Glycosylation of peptides via O-linked glycosylation sequences
First Claim
1. A method comprising expressing a sequon polypeptide in a host cell, wherein said sequon polypeptide corresponds to a parent polypeptide and comprises an exogenous O-linked glycosylation sequence comprising the amino acid sequence of SEQ ID NO:
- 1;
(X)mPO*U(B)p(Z)r(J)s(O)t(P)n
(SEQ ID NO;
1)whereinm, n, and p are integers independently selected from the group consisting of 0 and 1;
r, s, and t are 0;
P is proline;
O* is serine (S) or threonine (T);
U is a member selected from the group consisting of proline (P), glutamic acid (E), glutamine (Q), asparagine (N), threonine (T), glycine (G), and alanine (A);
X and B are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), and serine (S); and
Z, J and O are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), and methionine (M),wherein the O-linked glycosylation is at O*, andwherein the parent polypeptide is selected from the group consisting of 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, α
1-antitrypsin (α
-1 protease inhibitor), tissue-type plasminogen activator (TPA), human chorionic gonadotropin (hCG), 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, and Factor XIII.
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Abstract
The present invention provides sequon polypeptides with an amino acid sequence including one or more exogenous O-linked glycosylation sequence of the invention. In addition, the present invention provides methods of making polypeptide conjugates as well as methods of using such conjugates and their pharmaceutical compositions. The invention further provides libraries of sequon polypeptides, wherein each member of such library includes at least one exogenous O-linked glycosylation sequence of the invention. Also provided are methods of making and using such libraries.
362 Citations
25 Claims
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1. A method comprising expressing a sequon polypeptide in a host cell, wherein said sequon polypeptide corresponds to a parent polypeptide and comprises an exogenous O-linked glycosylation sequence comprising the amino acid sequence of SEQ ID NO:
- 1;
(X)mPO*U(B)p(Z)r(J)s(O)t(P)n
(SEQ ID NO;
1)wherein m, n, and p are integers independently selected from the group consisting of 0 and 1; r, s, and t are 0; P is proline; O* is serine (S) or threonine (T); U is a member selected from the group consisting of proline (P), glutamic acid (E), glutamine (Q), asparagine (N), threonine (T), glycine (G), and alanine (A); X and B are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), and serine (S); and Z, J and O are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), and methionine (M), wherein the O-linked glycosylation is at O*, and wherein the parent polypeptide is selected from the group consisting of 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, α
1-antitrypsin (α
-1 protease inhibitor), tissue-type plasminogen activator (TPA), human chorionic gonadotropin (hCG), 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, and Factor XIII. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
- 1;
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9. A method for making a library of sequon polypeptides,
wherein said library of sequon polypeptides comprises a plurality of different members, wherein each member of said library corresponds to a common parent polypeptide, wherein each member of said library comprises an exogenous O-linked glycosylation sequence, wherein each of said O-linked glycosylation sequence comprises the amino acid sequence of SEQ ID NO: - 1;
(X)mPO*U(B)p(Z)r(J)s(O)t(P)n
(SEQ ID NO;
1)wherein m, n, and p are integers independently selected from the group consisting of 0 and 1; r, s, and t are 0; P is proline; O* is serine (S) or threonine (T); U is a member selected from the group consisting of proline (P), glutamic acid (E), glutamine (Q), asparagine (N), threonine (T), glycine (G), and alanine (A); X and B are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), and serine (S); and Z, J and O are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), and methionine (M), wherein the O-linked glycosylation is at O*, wherein the parent polypeptide is selected from the group consisting of 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, α
1-antitrypsin (α
-1 protease inhibitor), tissue-type plasminogen activator (TPA), human chorionic gonadotropin (hCG), 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, and Factor XIII, andwherein said parent polypeptide has y 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)z, wherein z is a member selected from 1 to y; and (b) 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)z+x and (AA)z−
x, wherein x is a member selected from 1 to (y−
z),said method comprising; (i) recombinantly producing a first sequon polypeptide by introducing said O-linked glycosylation sequence at a first amino acid position (AA)z; 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)z+x and (AA)z−
x, wherein x is a member selected from 1 to (y−
z).
- 1;
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10. A method for identifying a lead polypeptide, said method comprising:
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(i) generating a library of sequon polypeptides, wherein said library of sequon polypeptides comprises a plurality of different members, wherein each member of said library corresponds to a common parent polypeptide, wherein each member of said library comprises an exogenous O-linked glycosylation sequence, and wherein each of said O-linked glycosylation sequence comprises the amino acid sequence of SEQ ID NO;
1;
(X)mPO*U(B)p(Z)r(J)s(O)t(P)n
(SEQ ID NO;
1)wherein m, n, and pare integers independently selected from the group consisting of 0 and 1; r, s, and t are 0; P is proline; O* is serine (S) or threonine (T); U is a member selected from the group consisting of proline (P), glutamic acid (E), glutamine (Q), asparagine (N), threonine (T), glycine (G), and alanine (A); X and B are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), and serine (S); and Z, J and O are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), and methionine (M), wherein the O-linked glycosylation is at O*, wherein the parent polypeptide is selected from the group consisting of 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, α
1-antitrypsin (α
-1 protease inhibitor), tissue-type plasminogen activator (TPA), human chorionic gonadotropin (hCG), 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, and Factor XIII; 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 sequences, wherein said glycosyl moiety is optionally derivatized with a modifying group, thereby identifying said lead polypeptide. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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Specification