BIVALENT, BISPECIFIC ANTIBODIES

• US 20120225071A1
• Filed: 01/31/2012
• Published: 09/06/2012
• Est. Priority Date: 12/21/2007
• Status: Active Grant

• Current Assignee: Christian Klein, Wolfgang Schaefer
• Original Assignee: Christian Klein, Wolfgang Schaefer
• Inventors: Klein, Christian, Schaefer, Wolfgang
• Granted Patent: US 10,138,293 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 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The present invention relates to nucleic acids which encode the heavy chains and light chains of a novel domain exchanged, bivalent, bispecific antibody, and vectors comprising the same.

Claims

1. An isolated nucleic acid encoding the heavy chain of an antibody wherein the VH domain of said heavy chain is replaced by the VL domain of the corresponding light chain of said antibody.

2. An isolated nucleic acid according to claim 1 wherein said antibody is an anti-IGF-1R antibody.

3. An isolated nucleic acid according to claim 2 wherein said heavy chain is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:

3 and SEQ ID NO;

10.

7. An isolated nucleic acid according to claim 1 wherein the constant heavy chain domain CH3 domain is replaced by the constant heavy chain domain CH1 of said heavy chain.

8. An isolated nucleic acid according to claim 1 wherein the constant heavy chain domain CH3 is replaced by the constant light chain domain CL of said antibody.

9. An isolated nucleic acid according to claim 1 wherein the constant heavy chain domain CH3 is altered so that an amino acid residue is replaced with an amino acid residue having a larger side chain volume.

10. An isolated nucleic acid according to claim 9, wherein 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).

11. An isolated nucleic acid according to claim 10, wherein said heavy chain contains an T366W substitution.

12. An isolated nucleic acid according to claim 11, wherein said CH3 domain has the amino acid sequence of SEQ ID NO:

8.

13. An isolated nucleic acid according to claim 1, wherein the constant heavy chain domain CH3 is altered so that an amino acid residue is replaced with an amino acid residue having a smaller side chain volume.

14. An isolated nucleic acid according to claim 13, wherein the 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).

15. An isolated nucleic acid according to claim 14 wherein said heavy chain contains a T3663, L368A, and Y407V substitutions.

16. An isolated nucleic acid according to claim 15 wherein said CH3 domain has the amino acid sequence of SEQ ID NO:

9.

17. An isolated nucleic acid according to claim 1 wherein the heavy chain constant domain CH3 is altered by the introduction of cysteine (C) as an amino acid.

21. A vector comprising a nucleic acid according to claim 1.

4. An isolated nucleic acid encoding the light chain of an antibody wherein the VL domain of said light chain is replaced by the VH domain of the corresponding heavy chain of said antibody.

5. An isolated nucleic acid according to claim 4 wherein said antibody is an anti-IGF-1 R antibody.

6. An isolated nucleic acid according to claim 5 wherein the said light chain is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:

4 and SEQ ID NO;

11.

22. A vector comprising a nucleic acid according to claim 4.

18. An isolated nucleic acid encoding the heavy chain of an anti-angiopoietin-2 antibody wherein the constant heavy chain domain CH3 is altered or replaced by the CH1 domain of said heavy chain or the CL domain of the light chain for said antibody.

19. An isolated nucleic acid according to claim 18 wherein said CH3 domain is modified by the replacement of an amino acid residue therein with an aurin acid residue having a larger side chain volume.

20. An isolated nucleic acid according to claim 18 wherein said CH3 domain is modified try the replacement of an amino acid residue therein with an amino acid residue having a smaller side chain volume.

23. A vector comprising a nucleic acid according to claim 18.

24. A bivalent, bispecific antibody, comprising:

a) the light chain and heavy chain of an antibody specifically binding to a first antigen; and

b) the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the variable domains VL and VH from the antibody specifically binding to a second antigen are replaced by each other.

25. The antibody according to claim 24, wherein the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each meet at an interface;

wherein the CH3 domain of one heavy chain is altered so that within the interface the CH3 domain of one heavy chain that meets the interface of the CH3 domain of the other heavy chain within the bivalent, bispecific 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

wherein the CH3 domain of the other heavy chain is altered, so that within the interface of the second CH3 domain that meets the interface of the first CH3 domain within the bivalent, bispecific 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.

26. The antibody according to claim 25, wherein 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).

27. The antibody according to claim 25, wherein the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (5), threonine (T), valine (V).

28. The antibody according to claim 25, wherein both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain.

29. The antibody according to claim 26, wherein both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain.

30. The antibody according to claim 27, wherein both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain.

31. The antibody according to claim 24, wherein one of the CH3 domains is replaced by a constant heavy chain domain CH1;

and the other CH3 domain is replaced by a constant light chain domain CL.

32. A method for the preparation of a bivalent, bispecific antibody according to claim 24 comprising the steps of

a) transforming a host cell with

vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a first antigen;

vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the variable domains VL and VH from the antibody specifically binding to a second antigen are replaced by each other;

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

c) recovering said antibody molecule from said culture.

33. A composition comprising a bivalent, bispecific antibody according to claim 24 and at least one pharmaceutically acceptable excipient.

34. A composition comprising a bivalent, bispecific antibody according to claim 25 and at least one pharmaceutically acceptable excipient.

35. A composition comprising a bivalent, bispecific antibody according to claim 28 and at least one pharmaceutically acceptable excipient.

36. A composition comprising a bivalent, bispecific antibody according to claim 31 and at least one pharmaceutically acceptable excipient.