GLYCOSYLATION OF PEPTIDES VIA O-LINKED GLYCOSYLATION SEQUENCES

1 . A covalent conjugate between a glycosylated or non-glycosylated sequon polypeptide and a polymeric modifying group, said sequon polypeptide corresponding to a parent polypeptide and comprising an exogenous O-linked glycosylation sequence, said polymeric modifying group being conjugated to said sequon polypeptide at said O-linked glycosylation sequence via a glycosyl linking group, wherein said glycosyl linking group is interposed between and covalently linked to both said sequon polypeptide and said polymeric modifying group, with the proviso that said parent polypeptide is not a member selected from human growth hormone (hGH), granulocyte colony stimulating factor (G-CSF), interferon-alpha (INF-alpha), glucagon-like peptide-1 (GLP-1) and fibroblast growth factor (FGF).

2 . The covalent conjugate of claim 1 , wherein said polymeric modifying group is a member selected from linear and branched and comprises one or more polymeric moiety, wherein each polymeric moiety is independently selected.

3 . The covalent conjugate of claim 2 , wherein said polymeric moiety is a member selected from poly(ethylene glycol) and methoxy-poly(ethylene glycol) (m-PEG).

4 . The covalent conjugate of claim 1 , wherein said glycosyl linking group is an intact glycosyl linking group.

5 . The covalent conjugate of claim 4 , comprising a moiety according to Formula (III):

wherein

R 9 is H, a negative charge or a salt counterion; and

R P is a member selected from:

wherein n is an integer selected from 1 to 20 and f and e are integers independently selected from 1-2500.

6 . The covalent conjugate according to claim 1 , wherein said parent-polypeptide is a member selected from bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease, erythropoietin (EPO), α 1 -antitrypsin (α-1 protease inhibitor), glucocerebrosidase, tissue-type plasminogen activator (TPA), leptin, hirudin, urokinase, human DNase, insulin, hepatitis B surface protein (HbsAg), chimeric diphtheria toxin-IL-2, human chorionic gonadotropin (hCG), thyroid peroxidase (TPO), alpha-galactosidase, alpha-L-iduronidase, beta-glucosidase, alpha-galactosidase A, acid □-glucosidase (acid maltase), anti-thrombin III (AT III), follicle stimulating hormone (FSH), glucagon-like peptide-2 (GLP-2), Factor VII, Factor VIII, B-domain deleted Factor VIII, Factor IX, Factor X, Factor XIII, prokinetisin, extendin-4, CD4, tumor necrosis factor receptor (TNF-R), □-CD20, P-selectin glycoprotein ligand-1 (PSGL-1), complement, transferrin, glycosylation-dependent cell adhesion molecule (GlyCAM), neural-cell adhesion molecule (N-CAM), TNF receptor-IgG Fc region fusion protein, anti-HER2 monoclonal antibody, monoclonal antibody to respiratory syncytial virus, monoclonal antibody to protein F of respiratory syncytial virus, monoclonal antibody to TNF-α, monoclonal antibody to glycoprotein Ilb/IIIa, monoclonal antibody to CD20, monoclonal antibody to VEGF-A, monoclonal antibody to PSGL-1, monoclonal antibody to CD4, monoclonal antibody to a-CD3, monoclonal antibody to EGF, monoclonal antibody to carcinoembryonic antigen (CEA) and monoclonal antibody to IL-2 receptor.

7 . The covalent conjugate of claim 1 , wherein said exogenous O-linked glycosylation sequence is a member selected from: (X) m PTP, (X) m PTEI(P) n , (X) m PTQA(P) n , (X) m PTINT(P) n , (X) m PTTVS(P) n , (X) m PTTVL(P) n , (X) m PTQGAM(P) n , (X) m TET(P) n , (X) m PTVL(P) n , (X) m PTLS(P) n , (X) m PTDA(P) n , (X) m PTEN(P) n , (X) m PTQD(P) n , (X) m PTAS(P) n , (X) m PTQGA(P) n , (X) m PTSAV(P) n , (X) m PTTLYV(P) n , (X) m PSSG(P) n and (X) m PSDG(P) n ,

wherein

m and n are integers independently selected from 0 and 1;

P is proline; and

X is a member independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids.

