Trispecific and/or trivalent binding proteins

1. A trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more target proteins, wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula:

V L2 -L 1 -V L1 -L 2 -C L   [I]

and a second polypeptide chain of the binding protein comprises a structure represented by the formula:

V H1 -L 3 -V H2 -L 4 -C H1 -hinge-C H2 -C H3   [II]

and a third polypeptide chain of the binding protein comprises a structure represented by the formula:

V H3 -C H1 -hinge-C H2 -C H3   [III]

and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula:

V L3 -C L   [IV]

wherein:

V L1 is a first immunoglobulin light chain variable domain;

V L2 is a second immunoglobulin light chain variable domain;

V L3 is a third immunoglobulin light chain variable domain;

V H1 is a first immunoglobulin heavy chain variable domain;

V H2 is a second immunoglobulin heavy chain variable domain;

V H3 is a third immunoglobulin heavy chain variable domain;

C L is an immunoglobulin light chain constant domain;

C H1 is an immunoglobulin C H1 heavy chain constant domain;

C H2 is an immunoglobulin C H2 heavy chain constant domain;

C H3 is an immunoglobulin C H3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and

L 1 , L 2 , L 3 and L 4 are each independently amino acid linkers or zero amino acids in length;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair of the binding protein; wherein V H1 and V L1 form a binding pair and a first antigen binding site, and V H2 and V L2 form a binding pair and a second antigen binding site; and wherein V H3 and V L3 form a binding pair and a third antigen binding site.

2. The binding protein of claim 1 , wherein the binding protein is trivalent and capable of specifically binding one antigen target.

3. The binding protein of claim 1 , wherein the binding protein is trivalent and capable of specifically binding two antigen targets.

4. The binding protein of claim 1 , wherein the binding protein is trivalent and capable of specifically binding three antigen targets.

5. The binding protein of claim 1 , wherein L 1 , L 2 , L 3 and L 4 each independently are the amino acid S or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:104), GGGGSGGGGSGGGGS (SEQ ID NO:105), RT, TKGPS (SEQ ID NO:106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO:148).

6. The binding protein of claim 1 , wherein

(a) L 1 comprises the sequence GQPKAAP (SEQ ID NO: 175), L 2 comprises the sequence TKGPS (SEQ ID NO:106), L 3 comprises the amino acid S, and L 4 comprises the sequence RT;

(b) L 1 comprises the sequence GGGGSGGGGS (SEQ ID NO:104), L 2 comprises the sequence GGGGSGGGGS (SEQ ID NO:104), L 3 is 0 amino acids in length, and L 4 is 0 amino acids in length;

(c) L 1 comprises the sequence GGSGSSGSGG (SEQ ID NO:148), L 2 comprises the sequence GGSGSSGSGG (SEQ ID NO:148), L 3 is 0 amino acids in length, and L 4 is 0 amino acids in length; or

(d) L 1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:105), L 2 is 0 amino acids in length, L 3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:105), and L 4 is 0 amino acids in length.

7. The binding protein of claim 1 , wherein the binding protein is capable of inhibiting the function of one or more target proteins.

8. The binding protein of claim 1 , wherein the binding protein comprises one, two, or three antigen binding sites that specifically bind a target protein selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MEW class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.

9. The binding protein of claim 1 , wherein the C H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the C H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

10. The binding protein of claim 1 , wherein the C H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the C H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.

11. The binding protein of claim 1 , wherein the C H3 domains of the second and the third polypeptide chains both comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are M428L and N434S.

12. The binding protein of claim 1 , wherein the CH3 domains of the second and the third polypeptide chains are human IgG1 or IgG4 C H3 domains, and wherein only one of the C H3 domains comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are H435R and Y436F.

13. The binding protein of claim 1 , wherein the C H3 domains of the second and the third polypeptide chains are human IgG4 C H3 domains, and wherein the C H3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.

14. The binding protein of claim 1 , wherein the C H3 domains of the second and the third polypeptide chains are human IgG4 C H3 domains, and wherein the C H3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.

15. The binding protein of claim 1 , wherein the C H3 domains of the second and the third polypeptide chains are human IgG1 C H3 domains, and wherein the C H3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A and L235A.

16. The binding protein of claim 1 , wherein:

(a) the C L domain of the first polypeptide chain is a human kappa C L domain, and the C L domain of the fourth polypeptide chain is a human lambda C L domain; or

(b) the C L domain of the first polypeptide chain is a human lambda C L domain, and the C L domain of the fourth polypeptide chain is a human kappa C L domain.

17. The binding protein of claim 1 , wherein the first polypeptide chain comprises a lambda C L domain; wherein the C H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to EU Index, wherein the amino acid substitutions are S354C and T366W; wherein the C H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and wherein the fourth polypeptide chain comprises a kappa C L domain.

18. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein of claim 1 .

19. An expression vector comprising the nucleic acid molecule of claim 18 .

20. An isolated host cell comprising the nucleic acid molecule of claim 18 .

21. An isolated host cell comprising the expression vector of claim 19 .

22. The isolated host cell of claim 20 , wherein the host cell is a mammalian cell or an insect cell.

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

24. A method of preventing or treating cancer in a patient comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 23 , wherein the binding protein comprises one antigen binding site that specifically binds a T-cell surface protein and another antigen binding site that specifically binds a tumor target protein.

25. The method of claim 24 , wherein the at least one binding protein is co-administered with a chemotherapeutic agent.

26. A method of preventing or treating an inflammatory disease or disorder in a patient comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 23 , wherein the binding protein comprises three antigen binding sites that each specifically bind a cytokine target protein.

27. The method of claim 26 , wherein the cytokine target proteins are selected from the group consisting of IL-4, IL-13 and TNFα.

28. The method of claim 26 , wherein the composition is co-administered with an anti-inflammatory agent.

29. The method of claim 24 , wherein the patient is a human.

30. The method of claim 24 , wherein the binding protein comprises one, two, or three antigen binding sites that specifically bind a target protein selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.