Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation

Abstract

The invention provides antibody-like binding proteins comprising four polypeptide chains that form four antigen binding sites, wherein each pair of polypeptides forming an antibody-like binding protein possesses dual variable domains having a cross-over orientation. The invention also provides methods for making such antigen-like binding proteins.

1 . An antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites, wherein two polypeptide chains have a structure represented by the formula:
   V L1 -L 1 -V L2 -L 2 -C L   [I]
   
       and two polypeptide chains have a structure represented by the formula:
   V H2 -L 3 -V H1 -L 4 -C H1 -Fc  [II]
 
 
       wherein:
 V L1  is a first immunoglobulin light chain variable domain; 
 V L2  is a second 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; 
 C L  is an immunoglobulin light chain constant domain; 
 C H1  is the immunoglobulin C H1  heavy chain constant domain; 
 Fc is the immunoglobulin hinge region and C H2 , C H3  immunoglobulin heavy chain constant domains; and 
 L 1 , L 2 , L 3 , and L 4  are amino acid linkers; 
 
       and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. 
     
     
         2 . The antibody-like binding protein of  claim 1 , wherein:
 L 1  is 3 to 12 amino acid residues in length;   L 2  is 3 to 14 amino acid residues in length;   L 3  is 1 to 8 amino acid residues in length; and   L 4  is 1 to 3 amino acid residues in length.   
     
     
         3 . The antibody-like binding protein of  claim 2 , wherein:
 L 1  is 5 to 10 amino acid residues in length;   L 2  is 5 to 8 amino acid residues in length;   L 3  is 1 to 5 amino acid residues in length; and   L 4  is 1 to 2 amino acid residues in length.   
     
     
         4 . The antibody-like binding protein of  claim 3 , wherein:
 L 1  is 7 amino acid residues in length;   L 2  is 5 amino acid residues in length;   L 3  is 1 amino acid residues in length; and   L 4  is 2 amino acid residues in length.   
     
     
         5 . The antibody-like binding protein of  claim 1 , wherein:
 L 1  is 1 to 3 amino acid residues in length;   L 2  is 1 to 4 amino acid residues in length;   L 3  is 2 to 15 amino acid residues in length; and   L 4  is 2 to 15 amino acid residues in length.   
     
     
         6 . The antibody-like binding protein of  claim 5 , wherein:
 L 1  is 1 to 2 amino acid residues in length;   L 2  is 1 to 2 amino acid residues in length;   L 3  is 4 to 12 amino acid residues in length; and   L 4  is 2 to 12 amino acid residues in length.   
     
     
         7 . The antibody-like binding protein of  claim 6 , wherein:
 L 1  is 1 amino acid residue in length;   L 2  is 2 amino acid residues in length;   L 3  is 7 amino acid residues in length; and   L 4  is 5 amino acid residues in length.   
     
     
         8 . The antibody-like binding protein of  claim 1 , wherein L 1  is 0 amino acid residues in length and L 3  is 2 or more amino acid residues in length. 
     
     
         9 . The antibody-like binding protein of  claim 1 , wherein L 3  is 0 amino acid residues in length and L 1  is 1 or more amino acid residues in length. 
     
     
         10 . The antibody-like binding protein of  claim 1 , wherein L 4  is 0 amino acid residues in length and L 2  is 3 or more amino acid residues in length. 
     
     
         11 . The antibody-like binding protein of  claim 1 , wherein the binding protein is capable of specifically binding one or more antigen targets. 
     
     
         12 . The antibody-like binding protein of  claim 11 , wherein the one or more antigen targets is selected from the group consisting of B7.1, B7.2, BAFF, BlyS, C3, C5, CCL11 (eotaxin), CCL15 (MIP-1d), CCL17 (TARC), CCL19 (MIP-3b), CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CD3, CD19, CD20, CD24, CD40, CD40L, CD80, CD86, CDH1 (E-cadherin), Chitinase, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA4, CX3CL1 (SCYD1), CXCL12 (SDF1), CXCL13, EGFR, FCER1A, FCER2, HER2, IGF1R, IL-1, IL-12, IL13, IL15, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL8, IL9, IL12/23, IL22, IL23, IL25, IL27, IL35, ITGB4 (b 4 integrin), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNFα, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5/CCXCR1), DNGR-1 (CLEC91), and HMGB1. 
     
     
         13 . The antibody-like binding protein of  claim 1 , wherein the binding protein is bispecific and capable of binding two different antigen targets. 
     
     
         14 . The antibody-like binding protein of  claim 13 , wherein the two different antigen targets are selected from the group consisting of IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1α and IL-1β, TNFα and IL12/23, TNFα and IL-12p40, TNFα and IL1β, TNFα and IL-23, and IL17 and IL23. 
     
     
         15 . The antibody-like binding protein of  claim 1 , wherein the binding protein is capable of inhibiting the function of one or more of the antigen targets. 
     
