Extension sequences for diabodies

1. A diabody comprising:

a heavy chain variable domain;

a light chain variable domain;

a linker; and

an extension sequence, wherein the extension sequence is selected from the group consisting of:

(SEQ ID NO: 1)

EPKSX n5 DKTHTCX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 2)

ERKX n5 CX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 3)

ELKTPLGDTTHTCX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 4)

ESKYGPPCX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 5)

CPPCPPC,

and

(SEQ ID NO: 11)

GGC(PPC) n ,

 wherein n is 2, 3, 4, 5, 6, 7, 8, or 9, and

wherein X n1 , X n2 , X n3 , X n4 , and X n5 can be any amino acid,

wherein the extension sequence connects either a C-terminus of the heavy chain variable domain to a C-terminus of a different heavy chain variable domain of the diabody, or a C-terminus of the light chain variable domain to a C-terminus of a different light chain variable domain of the diabody, through one or more disulfide bonds.

2. An extension sequence configured for use within an antigen binding construct, wherein the extension sequence is selected from the group consisting of:

(SEQ ID NO: 1)

EPKSX n5 DKTHTC n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 2)

ERKX n5 CX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 3)

ELKTPLGDTTHTCX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 4)

ESKYGPPCX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 5)

CPPCPPC,

and

(SEQ ID NO: 11)

GGC(PPC),

wherein n is 2, 3, 4, 5, 6, 7, 8, or 9, and

wherein X n1 , X n2 , X n3 , X n4 , and X n5 can be any amino acid,

wherein the extension sequence is located at or within 10 amino acids from the C-terminus of a polypeptide chain of an antigen binding construct, wherein the polypeptide chain comprises either (i) a heavy chain variable domain; or (ii) a heavy chain variable domain and a light chain variable domain, wherein the extension sequence covalently connects the polypeptide chain with a different polypeptide chain of the antigen binding construct through one or more disulfide bonds.

3. The extension sequence of claim 2 , wherein the extension sequence is located within an antibody.

4. The extension sequence of claim 3 , wherein the antibody is a mono-specific antibody.

5. The extension sequence of claim 3 , wherein the antibody is a bi-specific antibody.

6. The extension sequence of claim 2 , wherein the extension sequence is located within an antibody fragment.

7. The extension sequence of claim 2 , wherein the extension sequence is covalently attached to a detectable marker or a therapeutic agent.

8. The extension sequence of claim 2 , further comprising a detectable marker.

9. The extension sequence of claim 8 , wherein the detectable marker is a radionuclide.

10. The extension sequence of claim 9 , wherein the radionuclide is selected from the group consisting of 90 Y, 177 Lu, and 227 Ac.

11. The extension sequence of claim 5 , wherein the bi-specific antibody is assembled in a 1:1 ratio.

12. A pharmaceutical composition comprising the extension sequence of claim 2 .

13. A pharmaceutical composition comprising the diabody of claim 1 .

14. A diabody comprising two chains, comprising:

a first chain comprising:

a first heavy chain variable domain;

a first light chain variable domain;

a first linker that connects the first heavy chain variable domain and the first light chain variable domains; and

a first extension sequence; and

a second chain comprising:

a second heavy chain variable domain;

a second light chain variable domain;

a second linker that connects the second heavy and second light chain variable domains; and

a second extension sequence,

wherein the first extension sequence and the second extension sequence are covalently connected to one another, and connect either a) the first heavy chain variable domain to the second heavy chain variable or b) the first light chain variable domain to the second light chain variable domain, through one or more disulfide bonds, wherein the first extension sequence and the second extension sequence are selected from the group consisting of:

(SEQ ID NO: 1)

EPKSX n5 DKTHTCX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 2)

ERKX n5 CX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 3)

ELKTPLGDTTHTCX n1 X n2 CX n3 X n4 C,

(SEQ ID NO: 4)

ESKYGPPCX n1 X n2 CX n3 X n4 C, 

and

(SEQ ID NO: 5)

CPPCPPC,

and

(SEQ ID NO: 11)

GGC(PPC) n ,

 wherein n is 2, 3, 4, 5, 6, 7, 8, or 9, and

wherein X n1 , X n2 , X n3 , X n4 , and X n5 can be any amino acid.

15. The diabody of claim 1 , wherein one or more of X n1 , X n2 , X n3 , X n4 is a proline.

16. The extension sequence of claim 2 , wherein one or more of X n1 , X n2 , X n3 , X n4 is a proline.

17. The diabody of claim 14 , wherein one or more of X n1 , X n2 , X n3 , X n4 is a proline.

18. The diabody of claim 14 , wherein the extension sequence connects either a C-terminus of the first heavy chain variable domain to a C-terminus of the second heavy chain variable domain, or a C-terminus of the first light chain variable domain to a C-terminus of the second light chain variable domain.

19. The extension sequence of claim 2 , wherein the antigen binding construct is a diabody.

20. The extension sequence of claim 2 , wherein the polypeptide chain comprises a linker.

21. The extension sequence of claim 20 , wherein the polypeptide chain comprises the heavy chain variable domain and the light chain variable domain, and the linker connects the heavy chain variable domain to the light chain variable domain.

22. The diabody of claim 1 , wherein the extension sequence is covalently attached to a detectable marker or a therapeutic agent.

23. The diabody of claim 22 , wherein the detectable marker is a radionuclide.

24. The diabody of claim 23 , wherein the radionuclide is selected from the group consisting of 90 Y, 177 Lu, and 227 Ac.

25. The diabody of claim 14 , wherein the first chain and the second chain recognize different epitopes.