysis. 1. 1b Preparation of Interferon alfa-2ß-GalNAc (pH 7.4) Interferon alfa-2β was reconstituted as described by the manufacturer. Water, 50 μL, was added to 50 mg of IFN alpha-2β. When the solid dissolved, the reaction buffer (20 mM MES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 0.05% polysorbate, and 0.05% NaN3), 50 μL they added. Then the UDP-GalNAc (100 μg, 3 mM) and GalNAc T2 (8 mU, 8 μL) were added and the reaction mixture was incubated at 32 ° C under a slow rotary motion. The reaction was monitored using MALDI analysis and found to be complete within about 48 to 72 h. 1. 2 Preparation of Interferon-alpha-2ß-GalNAc-SA-PEG-20kilodalton using CMP-SA-PEG and ST6GalNAcI IFN-alpha-2β-GalNAc (1.0 mL, ~ 2mg, 0.1 μmol) of 1.1 (above) was exchanged for buffer (2x) using a cartridge MWCO Filter Centricon 5 kilodalton and a second buffer (20 mM MES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 0.05% of polysorbate, and 0.05% of NaN3). He IFN-alf a-2 ß-GalNAc was reconstituted from the spin cartridge using the second buffer, 1.0 mL, and both the CMP-SA-PEG-20kilodalton (10 mg, 0.5 micromoles) and the ST6GalNAcl (200 μL) were added to the reaction mixture. The reaction was incubated at 32 ° C for 96 h with slow rotary motion. The product, IFN-alpha-2β-GalNAc-SA-PEG-20-kilodalton was purified using SP Sepharose chromatography and SEC (Superdex 75). The addition of sialic acid-PEG was verified using the MALDI analysis. 1. 3 Preparation of Interferon-alpha-2β-GalNAc-SA-PEG-20kilodalton using CMP-SA-PEG, core-l-ßl, 3-galactosyl-transferase, and ST3Gal2 IFN-alpha-2β-GalNAc (1.0 mL, ~2 mg, 0.1 μmol) of the addition of GalNAc described above (pH 6.2) was exchanged for buffer solution (2x) using a 5 kilodalton MWCO Filter Centricon and a second buffer (20 mM MES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 0.05% polysorbate, and 0.05% NaN3 ). IFN-alpha-2β-GalNAc was reconstituted from the spin cartridge using 1.0 mL of the second buffer, containing CMP-SA-PEG-20-Kilodalton (10 mg, 0.5 micromoles), UDP-Galactose (1.8 mg, 3 mM) ), core-l-ßl, 3-galactosyl-transferase (200 U in resin) and ST3Gal2 (200 mU, a2, 3- (O) -sialyltransferase). The reaction mixture was incubated at 32 ° C for 96 h with slow rotary motion. The product, IFN-alpha-2β-GalNAc-SA-PEG-20-Kilodalton, was purified by SP Sepharose and SEC (Superdex 75) chromatography. The addition of sialic acid-PEG was verified using the MALDI analysis. 1. 9 Protein Concentration Assay The protein concentration was determined using a spectrophotometer at a fixed absorbance of 280 nm with a cell path length of 1 cm. The tripled readings were measured for a sample evaluated with water or buffer as controls. The protein concentration was determined using the extinction coefficient at 0.799 mL / mg protein. 1. 10 Final Product Formulation. The buffer solution of the formulation contained pyrogen-free PBS, pH 6.5, 2.5% mannitol, and 0.05% Polysorbate 80 which was vacuum degassed and sterile filtrate (0.2 μm). The endotoxin was removed using a Detoxi-Gel ™ equilibrated with 5 column beds of the buffer solution of the formulation (PBS, pH 6.5, 2.5% mannitol, and 0.05% Polysorbate 80). The flow rate was controlled by gravity at ~ 0.3 mL / min. The product samples were applied on the gel, and. the product was eluted using the buffer solution of the formulation. The volume of the collected product was adjusted with additional formulation buffer to provide a protein concentration of about 100 μg / mL. Peptide formulations were sterile filtered (0.2 μ) and the effluent was dispersed as 1 mL aliquots in 2.0 mL pirogen free flasks. In addition, aliquots were taken for endotoxin and protein analysis. All products were stored at 4 ° C. 1. 13 Pharmacokinetic study. The pharmacokinetic analysis was performed using radioiodinated protein. After administration of the interferons labeled by IV injections in the tail vein in the rats, the degree of depuration was measured as the reduction in radioactivity in the blood drained at specific intervals for 72 h. Each time point is a measurement of at least five rats. 1. 14 Results The reaction rate of GalNAc-T2 was measured at two pH, a neutral pH (7.4) and a slightly acidic pH (6.2). Glycosylation with GalNAc proceeded successfully at both pH 6.2 and pH 7.4. As can be seen in the MALDI analysis of the reaction progress, the reaction rate was faster at pH 7.4 than at pH 6.2. GalNAc-T2 and GalNAc were added to interferon alfa-2β quantitatively at any pH of 6.2 or 7.4. The reaction was followed by MALDI. During the enzymatic reaction, a new interferon alpha mass ion (IFN-alpha-2b 19,281 Da and IFN-alpha-2β-GalNAc, 19.485 Da) was formed. The product of the reaction at pH 6.2, IFN-alpha-2b-GalNAc, was subjected to analysis to determine the substitution position of GalNAc in the protein. Peptide mapping and site occupancy mapping were used for this purpose. Peptide mapping using LC-MS / MS TIC and a GluC digest of IFN-alpha-2b produced a peptide fragment of mass 1018.69. The MS / MS peptide amino acid sequencing of the peptide mass ion of 1018.69 containing GalNAc indicated that the sugar was attached to t106. The sialyl-PEGylation of IFN-alpha-2b-GalNAc was examined using ST6GalNAc-l and CMP-SA-PEG-20 kilodalton. The reaction of IFN-alpha-2b-GalNAc produced the PEG-ilated protein, which was visible by SDS PAGE. In general, the reaction proceeds at 32 ° C for 96 h. The reaction was monitored by SDS PAGE. The SDS PAGE indicated that about 70% - of IFN-alpha-2b-GalNAc was converted to IFN-alpha-2b-GalNAc-SA-PEG-20 kilodalton. The MALDI analysis of the new band indicated a mass ion of 41,500 Daltons, the mass of IFN-alpha-2b-GalNAc-SA-PEG-20 kilodalton. The glycoform of interferon alfa-2b PEG-ilada containing the GalNAc-Gal-SA-PEG structure was also produced. The reaction was carried out using the conditions described above. The desired product was detected by SDS PAGE. A two step reaction vessel was used to produce the desired product, starting with IFN-alpha-2β-GalNAc with core-l-β3-galactosyltransferase-1, ST3Gal2, UDP-galactose and CMP-SA-PEG-20 kilodalton. The reaction was incubated at 32 ° C for 96 h. The reaction was monitored by SDS PAGE. After 24 h, the reaction was around 70% complete. The MALDI of the product indicated a mass ion of 41,900 Da, which originated from the desired product IFN-alpha-2β-GalNAc-Gal-SA-PEG-20 kilodalton. Both glycoforms of PEG-γ interferon alfa-2b products were purified using a two-step process. In the first step, ion exchange chromatography was performed using Sp Sepharose. This procedure removed unreacted PEG materials and provided some separation of other proteins. The ion exchange step was followed by SEC separation. A Superdex 75 column was used to remove the smallest proteins that remained including the glycosyltransferases and the non-PEGylated alpha interferon. Both PEG-ilated glyco-forms of interferon alpha were purified to more than 90% as shown by SDS PAGE. The antiviral data indicate that the PEG-ilated glycoforms A and B retain their antiviral effects. The radioiodinated PEG-ilated proteins were injected into rats by their tail veins, the AUC for both proteins was 5-7 times larger than the non-PEG-γ-2β interferon. The glycoform A (IFN-alpha-2β-GalNAc-SA-PEG-20kilodaltons) and B (IFN-alpha-2β-GalNAc-Gal-SA-PEG-20kilodaltons) were both bioactive.

EXAMPLE 2 2.1 Preparation of G-CSF-GalNAc (pH 6.2) 960 μg of G-CSF in 3.2 mL of buffer solution was concentrated by ultrafiltration using a UF filter (5 kilodalton) and reconstituted with 1 mL of 25 mM MES buffer ( pH 6.2, 0.005% NaN3). UDP-GalNAc (6 mg, 9.24 mM), GalNAc-T2 (40 μL, 0.04 U), and 100 mM of MnCl2 (40 μL, 4 mM) were then added and the resulting solution was incubated at room temperature for 48 hours. hours. After 48 hours, the MALDI indicated that the reaction was completed (change of mass ion from 18800 to 19023 mass units).