8 . The covalent conjugate of claim 7 , wherein said exogenous O-linked glycosylation sequence is a member selected from: PTP, PTEI, PTEIP, PTQA, PTQAP, PTINT, PTINTP, PTTVS, PTTVL, PTQGAM, PTQGAMP and TETP.

9 . A pharmaceutical composition comprising a covalent conjugate according to claim 1 and a pharmaceutically acceptable carrier.

10 . A polypeptide conjugate comprising a sequon polypeptide, said sequon polypeptide corresponding to a parent polypeptide and having an exogenous O-linked glycosylation sequence, said polypeptide conjugate comprising a moiety according to Formula (V):

wherein

w is an integer selected from 0 and 1;

q is an integer selected from 0 and 1;

AA-O— is a moiety derived from an amino acid having a side chain substituted with a hydroxyl group, said amino acid positioned within said O-linked glycosylation sequence;

Z* is a member selected from a glycosyl moiety and a glycosyl linking group; and

X* is a member selected from a polymeric modifying group and a glycosyl linking group covalently linked to a polymeric modifying group,

with the proviso that said parent polypeptide is not a member selected from human growth hormone (hGH), granulocyte colony stimulating factor (G-CSF), interferon-alpha (INF-alpha), glucagon-like peptide- 1 (GLP-1) and fibroblast growth factor (FGF).

11 . The polypeptide conjugate according to claim 10 , wherein said amino acid is serine (S) or threonine (T).

12 . The polypeptide conjugate of claim 10 , wherein said exogenous O-linked glycosylation sequence is a member selected from:

(X) m PTP,

(X) m PTEI(P) n ,

(X) m PTQA(P) n ,

(X) m PTINT(P) n ,

(X) m PTTVS(P) n ,

(X) m PTTVL(P) n ,

(X) m PTQGAM(P) n ,

(X) m TET(P) n ,

(X) m PTVL(P) n ,

(X) m PTLS(P) n ,

(X) m PTDA(P) n ,

(X) m PTEN(P) n ,

(X) m PTQD(P) n ,

(X) m PTAS(P) n ,

(X) m PTQGA(P) n ,

(X) m PTSAV(P) n ,

(X) m PTTLYV(P) n ,

(X) m PSSG(P) n

and

(X) m PSDG(P) n ,

wherein

m and n are integers independently selected from 0 and 1;

P is proline; and

X is a member independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids.

13 . The polypeptide conjugate of claim 12 , wherein said exogenous O-linked glycosylation sequence is a member selected from: PTP, PTEI, PTEIP, PTQA, PTQAP, PTINT, PTINTP, PTTVS, PTTVL, PTQGAM, PTQGAMP and TETP.

14 . The polypeptide conjugate according to claim 10 , wherein Z* is a member selected from GalNAc, GalNAc-Gal, GalNAc-Gal-Sia and GalNAc-Sia.

15 . The polypeptide conjugate according to claim 10 , wherein said polymeric modifying group is a member selected from linear and branched and comprises one or more polymeric moiety, wherein each of said polymeric moiety is independently selected.

16 . The polypeptide conjugate according to claim 15 , wherein said polymeric moiety is a member selected from poly(ethylene glycol) and derivatives thereof.

17 . The polypeptide conjugate according to claim 10 , wherein w is 1.

18 . The polypeptide conjugate according to claim 17 , wherein X* comprises a moiety, which is a member selected from a sialyl (Sia) moiety, a galactosyl (Gal) moiety, a GalNAc moiety and a Gal-Sia moiety.

