Dual Variable Domain Immunoglobulin and Uses Thereof

Abstract

The present invention relates to engineered multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the prevention and/or treatment of acute and chronic inflammatory and other diseases.

1 . A binding protein comprising a polypeptide chain, wherein said polypeptide chain comprises 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 represents an amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1. 
     
     
         2 . The binding protein according to  claim 1 , wherein said VD1 and VD2 are heavy chain variable domains. 
     
     
         3 . The binding protein according to  claim 2 , wherein said heavy chain variable domain is a murine heavy chain variable domain, a human heavy chain variable domain, a CDR grafted heavy chain variable domain, or a humanized heavy chain variable domain. 
     
     
         4 . The binding protein according to  claim 2 , wherein VD1 and VD2 are capable of binding the same antigen. 
     
     
         5 . The binding protein according to  claim 2 , wherein VD1 and VD2 are capable of binding different antigens. 
     
     
         6 . The binding protein according to  claim 2 , wherein C is a heavy chain constant domain. 
     
     
         7 . The binding protein according to  claim 6 , wherein X1 is a linker with the proviso that X1 is not CH1. 
     
     
         8 . The binding protein according to  claim 7 , wherein the linker is AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA(G 4 S) 4 , SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP; GGGGSGGGGSGGGGS; GENKVEYAPALMALS; GPAKELTPLKEAKVS; or GHEAAAVMQVQYPAS. 
     
     
         9 . The binding protein according to  claim 7 , wherein X2 is an Fc region. 
     
     
         10 . The binding protein according to  claim 9 , wherein said Fc region is a variant Fc region. 
     
     
         11 . A binding protein comprising a polypeptide chain, wherein said polypeptide chain comprises VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 is an Fc region. 
     
     
         12 . The binding protein according to  claim 1 , wherein said VD1 and VD2 are light chain variable domains. 
     
     
         13 . The binding protein according to  claim 12 , wherein said light chain variable domain is a murine light chain variable domain, a human light chain variable domain, a CDR grafted light chain variable domain, or a humanized light chain variable domain. 
     
     
         14 . The binding protein according to  claim 12 , wherein VD1 and VD2 are capable of binding the same antigen. 
     
     
         15 . The binding protein according to  claim 12 , wherein VD1 and VD2 are capable of binding different antigens. 
     
     
         16 . The binding protein according to  claim 12 , wherein C is a light chain constant domain. 
     
     
         17 . The binding protein according to  claim 16 , wherein X1 is a linker with the proviso that X1 is not CL1. 
     
     
         18 . The binding protein according to  claim 17 , wherein the linker is AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; SAKTTPKLGG; AKTTPKLEEGEFSEARV; SAKTTP; SAKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA(G 4 S) 4 , SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP; GGGGSGGGGSGGGGS; GENKVEYAPALMALS; GPAKELTPLKEAKVS; or GHEAAAVMQVQYPAS. 
     
     
         19 . The binding protein according to  claim 17 , wherein the binding protein does not comprise X2. 
     
     
         20 . A binding protein comprising a polypeptide chain, wherein said polypeptide chain comprises VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region. 
     
     
         21 . A binding protein comprising four polypeptide chains, wherein:
 a first polypeptide chain and a second polypeptide chain each comprises 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 with the proviso that it is not a constant domain, X2 is an Fc region, and n is 0 or 1; and   a third polypeptide chain and a fourth polypeptide chain each comprises VD1-(X1)n-VD2-C, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, and X1 is a linker with the proviso that it is not a constant domain, and n is 0 or 1;   wherein either or each of VD1 and VD2 of said polypeptide chains is a non-immunoglobulin variable domain; and   wherein said four polypeptide chains of said binding protein form four functional binding domains.   
     
     
         22 . The binding protein according to  claim 21 , wherein:
 VD1 of said first polypeptide chain and of said second polypeptide chain is a non-immunoglobulin variable domain,   VD2 of said first polypeptide chain and of said second polypeptide chain is an immunoglobulin variable domain,   VD1 of said third polypeptide chain and of said fourth polypeptide chain is a non-immunoglobulin variable domain, and   VD2 of said third polypeptide chain and of said fourth polypeptide chain is an immunoglobulin variable domain.   
     
