Dual variable region antibody-like binding proteins having cross-over binding region orientation
View Patent ↗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 isolated nucleic acid molecule comprising a nucleotide sequence encoding 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;
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;
and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
2. The isolated nucleic acid molecule 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.
3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding 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 immuno globulin 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;
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;
and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding 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 immuno globulin 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;
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;
and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
5. The isolated nucleic acid molecule of claim 4 , 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.
6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding 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 immuno globulin 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;
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;
and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
7. An isolated nucleic acid molecule comprising a nucleotide sequence encoding 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;
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;
and wherein the first and second polypeptides form a cross-over light chain-heavy chain pair.
8. The isolated nucleic acid molecule of claim 7 , 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.
9. An isolated nucleic acid molecule comprising a nucleotide sequence encoding 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 immuno globulin 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;
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;
and wherein the first and second polypeptides form a cross-over light chain-heavy chain pair.
10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding 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 immuno globulin 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;
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.
11. The isolated nucleic acid molecule of claim 10 , 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. An isolated nucleic acid molecule comprising a nucleotide sequence encoding 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 immuno globulin 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;
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;
and wherein the first and second polypeptides form a cross-over light chain-heavy chain pair.
13. An isolated nucleic acid molecule comprising a nucleotide sequence encoding 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;
wherein L 2 and L 4 are both at least one amino acid in length, and
(a) the length of L 2 is at least twice the length of L 4 ; or
(b) the length of L 4 is at least twice the length of L 2 ;
and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
14. An isolated nucleic acid molecule comprising a nucleotide sequence encoding 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;
wherein L 2 and L 4 are both at least one amino acid in length, and
(a) the length of L 2 is at least twice the length of L 4 ; or
(b) the length of L 4 is at least twice the length of L 2 ;
and wherein the first and second polypeptides form a cross-over light chain-heavy chain pair.
15. An expression vector comprising the nucleic acid molecule of either claim 1 , 4 , 7 , 10 , 13 , or 14 .
16. An isolated host cell comprising the nucleic acid molecule of either claim 1 , 4 , 7 , 10 , 13 , or 14 .
17. The host cell of claim 16 , wherein the host cell is a mammalian cell.
18. An isolated host cell comprising the expression vector of claim 15 .
19. The host cell of claim 18 , wherein the host cell is a mammalian cell or an insect cell.
20. A method for making an antibody-like binding protein comprising expressing the nucleic acid molecule of claim 1 , 4 , 7 , 10 , 13 , or 14 in a cell.
21. The isolated nucleic acid molecule of claim 1 , 4 , 7 , 10 , 13 , or 14 , wherein the antibody-like binding protein is capable of specifically binding one or more antigen targets.
22. The isolated nucleic acid molecule of claim 21 , 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, CDHI (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, IL1FI1, 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.
23. The isolated nucleic acid molecule of claim 1 , 4 , 7 , 10 , 13 , or 14 , wherein the antibody-like binding protein is bispecific and capable of binding two different antigen targets.
24. The isolated nucleic acid molecule of claim 23 , 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.
25. The isolated nucleic acid molecule of claim 1 , 4 , 7 , 10 , 13 , or 14 , wherein the antibody-like binding protein is capable of inhibiting the function of one or more of the antigen targets.
26. The isolated nucleic acid molecule of claim 1 , 4 , 7 , 10 , 13 , or 14 , wherein at least one of the linkers selected from the group consisting of L1, L2, L3, and L4 contains at least one cysteine residue.