19 . The polypeptide conjugate according to claim 10 , wherein said parent-polypeptide is a member selected from bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease, erythropoietin (EPO), α 1 -antitrypsin (α-1 protease inhibitor), glucocerebrosidase, tissue-type plasminogen activator (TPA), leptin, hirudin, urokinase, human DNase, insulin, hepatitis B surface protein (HbsAg), chimeric diphtheria toxin-IL-2, human chorionic gonadotropin (hCG), thyroid peroxidase (TPO), alpha-galactosidase, alpha-L-iduronidase, beta-glucosidase, alpha-galactosidase A, acid □-glucosidase (acid maltase), anti-thrombin III (AT III), follicle stimulating hormone, glucagon-like peptide-2 (GLP-2), Factor VII, Factor VIII, B-domain deleted Factor VIII, Factor IX, Factor X, Factor XIII, prokinetisin, extendin-4, CD4, tumor necrosis factor receptor (TNF-R), □-CD20, P-selectin glycoprotein ligand-1 (PSGL-1), complement, transferrin, glycosylation-dependent cell adhesion molecule (GlyCAM), neural-cell adhesion molecule (N-CAM), TNF receptor-IgG Fc region fusion protein, anti-HER2 monoclonal antibody, monoclonal antibody to respiratory syncytial virus, monoclonal antibody to protein F of respiratory syncytial virus, monoclonal antibody to TNF-α, monoclonal antibody to glycoprotein IIb/IIIa, monoclonal antibody to CD20, monoclonal antibody to VEGF-A, monoclonal antibody to PSGL-1, monoclonal antibody to CD4, monoclonal antibody to a-CD3, monoclonal antibody to EGF, monoclonal antibody to carcinoembryonic antigen (CEA) and monoclonal antibody to IL-2 receptor.

20 . The polypeptide conjugate of claim 17 , wherein X* comprises a moiety according to Formula (VI):

wherein

E is a member selected from O, S, NR 27 and CHR 28 ,

wherein

R 27 and R 28 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;

E 1 is a member selected from O and S;

R 2 is a member selected from H, —R 1 , —CH 2 R 1 , and —C(X 1 )R 1 , wherein R 1 is a member selected from OR 9 , SR 9 , NR 10 R 11 , substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl

wherein

R 9 is a member selected from H, a negative charge, a metal ion, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

R 10 and R 11 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

X 1 is a member selected from substituted or unsubstituted alkenyl, O, S and NR 8

wherein

R 8 is a member selected from H, OH, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

Y is a member selected from CH 2 , CH(OH)CH 2 , CH(OH)CH(OH)CH 2 , CH, CH(OH)CH; CH(OH)CH(OH)CH, CH(OH), CH(OH)CH(OH), and CH(OH)CH(OH)CH(OH);

Y 2 is a member selected from H, OR 6 , R 6 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,

wherein

R 6 and R 7 are members independently selected from H, L a -R 6b , C(O)R 6b , C(O)-L a -R 6b , substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, wherein R 6b is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and a modifying group;

R 3 , R 3′ and R 4 are members independently selected from H, OR 3″ , SR 3″ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, -L a -R 6c , —C(O)-L a R 6c , —NH-L a -R 6c , ═N-L a -R 6c and —NHC(O)-L a -R 6c

wherein

R 3″ is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and

R 6c is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, NR 13 R 14 and a modifying group, wherein R 13 and R 14 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and

each L a is a member independently selected from a bond and a linker group.

21 . The polypeptide conjugate according to claim 20 , wherein X* comprises a moiety according to Formula (VII):

22 . The polypeptide conjugate according to claim 20 , wherein at least one of R 6b and R 6c is a member selected from:

wherein

s, j and k are integers independently selected from 0 to 20;

each n is an integer independently selected from 0 to 2500;

m is an integer from 1-5;

Q is a member selected from H and C 1 -C 6 alkyl;

R16 and R 17 are independently selected polymeric moieties;

X 2 and X 4 are independently selected linkage fragments joining polymeric moieties R 16 and R 17 to C;

X 5 is a non-reactive group other than a polymeric moiety; and

A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 8 , A 9 , A 10 and A 11 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —NA 12 A 13 , —OA 12 and -SiA 12 A 13

wherein

A 12 and A 13 are members independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

23 . A pharmaceutical composition comprising a polypeptide conjugate according to claim 10 and a pharmaceutically acceptable carrier.

