Methods for identifying safe and functional humanized antibodies

• US 9,221,900 B2
• Filed: 07/29/2011
• Issued: 12/29/2015
• Est. Priority Date: 07/30/2010
• Status: Active Grant

• Current Assignee: AC Immune SA, Genentech, Inc. (Roche Holding AG)
• Original Assignee: AC Immune SA, Genentech, Inc. (Roche Holding AG)
• Inventors: Pfeifer, Andrea, Muhs, Andreas, Adolfsson, Oskar, Watts, Ryan J.
• Primary Examiner(s): Ballard, Kimberly A
• Application Number:
  US13/136,435
• Publication Number:
  US 20120064065A1
• Time in Patent Office: 1,614 Days
• US Class Current: 1/1
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• 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 27/02 Ophthalmic agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 14/4711 Alzheimer's disease; Amyloid plaque core protein
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: G01N 2333/4709 Amyloid plaque core protein
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 2800/2821 Alzheimer
• CPC Class Codes: G01N 33/5055 involving macrophages
• CPC Class Codes: G01N 33/5058 Neurological cells

Abstract

The present invention is related to safe and functional antibodies for the therapeutic and diagnostic use in the treatment of an amyloidosis, a group of disorders and abnormalities associated with amyloid protein, such as Alzheimer'"'"'s disease.

Claims

1. A method for identifying an anti-beta amyloid antibody having neuroprotective activity, wherein the method comprises:

a. incubating microglia cells with beta amyloid oligomer and the anti-beta amyloid antibody; and

b. measuring the uptake of beta amyloid oligomer by microglia cells; and

c. measuring p38 MAP kinase activation in the microglia cells, and

d. identifying the uptake of beta amyloid oligomer into the microglia cells and an intermediate level of p38 MAP kinase activation in the microglia cells incubated with the anti-beta amyloid antibody as an indication of an anti-beta amyloid antibody having neuroprotective activity,

wherein the intermediate level of p38 MAP kinase activation is a level higher than a level of activation of p38 MAP kinase in the presence of beta amyloid oligomer without the anti-beta amyloid antibody, but lower than a level of activation of p38 MAP kinase in the presence of beta amyloid oligomer and an IgG1 antibody that specifically binds beta amyloid oligomer, and wherein the IgG1 antibody comprises a human IgG1 constant region.

4. The method of claim 1 or 2, wherein the intermediate activation of p38 MAP kinase in microglia cells is between 5%-15%;

10%-20%;

15%-25%;

20%-30%;

35%-45%;

40%-50%;

or 45%-55% above p38 MAP kinase activation in microglia cells in the presence of beta amyloid oligomer without the anti-beta amyloid antibody.

5. The method of claim 1 or 2, wherein the anti-beta amyloid antibody comprises:

a. a light chain variable region complementarity determining region (CDR)1 having the amino acid sequence of the light chain variable region CDR1 of an anti-beta amyloid antibody listed in Table 2;

b. a light chain variable region CDR2 having the amino acid sequence of the light chain variable region CDR2 of an anti-beta amyloid antibody listed in Table 2; and

c. a light chain variable region CDR3 having the amino acid sequence of the light chain variable region CDR3 of an anti-beta amyloid antibody listed in Table 2.

6. The method of claim 5, wherein the anti-beta amyloid antibody further comprises:

a. a heavy chain variable region CDR1 having the amino acid sequence of the heavy chain variable region CDR1 of an anti-beta amyloid antibody listed in Table 2;

b. a heavy chain variable region CDR2 having the amino acid sequence of the heavy chain variable region CDR2 of an anti-beta amyloid antibody listed in Table 2; and

c. a heavy chain variable region CDR3 having the amino acid sequence of the heavy chain variable region CDR3 of an anti-beta amyloid antibody listed in Table 2.

7. The method of claim 6, wherein the CDR1, CDR2, and CDR3 of the light chain variable region and the CDR1, CDR2, and CDR3 of the heavy chain variable region are all derived from the same anti-beta amyloid antibody listed in Table 2.

8. The method of claim 5, wherein the CDR1, CDR2, and CDR3 of the light chain variable region are all derived from the same anti-beta amyloid antibody listed in Table 2.

9. The method of claim 1 or 2, wherein the anti-beta amyloid antibody comprises:

a. a heavy chain variable region CDR1 having the amino acid sequence of the heavy chain variable region CDR1 of an anti-beta amyloid antibody listed in Table 2;

b. a heavy chain variable region CDR2 having the amino acid sequence of the heavy chain variable region CDR2 of an anti-beta amyloid antibody listed in Table 2; and

c. a heavy chain variable region CDR3 having the amino acid sequence of the heavy chain variable region CDR3 of an anti-beta amyloid antibody listed in Table 2.

