Dual variable domain immunoglobulins 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, diagnosis, and/or treatment of disease.

1 - 76 . (canceled) 
     
     
         77 . 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; and   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:
 (a) Tumor necrosis factor alpha (TNF) and nerve growth factor (NGF), wherein
 (1) the variable domains that form a functional target binding site for TNF comprise a sequence selected from the group consisting of SEQ ID NO: 38-43 and 46-49; 
 and 
 (2) the variable domains that form a functional target binding site for NGF comprise SEQ ID NO: 32 and/or SEQ ID NO: 33; 
 
 (b) TNF and prostaglandin E2 (PGE2), wherein
 (1) the variable domains that form a functional target binding site for TNF comprise a sequence selected from the group consisting of SEQ ID NO: 38-43 and 46-49; 
 and 
 (2) the variable domains that form a functional target binding site for PGE2 comprise SEQ ID NO: 36 and/or SEQ ID NO: 37, 
 
 or 
 (c) TNF and lysophosphatidic acid (LPA), wherein
 (1) the variable domains that form a functional target binding site for TNF comprise a sequence selected from the group consisting of SEQ ID NO: 38-43 and 46-49; 
 and 
 (2) the variable domains that form a functional target binding site for LPA comprise SEQ ID NO: 44 and/or SEQ ID NO: 45. 
 
 
     
     
         78 . 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; and   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:
 (a) TNF and NGF, wherein
 (1) the variable domains that form a functional target binding site for TNF comprise:
 CDRs 1-3 from SEQ ID NO: 38 and CDRs 1-3 from SEQ ID NO: 39, 
 CDRs 1-3 from SEQ ID NO: 40 and CDRs 1-3 from SEQ ID NO: 41, 
 CDRs 1-3 from SEQ ID NO: 42 and CDRs 1-3 from SEQ ID NO: 43, 
 CDRs 1-3 from SEQ ID NO: 46 and CDRs 1-3 from SEQ ID NO: 47, or 
 CDRs 1-3 from SEQ ID NO: 48 and CDRs 1-3 from SEQ ID NO: 49; 
 
 and/or 
 (2) the variable domains that form a functional target binding site for NGF comprise CDRs 1-3 from SEQ ID NO: 32 and CDRs 1-3 from SEQ ID NO: 33; 
 
 (b) TNF and PGE2, wherein
 (1) the variable domains that form a functional target binding site for TNF comprise:
 CDRs 1-3 from SEQ ID NO: 38 and CDRs 1-3 from SEQ ID NO: 39, 
 CDRs 1-3 from SEQ ID NO: 40 and CDRs 1-3 from SEQ ID NO: 41, 
 CDRs 1-3 from SEQ ID NO: 42 and CDRs 1-3 from SEQ ID NO: 43, 
 CDRs 1-3 from SEQ ID NO: 46 and CDRs 1-3 from SEQ ID NO: 47, or 
 CDRs 1-3 from SEQ ID NO: 48 and CDRs 1-3 from SEQ ID NO: 49; 
 
 and/or 
 (2) the variable domains that form a functional target binding site for PGE2 comprise CDRs 1-3 from SEQ ID NO: 36 and CDRs 1-3 from SEQ ID NO: 37; 
 
 or 
 (c) TNF and LPA, wherein
 (1) the variable domains that form a functional target binding site for TNF comprise:
 CDRs 1-3 from SEQ ID NO: 38 and CDRs 1-3 from SEQ ID NO: 39, 
 CDRs 1-3 from SEQ ID NO: 40 and CDRs 1-3 from SEQ ID NO: 41, 
 CDRs 1-3 from SEQ ID NO: 42 and CDRs 1-3 from SEQ ID NO: 43, 
 CDRs 1-3 from SEQ ID NO: 46 and CDRs 1-3 from SEQ ID NO: 47, or 
 CDRs 1-3 from SEQ ID NO: 48 and CDRs 1-3 from SEQ ID NO: 49; 
 
 and 
 (2) the variable domains that form a functional target binding site for LPA comprise CDRs 1-3 from SEQ ID NO: 44 and CDRs 1-3 from SEQ ID NO: 45. 
 