24 . A sequon polypeptide corresponding to a parent polypeptide, wherein said sequon polypeptide comprises an exogenous O-linked glycosylation sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2:

(X) m P O* U (B) p (Z) r (J) s (O) t (P) n ;

(SEQ ID NO: 1)

and

(X) m (B 1 ) p T U B (Z) r (J) s (P) n

(SEQ ID NO: 2)

wherein

m, n, p, r, s and t are integers independently selected from 0 and 1;

P is proline;

O* is a member selected from serine (S) and threonine (T);

U is a member selected from proline (P), glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids;

X, B and B 1 are members independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids; and

Z, J and O are members independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), methionine (M) and uncharged amino acids,

with the proviso that said parent polypeptide is not a member selected from human growth hormone (hGH), granulocyte colony stimulating factor (G-CSF), interferon-alpha (INF-alpha), glucagon-like peptide-1 (GLP-1) and fibroblast growth factor (FGF).

25 . The sequon polypeptide of claim 24 , wherein said exogenous O-linked glycosylation sequence is a member selected from: (X) m PTP, (X) m PTEI(P) n , (X) m PTQA(P) n , (X) m PTINT(P) n , (X) m PTTVS(P) n , (X) m PTTVL(P) n , (X) m PTQGAM(P) n , (X) m TET(P) n , (X) m PTVL(P) n , (X) m PTLS(P) n , (X) m PTDA(P) n , (X) m PTEN(P) n , (X) m PTQD(P) n , (X) m PTAS(P) n , (X) m PTQGA(P) n , (X) m PTSAV(P) n , (X) m PTTLYV(P) n , (X) m PSSG(P) n and (X) m PSDG(P) n , wherein

m and n are integers independently selected from 0 and 1;

P is proline; and

X is a member independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids.

26 . The sequon polypeptide of claim 25 , wherein said exogenous O-linked glycosylation sequence is a member selected from: PTP, PTEI, PTEIP, PTQA, PTQAP, PTINT, PTINTP, PTTVS, PTTVL, PTQGAM, PTQGAMP and TETP.

27 . The sequon polypeptide according to claim 24 , wherein said exogenous O-linked glycosylation sequence is a substrate for a GalNAc-transferase.

28 . The sequon polypeptide of claim 24 , wherein at least 3 amino acids are found between said O* and a lysine (K) or arginine (R) residue.

29 . The sequon polypeptide of claim 24 , wherein said parent polypeptide is a therapeutic polypeptide.

30 . The sequon polypeptide according to claim 24 , wherein said parent-polypeptide is a member selected from bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease, erythropoietin (EPO), α 1 -antitrypsin (α-1 protease inhibitor), glucocerebrosidase, tissue-type plasminogen activator (TPA), leptin, hirudin, urokinase, human DNase, insulin, hepatitis B surface protein (HbsAg), chimeric diphtheria toxin-IL-2, human chorionic gonadotropin (hCG), thyroid peroxidase (TPO), alpha-galactosidase, alpha-L-iduronidase, beta-glucosidase, alpha-galactosidase A, acid □-glucosidase (acid maltase), anti-thrombin III (AT III), follicle stimulating hormone, glucagon-like peptide-2 (GLP-2), Factor VII, Factor VIII, B-domain deleted Factor VIII, Factor IX, Factor X, Factor XIII, prokinetisin, extendin-4, CD4, tumor necrosis factor receptor (TNF-R), □-CD20, P-selectin glycoprotein ligand-1 (PSGL-1), complement, transferrin, glycosylation-dependent cell adhesion molecule (GlyCAM), neural-cell adhesion molecule (N-CAM), TNF receptor-IgG Fc region fusion protein, anti-HER2 monoclonal antibody, monoclonal antibody to respiratory syncytial virus, monoclonal antibody to protein F of respiratory syncytial virus, monoclonal antibody to TNF-α, monoclonal antibody to glycoprotein IIb/IIIa, monoclonal antibody to CD20, monoclonal antibody to VEGF-A, monoclonal antibody to PSGL-1, monoclonal antibody to CD4, monoclonal antibody to a-CD3, monoclonal antibody to EGF, monoclonal antibody to carcinoembryonic antigen (CEA) and monoclonal antibody to IL-2 receptor.

31 . An isolated nucleic acid encoding said sequon polypeptide of claim 24 .

32 . An expression vector comprising said nucleic acid of claim 31 .

33 . A cell comprising said nucleic acid of claim 31 .

