Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation
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 L1 -L 1 -V L2 -L 2 -C L , and a second polypeptide chain comprises the structure V H2 -L 3 -V H1 -L 4 -C H1 ; wherein
(a) V L1 is derived from a V H of a first immunoglobulin heavy chain variable domain, V L2 is derived from a V H of a second immunoglobulin heavy chain variable domain, V H2 is derived from a V L of a second immunoglobulin light chain variable domain, and V H1 is derived from a V L of a first immunoglobulin light chain variable domain; 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 CHi 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, CD4OL, 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, IL1(3, 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, TNF-α 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 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody-like binding protein of claim 1 .
14 . An expression vector comprising the nucleic acid molecule of claim 13 .
15 . An isolated host cell comprising the nucleic acid molecule of claim 13 .
16 . The host cell of claim 15 , wherein the host cell is a mammalian cell or an insect cell.
17 . An isolated host cell comprising the expression vector of claim 14 .
18 . The host cell of claim 17 , wherein the host cell is a mammalian cell or an insect cell.