BISPECIFIC ANTIBODIES COMPRISING A DISULFIDE STABILIZED - FV FRAGMENT

• US 20130267686A1
• Filed: 02/21/2013
• Published: 10/10/2013
• Est. Priority Date: 08/24/2010
• Status: Active Grant

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: BRINKMANN, ULRICH, METZ, SILKE, SCHANZER, JUERGEN MICHAEL, HAAS, ALEXANDER
• Granted Patent: US 9,879,095 B2
• Time in Patent Office: Days
• US Class Current: 530/387.3
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• 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 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 2317/56 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 2319/00 Fusion polypeptide

Abstract

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

Claims

1. A bispecific antibody comprising

a) a full length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains;

b) a Fv fragment specifically binding to a second antigen comprising a VH² domain and a VL² domain, wherein both domains are connected via a disulfide bridge, and

wherein only either the VH² domain or the VL² domain is fused via a peptide linker to the heavy or light chain of the full length antibody specifically binding to a first antigen.

2. The bispecific antibody according to claim 1 characterized in that the bispecific antibody is trivalent and

either the VH² domain or the VL² domain is fused via a peptide linker to the heavy chain of the full length antibody specifically binding to a first antigen.

3. The bispecific antibody according to claim 2 characterized in that the VH² domain or VL² domain is N-terminally fused via a peptide linker to the C-terminus of the full length antibody specifically binding to a first antigen.

4. The bispecific antibody according to claim 2, characterized in that the VH² domain or VL² domain is C-terminally fused via a peptide linker to the N-terminus of the full length antibody specifically binding to a first antigen.

5. The bispecific antibody according to claim 1, characterized in that the VH² domain or VL² domain is N-terminally fused via a peptide linker to the C-terminus of the heavy or light chain of the full length antibody specifically binding to a first antigen.

6. The bispecific antibody according to claim 1, characterized in that the VH² domain or VL² domain is C-terminally fused via a peptide linker to the N-terminus of the heavy or light chain of the full length antibody specifically binding to a first antigen.

7. The bispecific antibody according to claim 1, characterized in that the VH² domain and the VL² domain are connected via a disulfide bridge which is introduced between the following positions:

i) VH² domain position 44 and VL² domain position 100,

ii) VH² domain position 105 and VL² domain position 43, or

iii) VH² domain position 101 and VL² domain position 100.

8. The bispecific antibody according to claim 1, characterized in that the VH² domain and the VL² domain are connected via a disulfide bridge which is introduced between the positions VH² domain position 44 and VL² domain position 100.

9. The bispecific antibody according to claim 1 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.

10. The bispecific antibody according to claim 9 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).

11. The bispecific antibody according to claim 9 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.

12. A method for the preparation of the bispecific antibody according to claim 1 comprising the steps of

A) expressing in a mammalian cell nucleic acid encoding a bispecific antibody comprising

a) a full length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains;

b) a Fv fragment specifically binding to a second antigen comprising a VH² domain and a VL² domain, wherein both domains are connected via a disulfide bridge, and

wherein the Fv fragment is fused

via the N-termini of the VH² domain and the VL² domain to the both C-termini of the heavy chain of the full length antibody specifically binding to a first antigen via a first and second peptide linker to, or

via the C-termini of the VH² domain and the VL² domain to the both N-termini of one heavy and one light chain of the full length antibody specifically binding to a first antigen via a first and second peptide linker,

characterized in that

one of the linkers comprises a protease cleavage site cleavable by furin, and the other linker does not comprise a protease cleavage site;

B) recovering said antibody from said cell or the cell culture supernatant.

13. A method for the preparation of the trivalent, bispecific antibody according to claim 2 comprising the steps of

A) expressing in a mammalian cell nucleic acid encoding a bispecific antibody comprising

a) a full length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains;

b) a Fv fragment specifically binding to a second antigen comprising a VH² domain and a VL² domain, wherein both domains are connected via a disulfide bridge, and

wherein the Fv fragment is fused

via the N-termini of the VH² domain and the VL² domain to the both C-termini of the heavy chains of the full length antibody specifically binding to a first antigen via a first and second peptide linker to, or

via the C-termini of the VH² domain and the VL² domain to the both N-termini of the heavy chains of the full length antibody specifically binding to a first antigen via a first and second peptide linker,

characterized in that

one of the linkers comprises a protease cleavage site cleavable by Prescission protease, and the other linker does not comprise a protease cleavage site;

B) recovering said antibody from said cell or the cell culture supernatant.

14. The method according to claim 12 characterized in that the protease cleavage site cleavable by furin is SEQ ID NO:

13 or SEQ ID NO;

14.

15. The method according to claim 13 characterized in that the protease cleavage site cleavable by Prescission protease is SEQ ID NO:

15.

16. The method according to claim 12 characterized in that the mammalian cell is a CHO cell, NS0 cell, SP2/0 cell, HEK293 cell, COS cell or PER.C6 cell.

17. An antibody obtained by the methods according to claim 12.

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

19. The method according to claim 12, wherein the bispecific antibody is characterized in that the VH² domain and the VL² domain are connected via a disulfide bridge which is introduced between the following positions:

i) VH² domain position 44 and VL² domain position 100,

ii) VH² domain position 105 and VL² domain position 43, or

iii) VH² domain position 101 and VL² domain position 100.

20. The method according to claim 13, wherein the trivalent, bispecific antibody is characterized in that the VH² domain and the VL² domain are connected via a disulfide bridge which is introduced between the following positions:

i) VH² domain position 44 and VL² domain position 100,

ii) VH² domain position 105 and VL² domain position 43, or

iii) VH² domain position 101 and VL² domain position 100.