34 . A sequon polypeptide corresponding to a parent polypeptide, wherein said sequon polypeptide comprises an exogenous O-linked glycosylation sequence selected from:

XPO*P,

XPO*EI(P) n ,

(X) m PO*EI,

XPO*QA(P) n ,

XPO*TVS,

(X) m PO*TVSP,

XPO*QGA,

(X) m PO*QGAP,

XPO*QGAM(P) n ,

XTEO*P,

(X) m PO*VL,

XPO*VL(P) n ,

XPO*TVL,

(X) m PO*TVLP,

(X) m PO*TLYVP,

XPO*TLYV(P) n ,

(X) m PO*LS(P) n ,

(X) m PO*DA(P) n ,

(X) m PO*EN(P) n ,

(X) m PO*QD(P) n ,

(X) m PO*AS(P) n ,

XPO*SAV,

(X) m PO*SAYP,

(X) m PO*SG(P) n ,

XTEO*P

and

(X) m PO*DG(P) n

wherein

m and n are integers independently selected from 0 and 1;

0 * is a member selected from serine (S) and threonine (T);

X is a member selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids;

each S (serine) is optionally and independently replaced with T (threonine); and

each T (threonine) is optionally and independently replaced with S (serine).

35 . The sequon polypeptide according to claim 34 , wherein said O-linked glycosylation sequence is a substrate for GalNAc-transferase.

36 . The sequon polypeptide of claim 34 , wherein at least 3 amino acids are found between said 0 * and a lysine (K) or arginine (R) residue.

37 . The sequon polypeptide of claim 34 , wherein said parent polypeptide is a therapeutic polypeptide.

38 . The sequon polypeptide according to claim 34 , wherein said parent-polypeptide is a member selected from bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon alpha, interferon beta, interferon gamma, α 1 -antitrypsin (α-1 protease inhibitor), glucocerebrosidase, tissue-type plasminogen activator (TPA), interleukin-2 (IL-2), leptin, hirudin, urokinase, human DNase, insulin, hepatitis B surface protein (HbsAg), chimeric diphtheria toxin-IL-2, human growth hormone (hGH), human chorionic gonadotropin (hCG), thyroid peroxidase (TPO), alpha-galactosidase, alpha-L-iduronidase, beta-glucosidase, alpha-galactosidase A, acid □-glucosidase (acid maltase), anti-thrombin III (AT III), follicle stimulating hormone (FSH), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), fibroblast growth factor 7 (FGF-7), fibroblast growth factor 21 (FGF-21), fibroblast growth factor 23 (FGF-23), Factor VII, Factor VIII, B-domain deleted Factor VIII, Factor IX, Factor X, Factor XIII, prokinetisin, extendin-4, CD4, tumor necrosis factor receptor (TNF-R), □-CD20, P-selectin glycoprotein ligand-1 (PSGL-1), complement, transferrin, glycosylation-dependent cell adhesion molecule (GlyCAM), neural-cell adhesion molecule (N-CAM), TNF receptor-IgG Fc region fusion protein, anti-HER2 monoclonal antibody, monoclonal antibody to respiratory syncytial virus, monoclonal antibody to protein F of respiratory syncytial virus, monoclonal antibody to TNF-α, monoclonal antibody to glycoprotein IIb/IIIa, monoclonal antibody to CD20, monoclonal antibody to VEGF-A, monoclonal antibody to PSGL-1, monoclonal antibody to CD4, monoclonal antibody to a-CD3, monoclonal antibody to EGF, monoclonal antibody to carcinoembryonic antigen (CEA) and monoclonal antibody to IL-2 receptor.

39 . An isolated nucleic acid encoding said sequon polypeptide of claim 34 .

40 . An expression vector comprising said nucleic acid of claim 39 .

41 . A cell comprising said nucleic acid of claim 39 .