     
         16 . The antibody-like binding protein of  claim 1 , wherein at least one of the linkers selected from the group consisting of L 1 , L 2 , L 3 , and L 4  contains at least one cysteine residue. 
     
     
         17 . An antibody-like binding protein comprising two polypeptide chains that form two antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula:
   V L1 -L 1 -V L2 -L 2 -C L   [I]
   
       and a second polypeptide chain has a structure represented by the formula:
   V H2 -L 3 -V H1 -L 4 -C H1   [II]
 
 
       wherein:
 V L1  is a first immunoglobulin light chain variable domain; 
 V L2  is a second 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; 
 C L  is an immunoglobulin light chain constant domain; 
 C H1  is the immunoglobulin C H1  heavy chain constant domain; and 
 L 1 , L 2 , L 3 , and L 4  are amino acid linkers; 
 
       and wherein the first and second polypeptides form a cross-over light chain-heavy chain pair. 
     
     
         18 . The antibody-like binding protein of  claim 17 , wherein:
 L 1  is 3 to 12 amino acid residues in length;   L 2  is 3 to 14 amino acid residues in length;   L 3  is 1 to 8 amino acid residues in length; and   L 4  is 1 to 3 amino acid residues in length.   
     
     
         19 . The antibody-like binding protein of  claim 18 , wherein:
 L 1  is 5 to 10 amino acid residues in length;   L 2  is 5 to 8 amino acid residues in length;   L 3  is 1 to 5 amino acid residues in length; and   L 4  is 1 to 2 amino acid residues in length.   
     
     
         20 . The antibody-like binding protein of  claim 19 , wherein:
 L 1  is 7 amino acid residues in length;   L 2  is 5 amino acid residues in length;   L 3  is 1 amino acid residues in length; and   L 4  is 2 amino acid residue in length.   
     
     
         21 . The antibody-like binding protein of  claim 17 , wherein:
 L 1  is 1 to 3 amino acid residues in length;   L 2  is 1 to 4 amino acid residues in length;   L 3  is 2 to 15 amino acid residues in length; and   L 4  is 2 to 15 amino acid residues in length.   
     
     
         22 . The antibody-like binding protein of  claim 21 , wherein:
 L 1  is 1 to 2 amino acid residues in length;   L 2  is 1 to 2 amino acid residues in length;   L 3  is 4 to 12 amino acid residues in length; and   L 4  is 2 to 12 amino acid residues in length.   
     
     
         23 . The antibody-like binding protein of  claim 22 , wherein:
 L 1  is 1 amino acid residue in length;   L 2  is 2 amino acid residues in length;   L 3  is 7 amino acid residues in length; and   L 4  is 5 amino acid residues in length.   
     
     
         24 . The antibody-like binding protein of  claim 17 , wherein L 1  is 0 amino acid residues in length and L 3  is 2 or more amino acid residues in length. 
     
     
         25 . The antibody-like binding protein of  claim 17 , wherein L 3  is 0 amino acid residues in length and L 1  is 1 or more amino acid residues in length. 
     
     
         26 . The antibody-like binding protein of  claim 17 , wherein L 4  is 0 amino acid residues in length and L 2  is 3 or more amino acid residues in length. 
     
     
         27 . The antibody-like binding protein of  claim 17 , wherein the binding protein is capable of specifically binding one or more antigen targets. 
     
     
         28 . The antibody-like binding protein of  claim 27 , wherein the one or more antigen targets is selected from the group consisting of B7.1, B7.2, BAFF, BlyS, C3, C5, CCL11 (eotaxin), CCL15 (MIP-1d), CCL17 (TARC), CCL19 (MIP-3b), CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CD3, CD19, CD20, CD24, CD40, CD40L, CD80, CD86, CDH1 (E-cadherin), Chitinase, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA4, CX3CL1 (SCYD1), CXCL12 (SDF1), CXCL13, EGFR, FCER1A, FCER2, HER2, IGF1R, IL-1, IL-12, IL13, IL15, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL8, IL9, IL12/23, IL22, IL23, IL25, IL27, IL35, ITGB4 (b 4 integrin), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNFα, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5/CCXCR1), DNGR-1 (CLEC91), and HMGB1. 
     
     
         29 . The antibody-like binding protein of  claim 17 , wherein the binding protein is bispecific and capable of binding two different antigen targets. 
     
     
         30 . The antibody-like binding protein of  claim 29 , wherein the two different antigen targets are selected from the group consisting of IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1α and IL-β, TNFα and IL12/23, TNFα and IL-12p40, TNFα and IL1β, TNFα and IL-23, and IL17 and IL23. 
     
     
         31 . The antibody-like binding protein of  claim 17 , wherein the binding protein is capable of inhibiting the function of one or more of the antigen targets. 
     
     
         32 . The antibody-like binding protein of  claim 17 , wherein at least one of the linkers selected from the group consisting of L 1 , L 2 , L 3 , and L 4  contains at least one cysteine residue. 
     