 
     
     
         79 . The binding protein according to  claim 77  or  78 , wherein the first polypeptide chain comprises a first 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; 
 X2 is an Fc region; and 
 n is 0 or 1; 
 
       wherein the second polypeptide chain comprises a second 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; and 
 n is 0 or 1; 
 
       and 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. 
     
     
         80 . The binding protein according to  claim 77 , wherein the binding protein is capable of binding:
 (a) TNF and NGF, wherein
 (1) the variable domains that form a functional target binding site for TNF comprise:
 SEQ ID NO: 38 and SEQ ID NO: 39, 
 SEQ ID NO: 40 and SEQ ID NO: 41, 
 SEQ ID NO: 42 and SEQ ID NO: 43, 
 SEQ ID NO: 46 and SEQ ID NO: 47, or 
 SEQ ID NO: 48 and SEQ ID NO: 49; 
 
 and 
 (2) the variable domains that form a functional target binding site for NGF comprise SEQ ID NO: 32 and SEQ ID NO: 33; 
   (b) TNF and PGE2, wherein
 (1) the variable domains that form a functional target binding site for TNF comprise:
 SEQ ID NO: 38 and SEQ ID NO: 39, 
 SEQ ID NO: 40 and SEQ ID NO: 41, 
 SEQ ID NO: 42 and SEQ ID NO: 43, 
 SEQ ID NO: 46 and SEQ ID NO: 47, or 
 SEQ ID NO: 48 and SEQ ID NO: 49; 
 
 and 
 (2) the variable domains that form a functional target binding site for PGE2 comprise SEQ ID NO: 36 and SEQ ID NO: 37; 
   or   (c) TNF and LPA, wherein
 (1) the variable domains that form a functional target binding site for TNF comprise:
 SEQ ID NO: 38 and SEQ ID NO: 39, 
 SEQ ID NO: 40 and SEQ ID NO: 41, 
 SEQ ID NO: 42 and SEQ ID NO: 43, 
 SEQ ID NO: 46 and SEQ ID NO: 47, or 
 SEQ ID NO: 48 and SEQ ID NO: 49; 
 
 and 
 (2) the variable domains that form a functional target binding site for LPA comprise SEQ ID NO: 44 and SEQ ID NO: 45. 
   
     
     
         81 . The binding protein according to  claim 77  or  78 , comprising two first polypeptide chains and two second polypeptide chains, forming four functional target binding sites. 
     
     
         82 . The binding protein according to  claim 77  or  78 , wherein X1 is any one of SEQ ID NO: 1-29. 
     
     
         83 . The binding protein according to  claim 77  or  78 , wherein the Fc region is a variant sequence Fc region. 
     
     
         84 . The binding protein according to  claim 77  or  78 , wherein the Fc region is an Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. 
     
     
         85 . The binding protein according to  claim 77  or  78 , wherein the binding protein is a crystallized binding protein. 
     