42 . A library of sequon polypeptides comprising a plurality of different members, wherein each member of said library corresponds to a common parent polypeptide and wherein each member of said library comprises an exogenous O-linked glycosylation sequence, wherein each of said O-linked glycosylation sequence is a member independently selected from SEQ ID NO: 1 and SEQ ID NO: 2:

(X) m P O* U (B) p (Z) r (J) s (O) t (P) n ;

(SEQ ID NO: 1)

and

(X) m (B 1 ) p T U B (Z) r (J) s (P) n

(SEQ ID NO: 2)

wherein

m, n, p, r, s and t are integers independently selected from 0 and 1;

P is proline;

O* is a member selected from serine (S) and threonine (T);

U is a member selected from proline (P), glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids;

X, B and B 1 are members independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids; and

Z, J and O are members independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), methionine (M) and uncharged amino acids.

43 . The library of claim 42 , wherein said exogenous O-linked glycosylation sequence is a member selected from:

(X) m PTP,

(X) m PTEI(P) n ,

(X) m PTQA(P) n ,

(X) m PTINT(P) n ,

(X) m PTTVS(P) n ,

(X) m PTTVL(P) n ,

(X) m PTQGAM(P) n ,

(X) m TET(P) n ,

(X) m PTVL(P) n ,

(X) m PTLS(P) n ,

(X) m PTDA(P) n ,

(X) m PTEN(P) n ,

(X) m PTQD(P) n ,

(X) m PTAS(P) n ,

(X) m PTQGA(P) n ,

(X) m PTSAV(P) n ,

(X) m PTTLYV(P) n ,

(X) m PSSG(P) n

and

(X) m PSDG(P) n ,

wherein

m and n are integers independently selected from 0 and 1;

P is proline; and

X is a member independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids.

44 . The library of claim 43 , wherein said exogenous O-linked glycosylation sequence is a member selected from: PTP, PTEI, PTEIP, PTQA, PTQAP, PTINT, PTINTP, PTTVS, PTTVL, PTQGAM, PTQGAMP and TETP.

45 . The library of claim 42 , wherein each member of said library comprises the same O-linked glycosylation sequence at a different amino acid position within said parent polypeptide.

46 . The library of claim 42 , wherein each member of said library comprises a different O-linked glycosylation sequence at the same amino acid position within said parent polypeptide.

47 . The library of claim 42 , wherein said parent polypeptide has m amino acids, each amino acid corresponding to an amino acid position, said library comprising:

(a) a first sequon polypeptide having said O-linked glycosylation sequence at a first amino acid position (AA) n , wherein n is a member selected from 1 to m; and

(c) at least one additional sequon polypeptide, each additional sequon polypeptide having said O-linked glycosylation sequence at an additional amino acid position, which is a member selected from (AA) n+x and (AA) n−x , wherein x is a member selected from 1 to (m-n).

48 . The library of claim 47 , comprising a second sequon polypeptide having said O-linked glycosylation sequence at a second amino acid position selected from (AA) n+p and (AA) n−p , wherein p is selected from 1 to 10.

49 . The library of claim 47 , wherein each of said additional amino acid position is adjacent to a previously selected amino acid position.

50 . The library of claim 42 , wherein said O-linked glycosylation sequence is a substrate for a GalNAc-transferase.

51 . The library of claim 50 , wherein said GalNAc-transferase is a member selected from lectin-domain deleted GalNAc-T2 and lectin domain truncated GalNAc-T2.

52 . The library of claim 42 , wherein said parent polypeptide is a therapeutic polypeptide.