The reaction mixture was purified by CLAP using SEC (Superdex 75 and Superdex 200). The column was eluted using phosphate buffer solution saline, pH 4.9 and 0.005% Tween 80. The peak corresponding to G-CSF-GalNAc was collected and concentrated to about 150 μL using a Centricon 5 kilodalton filter and the volume was adjusted to 1 mL using PBS (phosphate buffer solution saline, pH 4.9 and 0.005% Tween 80); the concentration of the protein was 1 mg / mL A23o) • 2. 2 Preparation of G-CSF-GalNAc (pH 6.0) The G-CSF-GalNAc (100 μg) was added to about 100 μL of solution containing 25 mM of MES buffer, pH 6.0, 1.5 mM of UDP-GalNAc, 10 mM of MgCl2 and 80 mU of GalNAc-T2. The CMP-SA-PEG-20 kilodalton (0.5 mg, 0.025 μmole), UDP-galactose 75 μg (0.125 μmole), core-1-Gal-T 20 μL (10 μü) were then added and the solution which was in Slowly move to 32 ° C for 48 hours. MALDI indicated complete conversion of G-CSF-GalNAc into G-CCSF-GalNAc-Gal. 2. 3 Preparation of G-CSF-GalNAc-SA-PEG-20 kilodalton (C). 2. 3rd Sequential Process (pH 6. 2). A solution of G-CSF-GalNAc containing 1 mg of protein was exchanged from buffer in 25 mM MES buffer (pH 6.2, 0.005% NaN3) then 5 mg, (0.25 μmol) CPM-SA-PEG (20 kilodalton) were added. Finally, 100 μL of a solution of 100 mM MnCl2 and ST6GalNAc-I (100 μL) were added and the reaction mixture was slowly moving at 32 ° C. The aliquots were taken at moments (24, 48 and 72) and analyzed by SDS PAGE. After 24 h, no further reaction was observed. The reaction mixture was concentrated by spin filtration (5 kilodalton), the buffer was again exchanged 25 mM NaOAc (pH 4.9) and concentrated to 1 mL. The product was purified using ion exchange (SP-Sepharose, 25 mM NaOAc, pH 4.9) and SEC (Superdex 75; PBS-pH 7.2, 0.005% tween 80, 1 ml / min). The desired fraction was collected, concentrated to 0.5 mL and stored at 4 ° C. 2. 3b Process of a container using ST6GalNAc-l (pH 6. 0) 960 μg of G-CSF protein dissolved in 3.2 mL of product formulation buffer solution was concentrated by spin filtration (5 kilodalton) to 0.5 mL and reconstituted in 25 mM of MES buffer (pH 6.0, 0.005% NaN3) for a total volume of about 1 mL, or a protein concentration of 1 mg / mL. After reconstitution, UDP-GalNAc (6 mg, 9.21 μmol), GalNAc-T2 (80 μL, 80 mU), CPM-SA-PEG-20 kilodalton (6 mg, 0.3 μmol) and mouse enzyme ST6GalNAc-I were added. (120 μL). The solution was in motion at 32 ° C for 48 hours. After the reaction the product was purified using standard chromatography conditions on SP-Sepharose and SEC as described above. A total of 0.5 mg of protein (A28o) was obtained, for approximately an overall yield of 50%. The structure of the product was confirmed by analysis with both MALDI and SDS-PAGE. 2. 4 Preparation of G-CSF-GalNAc-SA-PEG-20 kilodalton (D). 2. 4th Start from G-CSF-GalNAc UDP-galactose (4 mg, 6.5 μmole), core-1-Gal-T? (320 μL, 160 mU), CPM-SA-PEG-20 kilodalton (8 mg, 0.4 μmole), ST3Gal2 (80 μL, 0.07 mU) and 80 μL of 100 mM MnCl2 were added directly to the crude 1.5 mL of reaction mixture of G-CSF-GalNAc (1.5 mg) in 25 mM MES buffer (pH 6.0) of Example 2.1 (above). The resulting mixture was incubated at 32 ° C for 60 hours, however, the reaction was completed after 24 h. The reaction mixture was centrifuged and the solution was concentrated to 0.2 mL using ultrafiltration (5 kilodalton) and then redissolved in 25 mM NaOAc (pH 4.5) to a volume of 1 mL. The product was purified using SP-Sepharose, the peak fractions were concentrated using a spin filter (5 kilodalton) and the purified residue using also SEC (Superdex 75). After concentration using a spin filter (5 kilodalton), the protein was diluted to 1 mL using formulation buffer consisting of PBS, 2.5% mannitol, 0.005% polysorbate, pH 6.5, and formulated at a protein concentration of 850 μg of protein per mL (A28o). The overall performance was 55%. The MALDI analysis is shown in FIG 28. 2. 4 Starting from G-CSF 960 μg of G-CSF (3.2 mL) were concentrated by spin filter (5 kilodalton) and reconstituted with 25 mM of MES buffer (pH 6.0, 0.005% NaN3). The total volume of the G-CSF solution was adjusted to around 1 mg / mL and UDP-GalNAc (6 mg), GalNAc-T2 (80 μL), UDP-galactose (6 mg), core-1-Gal were added. -T? (160 μL, 80 μü), CPM-SA-PEG (20 kilodalton) (6 mg), ST3Gal2 (160 μL, 120 μü) and MnCl2 (40 μL of a 100 mM solution). The resulting mixture was incubated at 32 ° C for 48 h. 2. 5 CLAP chromatography of SP Sepharose SP Sepharose was performed as described in Example 1.4. 2. 6 Size Exclusion Chromatography The SEC was performed as described in Example 1.5. The purified samples were stored at 4 ° C. 2. 6a Hydrophobic Interaction Chromatography (HIC) After the first step of chromatographic chromatography, HIC can be used as a second purification step to remove contaminants other than non-Pegylated G-CSF. Thus, a method is available for the purification of glycopegylated G-CSF which has been an initial purification through a gel permeation column. 2. 7 Analysis of SDS PAGE The SDS PAGE was performed as set forth in Example 1.6. 2. 8 MALDI analysis The MALDI analysis was performed as described in Example 1.7. 2.9 Peptide Mapping Analysis The protein mapping analysis was performed as illustrated in Example 1.8 2.10 Protein Concentration Assay. The protein concentration was determined as described in Example 1.9. 2.11 Product Formulation The product was formulated as set forth in Example 1.10 2.12 Determination of? Notoxin Endotoxin was determined as set forth in Example 1.11. 2. 13 Cell Proliferation Assay A G-CSF proliferation assay with an NFS-60 cell line and a Tf-1 cell line was performed according to standard procedures. The cells were plated in a 96-well plate at 25,000 cells / ml in the presence of different concentrations of G-CSF (51 nM, 25.5 nM, 12.75 nM, 3.2 nM, 1.6 nM, 0.8 nM, 0 nM), an analogue of G-CSF PEG-γ chemically, and PEGylated G-CSF C of Example 2.3 (above), and PEGylated G-CSF D of Example 2.4 (above). The cells were incubated at 37 ° C for 48 hours. A colorimetric MTT assay was used to determine the viability of the cells 2.14 In Vivo Activity: Production of White Blood Cells (WBC) in rats Two doses of drug (50 μg / kg, 250 μg / kg) were analyzed for Each of C, G-CSF and a G-CSF chemically PEGylated using mice were bled at different times of 2 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours and 96 hours. hours, and WBC and neutrophil counts were measured (FIG 4). 2. 15 Accelerated Stability Study An accelerated stability study of PEGylated G-CSF, C, of Example 2.3, and PEGylated G-CSF, D, of the Example 2.4, using a buffer solution at pH 8.0 heated to 40 ° C. 72 μg of PEGylated G-CSF C, were diluted to 8 mL with formulation buffer (PBS, 2.5% mannitol, 0.005% polysorbate 80). 1 mg of PEGylated G-CSF D was diluted with 16 mL of formulation buffer.

Both solutions were adjusted to pH 8.0 with NaOH and the resulting solution was filtered sterile in pyrogen-free tubes. The samples were slowly rotated at 40 ° C and the aliquots (0.8 mL) were taken at 0 hour and 168 hour moments. The analysis was performed using SEC (Superdex 200) as described above (FIG 6 and FIG 7). 2. 16 Protein radiolabelling. The G-CSF was radiolabelled using the Bolton Hunter reagent. This reaction was carried out at a pH of 7.4 for 15 minutes and was followed by a SEC purification (Superdex 200). Once purified, the pH of the formulation buffer was adjusted to 5.0 and the protein concentration is determined by A28o- 2. 17 ELISA Assay An ELISA assay was used to quantitate G-CSF derivatives in rat plasma. The pharmacokinetic results are shown in FIG. 9. 2. 18 Pharmacokinetic study. Two pharmacokinetic studies were performed. For the first pharmacokinetic study, the proteins were radiolabelled and administered by IV tail vein injections in rats. The degree of depuration was measured as the reduction in radioactivity in the blood drained at specific intervals over 48 hours. Each time interval was a measurement of at least five rats. Specifically, 10 μg of G-CSF derivative was injected per animal (~ 1 μg of labeled protein and 9 μg of unlabeled protein). In addition to the blood that is extracted and counted as described above, the plasma was also collected and the protein acid precipitated. The protein pellets were then counted for ~ -5 radioactivity. The data of these studies are shown in FIG. 2, FIG. 3 and FIG. 8. In the second pharmacokinetic study, non-labeled G-CSF derivatives (30 μg per animal) were administered by injections into the IV tail vein in rats. The samples Blood samples were collected at the indicated time intervals and samples analyzed by the G-CSF ELISA assay. The data is shown in FIG: 9. 2. 19 Results 15 The human T2 GalNAc transferred the GalNAc to G-CSF expressed in E. coli using UDP-GalNAc as the donor. Depending on the pH of the reaction buffer, one or two portions of GalNAc were added to the G-CSF as determined by MALDI. The addition of the second GalNAc proceeded slowly, equivalent to around 10-15% of the total product. A GalNAc could be selectively added to G-CSF, in conversion efficiencies of more than 90%, by adjusting the pH of the reaction solution to 6.0-6.2. The addition of the second GalNAc occurred when the reaction was performed at a pH of between about 7.2 and 7.4. Both Co + 2 and Mn + 2 are divalent metal ions useful in the reaction. Peptide mapping of the reaction products indicated that the predominant product of the reaction was the addition of GalNAc to threonine-133. the natural glycosylation site linked to 0 in mammalian systems. The second GalNAc was observed in the amino terminal peptide fragment of G-CSF and is postulated to occur in threonine-2. The reaction of G-CSF-GalNAc with ST6GalNAc-l (chicken or mouse) and CMP-SA-PEG-20kilodalton yielded the product G-CSF-GalNAc-SA-PEG-20 kilodalton, which was verified by MALDI, with Conversion yields of around 50% as determined by SDS-PAGE. The G-CSF-GalNAc could also be further elongated using core-1-Gal-T and UDP-galactose to provide complete conversion to G-CSF-GalNAc-Gal. The glyco-PEGylation of this intermediate with ST3Gal2 and CMP-SA-PEG-20kilodalton then gave the product G-CSF-GalNAc-Gal-SA-PEG-20 kilodalton in general yields of about 50%. These reactions were performed either sequentially in a container or simultaneously in a starting container of G-CSF or its glycosylated intermediates. In these studies, little or no difference was observed in the overall performance by the use of any approach. The products of the glycosylation reactions or glyco-PEGylation were purified using a combination of ion exchange and SEC. The ion exchange step removes the unreacted G-CSF or its glycosylated intermediates (GalNAc or GalNAc-Gal) in addition to "any unreacted CMP-SA-PEG-20 kilodalton." The SEC step removed the unreacted G-CSF remaining and Other protein contaminants of the glycosyltransferases used in the process The G-CSF containing the GalNAc-SA-PEG-20 kilodalton or the GalNAc-Gal-SA-PEG-20 kilodalton had identical properties and retention times using these methods. The final products had typical profiles as shown., PEG-ilated proteins were formulated in a buffer solution of PBS containing 2.5% mannitol and 0.005% Tween 80. Initially, pH 6.5 was used in the formulation but the accumulation of the glyco-PEG-ylated protein was a concern (see below) such that the pH of the buffer solution of the formulation was lowered to 5.0. Literature reports have indicated that the accumulation of G-CSF is prevented by maintaining a solution pH between 4-5. The endotoxin was removed using an endotoxin removal cartridge using sterile technique. Protein concentrations were commonly adjusted to concentrations between 100 μg / mL to 1 mg / mL as required for biological studies. The endotoxin calculations were usually below 3EU / ml through this process. The formulated products were stored at 4.

The products were evaluated - in an in vitro cell proliferation assay using NSF-60 cells sensitive to G-CSF. It was observed that both GalNAc-SA-PEG-20 kilodalton products and GalNAc-Gal-SA-PEG-20 kilodalton were effective in the initiation of cell proliferation (FIG 1). An accelerated stability study was performed on a PEG-chemically-gated G-CSF and C (G-CSF-GalNAc-SA-PEG-20 kilodalton). The pH of the buffer solution of the formulation was adjusted to 8.0 and the temperature was raised to 40 ° C. Samples were taken from each protein at time intervals of 0, 72 and 168 hours (FIG 6 and FIG 7). Chemically PEGylated G-CSF was observed to fully aggregate under these conditions within 168 h. The SEC using a Superdex 200 chromatography was used to separate the aggregates, the G-CSF-GalNAc-SA-PEG-20 kilodalton glucoconjugate also formed aggregates that were separable using SEC, • the accumulation occurred at a much slower rate. The glyco-PEG-ylated G-CSF was radioiodinated using the Bolton Hunter reagent. A cold labeling study was also carried out before the radio-labeling of the moment determined the extension of the aggregate and to stabilize a methodology for the removal of any aggregate formed. The use of the Bolton Hunter reagent (cold) provided some aggregates as shown in FIG. 5. The SEC using a Superdex 200 column removed the aggregates and provided the monomeric labeled material. Similar results were obtained using 125I labeled reagent. The use of the formulation minimized storage accumulation. The protein content was measured by absorbance measurement in A28o- The results of the rat pK study incorporating G-CSF, PEG-chemically bonded G-CSF and the PEG-G-CSF conjugate labeled with the Bolton Hunter reagent were measured. show in FIG. 3. In this study, the blood and protein precipitated from the plasma were counted for radioactivity after IV administration of 10 μg of G-CSF conjugate per rat. The data of both proteins in blood and plasma clearly indicated that the PEG conjugate and the PEG-chemically yielded G-CSF have identical degrees of clarity (FIG 3 and FIG 8). The ability of G-CSF derivatives to initiate WBC production was then examined in a mouse model. Each test compound was injected IV as a single bolus and the induction of WBC and neutrophils was monitored over time. The PEG-chemically chelated G-CSF was. the most potent protein evaluated when administered at 250 μg / kg. The P'EG conjugate (G-CSF-GalNAc-SA-PEG-20 kilodalton) induced the production of WBC to almost the same degree as G-CSF PEG-chemically yielded at 250 μg / kg, and considerably larger than G - CSF in a similar concentration.

