Tri- or Tetraspecific Antibodies

• US 20100322935A1
• Filed: 05/27/2010
• Published: 12/23/2010
• Est. Priority Date: 05/27/2009
• Status: Active Grant

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: SCHANZER, JUERGEN MICHAEL, SCHAEFER, WOLFGANG, KLEIN, CHRISTIAN, CROASDALE, REBECCA
• Granted Patent: US 8,796,424 B2
• Time in Patent Office: Days
• US Class Current: 424/136.1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/35 Valency
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/64 comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2317/66 comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The present invention relates to tri- or tetraspecific antibodies, their manufacture and use.

Claims

1. A trispecific or tetraspecific antibody, comprising:

a) a light chain and heavy chain of a full length antibody which specifically binds to a first antigen; and

b) a modified light chain and modified heavy chain of a full length antibody which specifically binds to a second antigen, wherein the variable domains VL and VH are replaced by each other, and/or wherein the constant domains CL and CH1 are replaced by each other; and

c) one to four antigen binding peptides fused via a peptide connector to the C- or N-terminus of the light chains or heavy chains of a) and/or b) wherein said antigen binding peptides specifically bind one or two further antigens.

2. The antibody according to claim 1, wherein the antigen binding peptides comprise one or two antigen binding peptides which specifically bind to one or two further antigens.

3. The antibody according to claim 1, wherein the antigen binding peptides comprise one or two antigen binding peptides which specifically bind to a third antigen.

4. The antibody according to claim 1, wherein the antigen binding peptides comprise two identical antigen binding peptides which specifically bind to a third antigen.

5. The antibody according to claim 1, wherein the antigen binding peptides comprise one antigen binding peptide which specifically binds to a third antigen and one antigen binding peptide which specifically binds to a fourth antigen.

6. The antibody according to claim 1, wherein the antigen binding peptides are selected from the group of a scFv fragment and a scFab fragment.

7. The antibody according to claim 1, wherein the antigen binding peptides are scFv fragments.

8. The antibody according to claim 1, wherein the antigen binding peptides are scFab fragments.

9. The antibody according to claim 1, wherein the antigen binding peptides are fused to the C-terminus of the heavy chains of a) and/or b).

10. The antibody according to claim 1, wherein the CH3 domain of the heavy chain of the full length antibody of a) and the CH3 domain of the modified heavy chain of the full length antibody of b) which meet at an interface which has been altered to promote the formation of the trispecific or tetraspecific antibody, wherein the interface of the first CH3 domain comprises a protuberance which is positionable in a cavity in the interface of the second CH3 domain, and wherein the amino acid sequence of an original interface has been altered so as to introduce the protuberance and/or cavity into the altered interface;

wherein said interface is altered to promote the formation of the trispecific or tetraspecific antibody, wherein the alteration comprises;

i) a CH3 domain of one heavy chain is altered, so that within the original interface the CH3 domain of one heavy chain that meets the original interface of the CH3 domain of the other heavy chain within the tri- or tetraspecific antibody, 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) a CH3 domain of the other heavy chain is altered, so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the tri- or tetraspecific antibody, 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.

11. The antibody according to claim 10, wherein said 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 said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).

12. The antibody of claim 10 wherein the protuberance comprises an introduced arginine (R) residue.

13. The antibody of claim 10 wherein the protuberance comprises an introduced phenylalanine (F) residue.

14. The antibody of claim 10 wherein the protuberance comprises an introduced tyrosine (Y) residue.

15. The antibody of claim 10 wherein the protuberance comprises an introduced tryptophan (W) residue.

16. The antibody of claim 10 wherein the cavity is formed by an introduced alanine (A) residue.

17. The antibody of claim 10 wherein the cavity is formed by an introduced serine (S) residue.

18. The antibody of claim 10 wherein the cavity is formed by an introduced threonine (T) residue.

19. The antibody of claim 10 wherein the cavity is formed by an introduced valine (V) residue.

20. The antibody according to claim 10 or 11, characterized in that both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.

21. A method for the preparation of a trispecific or tetraspecific antibody according to claim 1 or 10 comprising the steps of

a) transforming a host cell with vectors comprising nucleic acid molecules encoding

aa) the light chain and heavy chain of an antibody which specifically binds to a first antigen;

and

ab) the modified light chain and modified heavy chain of a full length antibody which specifically binds to a second antigen, wherein the variable domains VL and VH are replaced by each other, and/or wherein the constant domains CL and CH1 are replaced by each other; and

wherein the nucleic acid molecules further encode one to four antigen binding peptides which specifically bind to one or two further antigens fused via a peptide connector to the C- or N-terminus of the light chains or heavy chains of aa) and/or ab),

b) culturing the host cell under conditions that allow synthesis of said antibody; and

c) recovering said antibody from said culture.

22. A host cell comprising the vectors according to claim 21.

23. A composition, comprising the antibody according to claim 1.

24. The composition according to claim 23 wherein the composition is a pharmaceutical or diagnostic composition.

25. A pharmaceutical composition comprising an antibody according to claim 1 and at least one pharmaceutically acceptable excipient.

26. A method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of an antibody to claim 1.