Dual variable domain immunoglobulins and uses thereof

1. A binding protein comprising first and second polypeptide chains, each independently comprising VD1-(X1)n-VD2-C-(X2)n, wherein

VD1 is a first variable domain;

VD2 is a second variable domain;

C is a constant domain;

X1 is a linker;

X2 is an Fc region;

n is 0 or 1;

wherein the VD1 domains on the first and second polypeptide chains form a first functional target binding site and the VD2 domains on the first and second polypeptide chains form a second functional target binding site, and wherein the binding protein is capable of binding VEGF and DLL4, wherein the first and second polypeptide chains of the binding protein comprise:

SEQ ID NOs: 239 and 240,

SEQ ID NOs: 411 and 412,

SEQ ID NOs: 413 and 414,

SEQ ID NOs: 419 and 420,

SEQ ID NOs: 435 and 436,

SEQ ID NOs: 445 and 446,

SEQ ID NOs: 449 and 450,

SEQ ID NOs: 453 and 454,

SEQ ID NOs: 461 and 462,

SEQ ID NOs: 465 and 466,

SEQ ID NOs: 469 and 470,

SEQ ID NOs: 473 and 474,

SEQ ID NOs: 475 and 476,

SEQ ID NOs: 477 and 478,

SEQ ID NOs: 479 and 480,

SEQ ID NOs: 481 and 482,

SEQ ID NOs: 483 and 484,

SEQ ID NOs: 485 and 486,

SEQ ID NOs: 489 and 490,

SEQ ID NOs: 493 and 494,

SEQ ID NOs: 497 and 498,

SEQ ID NOs: 501 and 502,

SEQ ID NOs: 503 and 504,

SEQ ID NOs: 507 and 508,

SEQ ID NOs: 509 and 510,

SEQ ID NOs: 511 and 512,

SEQ ID NOs: 513 and 514,

SEQ ID NOs: 517 and 518,

SEQ ID NOs: 525 and 526,

SEQ ID NOs: 529 and 530,

SEQ ID NOs: 533 and 534,

SEQ ID NOs: 537 and 538,

SEQ ID NOs: 541 and 542,

SEQ ID NOs: 543 and 544,

SEQ ID NOs: 545 and 546,

SEQ ID NOs: 557 and 558,

SEQ ID NOs: 559 and 560,

SEQ ID NOs: 561 and 562,

SEQ ID NOs: 585 and 586,

SEQ ID NOs: 589 and 590, or

SEQ ID NOs: 593 and 594.

2. The binding protein according to claim 1 , wherein the first polypeptide chain is a heavy chain and n is 0 or 1 for (X2)n, and wherein the second polypeptide chain is a light chain and n is 0 for (X2)n.

3. The binding protein according to claim 1 , wherein the first and second polypeptide chains of the binding protein comprise:

SEQ ID NOs: 239 and 240,

SEQ ID NOs: 445 and 446,

SEQ ID NOs: 449 and 450,

SEQ ID NOs: 453 and 454,

SEQ ID NOs: 465 and 466,

SEQ ID NOs: 473 and 474,

SEQ ID NOs: 477 and 478,

SEQ ID NOs: 481 and 482,

SEQ ID NOs: 485 and 486,

SEQ ID NOs: 489 and 490,

SEQ ID NOs: 493 and 494,

SEQ ID NOs: 497 and 498,

SEQ ID NOs: 503 and 504,

SEQ ID NOs: 507 and 508,

SEQ ID NOs: 509 and 510,

SEQ ID NOs: 511 and 512,

SEQ ID NOs: 513 and 514,

SEQ ID NOs: 517 and 518,

SEQ ID NOs: 525 and 526,

SEQ ID NOs: 529 and 530,

SEQ ID NOs: 533 and 534,

SEQ ID NOs: 537 and 538,

SEQ ID NOs: 541 and 542,

SEQ ID NOs: 543 and 544,

SEQ ID NOs: 545 and 546,

SEQ ID NOs: 557 and 558,

SEQ ID NOs: 559 and 560,

SEQ ID NOs: 561 and 562,

SEQ ID NOs: 585 and 586,

SEQ ID NOs: 589 and 590, or

SEQ ID NOs: 593 and 594.

4. The binding protein according to claim 1 , comprising two first polypeptide chains and two second polypeptide chains and four functional target binding sites.

5. The binding protein according to claim 1 , wherein the Fc region is a variant sequence Fc region.

6. The binding protein according to claim 1 , wherein the Fc region is an Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

7. The binding protein according to claim 1 , wherein the binding protein is a crystallized binding protein.

8. A binding protein conjugate comprising a binding protein according to claim 1 , the binding protein conjugate further comprising an immunoadhesion molecule, an imaging agent, a therapeutic agent, or a cytotoxic agent.

9. The binding protein conjugate according to claim 8 , wherein the imaging agent is a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin.

10. The binding protein conjugate according to claim 9 , wherein the radiolabel is 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm.

11. The binding protein conjugate according to claim 8 , wherein the therapeutic or cytotoxic agent is an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, a toxin, or an apoptotic agent.

12. An isolated nucleic acid encoding the binding protein amino acid sequence according to claim 1 .

13. A vector comprising the isolated nucleic acid according to claim 12 .

14. A host cell comprising the vector according to claim 13 .

15. The host cell according to claim 14 , wherein the host cell is a prokaryotic cell, Escherichia coli , a eukaryotic cell, an animal cell, a plant cell, a fungal cell, a yeast cell, an Sf9 cell, a mammalian cell, an avian cell, an insect cell, a CHO cell or a COS cell.

16. A method of producing a binding protein, comprising culturing the host cell of claim 14 in culture medium under conditions sufficient to produce the binding protein.

17. A pharmaceutical composition comprising the binding protein according to claim 1 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17 , further comprising at least one additional therapeutic agent.

19. A method of determining the presence, amount, or concentration of VEGF and/or DLL4 in a test sample by an immunoassay comprising contacting the test sample with at least one binding protein of claim 1 and at least one detectable label.

20. A kit for assaying a test sample for the presence, amount, or concentration of VEGF and/or DLL4, said kit comprising (a) instructions for assaying the test sample for VEGF and/or DLL4 and (b) at least one binding protein comprising the binding protein of claim 1 .

21. The binding protein of claim 1 , wherein the first and second polypeptide chains of the binding protein comprise:

SEQ ID NOs: 489 and 490,

SEQ ID NOs: 517 and 518,

SEQ ID NOs: 503 and 504,

SEQ ID NOs: 507 and 508,

SEQ ID NOs: 525 and 526,

SEQ ID NOs: 511 and 512,

SEQ ID NOs: 529 and 530, or

SEQ ID NOs: 543 and 544.

22. The binding protein of claim 1 , wherein the first and second polypeptide chains of the binding protein comprise:

SEQ ID NOs: 411 and 412,

SEQ ID NOs: 413 and 414,

SEQ ID NOs: 475 and 476,

SEQ ID NOs: 477 and 478,

SEQ ID NOs: 479 and 480,

SEQ ID NOs: 481 and 482,

SEQ ID NOs: 483 and 484,

SEQ ID NOs: 485 and 486,

SEQ ID NOs: 511 and 512,

SEQ ID NOs: 513 and 514,

SEQ ID NOs: 543 and 544,

SEQ ID NOs: 545 and 546,

SEQ ID NOs: 559 and 560, or

SEQ ID NOs: 561 and 562.