EXAMPLE 3 This example describes amino acid sequence mutations that induce changes introduce glycosylation sites linked to 0, that is, serine or threonine residues, in a site preferably containing proline in the wild type sequence of amino acid 175 of G-CSF or any version of this modified. As a reference the sequence G-CSF wild type amino acid 175 shown below: MTPLGPASSLP QSFLLKCLEQ VRKIQGDGAA LQEKLCA TYKLCHPEEL VLLGHSLGIP WAPLSSCPSQ ALQLAGCLSQ LHSGLFLYQG LLQALEGISP ELGPTLDTLQ LDVADFATTI WQQMEELGMA PALQPTQGAM PAFASAFQRR AGGVLVASHL QSFLEVSYRV LRHLAQP (SEQ ID NO: 2) 3. 1 N-terminal mutations In the N-terminal mutants, the N-terminus of a wild-type G-CSF, M1TPLGPA (SEQ ID NO :), is replaced with either M ^ nTPLGPA or M ^? OPZmXnTPLGPA. Where n, o and m are integers selected from 0 to 3, and at least one of X, B and 0 is Thr or Ser. When more than one of X, B and 0 is Thr or Ser, the identity of these portions is select independently. When they appear, the superscripts denote the position of the amino acid in the wild type start sequence. Preferred examples include: M1VTPL4GPA (SEQ ID NO :) MXQTPL4GPA (SEQ ID NO :) 1ATPL4GPA (SEQ ID NO :) M1PTQGAMPL4GPA (SEQ ID NO :) MXVQTPL4GPA (SEQ ID NO :) 1QSTPL4GPA (SEQ ID NO :) M1GQTPL4GPA (SEQ ID NO :) M1APTSSSPL4GPA (SEQ ID NO :) M1APTPL4GPA (SEQ ID NO :) 3. 2 Internal Mutation Site 1 In these mutants, the N-terminus of a wild-type GCSF, M ^ PLGP (SEQ ID NO: 8), is replaced with M ^ PXnBoOrP. Where n, o and r are integers selected from 0 to 3, and at least one of X, B and O is Thr or Ser. When more than one of X, B and O is Thr or Ser, the identity of these portions is select independently. When superscripts appear, the position of the amino acid in the wild type start sequence is denoted. Preferred mutations include: MXTPTLGP (SEQ ID NO: 8) M1TPTQLGP (SEQ ID NO: 8) M1TPTSLGP (SEQ ID NO: 8) M1TPTQGP (SEQ ID NO: 8) XTPTSSP (SEQ ID NO: 8) M1TPQTP (SEQ ID NO : 8) MXTPTGP (SEQ ID NO: 8) M ^ -TPLTP (SEQ ID NO: 8) M ^ PNTGP (SEQ ID NO: 8) M ^ PVTP (SEQ ID NO: 8) M ^ -TPMVTP (SEQ ID NO : 8) MT1P2TQGL3G4P5AdS7 (SEQ ID NO: 8) 3. 3 Internal Mutation Site 2 This mutation is made for the purpose of maintaining G-CSF activity- In these mutants, the amino acid sequence containing H53, LGH53, SLGI (SEQ ID NO :) is mutated LGH53B0LGI, where T is H, S, R, E or Y, and B is any of Thr or Ser. Preferred examples include: LGHTLGI LGSSLGI LGYSLGI LGESLGI LGSTLGI 3.4 Internal Mutation Site 3 In this type of mutant, the amino acid sequence spanning P129, P129ALQPT (SEQ ID NO :), is mutated to P129ZmJqOrXnPT, where Z, J, 0 and X are independently selected from Thr or Ser , and m, q, r and n are integers selected from 0 to 3. Preferred examples include: P129TLGPT P129TQGPT P129TSSPT P129TQGAPT P129NTGPT P129ALTPT P129MVTPT P129ASSTPT P129TTQP P129NTLP P129TLQP MAP129ATQPTQGAM MP129ATTQPTQGAM 3. 5 Internal Mutation Site 4 In this type of mutant, the amino acid sequence surrounding P61, LGIPWAP61LSSC (SEQ ID NO :), is replaced with PZmUsJqP610rXnBoC, where m, s, q, r, nyo are integers selected from 0 to 3, and at least one of Z, J, O, X, B and U is selected as either Thr or Ser. When more than one of Z, J, 0, X, B and U are Thr or Ser, each one is independently selected. Preferred examples include: P61TSSC P61TSSAC LGIPTA P61LSSC LGIPTQ P61LSSC LGIPTQG 'P61LSSC LGIPQT P61LSSC LGIPTS P61LSSC LGIPTS P61LSSC LGIPTQP61LSSC LGTPWAP61LSSC LGTPFA P61LSSC P61FTP SLGAP58TAP61LSS 3 . 6 Mutations at the C-terminus In this type of mutant, the amino acid sequence at the C-terminus of a wild-type G-CSF, RHLAQP175 (SEQ ID NO:) is replaced with 0aGpJqOrP175XnBoZmUs? T, where a, p, q, r, n, o, m, s, and t are integers selected from 0 to 3, and at least one of Z, U, 0, J, G, 0, B and X is Thr or Ser and when more than one of Z, U, 0, J, G, 0, B and X are Thr or Being, they are selected independently. 0 optionally is R, and G is optionally H. The symbol ? represents any unloaded amino acid residue or E (glutamate). Preferred examples include: RHLAQTP175 RHLAGQTP175 QP175TQQGAMP RHLAQTP175AM QP175TSSAP QP175TSSAP QP175TQGAMP QP175TQGAM QP175TQGA QP175TVM QP175NTGP QP175QTLP 3. 7 Internal Mutations Surrounding P133 Additional G-CSF mutants include those with internal mutations surrounding the amino acid P133. Examples include: P133TQTAMP139 P133TQGTMP P133TQGTNP P133TQGTLP PALQP133TQTAMPA EXAMPLE 4 Mutations in the amino acid sequence of granulocyte colony stimulation factor (G-CSF) can introduce additional sites for 0-linked glycosylation, such that the protein can be modified at these sites using the method of the present invention. This example establishes selected representative mutants of the invention. 4.1 G-CSF (variant 178 aa wild type) mtplgpasslp qsfllkcleq vrkiqgdgaa Iqeklvseca tyklchpeel vllghslgip waplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlq Idvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp (SEQ ID N0: 1) 4. 2 G-CSF (variant 175 aa wild type) mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklca tyklchpeel vllghslgip waplsscpsq alqlagclsq Ihsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp (SEQ ID NO: 3) 4.9 G-CSF Mutant 1 (Terminal Mutation Amino) miatplgpasslp qsfllkcleq vrkiqgdgaa lqeklcatyk Ichpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp 4. 10 G-CSF Mutant 2 (Amino Terminal Mutation) mgvtetplgpasslp qsfllkcleq vrkiqgdgaa Iqeklcatyk Ichpeelvll ghslgipwap Isscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp 4. 11 G-CSF Mutant 3 (Amino Terminal Mutation) maptplgpasslp qsfllkcleq vrkiqgdgaa Iqeklcatyk Ichpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp 4. 12 G-CSF Mutant 4 (Site 1) mtp3tqglgpasslp qsfllkcleq vrkiqgdgaa Iqeklcatyk Ichpeelvll ghslgipwap Isscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp 4. 13 G-CSF Mutant 5 (Site 3) Mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklcatyk Ichpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmap12at qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp 4. 14 G-CSF mutant 6 (Site 4) Mtplgpasslp qsfllkcleq vrkiqgdgaa Iqeklcatyk Ichpeelvll ghslgip58ftp lsscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmapaL qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp EXAMPLE 5 GlicoPEGilación of G-CSF produced in CHO cells on the 5th. Preparation of Cologne Stimulation Factor Asialo-Granulocytes (G-CSF) The G-CSF produced in the CHO cells was dissolved in 2.5 mg / mL in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl2 and concentrated at 500 μL in a Centricon Plus 20 centrifugal filter. The solution was incubated with 300 mU / mL of Neuraminidase II. { Vibrio cholerae) for 16 hours at 32 ° C. To monitor the reaction a small aliquot of the reaction was diluted with the appropriate buffer and an IEF gel made. The reaction mixture was then added to prewashed N- (p-aminophenyl) oxamic acid agarose conjugate (800 μL / mL reaction volume) and the washed beads rotated gently for 24 hours at 4 ° C. The mixture was centrifuged at 10,000 rpm and the supernatant was collected. The beads were washed 3 times with Tris EDTA buffer, once with 0.4 mL of Tris EDTA buffer and once with 0.2 mL of the Tris EDTA buffer and all supernatants were accumulated. The supernatant was dialyzed at 4 ° C again against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN3 and then twice more against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN3. The dialyzed solution was then concentrated using a Centricon Plus 20 centrifugal filter and stored at -20 ° C. The conditions for the IEF gel were run according to the procedures and reagents provided by Invitrogen. Samples of the native and desialylated G-CSF were dialyzed against water and analyzed by MALDI-TOF MS. 5b. Preparation of G-CSF- (alpha2,3) -Sialyl-PEG The desialylated G-CSF was dissolved in 2.5 mg / mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3, pH 7.2. The solution was incubated with 1 mM of CMP-sialic acid-PEG and 0.1 U / mL of ST3Gall at 32 ° C for 2 days. To monitor the incorporation of sialic acid-PEG, a small aliquot of the reaction has fluorescent ligand of aggregated CMP-SA-PEG; The label incorporated in the peptide was separated from the free label by gel filtration on a Tosa Haas G3000SW analytical column using PBS buffer (pH 7.1). The incorporation of the fluorescent tag in the peptide was quantified using an in-line fluorescent detector. After 2 days, the reaction mixture was purified using a Toso Haas G3000SW preparative column using PBS buffer (pH 7.1) and collection fractions' based on UV absorption. The product of the reaction was analyzed using SDS-PAGE and IEF analysis according to the procedures and reagents supplied by Invitrogen. Samples of native and PEGylated G-CSF were dialyzed against water and analyzed by MALDI-TOF MS. 5c. Preparation of G-CSF- (alpha2, 8) -Sialyl-PEG The G-CSF produced in CHO cells, which contains a glycan linked to 0 alpha 2, 3-sialylated, was dissolved in 2.5 mg / mL in 50 mM of Tris -HCl, 0.15 M NaCl, 0.05% NaN3, pH 7.2. The solution was incubated with 1 mM of CMP-sialic acid-PEG and 0.1 U / mL of CST-II at 32 ° C for 2 days. To monitor the incorporation of sialic acid-PEG, a small aliquot of the reaction has fluorescent ligand of CMP-SA-PEG added; the label incorporated in the peptide was separated from the free label by gel filtration on a Tosa Haas G3000SW analytical column using PBS buffer (pH 7.1). The incorporation of the fluorescent tag in the peptide was quantified using an in-line fluorescent detector. After 2 days, the reaction mixture was purified using a Toso Haas G3000SW preparative column using PBS buffer (pH 7.1) and the collection fractions based on UV absorption. The product of the reaction was analyzed using SDS-PAGE and IEF analysis according to the procedures and reagents supplied by Invitrogen. Samples of native and PEGylated G-CSF were dialyzed against water and analyzed by MALDI-TOF MS. d. Preparation of G-CSF- (alpha2, 6) -Sialyl-PEG The G-CSF, contains only GalNAc bound to 0, was dissolved in 2/5 mg / mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3, pH 7.2. The solution was incubated with 1 mM of CMP-sialic acid-PEG and 0.1 U / mL of ST6GalNAcI or II at 32 ° C for 2 days. To monitor the incorporation of acid. Sialic-PEG, a small aliquot of the reaction has a fluorescent ligand. of CMP-SA-PEG added; The label incorporated in the peptide was separated from the free label by gel filtration on a Tosa Haas G3000SW analytical column using PBS buffer (pH 7.1). The incorporation of the fluorescent tag in the peptide was quantified using an in-line fluorescent detector. After 2 days, the reaction mixture was purified using a Toso Haas G3000SW preparative column using PBS buffer (pH 7.1) and the collection fractions based on UV absorption. The product of the reaction was analyzed using SDS-PAGE and IEF analysis according to the procedures and reagents supplied by Invitrogen. Samples of native and PEGylated G-CSF were dialyzed against water and analyzed by MALDI-TOF MS. The G-CSF produced in the CHO cells was treated with Arthrobacter sialidase and then purified by size exclusion in Superdex 75 and treated with ST3Gall or ST3Gal2 and then with CMP-SA-PEG 20 Kda. The resulting molecule was purified by ion exchange and gel filtration and analysis by SDS PAGE showed that PEGylation was complete. This was the first demonstration of the glycoPEGylation of a glycan ligand 0. EXAMPLE 6 Expression, refolding and purification of recombinant GCSF. Cell harvest by centrifugation, supernatant waste. The growth results of various media are shown in Figure 9. Resuspension of cell pellets in c-use 10 ml / g (lysis buffer) Microlized cell (French press Works as well) Centrifugation for 30 min, 4 ° C to 5,000 RPM-supernatant waste. Supernatant resuspended in lysis buffer and centrifuged as above. Wash the IBs in 25 mM Tris pH 8, 100 mM NaCl, 1% NaDOC, 5 mM EDTA. The pellets are resuspended by pipetting and vortices. Centrifugation 15 min at 4 ° C and 5,000 RPM. Repeat this step once more (total of two washes). Pellets washed twice in 25 mM Tris pH 8, 100 mM NaCl, 5 mM EDTA to remove detergents, centrifugation as previously Pelleted resuspended in dH20 for aliquot and centrifuged as above. The pellets are frozen at -20 ° C.

