Modified Glycoproteins and Uses Thereof

• US 20100081794A1
• Filed: 07/21/2009
• Published: 04/01/2010
• Est. Priority Date: 09/26/2008
• Status: Active Grant

• Current Assignee: Eureka Therapeutics Incorporated
• Original Assignee: Eureka Therapeutics Incorporated
• Inventors: Wang, Pei, Liu, Cheng, Yan, Su, Tai, Wei Chan, Xiang, Jingyi
• Granted Patent: US 8,025,879 B2
• Time in Patent Office: Days
• US Class Current: 530/387.300
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins

Abstract

The present disclosure provides compositions and methods comprising cells producing glycoproteins with variant glycosylation patterns. The methods and compositions may be used in producing antibodies and proteins of therapeutic value.

Claims

1. A glycoprotein modified to exhibit a variant glycosylation pattern, wherein said variant glycosylation pattern is characterized by a change in levels of at least two types of sugar molecules as compared to a corresponding wildtype glycoprotein.

2. The glycoprotein of claim 1, wherein said variant glycosylation pattern is evidenced by a change in level of glucose, galactose, or both.

3. The glycoprotein of claim 1, wherein said change in level of galactose is a decrease

4. The glycoprotein of claim 1, wherein said change in level of glucose is an increase.

5. The glycoprotein of claim 1, wherein said variant glycosylation pattern is evidenced by an increase in glucose level, a decrease in galactose level and a decrease in fucose level.

6. The glycoprotein of claim 1, said variant glycosylation pattern is evidenced by a terminal glucose moiety.

7. The glycoprotein of claim 1, said variant glycosylation pattern comprises an N-linked glycan having a structure of formula (I) or (II):

8. An antibody produced by a modified host cell, the antibody comprising an N-linked glycan, wherein the antibody has increased binding affinity to an Fc gamma receptor IIIa (Fcγ

RIIIa), and/or decreased binding affinity to an Fc gamma receptor IIb (Fcγ

RIIb), as compared to a corresponding antibody produced by an unmodified host cell, thereby enhancing ADCC (antibody-dependent cell-mediated cytotoxicity) activity against effector cells expressing Fcγ

RIIIa and/or Fcγ

RIIb.

10. The antibody of claim 8 or 9, wherein the N-linked glycan comprises one glucose molecule, four mannose molecules, and two N-acetylglycosamine molecules.

11. The antibody of claim 8 or 9, wherein the N-linked glycan comprising one or more fucose molecules.

12. The antibody of claim 8 or 9, wherein the N-linked glycan having a structure of formula (I) or (II):

13. The antibody of claim 8 or 9, wherein the N-linked glycan is attached to an Fc region of the antibody.

14. The antibody of claim 8 or 9, wherein the antibody binds to a cancer antigen.

15. The antibody of claim 14, wherein the cancer antigen is selected from the group consisting of HER2, Immunoglobulin epsilon Fc receptor II, Alk-1,CD20, EGF receptor, VEGF receptor, FGF receptor, NGF receptor, PDGF receptor, EpCam, CD3, CD4, CD11a, CD19, CD22, CD30, CD33, CD38, CD40, CD51, CD55, CD80, CD95, CCR2, CCR3, CCR4, CCR5, CTLA-4, Mucin 1, Mucin 16, Endoglin, Mesothelin receptor, Nogo receptor, folate receptor, CXCR4, insulin-like growth factor receptor, Ganglioside GD3, and alpha and beta Integrins.

16. The antibody of claim 8 or 9, wherein the antibody is an inhibitory antibody.

17. The antibody of claim 8 or 9, wherein the antibody is an IgG antibody.

18. The antibody of claim 8 or 9, wherein the antibody has an increased ADCC (antibody-dependent cell-mediated cytotoxicity) activity against effector cells expressing a low affinity FcR, Fcγ

RIIIa 158F/F or Fcγ

RIIIa 158 F/V, as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012).

19. The antibody of claim 8 or 9, wherein the antibody has an increased ADCC (antibody-dependent cell-mediated cytotoxicity) activity against effector cells expressing a high affinity FcR, Fcγ

RIIIa 158V/V, as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012).

20. The antibody of claim 8 or 9, wherein the host cell is a Chinese Hamster Ovarian (CHO) cell.

21. The antibody of claim 8 or 9, wherein the host cell is selected from the group consisting of NS0, SP2/0, HEK293, PER.C6 and YB2/0 cell.

22. The antibody of claim 8 or 9, wherein the host cell is a myeloma cell.

23. The antibody of claim 8 or 9, wherein the effector cell is a human peripheral blood mononuclear cell (PBMC).

24. The antibody of claim 8 or 9, wherein the antibody is an anti-Her2 antibody.

25. The antibody of claim 8 or 9, wherein the effector cell is an NK cell, a monocyte, a macrophage, or a polymorphonuclear neutraphils (PMN).

9. An antibody produced by a modified host cell, the antibody comprising an N-linked glycan, wherein the antibody exhibits an increased ADCC activity, as compared to a corresponding antibody produced by a corresponding unmodified host cell, and wherein the increased ADCC activity is against effector cells expressing a high affinity Fc gamma receptor, Fcγ

RIIIa 158V/V, and effector cells expressing a low affinity Fc gamma receptor, Fcγ

RIIIa 158F/F or Fcγ

RIIIa 158F/V.