Dual Variable Domain Immunoglobulin and Uses Thereof

1 - 76 . (canceled)

77 . A method of producing a plurality of dual specific tetravalent binding proteins, comprising culturing a host cell in culture medium under conditions such that at least 90% of binding proteins produced from the host cell are dual specific tetravalent binding proteins, each comprising two first polypeptide chains and two second polypeptide chains forming four functional binding sites, wherein the host cell comprises:

(a) a first nucleic acid encoding the first polypeptide chain, wherein the first 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,

X2 is an Fc region, and

n is 0 or 1; and

(b) a second nucleic acid encoding the second polypeptide chain, wherein the second polypeptide chain comprises VD1-(X1)n-VD2-C, 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

n is 0 or 1;

wherein the first nucleic acid is present on the same or a different expression vector as the second nucleic acid, and wherein at least 90% of the binding proteins expressed from the first and second nucleic acid form a dual specific tetravalent binding protein, 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 two first polypeptide chains and the two second polypeptides form four functional antigen binding sites.

78 . The method of claim 77 , wherein at least 95% of the binding proteins produced form a dual specific tetravalent binding protein.

79 . The method of claim 77 , wherein at least 98% of the binding proteins produced form a dual specific tetravalent binding protein.

80 . The method of claim 77 , wherein the dual specific tetravalent binding protein is capable of binding two or more different antigens.

81 . The method of claim 80 , wherein the dual specific tetravalent binding protein is capable of binding a pair of two targets selected from CD138 and CD20; CD138 and CD40; CD20 and CD3; CD38 and 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-1 beta; 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-β; 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; and TNFα and CTLA-4.

82 . The method of claim 80 , wherein the dual specific tetravalent binding protein is capable of binding IL-13 and IL-4.

83 . The method of claim 77 , wherein X1 is selected from AKTTPKLEEGEFSEAR (SEQ ID NO: 118); AKTTPKLEEGEFSEARV (SEQ ID NO: 119); AKTTPKLGG (SEQ ID NO: 120); SAKTTPKLGG (SEQ ID NO: 121); SAKTTP (SEQ ID NO: 122); RADAAP (SEQ ID NO: 123); RADAAPTVS (SEQ ID NO: 124); RADAAAAGGPGS (SEQ ID NO: 125); RADAAAA(G4S)4 (SEQ ID NO: 126); SAKTTPKLEEGEFSEARV (SEQ ID NO: 127); ADAAP (SEQ ID NO: 40); ADAAPTVSIFPP (SEQ ID NO: 103); TVAAP (SEQ ID NO: 44); TVAAPSVFIFPP (SEQ ID NO: 50); QPKAAP (SEQ ID NO: 88); QPKAAPSVTLFPP (SEQ ID NO: 92); AKTTPP (SEQ ID NO: 38); AKTTPPSVTPLAP (SEQ ID NO: 128); AKTTAP (SEQ ID NO: 129); AKTTAPSVYPLAP (SEQ ID NO: 99); ASTKGP (SEQ ID NO: 42); ASTKGPSVFPLAP (SEQ ID NO: 48); GGGGSGGGGSGGGGS (SEQ ID NO: 130); GENKVEYAPALMALS (SEQ ID NO: 131); GPAKELTPLKEAKVS (SEQ ID NO: 132); and GHEAAAVMQVQYPAS (SEQ ID NO: 133).

84 . The method of claim 77 , wherein X1 on the first and/or second polypeptide chain comprises a repeated sequence of (Gly-Gly-Gly-Gly-Ser) from SEQ ID NO: 130.

85 . The method of claim 77 , wherein the dual specific tetravalent binding protein is detected by size exclusion chromatography (SEC) and/or SDS-PAGE assay.

86 . The method of claim 85 , wherein the dual specific tetravalent binding protein is detected as a single peak by the SEC assay.

87 . The method of claim 85 , wherein the dual specific tetravalent binding protein migrates as a single species under non-reducing conditions in the SDS-PAGE assay.

88 . The method of claim 85 , wherein the dual specific tetravalent binding protein migrates as first and second species under reducing conditions in the SDS-PAGE assay.

89 . The method of claim 77 , wherein at least 50% of the binding proteins are dual specific tetravalent binding proteins after 8 weeks of incubation at 4° C., 25° C., or 40° C.

90 . The method of claim 89 , wherein less than 50% of the binding proteins are aggregated after the incubation.

91 . The method of claim 89 , wherein less than 15% of the binding proteins are fragmented after the incubation.

92 . The method of claim 89 , wherein the dual specific tetravalent binding protein is detected by SEC analysis.

93 . The method of claim 77 , wherein at least 50% of the binding proteins are dual specific tetravalent binding proteins after three freeze and thaw cycles.

94 . The method of claim 92 , wherein the dual specific tetravalent binding protein is detected after the freeze and thaw cycles by SEC analysis.

95 . A binding protein produced according to the method of claim 77 .

96 . A dual specific tetravalent binding protein produced according to the method of claim 77 .