IBs are re-suspended at 20 mg / ml in 6M guanidine HCl, 5 mM EDTA, 100 mM NaCl, 100 mM tris pH 8, 10 mM DTT using a pipettor, followed by rotation for 2-4 h at RT ambient. Centrifugation of IB for 1 min at room temperature and 14,000 RPM. Save supernatant. Dilute supernatant 1:20 with 50 mM MES pH6 quench buffer, 240 mM NaCl, 10 mM KCl, 0.3 mM lauryl maltoside, 0.055% PEG3350, lmM GSH, 0. M GSSG, 0.5 M arginine and Rotation in the rotator overnight at 4 ° C. Transfer retraction to snake skin Pierce 7Kda MWCO for dialysis. Dialysis of 20 mM buffer NaOAc pH4, 50 mM NaCl, 0.005% Tween-80, 0.1 mM EDTA. Dialyse a total of 3 times against at least one excess 200 multiples at 4 ° C. After dialysis pass material through a 0.45 μM filter. Balance column SP-sepharose with the dialysis buffer and apply sample. Wash column with dialysis buffer and flow with dialysis buffer containing a salt gradient up to 1M NaCl. The protein is commonly diluted to 300-400 mM NaCl. Verify material in SDS-PAGE (see for example, Figure 10).

EXAMPLE 7 The Two Methods of Enzyme in Two Vessels. The following example illustrates the preparation of G-CSF-GalNAc-SA-PEG in two sequential steps wherein each intermediate is purified before it is used in the next step. 7a. Preparation of G-CSF-GalNAc (pH 6.2) from G-CSF and UDP-GalNAc using GalNAc-T2. The G-CSF (960 μg) in 3.2 mL of packed buffer was concentrated by ultrafiltration using a UF filter (MWCO 5K) and then reconstituted with 1 mL of 25 mM MES buffer (pH 6.2, 0.005% NaN3). UDP-GalNAc (6 mg, 9.24 mM), GalNAc-T2 (40 μL, 0.04 U), and 100 mM of MnCl2 (40 μL, 4 mM) were then added and the resulting solution was incubated at room temperature. After 24 hrs, the MALDI indicated that the reaction was complete. The reaction mixture was subjected directly to purification by CLAP using SEC (Superdex 75 and Superdex 200) and an elution buffer comprising PBS (phosphate buffer solution, pH 4.9 and 0.005% Tween 80). The collected peak of G-CSF-GalNAc was concentrated using a Centricon 5 Kda MWCO filter to about 150 μL and the volume was adjusted to 1 ml using PBS (phosphate buffer solution saline, pH 4.9 and 0.005% Tween 80). Concentration of the final protein 1 mg / ml (A28o), 100% yield. The sample was stored at 4 ° C. 7b. Preparation of G-CSF-GalNAc-SA-PEG using G-CSF-GalNAc, CMP-SA-PEG (20 KDa) and mouse ST6GalNAc-TI (pH 6.2). The G-CSF-GalNAc solution containing 1 mg of protein was exchanged from buffer in 25 mM MES buffer (pH 6.2, 0.005% NaN3) and added CMP-SA-PEG (20KDa) (5 mg, 0.25 umol). After dissolution, the MnCl2 (100 mcL, 100 mM solution) and ST6GalNAc-I (100 mcL, mouse enzyme) was added and the reaction mixture moved slowly at 32 ° C for three days. The reaction mixture was concentrated by ultrafiltration (MWCO 5K) and the buffer solution exchanged with 25 mM 'of NaOAc (pH 4.9) once and then concentrated to 1 mL of the total volume. The product was then purified using SP-sepharose (A: 25 mM NaOAc + 0.005% tween-80 pH 4.5, B: 25 mM NaOAc + 0.005% tween-80 pH 4.5 + 2M NaCl) at retention time of 13-18 minutes and SEC (Superdex 75; PBS-pH 7.2, 0.005% Tween 80) at retention time of 8.6 minutes (superdex 75, flow of 1 ml / min). The desired fractions were collected, concentrated to 0.5 mL and stored at 4 ° C.

EXAMPLE 8 Method in a Vessel to Produce G-CSF-GalNAc-SA-PEG with Simultaneous Addition of Enzymes. The following example illustrates the preparation of G-CSF-GalNAc-SA-PEG in a container using simultaneous addition of enzymes. 8a. One-vessel process using mouse ST6GalNAc-I (pH 6.0) The G-CSF (960 μg of protein dissolved in 3.2 ml of the product formulation buffer) was concentrated by ultrafiltration (MWCO 5K) to 0.5 ml and reconstituted with 25 mM of MES buffer (pH 6.0, 0.005% NaN3) to a total volume of about 1 mL or a protein concentration of 1 mg / mL. UDP-GalNAc (6 mg, 9.21 μmol), GalNAc-T2 (80 μL, 80 mU), CMP-SA-PEG (20 KDa) (6 mg, 0.3 μmol) and ST6GalNAc-I mouse enzyme (120 μL) and 100 mM MnCl2 (50 μL) were then added. The solution was in motion at 32 ° C for 48 hours and purified using standard chromatography conditions in SP-Sepharose. A total of 0.5 mg of protein (A28o) were obtained or about 50% overall yield. The structure of the product was confirmed by analysis with MALDI and with SDS-PAGE. 8b. Process in One Vessel using chicken ST6GalNAc-I (pH 6.0) 14.4 mg of G-CSF; it was concentrated in 3 mL of final volume, the buffer solution was exchanged with 25 mM of MES buffer (pH 6.0, 0.05% of NaN3, = .004% of Tween 80) and the volume was adjusted to 13 mL. Then Udp-GalNAc (90 mg, 150 μmol), GalNAc-T2 (0.59 U), CMP-SA-PEG-20KDa (90 mg), chicken ST6GalNAc-I (0.44 U), and 100 mM MnCl2 ( 600 mcL). The resulting mixture was kept at room temperature for 60 hours. The reaction mixture was then concentrated using a UF (MWCO 5K) and centrifugation. The residue (about 2 mL) was dissolved in 25 mM NaOAc buffer (pH 4.5) and concentrated again to 5 mL of final volume. This sample was purified using SP-sepharose for about 10-23 min, SEC (Superdex75, 17 min, flow rate 0.5 ml / min) and an additional SEC (Superdex 200, 23 min, flow rate 0.5 ml / min), to produce 3.6 mg (25% overall yield) of G-CSF-GalNAc-SA-PEG-20KDa (A280 and BCA method).

EXAMPLE 9 Method in a Vessel to Produce G-CSF-GalNAc-Gal-SA-PEG with Simultaneous Addition of Enzymes. The following example illustrates a method for preparing G-CSF-GalNAc-SA-PEG in a vessel with simultaneous addition of enzymes. 9. 1 Start from GalNAc-G-CSF a. Preparation of G-CSF-GalNAc (pH 6.2) from G-CSF and UDP-GalNAC using GalNAc- 2. The G-CSF (960 mcg) in 3.2 mL of the packed buffer was concentrated by ultrafiltration using a UF filter (MWCO 5K) "and then reconstituted with 1 mL of 25 mM MES buffer (pH 6.2, 0.005% NaN3), then UDP-GalNAc (6 mg, 9.24 mM), GalNAc-T2 (40 μL, 0.04 U), and 100 mM MnCl2 (40 μL, 4mM) and the resulting solution was incubated at room temperature. b. Preparation of G-CSF-GalNAc-Gal-SA-PEG from G-CSF-GalNAc; UDP-Galactose, SA-PEG-20Kdalton, and the Appropriate Enzymes UDP-Galactose (4 mg, 6.5 μmoles), core-1-Gal-T (320 μL, 160 mU), CMP-SA-PEG-20Kda (8 mg, 0.4 μmole), ST3Gal2 (80 μL, 0.07 mU) and 100 mM MnCl2 (80 μL) were added directly to the crude reaction mixture of the G-CSF-GalNAc (1.5 mg) in 1.5 ml 25 mM MES buffer (pH 6.0) from step a, above. The resulting mixture was incubated at 32 ° C for 60 hours. The reaction mixture was centrifuged and the solution was concentrated using ultrafiltration (MWCO 5K) to 0.2 mL, and then redissolved with 25 mM NaOAc (pH 4.5) to a final volume of 1 mL. The product was purified using SP-sepharose (retention time between 10-15 min), the peak fraction was concentrated using a spin filter (MWCO 5K) and the residue purified further using SEC (Superdex 75, retention time of 10.2 min). After concentration using a spin filter (MWCO 5K), the "Protein was diluted to 1 mL using buffer solution of the formulation with PBS, 2.5% mannitol, 0.005% polysorbate, pH 6.5 and formulated at a protein concentration of 850 mcg protein per mL (A280) • The overall yield was 55%.

EXAMPLE 10 Method in a Vessel to Produce G-CSF-GalNAc-Gal-SA-PEG with Simultaneous Addition of Enzymes. to. Start from G-CSF. The G-CSF (960 mcg, 3.2 ml) was concentrated by ultrafiltration (MWCO 5K) and reconstituted with 25 mM MES buffer (pH 6.0, 0.005% NaN3). The total volume of the G-CSF solution was around 1 mg / ml. UDP-GalNAc (6 mg), GalNAc-T2 (80 μL, -80 μU), UDP-Gal (6 mg), Core 1 GalT (160 μL, 80 μU), CMP-SA-PEG (20K) were added ( 6 mg) 'and a 2, 3- (O) -sialyltransferase (160 μL, 120 μU), 100 mM MnCl2 (40 μL). The resulting mixture was incubated at 32 ° C for 48 h. The purification was performed as described below using IEX and SEC. The resulting fraction containing the product was concentrated using ultrafiltration (MWCO 5K) and the volume adjusted to about 1 mL with buffer. The concentration of the protein was determined to be 0.392 mg / ml per-A280, giving a general yield of 40% G-CSF.