53 . The library of claim 42 , wherein said parent-polypeptide is a member selected from bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon alpha, interferon beta, interferon gamma, α 1 -antitrypsin (α-1 protease inhibitor), glucocerebrosidase, tissue-type plasminogen activator (TPA), interleukin-2 (IL-2), leptin, hirudin, urokinase, human DNase, insulin, hepatitis B surface protein (HbsAg), chimeric diphtheria toxin-IL-2, human growth hormone (hGH), human chorionic gonadotropin (hCG), thyroid peroxidase (TPO), alpha-galactosidase, alpha-L-iduronidase, beta-glucosidase, alpha-galactosidase A, acid □-glucosidase (acid maltase), anti-thrombin III (AT III), follicle stimulating hormone (FSH), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), fibroblast growth factor 7 (FGF-7), fibroblast growth factor 21 (FGF-21), fibroblast growth factor 23 (FGF-23), Factor VII, Factor VIII, B-domain deleted Factor VIII, Factor IX, Factor X, Factor XIII, prokinetisin, extendin-4, CD4, tumor necrosis factor receptor (TNF-R), □-CD20, P-selectin glycoprotein ligand-1 (PSGL-1), complement, transferrin, glycosylation-dependent cell adhesion molecule (GlyCAM), neural-cell adhesion molecule (N-CAM), TNF receptor-IgG Fc region fusion protein, anti-HER2 monoclonal antibody, monoclonal antibody to respiratory syncytial virus, monoclonal antibody to protein F of respiratory syncytial virus, monoclonal antibody to TNF-α, monoclonal antibody to glycoprotein IIb/IIIa, monoclonal antibody to CD20, monoclonal antibody to VEGF-A, monoclonal antibody to PSGL-1, monoclonal antibody to CD4, monoclonal antibody to a-CD3, monoclonal antibody to EGF, monoclonal antibody to carcinoembryonic antigen (CEA) and monoclonal antibody to IL-2 receptor.

54 . A method comprising: expressing a sequon polypeptide in a host cell, said sequon polypeptide corresponding to a parent polypeptide and comprising an exogenous O-linked glycosylation sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2:

(X) m P O* U (B) p (Z) r (J) s (O) t (P) n ;

(SEQ ID NO: 1)

and

(X) m (B 1 ) p T U B (Z) r (J) s (P) n

(SEQ ID NO: 2)

wherein

m, n, p, r, s and t are integers independently selected from 0 and 1;

P is proline;

O* is a member selected from serine (S) and threonine (T);

U is a member selected from proline (P), glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids;

X, B and B 1 are members independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S) and uncharged amino acids; and

Z, J and O are members independently selected from glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), methionine (M) and uncharged amino acids,

with the proviso that said parent polypeptide is not a member selected from human growth hormone (hGH), granulocyte colony stimulating factor (G-CSF), interferon-alpha (INF-alpha), glucagon-like peptide-1 (GLP-1) and fibroblast growth factor (FGF).

55 . The method according to claim 54 , further comprising isolating said sequon polypeptide.

56 . The method according to claim 54 , further comprising enzymatically glycosylating said sequon polypeptide at said O-linked glycosylation sequence.

57 . The method according to claim 56 , wherein said enzymatically glycosylating is accomplished using a glycosyltransferase.

58 . The method according to claim 57 , wherein said glycosyltransferase is GalNAc-T2.

59 . The method of claim 58 , wherein said GalNAc-T2 is a member selected from lectin-domain deleted GalNAc-T2 and lectin domain truncated GalNAc-T2.

60 . The method according to claim 54 , further comprising generating an expression vector comprising a nucleic acid sequence encoding said sequon polypeptide.

61 . The method according to claim 60 , further comprising transfecting said host cell with said expression vector.

62 . The method according to claim 54 , wherein said parent polypeptide is a therapeutic polypeptide.

63 . The method according to claim 54 , wherein said parent-polypeptide is a member selected from bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease, erythropoietin (EPO), α 1 -antitrypsin (α-1 protease inhibitor), glucocerebrosidase, tissue-type plasminogen activator (TPA), leptin, hirudin, urokinase, human DNase, insulin, hepatitis B surface protein (HbsAg), chimeric diphtheria toxin-IL-2, human chorionic gonadotropin (hCG), thyroid peroxidase (TPO), alpha-galactosidase, alpha-L-iduronidase, beta-glucosidase, alpha-galactosidase A, acid □-glucosidase (acid maltase), anti-thrombin III (AT III), follicle stimulating hormone (FSH), glucagon-like peptide-2 (GLP-2), Factor VII, Factor VIII, B-domain deleted Factor VIII, Factor IX, Factor X, Factor XIII, prokinetisin, extendin-4, CD4, tumor necrosis factor receptor (TNF-R), □-CD20, P-selectin glycoprotein ligand-1 (PSGL-1), complement, transferrin, glycosylation-dependent cell adhesion molecule (GlyCAM), neural-cell adhesion molecule (N-CAM), TNF receptor-IgG Fc region fusion protein, anti-HER2 monoclonal antibody, monoclonal antibody to respiratory syncytial virus, monoclonal antibody to protein F of respiratory syncytial virus, monoclonal antibody to TNF-α, monoclonal antibody to glycoprotein IIb/IIIa, monoclonal antibody to CD20, monoclonal antibody to VEGF-A, monoclonal antibody to PSGL-1, monoclonal antibody to CD4, monoclonal antibody to a-CD3, monoclonal antibody to EGF, monoclonal antibody to carcinoembryonic antigen (CEA) and monoclonal antibody to IL-2 receptor.