     
         33 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody-like binding protein of either  claim 1  or  17 . 
     
     
         34 . An expression vector comprising the nucleic acid molecule of  claim 33 . 
     
     
         35 . An isolated host cell comprising the nucleic acid molecule of  claim 33 . 
     
     
         36 . An isolated host cell comprising the expression vector of  claim 34 . 
     
     
         37 . The host cell of either  claim 35  or  36 , wherein the host cell is a mammalian cell or an insect cell. 
     
     
         38 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antibody-like binding protein of either  claim 1  or  17 . 
     
     
         39 . A method for making the antibody-like binding protein of  claim 1 , comprising expressing in a cell one or more nucleic acid molecules encoding polypeptides having structures represented by the formulas [I] and [II] below:
   V L1 -L 1 -V L2 -L 2 -C L   [I]
     V H2 -L 3 -V H1 -L 4 -C H1 -Fc  [II]
   
       wherein:
 V L1  is a first immunoglobulin light chain variable domain; 
 V L2  is a second 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; 
 C L  is an immunoglobulin light chain constant domain; 
 C H1  is the immunoglobulin C H1  heavy chain constant domain; 
 Fc is the immunoglobulin hinge region and C H2 , C H3  immunoglobulin heavy chain constant domains; and 
 L 1 , L 2 , L 3 , and L 4  are amino acid linkers; 
 
       and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. 
     
     
         40 . A method for making the antibody-like binding protein of  claim 17 , comprising expressing in a cell one or more nucleic acid molecules encoding polypeptides having structures represented by the formulas [I] and [II] below:
   V L1 -L 1 -V L2 -L 2 -C L   [I]
     V H2 -L 3 -V H1 -L 4 -C H1   [II]
   
       wherein:
 V L1  is a first immunoglobulin light chain variable domain; 
 V L2  is a second 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; 
 C L  is an immunoglobulin light chain constant domain; 
 C H1  is the immunoglobulin C H1  heavy chain constant domain; and 
 L 1 , L 2 , L 3 , and L 4  are amino acid linkers; 
 
       and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair. 
     
     
         41 . A method of making an antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites, comprising:
 (a) identifying a first antibody variable domain that binds a first target antigen and a second antibody variable domain that binds a second target antigen, each containing a V L , and a V H ;   (b) assigning either the light chain or the heavy chain as template chain;   (c) assigning the V L  of the first antibody variable domain or the second antibody variable domain as V L1 ;   (d) assigning a V L2 ; a V H1 , and a V H2  according to formulas [I] and [II] below:
   V L1 -L 1 -V L2 -L 2 -C L   [I]
 
   V H2 -L 3 -V H1 -L 4 -C H1 -Fc  [II]
 
   (e) determining maximum and minimum lengths for L 1 , L 2 , L 3 , and L 4 ;   (f) generating polypeptide structures of formulas I and II;   (g) selecting polypeptide structures of formulas I and II that bind the first target antigen and the second target antigen when combined to form the antibody-like binding protein;   
       wherein:
 V L1  is a first immunoglobulin light chain variable domain; 
 V L2  is a second 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; 
 C L  is an immunoglobulin light chain constant domain; 
 C H1  is the immunoglobulin C H1  heavy chain constant domain; 
 Fc is the immunoglobulin hinge region and C H2 , C H3  immunoglobulin heavy chain constant domains; and 
 L 1 , L 2 , L 3 , and L 4  are amino acid linkers; 
 
       and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. 
     
     
         42 . The method of  claim 41 , wherein the first antibody variable domain and the second antibody variable domain are the same. 
     
     
         43 . A method of making an antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites, comprising:
 (a) identifying a first antibody variable domain that binds a first target antigen and a second antibody variable domain that binds a second target antigen, each containing a V L , and a V H ;   (b) assigning either the light chain or the heavy chain as template chain;   (c) assigning the V L  of the first antibody variable domain or the second antibody variable domain as V L1 ;   (d) assigning a V L2 ; a V H1 , and a V H2  according to formulas [I] and [II] below:
   V L1 -L 1 -V L2 -L 2 -C L   [I]
 
   V H2 -L 3 -V H1 -L 4 -C H1   [II]
 
   (e) determining maximum and minimum lengths for L 1 , L 2 , L 3 , and L 4 ;   (f) generating polypeptide structures of formulas I and II;   (g) selecting polypeptide structures of formulas I and II that bind the first target antigen and the second target antigen when combined to form the antibody-like binding protein;   
       wherein:
 V L1  is a first immunoglobulin light chain variable domain; 
 V L2  is a second 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; 
 C L  is an immunoglobulin light chain constant domain; 
 C H1  is the immunoglobulin C H1  heavy chain constant domain; and 
 L 1 , L 2 , L 3 , and L 4  are amino acid linkers; 
 
       and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. 
     
     
         43 . The method of claim  44 , wherein the first antibody variable domain and the second antibody variable domain are the same.