EXAMPLE 11 The following example illustrates an alternative ezimatic method for obtaining large quantities of GlycoPEGylated G-CSF. The Granulocyte Colony Stimulation Factor (G-CSF) protein was expressed in E. coli and re-multiplied from inclusion bodies as described in Example X (above). lia Priming the reaction by addition of GalNAc: The GalNAc-ilation of G-CSF was carried out at 33 ° C in 50 mM Bis-Tris pH 6.5 buffer solution containing 1 mM MnCl2 using GalNAcT2 re-multiplied in the presence of UDP-GalNAc. This step primes the reaction making it possible for both the GalNAc transferase and the sialyltransferase to work together in subsequent steps to very efficiently produce maximum amounts of GCSF-PEG in a short period of time. llb. PEGylation process: PEGylation was initiated 2 (+/- 1) hour after the GalNAc-ilation by direct addition of CMP-SA-PEG (20K) and ST6GalNAcI (chicken or human) for the cessation reaction. This step produces the substrate (GCSF-O-GalNAc) so that the sialyltransferases guide the reaction faster in a shorter period of time than what can be achieved in a two-step reaction where the GCSF-O-GalNAc is purified first of the UDP-GalNAc and other reaction components (see for example, Example X, above). In addition, the reaction of a primed container produces a higher yield of product than does a reaction of a container in which all the components are added simultaneously. Indeed, the comparison of several types of reactions from a single vessel shows that when all the components were added simultaneously and incubated for 23 hours, the GCSF-PEG produced was 77%. In contrast, when the addition of all the enzymes required for the PEGylation reaction was preceded by the 2 hour GalNAc-ylation step described above, the yield of the product was 85%. Therefore, the sequential addition of the reaction components resulted in a 10% yield greater than that which was obtained when all the reaction components were added simultaneously.

EXAMPLE 12 This example describes the results of GalNAc-illation linked to 0 of six mutant G-CSF proteins. 12. 1 GalNAcsylation of mutant G-CSF protein: All sequences of the mutant G-CSF proteins are listed below. Having these proteins, glycosylation bound to O were examined. Under the same condition for glycosylation of native G-CSF, GalNAc-T2 (BV) was used in vitro with UDP-GalNAc in 25 mM MES buffer (pH 6.0). The MALDI was used to monitor the reaction. The measurement of the molecular weight increase of proteins gave the addition number of GalNAc. For an addition of GalNAc, the increased molecular weight should be 204 Da. Based on the MALDI results, we found that the G-CSF-2, -3, -4 mutants accepted a GalNAc; and the mutant G-CSF-5 some addition was also observed, and the mutant G-CSF-1 accepted two GalNAc, forming MAPT-G-CSF (GalNAc) 2 (Increasing the molecular weight from 18965 to 19369 Daltons).

Table X. Addition of GalNAc of Mutant G-CSF (PM measured by MALDI) Peptide mapping and N-terminal analysis were used for the determination of the glycosylation sites of MAPT-G-CSF- (GalNAc) 2. In the digested peptide of C Glu that maps a peak of G-1 + GalNAc was found, indicating that a GalNAc was added to the G-1 sequence. The N-terminal Edman degdation analysis suggested that the normal T was lost indicating that the GalNAc was added to the T residue. 12.2 GlycoP? Gilation of mutant G-CSF sequences. to. GlycoP? Gilation of mutant G-CSF sequences and impact of the buffer on glycoPEGylation of MAPT-G-CSF An examination of the glycoPEGylation (20K) of 5 mutants was assumed. The glycoPEGylation was performed using the three enzyme / three nucleotide system. (UDP-GalNAc / GalNAc-T2 / UDP-Gal / core GalT / CMP-SA-PEG / O-sialyltransferase) in 25 mM of MES buffer (pH 6.0). All mutants can be monoglycolylated. No appreciable diPEGilation was detected in this condition by the SDS-PEGA gel by Comassie Blue Satin. Since the MAPT-G-CSF accepts two GalNAc, this mutant should receive two PEG in theory. Consequently, we examined the impact of the buffer solution on PEGylation of MAPT-G-CSF as a starting material. Four different buffer solutions were investigated for this reason (1.1M MES buffer, 2.25 mM MES buffer (pH 6.0), 3.50 mM Bis-tris buffer solution (pH 6.0), 4. HEM buffer (pH 7.4) MAPT-G was found. -CSF can be PEGylated in all buffer systems evaluated, however, the monoPEGylation product was still a higher one.In the case where ÍM of MES buffer and 1M of HEPS buffer were used, some diPEGylation product was formed , indicating that the high concentration of buffer increases glycoPEGylation. b. Comparison of the efficiency of glyco-pEGylation by the formation of MAPT-G-CSF (GalNAc-SA-PEG) 2 and MAPT-G-CSF (GalNAc-Gal-SA-EG) 2 In order to see the efficiency of the glycoPEGylation of G-CSF-1 mutant catalyzed by different enzymes, two enzymes (StßGalNAcI and 0-sialyltransferase) for sialylPEGylation were examined. Accordingly, MAPT-G-CSF was converted to MAPT-G-CSF (GalNAc) 2 and MAPT-G-CSF (GalNAc-Gal) 2 for sialylPEGylation. The former was treated with CMP-SA-lys-PEG (20K) / St6GalNAc I and the latter was treated with CMP-SA-PEG (20K) / O-sialyltransferase. Both reactions were performed in 25 mM of MES buffer (pH 6.0) and 1 mg / ml of protein concentration. The efficiency of PEGylation could be seen on SDS-PAGE gel. It was perceived that two enzymes were almost similar in the glycoPEGylation of this protein using CMP-SA-lys-PEG (20K) under the evaluated condition. c. The high protein concentration led to the formation of MAPT-G-CSF (GalNAc-SA-PEG (20KDa)) 2 as a major product. After examining the impact of the enzyme and buffer on glycoPEGylation, as described above, the influence of protein concentration on PEGylation by combination with a high buffering concentration factor using ST6GalNAcI as a glycoPEGylation enzyme. We then applied UDP-GalNAc / GalNAc-T2 and CMP-SA-PEG (20KDa) / ST6GalNAcI for glycoPEGylation of MAPT-G-CSF using 8 ~ 10 mg / ml protein concentration for reaction in 1 M of MES buffer (pH - 6.0). The result suggested that under this condition, the desired diPEGylation product becomes the main one. It was also achieved above 90% conversion by applying more CMP-SA-PEG (20K) and enzyme. The PEGylated G-CSF product, MAPT-G-CSF (GalNAc-SA-PEG (20KDa)) 2 was purified by purification combination SP-Sepharose and SEC in Supderdex 200. 12. 3 Proliferation activity of the MAPT-G-CSF- (GalNAc-SA-PEG) 2 Cells The proliferation assay of the MAPT-G-CSF- Cells (GalNAc-SA-PEG) 2 was performed with the line of NFS.60 cells and the Tf-1 cell line. The assay was performed, using protein concentration between 0 ng / ml up to 1000 ng / ml. MAPT-G-CSF- (GalNAc-SA-PEG (20K)) 2 was active in this assay. 12. 4 Experimental Details. 12. 4th General Procedure of GalNAcs ilation of G-CSF Mutant. A certain volume of mutant G-CSF solution (for 100 ug of protein) was exchanged from buffer solution with MES buffer (25 mM + 0.005% NaN3, pH 6.0). The final volume was adjusted to 100 ug / 100 ul. For this solution, 5ul of 100mM of MnCl2 and GalNAc-T2 (ImU) were added. The resulting mixture was in motion at room temperature for a period of time required for MALDI or QTOF analysis. 12. 4b Preparation of MAPT-G-CSF- (GalNAc) 2 5.4 mg MAPT-G-CSF (KJ-675-159, 0.18 mg / ml, 0.053 umol) were exchanged with MES buffer (25 mM + 0.005% NaN3, pH 6.0). The final volume was adjusted to . 4 ml. To this solution, UDP-GalNAc (5 mg, 0.15 umol), 100 mM MnCl2 0.25 ml and GalNAc-T2 (1.0 U / ml, 50 ul) were added. The resulting mixture was in motion at 32 ° C for 24 h. M + (MALDI): 19364 (MAPT-G-CSF- (GalNAc) 2 against 18951 (MVPTP-G-CSF). 22. 4c General procedure for glycoPEGylation of mutant G-CSF sequences by single-vessel reaction) The mutant G-CSF 100 ug (G-CSF-1, 2, 3, 4, 5 mutant) was mixed with UDP-GalNAc (0.6 mg, 0.923 umol), GalNAc-T2 (20 ul, 8 mü), UDP-Gal (0.6 mg, 0.923 umol), Core 1 Gal T (20 ul, 10 mU), CMP-SA-PEG (20K) (1 mg, 0.05 umol), St3GalII (20 ul, 28 mU), 100 mM MnC12 3 ul in 100 ul 25 mM MES buffer (pH 6.0 + 0.005% NaN3). The resulting mixture was in motion at room temperature for 24 hours. The glycoPEGylation was followed by SDS-PAGE. 12. 4d Comparison of glycoPEGylation (20KDa) of mutant G-CSF-1 in several buffer systems. The GalNAc2-MAPT-G-CSF- (54 ug) was exchanged for buffer solution by the following four buffer systems (1.1 M of MES buffer (pH 6.0), 2.25 mM of MES buffer (pH 6.0), 3.50 mM of Bis-Tris buffer (pH 6.5), 4.1M of HEPS buffer (pH 7.4), then CMP-SA-PEG (2OK) (216 ug) ST6GalNAcI (BV, 1 U / mL, 2.5 ul) were added. ), 100 mM MnCl2 2.5 ul The resulting mixture was in motion at room temperature for 24 hours The SDS-PAGE gel was used to follow the reaction. 12. 4e Comparison of the GlycoPEGylation of MAPT-G-CSF by the use of ST6GalNAcI and O-sialyltransferase (Wang 787-29 and 787-40) 12. 4.1 Use of ST6GalNAc I First step: 30 ml of solution KJ-675-159 ( 0.18 mg / ml, 5.4 mg of protein in total) was concentrated by ultrafiltration (MWCO 5K) to 3500 g, and then the buffer was exchanged with 25 mM of MES buffer (pH 6.0). The final volume was adjusted to 5.4 ml in a plastic tube. GalNAc-T2 (1.0 U / ml, 50 ul) was added, followed by the addition of 0.25 ml of MnCl2. The resulting mixture was in motion at 32 ° C for 24 hours. The MALDI suggested that the reaction was complete. The reaction mixture was concentrated by UF (MWCO 5K) and diluted with 25 mM MES buffer to 5 ml, when CMP-SA-PEG (20K) (2x25 mg), ST6GalNAc? (BV, lü / ml), 100 mM of 0.25 ml MnCl2 were added. The resulting mixture was in motion at 32 ° C during the night. The SDS-PAGE was used for the reaction. 12. 4e2 Use of O-silyltransferase (ST3Galt?): 200 ug of GalNAc2-MATP-G-CSF was mixed in 200 ul 25 mM of MES buffer (pH 6.0) with UDP-Gal 0.6 mg and GalT core (0.2U / ml, 10 ul) and 10 ul 100 mM MnCl2. The resulting mixture was in motion at 32 ° C for 24 h. The reaction mixture was concentrated by UF (MWCO 5K) and diluted with 25 mM of MES buffer up to 200 ul. CMP-SA-PEG (800 ug), ST3GalII (l.OU / ml, 10 ul), 10 ul 100 mM MnCl2 were added. The resulting mixture was in motion at room temperature for 24 h. The resulting mixture was in motion at 32 ° C during the night. The SDS-PAGE gel was used to follow the reaction. 12. 4 f MAPT-G-CSF- (GalNAc-SA-PEG (2 OK) 2 from glycoPEGylation of MAPT-G-CSF- (GalNAc) 2 (Wang 787-42) MAPT-G-CSF solution was concentrated ( 540 ug) and exchanged with 1 M of MES buffer (pH 6.0) and adjusted to 50 ul, then UDP-GalNAc (100 ug, 0.15 umol, 5 eq), GalNAcT2 (5U / ml, 5 ul) and 100 were added. mM MnCl2 (5 ul) The resulting mixture was in motion at ambient temperature • overnight. Then, 'CMP-SA-PEG (20K) (2.16 mg, 0.108 umol) and ST6GalNAcI (l.OU / ml) were added. 50 ul) The solution was in motion at room temperature for 60 hours, and CMP-SA-PEG (20K) (2.16 mg, 0.108 umol) and additional ST6GalNAcI (l.OU / ml, 50 ul) were added, followed by slow rotation at room temperature for 24 hours The reaction mixture was exchanged with buffer A (25 mM NaOAc, 0.005% polysorbate 80, pH 4.5), then it was purified on an Amersham Sp-FF column (5 mL) with a Isocratic elution of 100% A for 10 minutes followed by a linear gradient from 100% A to 20% B for 20 minutes at a flow rate of 3 mL min-1, where B = 25 mM NaOAc, 2 M NaCl 0.005% polysorbate 80, pH 4.5. The peak at the 17 minute retention time was accumulated and concentrated to 0.5 ml, which was also purified in an Amersham HiLoad Superdex 200 (16 x 600 mm, 34 μm) with phosphate buffer saline, pH 5.0, 0.005 % Tween 80, at a flow rate of 0.4 mL min-1. The product fractions at the time of retention of 160 minutes were accumulated, concentrated to provide 30 ug of MAPT-G-CSF (GalNAc-SA-PEG (20K)) 2 (BCA). The performance was not optimized. 12. 4g Sequences of mutant G-CSF G-CSF-1 Mutant: MAPTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPW APLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQ QMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP (SEQ ID NO: 9) G-CSF-2 Mutant: MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAP LSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQM EELGMAPATQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP (SEQ ID NO :) G-CSF-3 Mutant: MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAP LSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQM EELGMAPALQPTQTAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP (SEQ ID NO :) G-CSF-4 Mutant (C-terminal tag): MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAP LSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQM EELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQPTQGAMP (SEQ ID NO: 8) G-CSF-5 Mutant (MIATP N-terminal): MIATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPW APLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQ QMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP (SEQ ID NO: 10) G-CSF-6 Mutant (177 Mer): MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEELVLLGHSLGIP WAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIW QQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP (SEQ ID NO: l) Recombinant human G-CSF expressed in E. coli: MTPJLGPASSLPQSFLLKC EQVEKIQGDGAA QEK CATYKLCHPBELVLLGHS GIP APLSSCPSQA QLAGCLSQLHSG FLYQGL QALEGISPELGPTLDTLQ DYADFAT TG ^ QQMEELGMAPA QPTÍ3 QGAMPAFÁSAFQRRAGGV VASH QSFLEVSY AND R HLAQP (SEQ ID NO; 2) EXAMPLE 13 The following example illustrates the preparation of a GlycoPEGylated hGH protein. Wild type hGH does not have a natural glycosylation site, therefore a de novo O-glycosylation site was engineered into a hGH mutant protein which was then glycosylated with a GalNAc transferase and sialylPEGylated at the mutant site. Five hGH mutant proteins were designed to incorporate an O-glycosylation site in either the amin-o terminus or in the loop region of the protein molecule. The 5 mutant proteins were produced and each was tested for its hGH activity in an Nb2-ll cell proliferation assay. 13. 1 hGH mutant amino acid sequences. hGH derived from the wild-type pituitary of 192 amino acids comprising a methionine at the N-terminus.

