Antibodies Against Amyloid Beta 4 With Glycosylated in the Variable Region

• US 20090252724A1
• Filed: 12/11/2006
• Published: 10/08/2009
• Est. Priority Date: 12/12/2005
• Status: Active Grant

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Bohrmann, Bernd, Weyer, Karl, Schuhbauer, Diana, Schaubmar, Andreas, Huber, Walter, Lang, Kurt, Brockhaus, Manfred, Koll, Hans, Loetscher, Hansruedi
• Granted Patent: US 8,906,370 B2
• Time in Patent Office: Days
• US Class Current: 424/133.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/77 Internalization into the cell

Abstract

The present invention relates to a purified antibody molecule preparation being characterized in that at least one antigen binding site comprises a glycosylated asparagine (Asn) in the variable region of the heavy chain (V_H). More specifically, a pharmaceutical and a diagnostic composition comprising said antibody molecule and antibody mixtures are provided which is/are capable of specifically recognizing the β-A4 peptide/Aβ4. The present invention relates in particular to a mixture of antibodies comprising one or two glycosylated antigen binding sites with a glycosylated asparagine (Asn) in the variable region of the heavy chain, i.e. mixtures of isoforms of antibodies which comprise a glycosylated Asn in the variable region of the heavy chain (V_H). Also disclosed are compositions or antibody preparations comprising the specifically glycosylated antibody isoforms. Furthermore, the pharmaceutical and diagnostic uses for these antibodies are provided. The antibody isoforms may for example be used in the pharmaceutical intervention of amyloidogenesis or amyloid-plaque formation and/or in the diagnosis of the same.

Claims

1. A purified antibody molecule which comprises at least one antigen binding site comprising a glycosylated asparagine (Asn) in the variable region of a heavy chain (V_H).

2. The purified antibody molecule of claim 1, wherein said glycosylated asparagine (Asn) in the variable region of the heavy chain (V_H) is in the CDR2 region.

3. The purified antibody molecule of claim 1, wherein said glycosylated asparagine (Asn) in the variable region of the heavy chain (V_H) is in position 52 of SEQ ID NO:

2 or position 52 of SEQ ID NO;

6.

4. The purified antibody molecule of claim 1, wherein said antibody molecule is capable of specifically recognizing the β

-A4 peptide/Aβ

4.

5. The purified antibody molecule of claim 4, wherein said β

-A4 peptide/Aβ

4 has the following sequence;

6. The antibody molecule of claim 1, comprising a heavy chain (V_H) encoded by:

(a) a nucleic acid molecule comprising the nucleotide sequence as shown in SEQ ID NO;

1

7. The antibody molecule of claim 1, wherein the variable region comprising a glycosylated asparagine (Asn) is comprised in a heavy chain selected from the group consisting of:

(a) a heavy chain polypeptide encoded by a nucleic acid molecule as shown in SEQ ID NOS;

5, 23 or 25;

(b) a heavy chain polypeptide having the amino acid sequence as shown in SEQ ID NO;

6 or 26;

(c) a heavy chain polypeptide encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule defined in (a) and which encodes a polypeptide which is capable of binding to the β

-A4 peptide/Aβ

4 as shown in the following amino acid sequence

8. The antibody molecule of claim 4, comprising two antigen binding sites, each antigen binding site comprising a glycosylated Asn in the respective variable region of the heavy chain (V_H).

9. The antibody molecule of claim 8, wherein the CDR2 region of each of the heavy chains (V_H) comprises the glycosylated Asn.

10. The antibody molecule of claim 9, wherein said Asn is Asn on position 52 of SEQ ID NO:

2 or is the Asn on position 52 of SEQ ID NO;

6.

11. The antibody molecule of claim 1, wherein said glycosylation on Asn in the V_H region is selected from the group consisting of

(a) a sugar structure of the biantennary complex type;

(b) a sugar structure of the biantennary hybrid type;

(c) a sugar structure of the biantennary oligomannose type; and

(d) a sugar bi-antennary structure of the any of the structures as provided in FIG. 5 or FIG. 27.

12. The antibody molecule of claim 11, wherein said sugar structure does not comprise a core fucosylation.

13. The antibody molecule of claim 2, wherein said CDR-2 region has an amino acid sequence as shown in SEQ ID NO:

12.

14. The antibody molecule of claim 4, which is recombinantly produced.

15. The antibody molecule of claim 14 which is produced in a CHO-cell.

16. The antibody molecule of claim 15, whereby wherein said CHO cell is CHO K1 or CHO K1 SV.

17. A method for the preparation of an antibody molecule according to claim 1 comprising the steps of

(a) recombinantly expressing a heterologous nucleic acid molecule encoding an antibody molecule as defined in claim 1 in a mammalian cultured cell;

(b) purifying said recombinantly expressed antibody molecule by a method comprising the steps of

(b1) protein A column purification;

(b2) ion exchange column purification; and

(b3) size exclusion column purification.

