Binding proteins having tethered light chains

1. A binding protein comprising an inside-out tethered light chain, a first inside-out heavy chain and a second inside-out heavy chain that specifically binds at least one antigen, wherein the inside-out tethered light chain comprises VH1-CL-linker-VH2-CL, the first inside-out tethered heavy chain comprises VL1-CH, and the second inside-out tethered heavy chain comprises VL2-CH, wherein

i) VL1 is a first light chain variable region;

ii) VL2 is a second light chain variable region;

iii) CL is a light chain constant region;

iv) VH1 is a first heavy chain variable region;

v) VH2 is a second heavy chain variable region;

vi) CH is a heavy chain constant region comprising a C H 1 domain, hinge, a C H 2 domain, and C H 3 domain; and

vii) linker is a polypeptide linker.

2. The binding protein of claim 1 , wherein the VL1 and the VL2 comprise identical or substantially identical amino acid sequences.

3. The binding protein of claim 1 , which is bispecific.

4. The binding protein of claim 1 , wherein the VL1, the VL2, the VH1 and the VH2 comprise human, humanized, human-adapted or murine polypeptide sequences.

5. The binding protein of claim 1 , wherein the CL is of kappa (κ) or lambda (λ) type and the CH is of IgG1, IgG2, IgG3, or IgG4 type.

6. The binding protein of claim 1 , wherein the CL and CH are of human origin.

7. The binding protein of claim 1 , wherein the linker is about 5-50 amino acids long.

8. The binding protein of claim 7 , wherein the linker is (G 4 S) 4 (SEQ ID NO: 1), (G 4 S) 6 (SEQ ID NO: 2), (G 4 S) 8 (SEQ ID NO: 3) or (G 4 S) 10 (SEQ ID NO: 4).

9. The binding protein of claim 1 , wherein the binding protein binds at least one antigen with a dissociation constant (K D ) of at most about 10 −9 M, at most about 10 −10 M, at most about 10 −11 M, at most about 10 −12 M, or at most about 10 −13 M.

10. The binding protein of claim 1 , wherein the binding protein binds at least one antigen with an on rate constant (K on ) of at least about 10 4 M −1 s −1 , at least about 10 5 M −1 s −1 , or at least about 10 4 M −1 s −1 .

11. The binding protein of claim 1 , wherein the binding protein binds at least one antigen with an off rate constant (K off ) of at most about 10 4 s −1 , at most about 10 −5 s −1 , or at most about 10 −6 s −1 .

12. A binding protein of claim 1 produced according to a method comprising culturing a host cell under conditions sufficient to produce the binding protein, wherein the host cell comprises at least one vector, the at least one vector comprising a nucleic acid encoding the inside-out tethered light chain, the first inside-out heavy chain and the second inside-out heavy chain.

13. An isolated polynucleotide encoding the binding protein of claim 1 .

14. A vector comprising an isolated polynucleotide of claim 13 .

15. A host cell comprising a vector of claim 14 .

16. A method of making the binding protein of claim 1 comprising culturing the host cell of claim 17 under conditions sufficient to produce the binding protein.

17. A method of making a bispecific binding protein that binds a first antigen and a second antigen comprising an inside-out tethered light chain, a first inside-out heavy chain and a second inside-out heavy chain, comprising

a) providing an antibody that binds the first antigen having a first light chain comprising a first light chain variable region (VL1) and a first light chain constant region (CL1), and a first heavy chain comprising a first heavy chain variable region (VH1) and a first heavy chain constant region (CH1), wherein CH1 comprises a C H 1 domain, hinge, C H 2 domain, and a C H 3 domain;

b) providing an antibody that binds the second antigen having a second light chain comprising a second light chain variable region (VL2) and a second light chain constant region (CL2), and a second heavy chain comprising a second heavy chain variable region (VH2) and a second heavy chain constant region (CH2), wherein CH2 comprises a C H 1 domain, hinge, a C H 2 domain, and a C H 3 domain;

c) providing a linker;

d) operably linking VH1-CL1-linker-VH2-CL2 from the N-terminus to the C-terminus to generate the inside-out tethered light chain;

e) operably linking VL1-CH1 from the N-terminus to the C-terminus to generate the first inside-out heavy chain;

f) operably linking VL2-CH2 from the N-terminus to the C-terminus to generate the second inside-out heavy chain;

g) expressing the inside-out tethered light chain, the first inside-out heavy chain and the second inside-out heavy chain; and

h) recovering the bispecfic binding protein.

18. The method of claim 17 , wherein the VL1 and the VL2 comprise identical or substantially identical amino acid sequences.

19. A bispecific binding protein produced by a method of claim 17 .

20. A pharmaceutical composition comprising the binding protein of claim 1 and a pharmaceutically acceptable carrier.