MFPTIPI ^ IlLFDNAK R? RLHQLAFD'ryQEFEEAYIP EQKYSFLQNP QTS CFSESIPTPSNREETQQKSNLELLRISL LIQSWL1PVQFLRSVFANSLYYGASDS VYD LKDLEEGIQTLMGRLEDGSPRTGQ1FKQTYSIFDTNSHNDI5ALLKNYGLLYC FK? CDMDKVETFLRIVQCRSVEGSCGF (SEQ IB NO :) HGH derived from the wild-type pituitary of 191 amino acids that lacks a methionine at the N-terminus FPTIPLSRLFDNÁMLRAHRLHQ AFDTYQEEEEAYIPKEQKYSFLQNPQ TSLCFSESIPWSNHBETQQ SNLELljaS LLIQSWLEPYQFlJlSVFANS VYGÁSDSN VYD LEGEND EEGIQT GRLEDGSPRTGQIF QTYSKFD 'SHNDDA LKNYGL YCF RICDMDKVETF RIYQCRSVEGSCGF (SEQ ID NO;) mutant MYTP: (M) YTPT? PLSRLFDNA ^ IL1AHRLHQ AFDTYQEFEEA? IP EQKYSPL QKPQTSLCFSESIFGPSKREETQQKS LELLRISLLLIQSWLEPVQF RSVFANSLVYGA SDSNYYDLLKDLEEGIQTLMGRLEDGSPRTGQ? FKQ YSKFDI? ÍSHNDDA L? CHYG LYCFR DMDKVETFLRIVQCRS VEGSCGF (SEQ IB NO;) mutant PTOGAMP: 1VIFPTIP SRLFDNÁ1VILRAHRLHQLAFDTYQEFEEAYIP EQKYSFLQNP QTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQS ^ TEPYQFLRSYFANSLYYGASDS í rDLIJaLBEG10TLMGRLEDGSPTOGA ^ CFRKDMDICVETFLRIVQCRSVEGSGGF (SEQIDNO:) mutant TTT: MFP11PLSRLFDNA LRAÍ? RLHQLAFDTYQEFEEAYIPKEQKYSFLQNP QTSLCFSESffTPSNREETQQKSNLEL RISIX IQS LEPYQFLRSVF? NSL \ ryGASDS lWYDLLKDLEEGIOTLMGRLEOGSPTI ?? tFKOTYSKFDTNßHM3DALLEMYGLLYC FREBIvIDKYETFLRIVQCRSVEGSCGF (SEQp NO:) mutant MAPTt MAPTSSPTDP SBLFDN ÍLR? HRLHQ AEDTYQEFEEAYIP EQKYSF LQNPQTSLCFSESIPTPSNREETQQKSN ELLRISLLLIQSVLEPYQFLRSYFANSLYYG ASDSNVYDLLKDLEEGIQTLMGRUEDGSPRTGQIFKQTYSKPDTNSHHDDALLKNYG mutant NTG; MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAY1PKEQKYSFLQNP QTSLCFSESIPTPSNREETQQKSNLELLRISLL IQSW EPYQFLRSVFANS YYGASDS OTYDLLKDLEBG? QTLMGRLEDGSPOT ^ CFRKDMDKYETF R1VQCRSY1GSCGF The four hGH mutants were tested for their ability to act as substrates for glycosyltransferase GalNAcT2. Of the 4 hGH mutants, 2 were found to be glycosylated by GalNAcT2 by a MALDI-MS analysis.13. 2 Preparation of hGH- (TTT) -GalNAc-SA-PEG-30KDa. For the mutant TTT, the addition of GalNAc resulted in a complex mixture of non-glycosylated species and 1-GalNAc and 1-GalNAc. Peptide mapping experiments (digested with trypsin) showed that 2 GalNAc was added to the T12 peptide (L129-K141) containing the TTT mutation. The mutant (M) VTP showed only one trace of GalNAc added by MALDI-MS. The mutant hGH-TTT (4.0 mL, 6.0 mg, 0.27 micromoles) was exchanged in buffer solution 2 times with 15 mL with wash buffer (20 mM HEPES, 150 mM NaCl, 0.02% NaN3, pH 7.4) and once with reaction buffer (20 mM HEPES, 150 mM NaCl, 5 mM MnCl2, 5 mM MgCl2, 0.02% NaN3, pH 7.4) was then concentrated to 2.0 mL using a Centricon 5 KDa MWCO centrifugal filter. The hGH-TTT mutant was combined with UDP-GalNAc (1.38 micromoles, 0.90 mg) and GalNAc-T2 (0.12 mL, 120 mU). The reaction was incubated at 32 ° C with a gentle shaking for 19 hours. The reaction was analyzed by MALDI-MS and the partial addition of GalNAc to the mutant of hGH-TTT was observed (approximately 40%). CMP-SA-PEG-30K (16 mg, 0.533 micromoles) and ST6GalNAcl (0.375 mL, 375 mU) were added to the reaction mixture to bring the total volume to 2.85 mL. The reaction was incubated at 32 ° C with gentle shaking for 22 hours. The reaction was observed by SDS PAGE at 0 h and 22 h. The degree of reaction was determined by an SDS-PAGE gel. The product, hGH- (TTT) -GalNAc-SA-PEG-30 KDa, was purified using SP Sepharose and analyzed by SDS-PAGE. A very low yield of the desired hGH- (TTT) -GalNAc-SA-PEG-30 KDa was observed. 13. 3 Preparation of hGH- (PTQGAMP) - GalNAc -SA-PEG-3 OKDa. The PTQGMP mutant was easily glycosylated with UDP-GalNAc and GalNAc T2, then GlycoPEGylated using CMP-SA-PEG-30KDa and STdGalNAcl on a 10 mg scale to produce 1.45 mg of hGH- (PTQGAMP) -GalNAc-SA-PEG- 30Kda purified. The peptide mapping experiments (product of digestion with tripsiona) located the GalNAc in the trypsin peptide T12 (L129-K141) which contains the mutation of PTQGAMP. The mutant hGH-PTQGAMP (4.55 mL, 10.0 mg, 0.45 micromoles) was exchanged in buffer solution 2 times with 15 L of wash buffer (20 mM HEPES, 150 mM NaCl, 0.02% NaN3, pH 7.4) and once with reaction buffer (20 mM HEPES, 150 mM NaCl, 5 mM MnCl2, 5 mM MgCl2, 0.02% NaN3, pH 7.4) was then concentrated to 3 mL using a Centricon centrifugal filter, 5 KDa MWCO. The hGH-PTQGAMP mutant was combined with UDP-GalNAc (2.26 micromoles, 1.47 mg) and GalNAc-T2 (0.1 mL, 100 MU). The reaction was incubated at 32 ° C with gentle shaking for 22 hours. The reaction was analyzed by MALDI-MS and the complete addition of GalNAc to the mutant hGH-PTQGAMP was observed. CMP-SA-PEG-30 K (27 mg, 0.9 micromoles) and STdGalNAcl (0.350 mL, 350 mU) were added to the reaction mixture to bring the total volume to 3.4 mL. The reaction was incubated at 32 ° C with gentle shaking for 24 hours. The reaction was observed by SDS PAGE at 0 hours and 16.5 hours. The degree of reaction was determined by SDS-PAGE gel. The product, hGH- (PTQGAMP) -GalNAc-SA-PEG-30 KDa, was purified using SP Sepharose and SEC chromatography (Superdex 200) and then it was formulated. The final product was then analyzed by MALDI, peptide mapping and SDS-PAGE (silver staining). The protein was determined by the BCA vs. BSA The isolated overall yield (1.45 mg) was 12.5% based on the protein.

EXAMPLE 14 This example establishes the preparation of a glucoconjugate of GM-CSF PEG of the invention. 14. 1 Preparation of (PEG (20K) -SA-Gal-GalNAc) 2-GM-CSF and PEG (20 k) -SA-Gal-GalNAc-GM-CSF GM-CSF (1 mg) was dissolved in buffer solution from mM MES (1 mL) (pH 6.0, 0.005% NaN3), then UDP-GalNAc (1 mg), GalNAc-T2 (200 μL, 0.38 U / mL, 0.076 U), 100 mM MnCl2 (80 μL) were added . The resulting mixture was incubated at room temperature for 72 hours. MALDI indicated that GalNAc2-GM-CSF was formed. We added UDP-Gal (6 mg, 9.8 mmol), core-1-Gal-T? (0.5 U / mL, 80 μL), CMP-SA-PEG (20 Kilodalton) (6 mg, 0.3 μmol), a- (O) -sialyltransferase (1 U / mL, 120 μL), 100 mM MnCl2 (50 μL). The resulting mixture was slowly rotated at 32 ° C for 48 h. The reaction mixture was centrifuged at 2 rpm for 5 minutes. The protein solution was taken. The remaining resin was mixed with 1 mL 25 mM MES buffer (pH 6.0) and vibrated for 30 sec. The suspension was concentrated again, the protein solutions were combined and concentrated to 200 mcL. Purification by HPLC provides glyco-PEG-yielded GM-CSF.