     
         23 . The binding protein according to  claim 21 , wherein:
 VD1 of said first polypeptide chain and of said second polypeptide chain is an immunoglobulin variable domain,   VD2 of said first polypeptide chain and of said second polypeptide chain is a non-immunoglobulin variable domain,   VD1 of said third polypeptide chain and of said fourth polypeptide chain is an immunoglobulin variable domain, and   VD2 of said third polypeptide chain and of said fourth polypeptide chain is a non-immunoglobulin variable domain.   
     
     
         24 . The binding protein according to  claim 21 , wherein:
 VD1 of said first polypeptide chain and of said second polypeptide chain is a non-immunoglobulin variable domain,   VD2 of said first polypeptide chain and of said second polypeptide chain is a non-immunoglobulin variable domain,   VD1 of said third polypeptide chain and of said fourth polypeptide chain is a non-immunoglobulin variable domain, and   VD2 of said third polypeptide chain and of said fourth polypeptide chain is a non-immunoglobulin variable domain.   
     
     
         25 . The binding protein according to  claim 22 , wherein:
 non-immunoglobulin variable domain VD1 of said first polypeptide chain and of said second polypeptide chain, and   non-immunoglobulin variable domain VD1 of said third polypeptide chain and of said fourth polypeptide chain,   together form two functional ligand binding domains of a receptor.   
     
     
         26 . The binding protein according to  claim 23 , wherein:
 non-immunoglobulin variable domain VD2 of said first polypeptide chain and of said second polypeptide chain, and   non-immunoglobulin variable domain VD2 of said third polypeptide chain and of said fourth polypeptide chain,   together form two functional ligand binding domains of a receptor.   
     
     
         27 . The binding protein according to  claim 24 , wherein:
 non-immunoglobulin variable domain VD1 of said first polypeptide chain and of said second polypeptide chain, and   non-immunoglobulin variable domain VD1 of said third polypeptide chain and of said fourth polypeptide chain,   together form two functional ligand binding domains of a receptor; and wherein:   non-immunoglobulin variable domain VD2 of said first polypeptide chain and of said second polypeptide chain, and   non-immunoglobulin variable domain VD2 of said third polypeptide chain and of said fourth polypeptide chain,   together form two functional ligand binding domains of a receptor.   
     
     
         28 . The binding protein according to  claim 21 , wherein said Fc region, X2, is a variant of a native sequence Fc region, containing mutations to disrupt dimerization of the CH3 domain. 
     
     
         29 . The binding protein according to  claim 21 , wherein X1 is AKTTPKLEEGEFSEAR; AKTTPKLEEGEFSEARV; AKTTPKLGG; RADAAP; RADAAPTVS; RADAAAAGGPGS; RADAAAA(G 4 S) 4 , SAKTTP; SAKTTPKLGG; SAKTTPKLEEGEFSEARV; ADAAP; ADAAPTVSIFPP; TVAAP; TVAAPSVFIFPP; QPKAAP; QPKAAPSVTLFPP; AKTTPP; AKTTPPSVTPLAP; AKTTAP; AKTTAPSVYPLAP; ASTKGP; ASTKGPSVFPLAP; GGGGSGGGGSGGGGS; GENKVEYAPALMALS; GPAKELTPLKEAKVS; or GHEAAAVMQVQYPAS. 
     
     
         30 . The binding protein according to  claim 21 , wherein, for (X2)n, n is 0. 
     
     
         31 . The binding protein according to  claim 21 , wherein said binding protein is capable of binding one or more targets. 
     
     
         32 . The binding protein according to  claim 31 , wherein said one or more targets is selected from the group consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA);CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1/Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A (p161NK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CERT; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (1-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB21P; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENOL; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2;IL1RL1;IL1RL2 IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR112; NR113; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Sprl); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB111; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase Iia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YY1; and ZFPM2. 
     
