Site-specific antibody-drug conjugation through glycoengineering

Patent metadata

Field	Value
Publication number	US-9580511-B2
Application number	US-201414203479-A
Country	US
Kind code	B2
Filing date	Mar 10, 2014
Priority date	Mar 11, 2013
Publication date	Feb 28, 2017
Grant date	Feb 28, 2017

Abstract

The current disclosure provides binding polypeptides (e.g., antibodies), and effector moiety conjugates thereof (e.g., antibody-drug conjugates or ADCs), comprising a site-specifically engineered drug-glycan linkage within native or engineered glycans of the binding polypetpide. The current disclosure also provides nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. Methods of using the antigen-binding polypeptides disclosed herein to treat disease are also provided.

First claim

We claim:

1. A binding polypeptide comprising at least one glycan comprising at least one moiety of Formula (IV):
-Gal-Sia-C(H)=N-Q-CON-X  Formula (IV),

wherein:
A) Q is NH or O;
B) CON is a connector moiety;
C) X is a cytotoxin;
D) Gal is a galactose moiety;
E) Sia is a sialic acid moiety.

2. The binding polypeptide of claim 1 , wherein the glycan is a biantennary glycan.

3. The binding polypeptide of claim 2 , wherein the biantennary glycan is fucosylated or non-fucosylated.

4. The binding polypeptide of claim 1 , wherein said glycan comprises at least two moieties of Formula (IV), and wherein Sia is present in the cytotoxin of Formula (IV).

5. The binding polypeptide of claim 3 , wherein said glycan comprises at least two moieties of Formula (IV), and wherein Sia is present in both the connector moiety and the cytotoxin of Formula (IV).

6. The binding polypeptide of claim 1 , wherein the glycan is N-linked to the binding polypeptide.

7. The binding polypeptide of claim 1 , wherein the binding polypeptide comprises an Fc domain.

8. The binding polypeptide of claim 7 , wherein the glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 297 of the Fc domain, according to EU numbering.

9. The binding polypeptide of claim 7 , wherein the glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 298 of the Fc domain, according to EU numbering.

10. The binding polypeptide of claim 7 , wherein the Fc domain is human.

11. The binding polypeptide of claim 1 , wherein the binding polypeptide comprises a CH1 domain.

12. The binding polypeptide of claim 11 , wherein the glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 114 of the CH1 domain, according to Kabat numbering.

13. The binding polypeptide of claim 1 , wherein the cytotoxin is selected from the group consisting of the cytotoxins listed in Table 1.

14. The binding polypeptide of claim 1 , wherein the connector moiety comprises a pH-sensitive linker, disulfide linker, enzyme-sensitive linker or other cleavable linker moiety.

15. The binding polypeptide of claim 1 , wherein the connector moiety comprises a linker moiety selected from the group consisting of:

and wherein R 1 -R 5 are each independently selected from a group consisting of: H, an alkyl moiety, or an aryl moiety.

16. The binding polypeptide of claim 1 , wherein the binding polypeptide is an antibody or immunoadhesin.

17. A composition comprising a binding polypeptide of claim 1 and a pharmaceutically acceptable carrier or excipient.

18. The composition of claim 17 , wherein the ratio of cytokine to binding polypeptide is less than 4.

19. The composition of claim 18 , wherein the ratio of cytokine to binding polypeptide is about 2.

20. A method of making a binding polypeptide of claim 1 , wherein the method comprises reacting an effector moiety of Formula (I):
NH 2 -Q-CON-X  Formula (I),

wherein:
A) Q is NH or O;
B) CON is a connector moiety; and
C) X is a cytotoxin, with a binding polypeptide comprising an oxidized glycan.

21. The method of claim 20 , wherein the binding polypeptide comprising an oxidized glycan is generated by reacting a binding polypeptide comprising a glycan with a mildly oxidizing agent.