Example 15 An O-linked glycosylation site similar to that of interferon alfa-2 can be incorporated into any alpha interferon protein in the same relative position. This can be done by aligning the amino acid sequence of interest with the IFN-alpha-2b sequence (10 to 20 amino acids in length) and modifying the amino acid sequence to incorporate the glycosylation site. The mutation can be used with any amino acid, deletion or insertion to create the site. Exemplary mutants maintain a homology as high as possible with an IFN-alpha-2 sequence in this region with an emphasis on T at position 106 (shown below in bold). An example of how this is done is shown below.

Alignment of the alpha interferons in the protein database NCBI GW AÁ # AA Sequence Name IPN-a-2ß 1 CVIQGVGVTBTPLMKEDSI? twenty . { SEQ ID ®Q: X} 124449 98. 117 IFN-alpha 2 (a, b, c) 20178265 99 1? . ?IN. * * * 118 IFN- -alfa 14 124453 99 ... E. ..S .. ... N, US IFN- -alpha 10 585316 99 ... E. .ME .. ... N. 118 IFN- • alpha 17 124442 99 .. - E. ..E ,,,., N. .F. 118 IFN-alpha 7 124438 '99 S MV ... 118 IFN- alpha 4 417188 99 X * * * * -X * * * * 118 IFN- -alpha 8 20178289 99 .... B .. E. NV. .. 118 IFN- -alfa 21 124457 99"MM.E". ED .., .NV .. »118 IFN - alpha 5 124463 99, .T.E .. E.1A..N .... 118 IFN- • alpha 16 124460 99 .. .E. 118 IFN- -alpha 6 124455 99 ..M-EER.G. NA 118 IFN- • alpha 1/13 Glycosylation / Glyco-PEGylation occurs in T106 (IFN-alpha-2). The protein numbering begins with the first amino acid after the removal of the protein leader sequence of the naturally expressed pre-pro form. Interferon alpha mutations to introduce glycosylation sites bound in O in IFN-alpha that lack this site.

GI # AA # AA Sequence Name IPN ~ a ~ 2ß 1 CVIQGVGVTETPL KEDS? L 20 (SEQ D XO-.X) 124449 98 117 IFN - alpha 2 (a, brc) 20178265 99 .... E ...? .H ..... 118 IFN-alpha 14 (E107T) 20178265 99 .... G ... N ..... 118 IFN-alpha 14 (E103G; E107! 1) 124453 99 • E ... T. .N. 118 IFN-alpha 10 (E1C > 7T) 124453 99 ß ... X ..... N ..... 118 IFN-alpha 10. { Ei03G? EW7T) 585316 99 .... E..MT H ..... 118 IFN - alpha 17 . { E107T5 585316 99 E..VT N ..... 118 IFN - alpha 17 . { MEM7VT) 585316 99 T..ME ..... N ..... 118 IFN - alpha 17 £? S? G; E? N7T) ± AH.'i'i ?. W .... U ... X .... • i »•. £. • ixo ?? I - euia 1 (E107T) 124442 99. ,., G. , .T ...., N.F .. 118 IFN - alpha 1 (Ea03G;?, 1 &1T) 124438 99 .... E ... T NV .... 118 IFN-alpha 4 (E107T) 124438 99 .... G ... T HV 118 IFN-alpha 4 (E103G;? 107T.}. 417188 99 ..M.E ... 7.S ... Y. 118 IFN-alpha 8 (Ia0T) 417188 99 ..M.G ...?. S ... Y ..... 118 IFN-alpha 8 (EMG; 11D7T) 20178289 99 ... -E ... T ..... NV .... 118 IFN-alpha 21 (E107T) 20178289 99 .... 6 ... T ..... MV .... 118 | FN-alpha 21 . { B103G; E10T) 124457 99 .MM.B ... TD MV .... 118 IFN-alpha 5 (EW7T) 124457 99 -MM.B ... TB .... NV .... 118 IFN-alpha 5 (ED108TE) 124457 99 .MM.-G ... XD .... NV 118 IFN-alpha 5 (E103G; E107T) 124463 99 ..T.E ... T.IP..N., ... 118 IFN-alpha 16 (EX07T; AU0P) 124463 99 ..T.E ... T.?P .. ». 118 IFN-alpha 16 E107T; m110 P.}. 124463 99 ..T.G ... T.T ».. N 118 IFN-alpha 16 (E103G, -E107T; IAU0? P) 124460 99 ..M.E.W.TG .... N ..... 118 IFN-alpha 6 CG 107p 124460 99 ..M; E.G. G., N. 118 IFN-alpha (105G? G10? T) 124460 99 ..M.ß.ß.TE .... N. 118 IFN-alpha 6 (E103G; W105G; GG10aTE.) .1244B5 99 ..M. ER.T ..... NA .... 118 IFN-alpha 1/13 (G107T) 124455 99 ..M.EEG .T ..... NA .... 118 IFN-alpha 1/13 (R105G; Gl? 7T) 124455 99 ..M.GVG.T ..... HA .... 118 IFN-alpha 1 / 13 (EER10GVG; GM7T) The numbers Gl in the above table except the first number 124449, refer to those of the unmodified wild-type proteins. The O-linked glycosylation site can be created in any alpha interferon isoform by placing a T or S at the appropriate amino acid site as shown above. The substitution is T as shown in the previous table. The amino acid sequences between the various forms of alpha interferon are similar. Any mutation, inson and deletion of amino acids can be done in this region provided that T or S are in the proper position for glycosylation / glyco-PEG-illation relative to P109 (IFN-alpha-2) in the sequence of alignment shown above. Although this invention has been described with reference to the specific embodiments, it is. it is evident that other embodiments and variations of this invention can be dazzled by others skilled in the art without departing from the true spirit and scope of the invention. All patents, patent applications and publications cited in this application are incorporated by reference in their entirety. It is noted that with this date, the best method known to the applicant to carry out the practice of said invention, is that which is clear from the present description of the invention.