64 . A method for making a polypeptide conjugate according to claim 10 , comprising the steps of:

(i) recombinantly producing said sequon polypeptide; and

(ii) enzymatically glycosylating said sequon polypeptide at said O-linked glycosylation sequence.

65 . The method according to claim 64 , wherein said enzymatically glycosylating of step (ii) is accomplished using a GalNAc transferase.

66 . The method according to claim 65 , wherein said GalNAc transferase is human GalNAc-T2.

67 . The method of claim 66 , wherein said GalNAc-T2 is a member selected from lectin-domain deleted GalNAc-T2 and lectin domain truncated GalNAc-T2.

68 . A method for making a library of sequon polypeptides according to claim 47 , said method comprising:

(i) recombinantly producing a first sequon polypeptide by introducing said O-linked glycosylation sequence at a first amino acid position (AA) n ; and

(ii) recombinantly producing at least one additional sequon polypeptide by introducing said O-linked glycosylation sequence at an additional amino acid position selected from (AA) n+x and (AA) n−x , wherein x is a member selected from 1 to (m-n).

69 . A method for identifying a lead polypeptide, said method comprising:

(i) generating a library of sequon polypeptides according to claim 42 ; and

(ii) subjecting at least one member of said library to an enzymatic glycosylation reaction, transferring a glycosyl moiety from a glycosyl donor molecule onto at least one of said O-linked glycosylation sequence, wherein said glycosyl moiety is optionally derivatized with a modifying group,

thereby identifying said lead polypeptide.

70 . The method according to claim 69 , further comprising measuring yield for said enzymatic glycosylation reaction for at least one member of said library.

71 . The method according to claim 70 , wherein said measuring is accomplished by a member selected from mass spectroscopy, gel electrophoresis, nuclear magnetic resonance (NMR) and HPLC.

72 . The method according to claim 70 , wherein said yield for said lead polypeptide is between about 50% and about 100%.

73 . The method according to claim 69 , further comprising, prior to step (ii), purifying at least one member of said library.

74 . The method according to claim 69 , wherein said glycosyl moiety of step (ii) comprises a member selected from a galactose moiety and a GalNAc moiety.

75 . The method according to claim 69 , wherein said enzymatic glycosylation reaction of step (ii) occurs within a host cell, in which said at least one member of said library is expressed.

76 . The method according to claim 69 , further comprising:

(iii) subjecting the product of step (ii) to a PEGylation reaction, wherein said PEGylation reaction is a member selected from a chemical PEGylation reaction and an enzymatic glycoPEGylation reaction.

77 . The method according to claim 76 , wherein step (ii) and step (iii) are performed in a single reaction vessel.

78 . The method according to claim 76 , further comprising measuring yield of said PEGylation reaction.

79 . The method according to claim 78 , wherein said measuring is accomplished by a member selected from mass spectroscopy, gel electrophoresis, nuclear magnetic resonance (NMR) and HPLC.

80 . The method according to claim 78 , wherein said yield of said PEGylation reaction for said lead polypeptide is between about 50% and about 100%.

81 . The method according to claim 76 , wherein said lead polypeptide upon said PEGylation reaction has a therapeutic activity essentially the same as the therapeutic activity of said parent polypeptide.

82 . The method according to claim 76 , wherein said lead polypeptide upon said PEGylation reaction has a therapeutic activity distinct from the therapeutic activity of said parent polypeptide.

83 . The method according to claim 69 , further comprising generating an expression vector comprising a nucleic acid sequence encoding said sequon polypeptide.

84 . The method according to claim 83 , further comprising transfecting said host cell with said expression vector.