22. The method of claim 21 , wherein the mildly oxidizing agent is sodium periodate.

23. The method of claim 22 , wherein less than 1 mM sodium periodate is employed.

24. The method of claim 21 , wherein the mildly oxidizing agent is galactose oxidase.

25. The method of claim 20 , wherein the binding polypeptide comprising the glycan comprises one or two terminal sialic acid residues.

26. The method of claim 25 , where the terminal sialic acid residues are introduced by treatment of the binding polypeptide with a sialyltransferase or combination of sialyltransferase and galactosyltransferase.

27. The binding polypeptide of claim 1 , comprising an anti-human epidermal growth factor receptor 2 (HER2) antibody or an anti-fibroblast activation protein (FAP) antibody.

28. The binding polypeptide of claim 1 , wherein the glycan is selected from the group consisting of one or any combination of a biantennary glycan, a glycan comprising at least two moieties of Formula (IV), and a glycan that is N-linked to the binding polypeptide.

29. A binding polypeptide comprising at least one modified glycan comprising at least one moiety of Formula (IV):
-Gal-Sia-C(H)=N-Q-CON-X  Formula (IV),

wherein:
A) Q is O;
B) CON is Cys-MC-VC-PABC;
C) X is MMAE;
D) Gal is a galactose moiety;
E) Sia is a sialic acid moiety; and

wherein Sia is present or absent.

30. A binding polypeptide comprising at least one modified glycan comprising at least one moiety of Formula (IV):
-Gal-Sia-C(H)=N-Q-CON-X  Formula (IV),

wherein:
A) Q is NH or O;
B) CON is a connector moiety;
C) X is a diagnostic or therapeutic effector moiety;
D) Gal is a galactose moiety;
E) Sia is a sialic acid moiety;

and
wherein the glycan is selected from the group consisting of one or any combination of a biantennary glycan, a glycan comprising at least two moieties of Formula (IV), and a glycan that is N-linked to the binding polypeptide.

31. A binding polypeptide comprising at least one glycan comprising at least one moiety of Formula (IV):
-Gal-Sia-C(H)=N-Q-CON-X  Formula (IV),

wherein:
A) Q is NH or O;
B) CON is a connector moiety;
C) X is a diagnostic or therapeutic effector moiety;
D) Gal is a galactose moiety;
E) Sia is a sialic acid moiety;

and
wherein the binding polypeptide comprises an anti-HER2 antibody or an anti-FAP antibody.

32. A binding polypeptide comprising at least one modified glycan comprising at least one moiety of Formula (IV):
-Gal-Sia-C(H)=N-Q-CON-X  Formula (IV),

wherein:
A) Q is NH or O;
B) CON is a linker moiety;
C) X is a cytotoxin;
D) Gal is a galactose moiety;
E) Sia is a sialic acid moiety.

33. The binding polypeptide of claim 32 , wherein one or more of the following conditions are met:
a) the modified glycan is a biantennary glycan;
b) the biantennary glycan is fucosylated or non-fucosylated;
c) the modified glycan comprises at least two moieties of Formula (IV);
d) the modified glycan is N-linked to the binding polypeptide;
e) the binding polypeptide comprises an Fc domain;
f) the binding polypeptide comprises a human Fc domain;
g) the sialic acid moiety is present at a position that did not have a sialic acid moiety prior to modification;
h) the binding polypeptide is an antibody or immunoadhesin; and
i) the binding polypeptide is an IgG antibody.

34. The binding polypeptide of claim 32 , wherein the binding polypeptide comprises an Fc domain and wherein the modified glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 297 of the Fc domain or at amino acid position 298 of the Fc domain, according to EU numbering.

35. The binding polypeptide of clai

Assignees

• Genzyme Corp

Inventors

• Pan Clark
• Zhou Qun
• Stefano James
• Dhal Pradeep
• Chen Bo
• Gianolio Diego
• Miller Robert
• Qiu Huawei

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61K47/6889

  Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment · CPC title

• C07K16/2809

  against the T-cell receptor (TcR)-CD3 complex · CPC title

• C07K16/2893 Primary

  against CD52 · CPC title