SINGLE-CHAIN MULTIVALENT BINDING PROTEINS WITH EFFECTOR FUNCTION
The present disclosure relates to single-chain multivalent binding proteins, compositions comprising the single-chain multivalent binding proteins, and methods of making and using the single chain-multivalent binding proteins.
1 . A single-chain multivalent binding protein comprising from amino-terminus to carboxyl-terminus:
(a) a first binding domain comprising a first light chain immunoglobulin variable region (VL1) and a first heavy chain immunoglobulin variable region (VH1), wherein said variable regions are positioned in a VH1-VL1 or a VL1-VH1 orientation and separated by a first peptide linker of 2-50 amino acids;
(b) an immunoglobulin hinge domain;
(c) an immunoglobulin C H2 domain
(d) an immunoglobulin C H3 domain
(e) a scorpion linker; and
(f) a second binding domain comprising a second light chain immunoglobulin variable region (VL2) and a second heavy chain immunoglobulin variable region (VH2), wherein said variable regions are positioned in a VH2-VL2 or a VL2-VH2 orientation and separated by a second peptide linker of 2-50 amino acids,
wherein the multivalent binding protein is capable of forming a dimer in solution.
2 . The binding protein of claim 1 , wherein VL1 and VH1 are from the same immunoglobulin.
3 . The binding protein of claim 1 , wherein VL1 and VH1 are from different immunoglobulins.
4 . The binding protein of claim 1 , wherein VL2 and VH2 are from the same immunoglobulin.
5 . The binding protein of claim 1 , wherein VL2 and VH2 are from different immunoglobulins.
6 . The binding protein of claim 1 , wherein the first and second binding domains are derived from different immunoglobulins.
7 . The binding protein of claim 1 , wherein the first and second binding domains specifically bind different target molecules located on the same cell.
8 . The binding protein of claim 1 , wherein the first and second binding domains specifically bind different target molecules located on physically distinct cells.
9 . The binding protein of claim 1 , wherein at least one binding domain specifically binds a cell-free molecular target.
10 . The binding protein of claim 1 , wherein at least one binding domain comprises a sequence selected from SEQ ID NO: 2, 4, 6, 103, 105, 107, and 109.
11 . The binding protein of claim 1 , wherein the first and/or second peptide linker is 15 amino acids in length.
12 . The binding protein of claim 1 , wherein the first and/or second peptide linker is 20 amino acids in length.
13 . The binding protein of claim 1 , wherein the first and/or second peptide linker comprises a Gly 4 Ser sequence.
14 . The binding protein of claim 1 , wherein the first and/or second peptide linker comprises the sequence Asp-Gly 3 -Ser-(Gly 4 Ser) 2 .
15 . The binding protein of claim 1 , wherein the first and/or second peptide linker comprises the sequence (Gly 4 Ser) 4 .
16 . The binding protein of claim 1 , wherein the immunoglobulin hinge domain is a hinge domain selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgE, IgA2, synthetic hinge and the hinge-like C H2 domain of IgM.
17 . The binding protein of claim 16 , wherein the hinge domain is an IgG1 hinge domain.
18 . The binding protein of claim 17 , wherein the hinge domain is a human IgG1 hinge domain with a mutation at one or two cysteine residues.
19 . The binding protein of claim 1 , wherein the immunoglobulin C H2 domain is a IgG1 immunoglobulin C H2 domain.
20 . The binding protein of claim 1 , wherein the immunoglobulin C H3 domain is a IgG1 immunoglobulin C H3 domain.
21 . The binding protein of claim 20 , wherein the C H3 domain is truncated and comprises a C-terminal sequence selected from the group consisting of SEQ ID NO: 366-371.
22 . The binding protein of claim 1 , wherein the binding protein does not comprise a C H1 domain.
23 . The binding protein of claim 1 , wherein the scorpion linker is at least 5 amino acids in length.
24 . The binding protein of claim 23 , wherein the scorpion linker is between 5 and 45 amino acids in length.
25 . The binding protein of claim 1 , wherein the scorpion linker is resistant to proteolytic cleavage.
26 . The binding protein of claim 1 , wherein the scorpion linker comprises an amino acid sequence derived from an immunoglobulin hinge.
27 . The binding protein of claim 1 , wherein the scorpion linker comprises an amino acid sequence derived from an interdomain region of an immunoglobulin superfamily member.
28 . The binding protein of claim 27 , wherein the immunoglobulin superfamily member is CD2, CD80, CD86, or CD22.
29 . The binding protein of claim 1 , wherein the scorpion linker comprises an amino acid sequence derived from a stalk region of a C-type lectin.
30 . The binding protein of claim 29 , wherein the C-type lectin is selected from the group consisting of CD69, CD72, CD94, NKG2A, and NKG2D.
31 . The binding protein of claim 1 , wherein the scorpion linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 287, 289, 297, 305, 307, 309, 310, 311, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 346, 373, 374, 375, 376, 377, 380, and 381.
32 . The binding protein of claim 1 , wherein the first and/or second binding domains comprise chimeric, humanized, or human immunoglobulin variable domains.
33 . The binding protein of claim 1 , wherein at least one binding domain specifically binds a target selected from a tumor antigen, a B-cell target, a TNF receptor superfamily member, a Hedgehog family member, a receptor tyrosine kinase, a proteoglycan-related molecule, a TGF-beta superfamily member, a Wnt-related molecule, a receptor ligand, a T-cell target, a Dendritic cell target, an NK cell target, a monocyte/macrophage cell target, a myeloid target, and an angiogenesis target.
34 . The binding protein of claim 1 , wherein the binding protein is capable of cross-linking two or more targets.
35 . The binding protein of claim 1 , wherein the first binding domain and the second binding domain are about 20-30 Å apart to about 150-180 Å apart.
36 . The binding protein of claim 1 , wherein the first binding domain and the second binding domain are about 20-30 Å apart to less than about 50 Å apart.
37 . The binding protein of claim 1 , wherein the first binding domain and the second binding domain are about 50 Å apart.
38 . The binding protein of claim 1 , wherein the binding protein exhibits a binding affinity of less than 10 −9 M or at least 10 −6 M for at least one of the first binding domain and the second binding domain.
39 . A protein dimer comprising two multivalent binding proteins of claim 1 .
40 . A nucleic acid encoding the binding protein of claim 1 .
41 . A vector comprising the nucleic acid of claim 40 .
42 . A host cell comprising the nucleic acid of claim 40 or vector of claim 41 .
43 . A composition comprising the binding protein of claim 1 and a pharmaceutically acceptable carrier.