     
         33 . The binding protein according to  claim 31 , wherein said binding protein is capable of binding a two targets, wherein the two targets are CD138 and CD20; CD138 and CD40; CD20 and CD3; CD38 & CD138; CD38 and CD20; CD38 and CD40; CD40 and CD20; CD19 and CD20; CD-8 and IL-6; PDL-1 and CTLA-4; CTLA-4 and BTNO2; CSPGs and RGM A; IGF1 and IGF2; IGF1/2 and Erb2B; IL-12 and IL-18; IL-12 and TWEAK; IL-13 and ADAMS; IL-13 and CL25; IL-13 and IL-1beta; IL-13 and IL-25; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-9; IL-13 and LHR agonist; IL-13 and MDC; IL-13 and MIF; IL-13 and PED2; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and TARC; IL-13 and TGF-13; IL-1α and IL-1β; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; RGM A and RGM B; Te38 and TNFα; TNFα and IL-12; TNFα and IL-12p40; TNFα and IL-13; TNFα and IL-15; TNFα and IL-17; TNFα and IL-18; TNFα and IL-1beta; TNFα and IL-23; TNFα and MIF; TNFα and PEG2; TNFα and PGE4; TNFα and VEGF; VEGFR and EGFR; TNFα and RANK ligand; TNFα and Blys; TNFα and GP130; TNFα and CD-22; or TNFα and CTLA-4. 
     
     
         34 . A binding protein conjugate comprising a binding protein described in  claim 21 , said binding protein conjugate further comprising an agent that is an immunoadhension molecule, an imaging agent, a therapeutic agent, or a cytotoxic agent. 
     
     
         35 . The binding protein conjugate according to  claim 34 , wherein said imaging agent is a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or a biotin. 
     
     
         36 . The binding protein conjugate according to  claim 35 , wherein said imaging agent is a radiolabel that is  3 H,  14 C,  35 S,  90 Y,  99 Tc,  111 In,  125 I,  131 I,  177 Lu,  166 Ho, or  153 Sm. 
     
     
         37 . The binding protein conjugate according to  claim 34 , wherein said agent is a therapeutic or cytotoxic agent that 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. 
     
     
         38 . The binding protein according to  claim 21 , wherein said binding protein is a crystallized binding protein. 
     
     
         39 . The crystallized binding protein according to  claim 38 , wherein said crystal is a carrier-free pharmaceutical controlled release crystal. 
     
     
         40 . The crystallized binding protein according to  claim 38 , wherein said binding protein has a greater half-life in vivo than the soluble counterpart of said binding protein. 
     
     
         41 . The crystallized binding protein according to  claim 38 , wherein said binding protein retains biological activity. 
     
     
         42 . An isolated nucleic acid comprising a coding sequence for a polypeptide chain comprising the structure VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a variable domain, VD2 is a second variable domain, C is a constant domain, X1 is a linker with the proviso that it is not a constant domain, X2 is an Fc region, and n is 0 or 1. 
     
     
         43 . The isolated nucleic acid according to  claim 42 , wherein said encoded polypeptide chain is capable of pairing with a polypeptide chain comprising the structure VD1-(X1)n-VD2-C, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 is a linker with the proviso that it is not a constant domain, and n is 0 or 1; to form a two-polypeptide chain binding protein comprising two functional binding domains. 
     
     
         44 . The isolated nucleic acid according to  claim 43 , wherein two of said two-polypeptide chain binding proteins are capable of associating to form a four-polypeptide chain binding protein comprising four functional binding domains. 
     
     
         45 . A vector comprising the nucleic acid according to  claim 42 . 
     
     
         46 . The vector according to  claim 45 , wherein said vector is an expression vector wherein said nucleic acid is operably linked to one or more expression control sequences. 
     
     
         47 . The expression vector according to  claim 46 , wherein said expression vector is a pcDNA vector, a pTT vector, a pTT3 vector, a pEFBOS vector, a pBV vector, a pJV vector, a pcDNA3.1 TOPO® vector, a pEF6 TOPO® vector, or a pBJ vector. 
     
     
         48 . A host cell comprising a vector according to  claim 45 . 
     
     
         49 . An isolated nucleic acid comprising a coding sequence for a polypeptide chain comprising the structure VD1-(X1)n-VD2-C, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 is a linker with the proviso that it is not a constant domain, and n is 0 or 1. 
     
     
         50 . The isolated nucleic acid according to  claim 49 , wherein said encoded polypeptide chain is capable of pairing with a polypeptide chain comprising the structure 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 with the proviso that it is not a constant domain, X2 is an Fc region, and n is 0 or 1; to form a two-polypeptide chain binding protein comprising two functional binding domains. 
     
