ACTIVATABLE BISPECIFIC ANTIBODIES

• US 20130266568A1
• Filed: 02/21/2013
• Published: 10/10/2013
• Est. Priority Date: 08/24/2010
• Status: Abandoned Application

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Roche Glycart AG (Roche Holding AG)
• Inventors: BRINKMANN, ULRICH, CROASDALE, REBECCA, SCHANZER, JUERGEN MICHAEL, METZ, SILKE, SUSTMANN, CLAUDIO, UMANA, PABLO
• Application Number:
  US13/773,013
• Publication Number:
  US 20130266568A1
• Time in Patent Office: Days
• US Class Current: 424/136.1
• 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 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/624 Disulfide-stabilized antibody (dsFv)
• CPC Class Codes: C07K 2317/64 comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The current invention relates to bispecific antibodies wherein the binding affinity to one of the two antigens is reduced and which can be activated by tumor- or inflammation-specific proteases, and the preparation and use of such bispecific antibodies.

Claims

1. A bispecific antibody comprising

a) a first antibody that binds to a first antigen comprising a VH¹ domain and a VL¹ domain, and

b) a second antibody that binds to a second antigen,

wherein the VH¹ domain is fused N-terminally via a first peptide linker to the second antibody, and the VL¹ domain is fused N-terminally via a second peptide linker to the second antibody, and

characterized in that

one of the linkers comprises a tumor- or inflammation-specific protease cleavage site, and the other linker does not comprise a protease cleavage site;

and

the binding affinity of the bispecific antibody to the first antigen is reduced 5 times or more compared to the corresponding bispecific antibody in which the protease cleavage site is cleaved.

2. The bispecific antibody according to claim 1 characterized in that the second antibody is a whole antibody;

and

the VH¹ domain is fused N-terminally via the first linker to the C-terminus of the first heavy chain of the second antibody, and

the VL¹ domain is fused N-terminally via the second linker to the C-terminus of the second heavy chain of the second antibody.

3. The bispecific antibody according to claim 2 characterized in comprising from C- to N-terminus the following polypeptide chains

one VH¹-peptide linker-CH3-CH2-CH1-VH² chain

two CL-VL² chains

one VL¹-peptide linker-CH3-CH2-CH1-VH² chain.

4. The bispecific antibody according to claim 2 characterized in that

the first CH3 domain of the heavy chain of the whole antibody and the second CH3 domain of the whole antibody each meet at an interface which comprises an alteration in the original interface between the antibody CH3 domains;

wherein i) in the CH3 domain of one heavy chain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain and

ii) in the CH3 domain of the other heavy chain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable.

5. The bispecific antibody according to claim 4 characterized in that said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W) and said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

6. The bispecific antibody according to claim 5 characterized in that both CH3 domains are further altered by the introduction of a cysteine (C) residue in positions of each CH3 domain such that a disulfide bridge between the CH3 domains can be formed.

7. The bispecific antibody according to claim 1, characterized in that

the VH¹ domain and the VL¹ domain are stabilized

a) by a disulfide bridge;

and/or

b) by a CH1 domain and a CL domain.

8. The bispecific antibody according to claim 1 characterized in that the second antibody is a Fv fragment;

and

the VH¹ domain is fused N-terminally via the first linker to the C-terminus of the first chain of the second antibody Fv fragment, and

the VL¹ domain is fused N-terminally via a second linker to the C-terminus of the second chain of the second antibody Fv fragment.

9. The bispecific antibody according to claim 8 characterized in that the first antibody is a whole antibody.

10. The bispecific antibody according to claim 9 characterized in comprising from C- to N-terminus the following polypeptide chains

a)

two CH3-CH2-CH1-VH¹-peptide linker-VH² chains

two CL-VL¹-peptide linker-VL²-chains;

or

b)

two CH3-CH2-CH1-VH¹-peptide linker-VL² chains

two CL-VL¹-peptide linker-VH² chains.

11. The bispecific antibody according to claim 8, characterized in that

the VH² domain and the VL² domain are stabilized by a disulfide bridge.

12. The bispecific antibody according to claim 1 characterized in that the second antibody is a Fab fragment;

and

the VH¹ domain is fused N-terminally via the first linker to the C-terminus of the first chain of the second antibody Fab fragment, and

the VL¹ domain is fused N-terminally via a second linker to the C-terminus of the second chain of the second antibody Fab fragment.

13. The bispecific antibody according to claim 12 characterized in that the first antibody is a whole antibody.

14. The bispecific antibody according to claims 13 characterized in comprising from C- to N-terminus the following polypeptide chains

a)

two CH3-CH2-CH1-VH¹-peptide linker-CH1-VH² chains

two CL-VL¹-peptide linker-CL-VL²-chains;

or

b)

two CH3-CH2-CH1-VH¹-peptide linker-CL-VL² chains

two CL-VL¹-peptide linker-CH1-VH²-chains.

15. The bispecific antibody according to claim 12 characterized in that the first antibody is a Fv fragment.

16. The bispecific antibody according to claim 15 characterized in comprising from C- to N-terminus the following polypeptide chains

a)

one VH¹-peptide linker-CH1-VH² chain

one VL¹-peptide linker-CL-VL² chains;

or

b)

one VH¹-peptide linker-CL-VL² chain

one VL¹-peptide linker-CH1-VH² chains.

17. The bispecific antibody according to claim 1 characterized in that

the binding affinity of the bispecific antibody to the first antigen is reduced 10 times or more compared to the corresponding bispecific antibody in which the protease cleavage site is cleaved.

18. A pharmaceutical composition comprising the antibody according to claim 1.

23. A method of treatment of patient suffering from cancer or inflammation by administering an antibody according to claim 1 to a patient in need of such treatment.

25. The method of claim 23 wherein the bispecific antibody is characterized in that the binding affinity of the bispecific antibody to the first antigen is reduced 10 times or more compared to the corresponding bispecific antibody in which the protease cleavage site is cleaved.

26. The pharmaceutical composition of claim 18, wherein the bispecific antibody is characterized in that the binding affinity of the bispecific antibody to the first antigen is reduced 10 times or more compared to the corresponding bispecific antibody in which the protease cleavage site is cleaved.

19-22. -22. (canceled)

24. (canceled)