     
         86 . A binding protein capable of binding:
 (a) TNF and NGF, wherein the binding protein comprises:
 SEQ ID NOs: 54 and 55, 
 SEQ ID NOs: 56 and 57, 
 SEQ ID NOs: 58 and 59, 
 SEQ ID NOs: 60 and 61, 
 SEQ ID NOs: 62 and 63, 
 SEQ ID NOs: 64 and 65, 
 SEQ ID NOs: 66 and 67, 
 comprising SEQ ID NOs: 68 and 69, 
 comprising SEQ ID NOs: 70 and 71, or 
 comprising SEQ ID NOs: 72 and 73; 
   (b) TNF and PGE2, wherein the binding protein comprises:
 comprising SEQ ID NOs: 94 and 95, 
 comprising SEQ ID NOs: 96 and 97, 
 comprising SEQ ID NOs: 98 and 99, 
 comprising SEQ ID NOs: 100 and 101, 
 comprising SEQ ID NOs: 102 and 103, 
 comprising SEQ ID NOs: 104 and 105, 
 comprising SEQ ID NOs: 106 and 107, 
 comprising SEQ ID NOs: 108 and 109, 
 comprising SEQ ID NOs: 110 and 111, or 
 comprising SEQ ID NOs: 112 and 113; 
   or   (c) TNF and LPA, wherein the binding protein comprises:
 comprising SEQ ID NOs: 114 and 115, 
 comprising SEQ ID NOs: 116 and 117, 
 comprising SEQ ID NOs: 118 and 119, 
 comprising SEQ ID NOs: 120 and 121, 
 comprising SEQ ID NOs: 122 and 123, 
 comprising SEQ ID NOs: 124 and 125, 
 comprising SEQ ID NOs: 126 and 127, 
 comprising SEQ ID NOs: 128 and 129, 
 comprising SEQ ID NOs: 130 and 131, 
 comprising SEQ ID NOs: 132 and 133, 
 comprising SEQ ID NOs: 134 and 135, or 
 comprising SEQ ID NOs: 136 and 137. 
   
     
     
         87 . A binding protein conjugate comprising a binding protein according  claim 77 , the binding protein conjugate further comprising an immunoadhesion molecule, an imaging agent, a therapeutic agent, or a cytotoxic agent. 
     
     
         88 . The binding protein conjugate of  claim 87 , wherein the imaging agent is a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin. 
     
     
         89 . The binding protein conjugate of  claim 88 , wherein said 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. 
     
     
         90 . The binding protein conjugate of  claim 87 , wherein said 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. 
     
     
         91 . An isolated nucleic acid encoding a binding protein amino acid sequence according to  claim 77 . 
     
     
         92 . A vector comprising the isolated nucleic acid according to  claim 91 . 
     
     
         93 . A host cell comprising the vector according to  claim 92 , wherein the host cell is optionally selected from the group consisting of 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, and a COS cell. 
     
     
         94 . A method of producing a binding protein, comprising culturing the host cell of  claim 93  in culture medium under conditions sufficient to produce the binding protein. 
     
     
         95 . A pharmaceutical composition comprising the binding protein according to any one of  claims 77 ,  78 , and  86 , and a pharmaceutically acceptable carrier. 
     
     
         96 . The pharmaceutical composition according to  claim 95 , further comprising at least one additional therapeutic agent. 
     
     
         97 . A method of determining the presence, amount, or concentration of TNF, NGF, PGE2, and/or LPA in a test sample by an immunoassay,
 wherein the immunoassay comprises contacting the test sample with at least one binding protein and at least one detectable label; and   wherein the at least one binding protein comprises the binding protein of  claim 77 .   
     
     
         98 . A kit for assaying a test sample for the presence, amount, or concentration of TNF, NGF, PGE2, and/or LPA in the sample, said kit comprising:
 (a) instructions for assaying the test sample for TNF, NGF, PGE2, and/or LPA; and   (b) at least one binding protein comprising the binding protein of  claim 77 .   
     
     
         99 . The binding protein according to  claim 77  or  78 , wherein
 (a) the binding protein is capable of binding to TNF and NGF, and
 (1) is capable of binding TNF with a K D  of at most about 3.42×10 −9  M, as measured by surface plasmon resonance; and/or 
 (2) is capable of binding NGF with a K D  of at most about 2.93×10 −9  M, as measured by surface plasmon resonance; 
 
 (b) the binding protein is capable of binding to TNF and PGE2, and
 (1) is capable of binding TNF with a K D  of at most about 2.70×10 −9  M, as measured by surface plasmon resonance; and/or 
 
 (2) is capable of inhibiting PGE2 with an EC50 of at most about 195 pM, as measured in a PGE2 inhibition assay, 
 or 
 (c) the binding protein is capable of binding to TNF and LPA, and is capable of binding TNF with a K D  of at most about 3.95×10 −9  M, as measured by surface plasmon resonance.