10. The method of claim 9, wherein the CDR1, CDR2, and CDR3 of the heavy chain variable region are all derived from the same anti-beta amyloid antibody listed in Table 2.

11. The method of claim 1 or 2, wherein the intermediate level of p38 MAP kinase activation is determined by the step of:

comparing the levels of p38 MAP kinase activation in microglia cells by beta amyloid oligomer in the presence and absence of the anti-beta amyloid antibody, wherein if the level of p38 MAP kinase activation by beta amyloid oligomer in the presence of the anti-beta amyloid antibody is greater than the level of p38 MAP kinase activation by beta-amyloid oligomer in the absence of the anti-beta amyloid antibody, but less than the level of p38 MAP kinase activation by beta-amyloid oligomer and an IgG1 anti-beta amyloid antibody comprising a human IgG1 constant region, then the level of p38 MAP kinase activation is determined to be an intermediate level.

12. The method of claim 1 or 2, wherein the anti-beta amyloid antibody is safe and functional for the treatment of an amyloidosis associated with beta-amyloid deposits.

13. The method of claim 12, wherein the amyloidosis is Alzheimer'"'"'s Disease.

14. The method of claim 1 or 2, wherein the anti-beta amyloid antibody has the effector region of an IgG4 antibody.

15. The method of claim 1 or 2, wherein the anti-beta amyloid antibody is a non-IgG1 antibody which does not comprise a human IgG1 constant region.

16. The method of claim 1 or 2, wherein the anti-beta amyloid antibody comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:

1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO;

2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO;

3, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO;

4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO;

5, SEQ ID NO;

23 or SEQ ID NO;

24, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO;

6.

17. The method of claim 1 or 2, wherein the anti-beta amyloid antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

7.

18. The method of claim 1 or 2, wherein the anti-beta amyloid antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

10.

19. The method of claim 1 or 2, wherein the anti-beta amyloid antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO;

10.

26. The method of claim 1, 2, or 20, wherein the beta amyloid oligomer in step a. is beta amyloid 1-42 oligomers.

2. A method for testing the safety of an anti-beta amyloid antibody, wherein the method comprises:

a. incubating microglia cells with beta amyloid oligomer in the presence and the absence of a anti-beta amyloid antibody; and

b. measuring p38 MAP kinase activation in microglia cells in the presence and the absence of the anti-beta amyloid antibody; and

c. identifying the anti-beta amyloid antibody as safe if the anti-beta amyloid antibody is capable of inducing an intermediate level of p38 MAP kinase activation in the microglia cells, wherein the intermediate level of p38 MAP kinase activation is a level higher than a level of activation of p38 MAP kinase in the presence of beta amyloid oligomer without the anti-beta amyloid antibody, but lower than a level of activation of p38 MAP kinase in the presence of beta amyloid oligomer and an IgG1 antibody that specifically binds beta amyloid oligomer, and wherein the IgG1 antibody comprises a human IgG1 constant region.

3. The method of claim 2, wherein the IgG1 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

10.

20. A method for testing the safety and neuroprotective activity of an anti-beta amyloid antibody, wherein the method comprises:

a. incubating microglia cells with beta amyloid oligomer in the presence and absence of the anti-beta amyloid antibody;

b. measuring uptake of beta amyloid oligomer by the microglia cells in the presence and absence of the anti-beta amyloid antibody;

c. measuring p38 MAP kinase activation in microglia cells in the presence and absence of the anti-beta amyloid antibody,

wherein if (i) the presence of the anti-beta amyloid antibody increases uptake of beta amyloid oligomer by microglia cells as compared to uptake of beta amyloid oligomer by microglia cells in the absence of the anti-beta amyloid antibody, and (ii) the level of p38 MAP kinase activation in microglia cells by beta-amyloid oligomer in the presence of the anti-beta amyloid antibody is greater than the level of p38 MAP kinase activation by beta-amyloid oligomer in the absence of the anti-beta amyloid antibody, but less than the level of p38 MAP kinase activation by beta amyloid oligomer and an IgG1 anti-beta amyloid antibody comprising a human IgG1 constant region, then the anti-beta amyloid antibody is identified as a safe and neuroprotective anti-beta amyloid antibody.

21. The method of claim 20, wherein the anti-beta amyloid antibody is safe and functional for the treatment of an amyloidosis associated with beta-amyloid deposits.

22. The method of claim 21, wherein the antibody is a non-IgG1 antibody which does not comprise a human IgG1 constant region.

23. The method of claim 20, wherein the amyloidosis is Alzheimer'"'"'s Disease.

24. The method of claim 20, wherein the anti-beta amyloid antibody has the effector region of an IgG4 antibody.

25. The method of claim 20, wherein the anti-beta amyloid antibody is a human IgG4 isotype and the IgG1 antibody is the same anti-beta amyloid antibody having a human IgG1 isotype.