Claims

CLAIMS Having described the invention as above, the content of the following claims is claimed as property. An isolated polypeptide characterized in that it comprises a mutant peptide sequence, wherein the mutant peptide sequence encodes an O-linked glycosylation site that does not exist in a wild-type polypeptide corresponding to the isolated polypeptide.
2. The polypeptide according to claim 1, characterized in that it is a G-CSF polypeptide.
3. The polypeptide according to claim 2, characterized in that the G-CSF polypeptide comprises a mutant peptide sequence with the formula of M ^ nTPLGP or M ^ oPZm nTPLGP, and wherein the supra index denotes the position of the amino acid in the amino acid sequence Wild type G-CSF (SEQ ID NO: 3), supra indexes n and m are integers selected from 0 to 3, and at least one of X and B is Thr or Ser, and when more than one of X and B is Thr or Ser Ser, the identity of these portions is independently selected, and Z is selected from glutamate, or any unloaded amino acid.
4. The mutant G-CSF polypeptide according to claim 3, characterized in that the mutant peptide sequence is selected from the sequences consisting of MVTPLGP, MQTPLGP, MIATPLGP), MATPLGP, MPTQGAMPLGP, MVQTPLGP, MQSTPLGP, MGQTPLGP, MAPTSSSPLGP, and MAPTPLGPA .
5. The polypeptide according to claim 2, characterized in that the G-CSF polypeptide comprises a mutant peptide sequence with the formula M1TPXnBo0rP wherein the supra index denotes the position of the amino acid in SEQ ID NO: 3, and the supra indices n, o, and r are integers selected from 0 to 3, and at least one of X, B and 0 is Thr 'or Ser, and when more than one of X, B and 0 is Thr or Ser, the identity of these portions is independently selected.
6. The polypeptide according to claim 5, characterized in that the mutant peptide sequence is selected from the sequences consisting of: MTPTLGP, MTPTQLGP, MTPTSLGP, MTPTQGP, MTPTSSP, MXTPQTP, M ^ PTGP, M1TPLTP, M1TPNTGP, MTPLGP (G-CSF) mut # 4), M1TPVTP, MaTPMVTP, and MT1P2TQGL3G4P5 A6S7.
7. The polypeptide according to claim 2, characterized in that the G-CSF polypeptide comprises a mutant peptide sequence with the formula of LGX53B0LGI wherein the supra index denotes the position of the amino acid in the wild-type amino acid sequence G-CSF (SEQ. DO NOT: 3), and X is histidine, serine, arginine, glutamic acid or tyrosine, and B is either threonine or serine, and o is an integer from 0 to 3.
8. The polypeptide according to claim 7, characterized in that the mutant peptide sequence is selected from the sequences consisting of: LGHTLGI, LGSSLGI, LGYSLGI, LGESLGI, and LGSTLGI.
9. The polypeptide according to claim 2, characterized in that the G-CSF polypeptide comprises a sequence of mutant peptide with the formula of p129ZmJqOrXnPT wherein the supra index denotes the position of the amino acid in the wild-type amino acid sequence G-CSF (SEQ. ID No. 3), Z, J, O and X are independently selected from Thr or Ser, and m, q, r, and n are independently selected integers from 0 to 3.
10. The polypeptide according to claim 9, characterized in that the mutant peptide sequence is selected from the sequences consisting of: p129ATQPT, P129TLGPT, P129TQGPT, P129TSSPT, P129TQGAPT, P129NTGPT, PALQPTQT, P129 ALTPT, P129MVTPT, P129 ASSTPT, P129TTQP, P129NTLP , P129TLQP, MAP129 ATQPTQGAM, and MP129 ATTQPTQGAM.
11. The polypeptide according to claim 2, characterized in that the G-CSF polypeptide comprises a mutant peptide sequence with the formula of PZmUsJqP610rXnBoC wherein the supra index denotes the position of the amino acid in the wild-type amino acid sequence G-CSF (SEQ ID NO. NO. 3), at least one of Z, J, O, and U is selected from threonine or serine, and when more than one of Z, J, O and U is threonine or serine, each is independently selected, and m, s, q, r, n, i are integers independently selected from 0 to 3.
12. The polypeptide according to claim 11, characterized in that the mutant peptide sequence is selected from the sequences consisting of: P61TSSC, P61TSSAC, LGIPTA P61LSSC, LGIPTQ P61LSSC, LGIPTQG P61LSSC, LGIPQT P61LSSC, LGIPTS P61LSSC, LGIPTS P61LSSC, LGIPTQP61LSSC, LGTPWAP61LSSC , LGTPFA P61LSSC, P61FTP, and SLGAP58TAPS1LSS.
13. The polypeptide according to claim 2, characterized in that the G-CSF polypeptide comprises a mutant peptide sequence with the formula of '0aGpJqOrP175XnBoZmUs? T wherein the supra index denotes the position of the amino acid in the wild-type amino acid sequence G-CSF (SEQ ID NO.3), at least one of Z, U, O, J, G, 0, B and X is threonine or serine, and when more than one of Z, U, O, J, G, 0, B and X are threonine or serine, are independently selected; 0 is optionally R, and G is optionally H; the symbol ? represents any unloaded amino acid residue or glutamate and a, p, q, r, n, o, m, s, and t are independently selected integers from 0 to 3.
14. The polypeptide according to claim 13, characterized in that the mutant peptide sequence is selected from the sequences consisting of: RHLAQTP175, RHLAGQTP175, QP175TQGAMP, RHLAQTP175 AM, QP175TSSAP, QP175TSSAP, QP175TQGAMP, QP175TQGAM, 'QP175TQGA, QP175TVM, QP175NTGP, and QP175QTLP. fifteen .
The polypeptide according to claim 2, characterized in that it comprises a mutant peptide sequence selected from the sequences' P133TQTAMP139, P133TQGTMP, P133TQGTNP, P133TQGTLP, and PALQP133TQTAMPA.
16. The polypeptide according to claim 1, characterized in that the polypeptide is an hGH polypeptide.
17. The polypeptide according to claim 16, characterized in that the mutant peptide sequence comprises a sequence selected from: M1APTSSPTIPL7SR9 and DGSP133NTGQIFK140.
18. The polypeptide according to claim 15, characterized in that the hGH polypeptide comprises a mutant peptide sequence with a formula of P133JXBOZUK10QTYS, and wherein the supra index denotes the position of the amino acid in the wild type hGH amino acid sequence (SEQ ID NO: 20) ', and J is selected from threonine and arginine; X is selected from alanine, glutamine, isoleucine, and threonine; B is selected from glycine, alanine, leucine, valine, asparagine, glutamine, and threonine; 0 is selected from tyrosine, serine, alanine, and threonine; Z is selected from isoleucine and methionine; and U is selected from phenylalanine and proline.
19. The polypeptide according to claim 18, characterized in that the mutant peptide sequence is selected from the group consisting of PTTGQIFK, PTTAQIFK, PTTLQIFK, PTTLYVFK, PTTVQIFK, PTTVSIFK, PTTNQIFK, PTTQQIFK, PTATQIFK, PTQGQIFK, PTQGAIFK, PTQGAMFK, PTIGQIFK, PTINQIFK , PTINTIFK, PTILQIFK, PTIVQIFK, PTIQQIFK, PTIAQIFK, P133TTTQIFK1 0QTYS, and P133TQGAMPK140QTYS.
20. The polypeptide according to claim 15, characterized in that the hGH polypeptide comprises a mutant peptide sequence with a formula of P133RTGQIPTQBYS wherein the supra index denotes the position of the amino acid in the wild type hGH amino acid sequence (SEQ ID NO: 20) , and B is selected from alanine and threonine.
21. The polypeptide according to claim 20, characterized in that the mutant peptide sequence is selected from the group consisting of PRTGQIPTQTYS and PRTGQIPTQAYS.
22. The polypeptide according to claim 16, characterized in that the hGH polypeptide comprises a mutant peptide sequence with a formula of L128XTBOP133UTG wherein the supra indices denote the position of the amino acid 'in the wild type hGH amino acid sequence; and wherein X is selected from glutamic acid, valine and alanine; B is selected from glutamine, glutamic acid, and glycine; 0 is selected from serine and threonine; and U is selected from arginine, serine, alanine and leucine
23. The mutant hGH polypeptide according to claim 22, characterized in that the mutant peptide sequence is selected from the group consisting of: LETQSP133RTG, LETQSP133STG, LETQSP133ATG, LETQSP133LTG, LETETP133R, LETETP133A, LVTQSP133RTG, LVTETP133RTG, LVTETP133ATG, and LATGSP133RTG.
24. The polypeptide according to claim 16, characterized in that the hGH polypeptide comprises a mutant peptide sequence with a formula of M ^ PTXnZaOPLSRL wherein the supra index denotes the position of the amino acid in the wild type hGH amino acid sequence (SEQ ID NO: 19); and B is selected from phenylalanine, valine and alanine or a combination thereof; X is selected from glutamate, valine and proline Z is threonine; Or it is selected from leucine and isoleucine; and when X is proline, Z is threonine; and where n and m are integers selected from 0 and 2.
25. The polypeptide according to claim 24, characterized in that the mutant peptide sequence is selected from the group consisting of M1FPTE IPLSRL, MXFPTV LPLSRL, and M ^ PTPTIPLSRL.
26. The polypeptide according to claim 24, characterized in that the mutant peptide sequence is M1VTPTIPLSRL, wherein the supra-index 1, denotes the amino acid in the first position in the wild-type hGH amino acid sequence (SEQ ID NO: 19)
27. The polypeptide according to claim 15, characterized in that the mutant peptide sequence is selected from the group consisting of: LEDGSPTTGQIFKQTYS, LEDGSPTTAQIFKQTYS, LEDGSPTATQIFKQTYS, LEDGSPTQGAMFKQTYS, LEDGSPTQGAIFKQTYS, LEDGSPTQGQIFKQTYS, LEDGSPTTLYVFKQTYS, LEDGSPTINTIFKQTYS, LEDGSPTTVSIFKQTYS, LEDGSPRTGQIPTQTYS, LEDGSPRTGQIPTQAYS, LEDGSPTTLQIFKQTYS, LETETPRTGQIFKQTYS, LVTETPRTGQIFKQTYS, LETQSPRTGQIFKQTYS, LVT.QSPRTGQIFKQTYS, LVTETPATGQIFKQTYS, LEDGSPTQGAMPKQTYS, and LEDGSPTTTQIFKQTYS.
28. The polypeptide according to claim 1, characterized in that the polypeptide is an IFN alpha polypeptide.
29. The polypeptide according to claim 28, characterized in that the INF alpha polypeptide has a peptide sequence comprising a mutant amino acid sequence, and the peptide sequence corresponds to a region of INF alpha 2 having a sequence as shown in SEQ NO: 22, and wherein the mutant amino acid sequence contains a mutation to an amino threonine or serine acid in a position corresponding to T106 of INF alpha 2.
30. The polypeptide according to claim 29, characterized in that the IFN alpha polypeptide is selected from the group consisting of IFN alpha, IFN alpha 4, IFN alpha 5, IFN alpha 6, IFN alpha 7, IFN alpha 8, IFN alpha 10, IFN alpha 14, IFN alpha 16, IFN alpha 17, and IFN alpha 21.
31. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVMQEERVTETPLMNADSIL118, 99CVMQEEGVTETPLMNADSIL118, and" CVMQGVGVTETPLMNADSIL118.
32. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 4 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVIQEVGVTETPLMNVDSIL118, and 99CVIQGVGVTETPLMKEDSIL118.
33. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 5 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CMMQEVGVTDTPLMNVDSIL118, 99CMMQEVGVTETPLMNVDSIL118 and" CMMQGVGVTDTPLMNVDSIL118.
34. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 6 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVMQEVWVTGTPLMNEDSIL118, 99CVMQEVGVTGTPLMNEDSIL118," and 99CVMQGVGVTETPLMNEDSIL118.
35. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 7 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVIQEVGVTETPLMNEDFIL118, and 99CVIQGVGVTETPLMNEDFIL118.
36. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 8 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVMQEVGVTESPLMYEDSIL118, and 99CVMQGVGVTESPLMYEDSIL118.
37. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 10 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: 99CVIQEVGVTETPLMNEDSIL118, and "CVIQGVGVTETPLMNEDSIL118
38. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 14 polypeptide comprising a mutant amino acid sequence selected from the group consisting of:" CVIQEVGVTETPLMNEDSIL118, and 99CVIQGVGVTETPLMNEDSIL118.
39. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 16 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVTQEVGVTEIPLMNEDSIL118, 99CVTQEVGVTETPLMNEDSIL118, and" CVTQGVGVTETPLMNEDSIL118.
40. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 17 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVIQEVGMTETPLMNEDSIL118," CVIQEVGVTETPLMNEDSIL118, and "CVIQGVGMTETPLMNEDSIL118,
41. The polypeptide according to claim 30, characterized in that the IFN alpha polypeptide is an IFN alpha 21 polypeptide comprising a mutant amino acid sequence selected from the group consisting of: "CVIQEVGVTETPLMNVDSIL118, and 99CVIQGVGVTETPLMNVDSIL118.
42. An isolated nucleic acid characterized in that it encodes the polypeptide according to claim 1.
43. An expression cassette characterized in that it comprises the nucleic acid according to claim 42.
44. A cell characterized in that it comprises the nucleic acid according to claim 42.
45. The polypeptide according to claim 1, characterized in that it has a formula selected from: O-GalNA-X wherein AA is an amino acid side chain comprising a hydroxyl portion that is within the mutant peptide sequence; and X a modification group or a saccharyl moiety.
46. The polypeptide according to the claim 45, characterized in that X comprises a selected group of sialyl, galactosyl and Gal-Sia portions, wherein at least one of the sialyl, galactosyl and Gal-Sia comprise a modification group.
47 The polypeptide according to claim 45, characterized in that X comprises the portion: wherein D is a member selected from -OH and R1-L-HN-; G is a member selected from R1-L- and -C (O) (C? -C6) alkyl; R1 is a portion comprising a member selected from a portion comprising a straight or branched chain poly (ethylene glycol) residue; and L is a bond which is a selected member of a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, such that when D is OH, G is R1-L-, and when G is -C (O) (d-Ce) alkyl, D is R1-L-NH-.
48. The polypeptide according to claim 45, characterized in that. X includes the structure: wherein L is a substituted or unsubstituted alkyl group or substituted or unsubstituted heteroalkyl; and n is selected from the integers from 0 to about 500.
49. The polypeptide according to claim 45, characterized in that X comprises the structure: where s is selected from the integers from 0 to 20.
50. A method for making a glycoconjugate of the polypeptide according to claim 1, characterized in that it comprises the steps of: (a) recombinantly producing the polypeptide, and (b) enzymatically glycosylating the polypeptide with a modified sugar at the glycosylation site bound to the polypeptide. 0
51. A pharmaceutical composition of a granulocyte colony stimulation factor (G-CSF) characterized in that it comprises: an effective amount of the polypeptide according to claim 2, wherein the polypeptide is glycoconjugated with a modified sugar.
52. The pharmaceutical composition according to claim 51, characterized in that the modified sugar is modified with a member from poly (ethylene glycol) and methoxy-poly (ethylene glycol) (m-PEG).
53. A human growth hormone (hGH) pharmaceutical composition characterized in that it comprises an effective amount of the polypeptide according to claim 16, wherein the polypeptide is glycoconjugated with a modified sugar.
54. The pharmaceutical composition according to claim 53, characterized in that the modified sugar is modified with a member selected from poly (ethylene glycol) and methoxy-poly (ethylene glycol) (m-PEG).
55. A pharmaceutical composition of a granulocyte macrophage colony stimulation factor (GM-CSF) characterized in that it comprises an effective amount of GM-CSF polypeptide comprising a mutant peptide sequence, wherein the mutant sequence comprises a glycosylation site linked to 0 that does not exist in a wild type GM-CSF polypeptide, and wherein the polypeptide peptide is glycoconjugated with a modified sugar.
56. The pharmaceutical composition according to claim 55, characterized in that the modified sugar is modified with a member from poly (ethylene glycol) and methoxy-poly (ethylene glycol) (m-PEG).
57. A pharmaceutical composition of an interferon alpha-2b characterized in that it comprises an effective amount of the polypeptide • according to claim 28, wherein the polypeptide is glycoconjugated with a modified sugar.
58. The pharmaceutical composition according to claim 57, characterized in that the modified * sugar is modified with a member from poly (ethylene glycol) and methoxy-poly (ethylene glycol) (m-PEG).
59. A method for providing G-CSF therapy to a subject in need of therapy, characterized in that it comprises administering to the subject an effective amount of the pharmaceutical composition according to claim 51.
60. A method for providing stimulation factor therapy of the granulocyte macrophage colony to a subject in need of therapy, characterized in that it comprises: administering to the subject an effective amount of the pharmaceutical composition according to claim 55.
61. A method for providing interferon therapy to a subject in need of therapy, characterized in that it comprises: administering to the subject an effective amount of the pharmaceutical composition according to claim 57.
62. A method for providing Growth Hormone Therapy to a subject in need of therapy, characterized in that it comprises: administering to the subject an effective amount of the pharmaceutical composition according to claim 53.