TRIVALENT, BISPECIFIC ANTIBODIES

• US 20130022601A1
• Filed: 08/07/2012
• Published: 01/24/2013
• Est. Priority Date: 04/07/2009
• Status: Active Grant

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Brinkmann, Ulrich, Croasdale, Rebecca, Hoffmann, Eike, Klein, Christian, Moessner, Ekkehard, Schanzer, Juergen Michael, Sustmann, Claudio, Umana, Pablo
• Granted Patent: US 9,890,204 B2
• Time in Patent Office: Days
• US Class Current: 424/136.1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The present invention relates to trivalent, bispecific antibodies, methods for their production, pharmaceutical compositions containing the antibodies, and uses thereof.

Claims

1. A trivalent, bispecific antibody comprising

a) a full length antibody that specifically binds to a first antigen wherein the full length antibody consists of two antibody heavy chains and two antibody light chains;

b) a polypeptide consisting of

ba) an antibody heavy chain variable domain (VH);

or

bb) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), wherein the N-terminus of the VH domain of the polypeptide is fused via a peptide connector to the C-terminus of one of the two heavy chains of the full length antibody;

c) a polypeptide consisting of

ca) an antibody light chain variable domain (VL), or

cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL);

wherein N-terminus of the VL domain of the polypeptide is fused via a peptide connector to the C-terminus of the other of the two heavy chains of the full length antibody;

and wherein the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) together form an antigen-binding site specifically binding to a second antigen.

2. The trivalent, bispecific antibody according to claim 1, wherein

the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain 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 wherein

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.

3. The trivalent, bispecific antibody according to claim 2, wherein

i) the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W);

and

ii) the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).

4. The trivalent, bispecific antibody according to claim 3, wherein

both CH3 domains are further altered by the introduction of cysteine as an amino acid in each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.

5. The trivalent, bispecific antibody according to claim 4, wherein

the CH3 domain under i) comprises a T366W mutation;

and

the CH3 domain under ii) comprises T366S, L368A, and Y407V mutations.

6. The trivalent, bispecific antibody according to claim 5, wherein

the CH3 domain under i) comprises Y349C and T366W mutations;

and

the CH3 domain under ii) comprises S354C, T366S, L368A, and Y407V mutations.

7. The trivalent, bispecific antibody according to claim 4, wherein

the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via a interchain disulfide bridge by introduction of a disulfide bond between the following positions:

i) heavy chain variable domain position 44 to light chain variable domain position 100,

ii) heavy chain variable domain position 105 to light chain variable domain position 43, or

iii) heavy chain variable domain position 101 to light chain variable domain position 100.

8. The trivalent, bispecific antibody according to claim 7, wherein

the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via a interchain disulfide bridge by introduction of a disulfide bond between the following positions:

i) heavy chain variable domain position 44 to light chain variable domain position 100.

9. The trivalent, bispecific antibody according to claim 6, wherein the peptide connectors under b) and c) are identical peptides with a length between 25 and 50 amino acids.

10. A pharmaceutical composition comprising a trivalent, bispecific antibody according to claim 1.

11. A nucleic acid encoding a trivalent, bispecific antibody according to claim 2.

12. A nucleic acid encoding a trivalent, bispecific antibody according to claim 3.

13. A nucleic acid encoding a trivalent, bispecific antibody according to claim 4.

14. A nucleic acid encoding a trivalent, bispecific antibody according to claim 5.

15. A nucleic acid encoding a trivalent, bispecific antibody according to claim 6.

16. A nucleic acid encoding a trivalent, bispecific antibody according to claim 7.

17. A nucleic acid encoding a trivalent, bispecific antibody according to claim 8.

18. A method for treating a human suffering from cancer by administering an effective amount of the trivalent, bispecific antibody according to claim 8 to a human in need of such treatment.