     
         51 . The isolated nucleic acid according to  claim 50 , wherein two of said two-polypeptide chain binding proteins are capable of associating to form a four-polypeptide chain binding protein comprising four functional binding domains. 
     
     
         52 . A vector comprising the nucleic acid according to  claim 49 . 
     
     
         53 . The vector according to  claim 52 , wherein said vector is an expression vector wherein said nucleic acid is operably linked to one or more expression control sequences. 
     
     
         54 . The expression vector according to  claim 53 , wherein said expression vector is a pcDNA vector, a pTT vector, a pTT3 vector, a pEFBOS vector, a pBV vector, a pJV vector, a pcDNA3.1 TOPO® vector, a pEF6 TOPO® vector, or a pBJ vector. 
     
     
         55 . A host cell comprising a vector according to  claim 52 . 
     
     
         56 . A host cell comprising a nucleic acid sequence encoding a polypeptide chain comprising the structure VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first immunoglobulin heavy chain variable domain, VD2 is a second immunoglobulin heavy chain variable domain, C is a constant domain, X1 is a linker with the proviso that it is not constant domain, X2 is an Fc region, and n is 0 or 1. 
     
     
         57 . A host cell comprising a nucleic acid sequence encoding a polypeptide chain comprising the structure VD1-(X1)n-VD2-C, wherein VD1 is a first immunoglobulin light chain variable domain, VD2 is a second immunoglobulin light chain variable domain, C is a constant domain, X1 is a linker with the proviso that it is not a constant domain, and n is 0 or 1. 
     
     
         58 . A host cell comprising:
 a) a first expression vector comprising a nucleic acid comprising a coding sequence for a first polypeptide chain comprising VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first immunoglobulin heavy chain variable domain, VD2 is a second immunoglobulin heavy chain variable domain, C is a constant domain, X1 is a linker with the proviso that it is not a constant domain, X2 is an Fc region, and n is 0 or 1; and   b) a second expression vector comprising a nucleic acid comprising a coding sequence for a second polypeptide chain comprising VD1-(X1)n-VD2-C, wherein VD1 is a first immunoglobulin light chain variable domain, VD2 is a second immunoglobulin light chain variable domain, C is a constant domain, X1 is a linker with the proviso that it is not a constant domain; and n is 0 or 1.   
     
     
         59 . The host cell according to  claim 58 , wherein co-expression of said first polypeptide chain and said second polypeptide chain in said host cell yields a binding protein comprising two first polypeptide chains in association with two second polypeptide chains, wherein the four polypeptide chains form a four-polypeptide chain binding protein comprising four functional antigen binding domains. 
     
     
         60 . The host cell according to  claim 48  or  claim 55 , wherein said host cell is a prokaryotic cell, a eukaryotic cell, or a protist cell. 
     
     
         61 . The host cell according to  claim 60 , wherein said prokaryotic cell is  Escherichia coli.    
     
     
         62 . The host cell according to  claim 60 , wherein said eukaryotic cell is an animal cell, a plant cell, a fungal cell, a mammalian cell, an avian cell, or an insect cell. 
     
     
         63 . The host cell according to  claim 62 , wherein said mammalian cell is a COS cell or a CHO cell. 
     
     
         64 . The host cell according to  claim 62 , wherein said fungal cell is a yeast cell. 
     
     
         65 . The host cell according to  claim 60 , wherein said insect cell is an Sf9 cell. 
     
     
         66 . A method of producing a four-polypeptide chain binding protein having four functional antigen binding domains comprising culturing a host cell according to  claim 58  or  claim 59  in culture medium under conditions sufficient to produce the binding protein. 
     
     
         67 . A method of producing a polypeptide chain comprising the structure 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 with the proviso that it is not a constant domain, X2 is an Fc region, and n is 0 or 1; comprising the steps of:
 a) transforming a host cell with the expression vector according to  claim 46 ; and 
 b) culturing the transformed host cell in culture medium under conditions sufficient to produce the polypeptide chain. 
 
