MULTISPECIFIC ANTIGEN-BINDING MOLECULES
Novel antigen-binding molecules are provided, with the ability to target different antigens with different valency, e.g. one antigen monovalently and another antigen bivalently.
1 . An antigen-binding molecule, comprising
a) a first Fab comprising a first Fv region, which specifically binds to a first antigen,
b) a second Fv region which specifically binds to a second antigen and
c) a second Fab comprising a third Fv region, which specifically binds to a third antigen, and
d) a Fc region composed of a first and second Fc region subunit; wherein
i. the C-terminus of the heavy or light chain of the first Fab is fused to the N-terminus of the VH or VL of the second Fv region, and wherein
ii. the C-terminus of the VH or VL of the second Fv region is fused to the N-terminus of the first Fc region subunit and the N-terminus of the second Fc subunit is fused to the C-terminus of the complementary variable domain of the second Fv region, and wherein
iii. the C-terminus of the heavy or light chain the second Fab is fused to the N-terminus of the VH or VL of the second Fv region with the proviso that the first and second Fab are fused to distinct variable domains of the second Fv region, and wherein
iv. in the CH3 domain of the first Fc region subunit, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) and in the CH3 domain of the second Fc region subunit the tyrosine residue at position 407 is replaced with a valine residue (Y407V), the threonine residue at position 366 is replaced with a serine residue (T366S), the leucine residue at position 368 is replaced with an alanine residue (L368A) and the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) with numbering according EU index.
2 . The antigen-binding molecule according to claim 1 , wherein each fusion occurs via a peptide linker.
3 . The antigen-binding molecule according to claim 1 , wherein the antigen-binding molecule is composed of at least 4 polypeptides, wherein
a. a first polypeptide comprises the light or heavy chain of the first Fab,
b. a second polypeptide comprises from its N-terminus to its C-terminus
i. the complementary light or heavy chain of the first Fab,
ii. the VH or VL of the second Fv region and
iii. the first or second Fc region subunit
c. a third polypeptide comprises from its N-terminus to its C-terminus
i. the light or heavy chain of the second Fab,
ii. the complementary VH or VL of the second Fv region and
iii. the complementary first or second Fc region subunit
d. a fourth polypeptide comprises the complementary light or heavy chain of the second Fab.
4 . The antigen-binding molecule according to claim 1 , wherein the third antigen is identical to the first antigen.
5 . The antigen-binding molecule according to claim 1 , wherein the antigen-binding molecule provides bivalent binding to the first antigen and monovalent binding to the second antigen.
6 . The antigen-binding molecule according to claim 1 wherein the antigen-binding molecule is a trivalent bispecific antigen-binding molecule.
7 . The antigen-binding molecule according to claim 1 , wherein the second antigen is expressed on an immune effector cell.
8 . The antigen-binding molecule according to claim 1 , wherein the first antigen is a member of the T-cell receptor complex.
9 . The antigen-binding molecule according to claim 1 , wherein the second antigen is CD3.
10 . The antigen-binding molecule according to claim 1 , wherein the Fc region is a human IgG1 Fc region.
11 . The antigen-binding molecule according to claim 1 , wherein the Fc region comprises one or more amino acid modification promoting the association of the first and second Fc region subunit.
12 . The antigen-binding molecule according to claim 1 , wherein in each of the Fc region subunit at least 5 amino acid residues in the positions corresponding to positions L234, L235, G237, A330, P331 with numbering according EU index in a human IgG1 are mutated to A, E, A, S, and S, respectively.
13 . A pharmaceutical composition comprising the antigen-binding molecule according to claim 1 and a pharmaceutically acceptable carrier or excipient.
14 . (canceled)
15 . A method for re-directing cytotoxic activity of a T-cell to a cancer cell comprising contacting said cancer cell in the presence of a T-cell with a antigen-binding molecule according to claim 1 .
16 . A method for treating a disease in an individual, said method comprising administering an effective amount of the pharmaceutical composition of claim 13 to the individual.
17 . The method of claim 16 wherein the disease is an autoimmune disease, an inflammatory disease, cancer, a vascular disease, an infectious disease, thrombosis, myocardial infarction or diabetes.
18 . The method of claim 16 wherein the disease is a proliferative disease.
19 . The method of claim 16 wherein the disease is cancer.