Covalent diabodies and uses thereof
The present invention is directed to diabody molecules and uses thereof in the treatment of a variety of diseases and disorders, including immunological disorders, infectious disease, intoxication and cancers. The diabody molecules of the invention comprise two polypeptide chains that associate to form at least two epitope binding sites, which may recognize the same or different epitopes on the same or differing antigens. Additionally, the antigens may be from the same or different molecules. The individual polypeptide chains of the diabody molecule may be covalently bound through non-peptide bond covalent bonds, such as, but not limited to, disulfide bonding of cysteine residues located within each polypeptide chain. In particular embodiments, the diabody molecules of the present invention further comprise an Fc region, which allows antibody-like functionality to engineered into the molecule.
1. A diabody molecule comprising a first and a second polypeptide chain, said chains each having an N-terminal end and a C-terminal end and being covalently bonded to one another,
which first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, and (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope, which first domain and second domain are covalently linked such that the first domain and second domain do not associate to form an epitope binding site;
which second polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a third domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), and (ii) a fourth domain comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1), which third domain and fourth domain are covalently linked such that the third domain and fourth domain do not associate to form an epitope binding site;
wherein:
the first domain and the fourth domain associate to form a first binding site (VL1)(VH1) that binds the first epitope;
the second domain and the third domain associate to form a second binding site (VH2)(VL2) that binds the second epitope; and
the first polypeptide chain and the second polypeptide chain are covalently linked via a disulfide bond between at least one cysteine residue outside of the first domain and the second domain on the first polypeptide chain and at least one cysteine residue outside of the third domain and the fourth domain on the second polypeptide chain, which cysteine residue on the first polypeptide chain is not at the C-terminus of the first polypeptide chain and cysteine residue on the second polypeptide chain is not at the C-terminus of the second polypeptide chain; and
wherein said first or second polypeptide chain additionally comprises either an E-coil comprising the amino acid sequence of SEQ ID NO:299 or a K-coil domain comprising the amino acid sequence of SEQ ID NO:300, wherein said domain promotes heterodimer formation and is positioned C-terminally to said second domain or said fourth domain.
2. The diabody of claim 1 wherein one of said first and second polypeptide chains of said diabody comprises said E-coil domain and the other of said first and second polypeptide chains comprises said K-coil domain.
3. The diabody of claim 1 wherein said E-coil or K-coil domain is linked to an Fc fragment.
4. The diabody of claim 2 wherein said E-coil or K-coil domain is linked to an Fc fragment.
5. The diabody of claim 1 , wherein said E-coil or K-coil domain is linked to a polypeptide portion of a serum-binding protein, said polypeptide portion being capable of binding to said serum protein.
6. The diabody of claim 5 , wherein said serum-binding protein is an albumin-binding protein.
7. The diabody of claim 6 , wherein said albumin-binding protein is streptococcal protein G and said polypeptide is an albumin-binding domain (ABD) of said streptococcal protein G.
8. The diabody of claim 7 , wherein albumin-binding domain (ABD) of said streptococcal protein G is albumin-binding domain 3 (ABD3) of protein G of Streptococcus strain G148.
9. The diabody molecule of claim 1 that binds the T-cell receptor (TCR) or the Natural Killer Group 2D (NKG2D) receptor.
10. The diabody molecule of claim 9 , wherein said molecule additionally binds to a tumor-associated antigen selected from the group consisting of a breast cancer antigen, an ovarian cancer antigen, a prostate cancer antigen, a cervical cancer antigen, a pancreatic carcinoma antigen, a lung cancer antigen, a bladder cancer antigen, a colon cancer antigen, a testicular cancer antigen, a glioblastoma cancer antigen, an antigen associated with a B cell malignancy, an antigen associated with multiple myeloma, an antigen associated with non-Hodgkin's lymphoma, and an antigen associated with chronic lymphocytic leukemia.
11. The diabody molecule of claim 10 , wherein said tumor-associated antigen is the breast cancer antigen HER-2/neu.
12. The diabody of claim 1 , wherein said first epitope is an epitope of CD32B and said second epitope is an epitope of CD16, or said first epitope is an epitope of CD16 and said second epitope is an epitope of CD32B.