18. The method of claim 17, wherein said ion exchange column purification comprises a cation exchange chromatography.

19. The method of claim 17, further comprising as an additional step (c) an analytical chromatography and/or a further concentration step.

23. A method for the prevention and/or treatment of a disease associated with amyloidogenesis and/or amyloid-plaque formation comprising administering to a subject in need thereof an effective amount of an antibody molecule according to claim 4.

24. A diagnostic kit for the detection of a disease associated with amyloidogenesis and/or amyloid-plaque formation comprising the antibody molecule according to claim 4.

25. A method for the disintegration of β

-amyloid plaques comprising administering to a subject in need thereof an effective amount of an antibody molecule according to claim 4.

26. A method for passive immunization against β

-amyloid plaque formation comprising administering to a subject in need thereof an effective amount of an antibody molecule according to claim 4.

27. A method for the preventive treatment against a disease associated with amyloidogenesis and/or amyloid-plaque formation comprising administering to a subject in need thereof an effective amount of an antibody molecule according to claim 4.

28. The method according to claim 27, wherein pre-existing plaques or aggregation intermediates of amyloid-β

are to be reduced.

29. A diagnostic kit for the diagnosis of a disease associated with amyloidogenesis and/or amyloid-plaque formation in a patient or for the diagnosis of the susceptibility of a patient for the development of a disease associated with amyloidogenesis and/or amyloid-plaque formation comprising the antibody molecule according to claim 8.

30. The method according to claim 23, wherein said disease is dementia, Alzheimer'"'"'s disease, motor neuropathy, Down'"'"'s syndrome, Creutzfeld Jacob disease, hereditary cerebral hemorrhage with amyloidosis Dutch type, dementia associated with Lewy body formation, Parkinson'"'"'s disease, HIV-related dementia, ALS or neuronal disorders related to aging.

31. A kit comprising an antibody molecule of claim 1.

20. A composition comprising a first antibody molecule wherein one antigen binding site of said first antibody molecule comprises a glycosylated asparagine (Asn) in the variable region of the heavy chain (V_H) and further comprising a second antibody molecule wherein the two antigen binding sites of said second antibody molecule comprise a glycosylated asparagine (Asn) in the variable region of the heavy chains (V_H), and wherein said composition comprises less than 5% of an antibody molecule wherein none of the antigen binding sites of said antibody molecule comprises a glycosylated asparagine (Asn) in the variable region of the heavy chain (V_H).

21. The composition of claim 20 comprising an antibody molecule according to claim 4.

22. The composition of claim 20, which is a pharmaceutical composition, and further comprises a pharmaceutically acceptable carrier or diluent.

32. A method for the prevention, treatment or amelioration of a disease associated with amyloidogenesis and/or amyloid-plaque formation, comprising administering an effective amount of the composition of claim 20 to a mammal in need thereof.

33. The method of claim 32, wherein said disease is dementia, Alzheimer'"'"'s disease, motor neuropathy, Down'"'"'s syndrome, Creutzfeld Jacob disease, hereditary cerebral hemorrhage with amyloidosis Dutch type, dementia associated with Lewy body formation, Parkinson'"'"'s disease, HIV-related dementia, ALS or neuronal disorders related to aging.

34. The method of claim 32, wherein said mammal is a human.

35. An antibody molecule comprising two light chains and two heavy chains, wherein each of the light chains comprises SEQ ID NO:

8 and each of the heavy chains comprises SEQ ID NO;

6, and wherein position 52 of SEQ ID NO;

6 in one of the heavy chains, but not both, is glycosylated.

37. A pharmaceutical composition comprising the antibody molecule of claim 35 and a pharmaceutically acceptable carrier or diluent.

39. A pharmaceutical composition comprising the antibody molecules of claims 35 and 36 and a pharmaceutically acceptable carrier or diluent.

40. The pharmaceutical composition of claim 39, wherein less than 5% of the antibody molecules present in the composition are non-glycosylated in the variable region.

36. An antibody molecule comprising two light chains and two heavy chains, wherein each of the light chains comprises SEQ ID NO:

8 and each of the heavy chains comprises SEQ ID NO;

6, and wherein position 52 of SEQ ID NO;

6 in both of the heavy chains is glycosylated.

38. A pharmaceutical composition comprising the antibody molecule of claim 36 and a pharmaceutically acceptable carrier or diluent.