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 two polypeptide chains that form two antigen binding sites, wherein a first polypeptide chain comprises the structure V 1 -L 1 -V 2 -L 2 -C L , and a second polypeptide chain comprises the structure V 3 -L 3 -V 4 -L 4 -C H1 ;
 wherein:
 (a) V 1  is a first immunoglobulin heavy chain variable domain (V H1 ), V 2  is a second immunoglobulin light chain variable domain (V L2 ), V 3  is a second immunoglobulin heavy chain variable domain (V H2 ), and V 4  is a first immunoglobulin heavy chain variable domain (V L1 ); or
 V 1  is a first immunoglobulin light chain variable domain (V L1 ), V 2  is a second immunoglobulin heavy chain variable domain (V H2 ), V 3  is a second immunoglobulin light chain variable domain (V L2 ), and V 4  is a first immunoglobulin heavy chain variable domain (V H1 ); or 
 V 1  is a first immunoglobulin heavy chain variable domain (V H1 ), V 2  is a second immunoglobulin heavy chain variable domain (V H2 ), V 3  is a second immunoglobulin light chain variable domain (V L2 ), and V 4  is a first immunoglobulin light chain variable domain (V L1 ); 
 wherein V L1  and V H1  associate to form a first antigen binding site, and V L2  and V H2  associate to form a second antigen binding site; 
 
 (b) C L  is an immunoglobulin light chain constant domain; 
 (c) C H1  is the immunoglobulin C H1  heavy chain constant domain; and 
 (d) L 1 , L 2 , L 3 , and L 4  are amino acid linkers;
 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; or 
 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; and 
 
   wherein the first and second polypeptides form a cross-over light chain-heavy chain pair.   
     
     
         2 . The antibody-like binding protein of  claim 1 , wherein the binding protein is capable of specifically binding one or more antigen targets. 
     
     
         3 . The antibody-like binding protein of  claim 2 , 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. 
     
     
         4 . The antibody-like binding protein of  claim 1 , wherein the binding protein is bispecific and capable of binding two different antigen targets. 
     
     
         5 . The antibody-like binding protein of  claim 4 , 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. 
     
     
         6 . 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. 
     
     
         7 . 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. 
     
     
         8 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antibody-like binding protein of  claim 1 . 
     
     
         9 . The antibody-like binding protein of  claim 1 , 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.   
     
     
         10 . The antibody-like binding protein of  claim 1 , 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 residue in length; and   L 4  is 2 amino acid residues in length.   
     
     
         11 . The antibody-like binding protein of  claim 1 , 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.   
     
     
         12 . The antibody-like binding protein of  claim 1 , 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.   
     
     
         13 . The antibody-like binding protein of  claim 1 , wherein at least one of the polypeptide chains further comprises a Fc domain. 
     
     
         14 . The antibody-like binding protein of  claim 13 , wherein both the first and second polypeptides further comprise a Fc domain. 
     
     
         15 . The antibody-like binding protein of  claim 1 , wherein at least one of the polypeptide chains further comprises a Fc domain at the carboxyl terminal region of the polypeptide chain. 
     
     
         16 . The antibody-like binding protein of  claim 13 , wherein the Fc domain is linked to C L  or C H1 . 
     
     
         17 . The antibody-like binding protein of  claim 16 , wherein the first polypeptide comprises the structure V 1 -L 1 -V 2 -L 2 -C L -Fc. 
     
     
         18 . The antibody-like binding protein of  claim 16 , wherein the second polypeptide comprises the structure V 3 -L 3 -V 4 -L 4 -C H1 -Fc. 
     
     
         19 . The antibody-like binding protein of  claim 14 , wherein the Fc domain of the first polypeptide is linked to C L  and the Fc domain of the second polypeptide is linked to the C H1 . 
     
     
         20 . The antibody-like binding protein of  claim 19 , wherein the first polypeptide chain comprises the structure V 1 -L 1 -V 2 -L 2 -C L -Fc, and the second polypeptide chain comprises the structure V 3 -L 3 -V 4 -L 4 -C H1 -Fc.