     
     
         68 . A method of producing a polypeptide chain comprising the structure VD1-(X1)n-VD2-C, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 is a linker with the proviso that it is not a constant domain; and n is 0 or 1; comprising the steps of:
 a) transforming a host cell with the expression vector according to  claim 53 ; and   b) culturing the transformed host cell in culture medium under conditions sufficient to produce the polypeptide chain.   
     
     
         69 . A method of producing a four-polypeptide chain binding protein having four functional antigen binding sites comprising:
 a) transfecting a host cell with a first expression vector and a second expression vector, wherein:
 the first expression vector comprises a nucleic acid comprising a coding sequence for a first polypeptide chain comprising VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first immunoglobulin heavy chain variable domain, VD2 is a second immunoglobulin heavy chain variable domain, C is a constant domain, X1 is a linker with the proviso that it is not a constant domain, X2 is an Fc region, and n is 0 or 1; and 
 the second expression vector comprises a nucleic acid comprising a coding sequence for a second polypeptide chain comprising VD1-(X1)n-VD2-C, wherein VD1 is a first immunoglobulin light chain variable domain, VD2 is a second immunoglobulin light chain variable domain, C is a constant domain, X1 is a linker with the proviso that it is not a constant domain, and n is 0 or 1; and 
   b) culturing the transfected host cell in (a) in culture medium under conditions sufficient to produce a four polypeptide chain binding protein comprising two first polypeptide chains in association with two second polypeptide chains, wherein the four polypeptide chains of the binding protein form four functional antigen binding domains.   
     
     
         70 . A pharmaceutical composition comprising the binding protein described in  claim 21 , and a pharmaceutically acceptable carrier. 
     
     
         71 . The pharmaceutical composition of  claim 70  further comprising at least one additional agent. 
     
     
         72 . The pharmaceutical composition of  claim 71 , wherein said additional agent is a therapeutic agent, an imaging agent, a cytotoxic agent, an angiogenesis inhibitors, a kinase inhibitor, a co-stimulation molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or functional fragment thereof, methotrexate, a cyclosporin, a rapamycin, FK506, a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine, an epinephrine analog, a cytokine, or a cytokine antagonist. 
     
     
         73 . A method for treating a subject for a disease or a disorder by administering to the subject the binding protein according to  claim 21 , such that treatment is achieved. 
     
     
         74 . The method of  claim 73 , wherein said disease or disorder is rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis, scleroderma, graft versus host disease, organ transplant rejection, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic polyglandular deficiency type I, polyglandular deficiency type II (Schmidt's syndrome), adult (acute) respiratory distress syndrome, alopecia, alopecia greata, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis,  Chlamydia -,  Yersinia -, and  Salmonella -associated arthropathy, spondyloarthropathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency syndrome, acquired immunodeficiency related diseases, hepatitis B, hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjögren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasculitis of the kidneys, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjörgren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergy, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Th1 Type mediated diseases, acute and chronic pain (different forms of pain), lung cancer, breast cancer, stomach cancer, bladder cancer, colon, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, hematopoietic malignancies (leukemia and lymphoma), abetalipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, atrial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti-CD3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and peripheral aneurysms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders, dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatologic conditions, diabetes, diabetes mellitus, diabetic arteriosclerotic disease, diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's syndrome in middle age, drug-induced movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hemophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease, Hashimoto's thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis A, His bundle arrhythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitivity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity, asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza A, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection,  legionella , leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic migraine headache, idiopathic migraine headache, mitochondrial multisystem disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Menzel, Dejerine-Thomas, Shy-Drager, and Machado-Joseph), myasthenia gravis,  mycobacterium avium  intracellulare,  mycobacterium tuberculosis , myelodysplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic muscular atrophies, neutropenic fever, non-Hodgkin's lymphoma, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, OKT 3 therapy, orchitis/epididymitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia,  pneumocystis carinii  pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, progressive supranucleo palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon, Raynaud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, senile chorea, senile dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrhythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, subacute sclerosing panencephalitis, syncope, syphilis of the cardiovascular system, systemic anaphylaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, viral encephalitis/aseptic meningitis, viral-associated hemophagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, or xenograft rejection of any organ or tissue. 
     
     
         75 . The method according to  claim 73 , wherein said step of administering to the subject is by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.