Covalent Diabodies and Uses Thereof

Abstract

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 polypeptide chain and a second polypeptide chain, said chains each having an N-terminus and a C-terminus and being covalently bonded to one another, wherein:
 (A) said first polypeptide chain comprises, in the N-terminal to C-terminal direction:
 (i) a Domain A comprising an epitope-binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, 
 (ii) a Domain B comprising an epitope-binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope, 
 wherein said Domain A and said Domain B are covalently linked such that said Domain A and said Domain B do not associate with one another to form an epitope binding site capable of specifically binding said first or second epitope; and 
 wherein said first polypeptide chain additionally comprises a Domain C that comprises, in the N-terminus to C-terminus direction, a CH 2  domain and a CH 3  domain of an Fc domain, and wherein a cysteine residue (Cys1)-containing linker domain covalently links said Domain C to the N-terminus of said Domain A or to the C-terminus of said Domain B; and 
   (B) said second polypeptide chain comprises, in the N-terminal to C-terminal direction:
 (i) a Domain D comprising an epitope-binding region of a light chain variable domain of the second immunoglobulin (VL2) specific for said second epitope, 
 (ii) a Domain E comprising an epitope-binding region of a heavy chain variable domain of the first immunoglobulin (VH1) specific for said first epitope, 
 wherein said Domain D and said Domain E are covalently linked such that said Domain D and said Domain E do not associate with one another to form an epitope binding site capable of specifically binding said first or second epitope; and 
 wherein said second polypeptide chain additionally comprises a Domain F that comprises, in the N-terminus to C-terminus direction, a CH 2  domain and a CH 3  domain of an Fc domain; wherein a cysteine residue (Cys2)-containing linker domain covalently links said Domain F to the N-terminus of said Domain D or to the C-terminus of said Domain E; 
   wherein said Domain A and said Domain E associate with one another to form a (VL1)(VH1) binding site capable of specifically binding said first epitope;   wherein said Domain B and said Domain E associate with one another to form a (VL2)(VH2) binding site capable of specifically binding said second epitope;   wherein said first and second epitopes are different;   wherein said first polypeptide chain and said second polypeptide chain are covalently bonded to one another to one another via a disulfide bond between said cysteine residue Cys1 and said cysteine residue Cys2; and   wherein said CH2 and CH3 domains of said Domain C and said Domain F associate with one another to form an Fc domain.   
     
     
         2 - 113 . (canceled) 
     
     
         114 . The diabody molecule of  claim 1 , wherein said Domain C additionally comprises a hinge domain N-terminal to said CH 2  domain. 
     
     
         115 . The diabody molecule of  claim 1 , wherein said cysteine residue (Cys1)-containing linker domain covalently linked said Domain C to the N-terminus of said Domain A. 
     
     
         116 . The diabody molecule of  claim 1 , wherein said cysteine residue (Cys1)-containing linker domain covalently linked said Domain C to the C-terminus of said Domain B. 
     
     
         117 . The diabody molecule of  claim 1 , wherein the first immunoglobulin or second immunoglobulin is a human or humanized immunoglobulin, which human or humanized immunoglobulin is an IgA, IgE, IgD, IgG or IgM. 
     
     
         118 . The diabody molecule of  claim 117 , wherein the human or humanized immunoglobulin is an IgG, which IgG is selected from the list consisting of IgG1, IgG2, IgG3 and IgG4. 
     
     
         119 . The diabody molecule of  claim 1 , wherein the Fc domain is a human or humanized Fc domain. 
     
     
         120 . The diabody molecule of  claim 1 , wherein at least one epitope binding site is specific for an FcγRI, FcγRII or FcγRIII receptor. 
     
     
         121 . The diabody molecule of  claim 120 , wherein the Fcγ receptor is a FcγRIII receptor, which FcγRIII receptor is FcγRIIIA (CD16A) receptor or FcγRIIIB (CD16B) receptor. 
     
     
         122 . The diabody molecule of  claim 121 , wherein the FcγRIII receptor is FcγRIIIA (CD16A) receptor. 
     
     
         123 . The diabody molecule of  claim 122 , wherein the Fcγ receptor is a FcγRII receptor, which FcγRII receptor is FcγRIIA (CD32A) receptor or FcγRIIB (CD32B) receptor. 
     
     
         124 . The diabody molecule of  claim 123 , wherein the FcγRII receptor is the FcγRIIB (CD32B) receptor. 
     
     
         125 . The diabody molecule of  claim 1 , wherein at least one epitope binding site is specific for a pathogenic antigen, a cancer antigen, a toxin or a drug. 
     
     
         126 . The diabody molecule of  claim 120 , wherein said diabody has an epitope binding site specific for CD32B and an epitope binding site specific for CD16A. 
     
     
         127 . The diabody molecule of  claim 1 , wherein Domain C comprises a CH2 domain and a CH3 domain of a variant Fc domain, which variant Fc domain comprises at least one amino acid modification relative to the wild-type Fc domain. 
     
     
         128 . A pharmaceutical composition that comprises the diabody molecule of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         129 . A method for the treatment of a disease or disorder characterized by the presence of a pathogenic antigen, wherein said method comprises administering a therapeutically effective amount of the pharmaceutical composition of  claim 128  to a subject in need thereof. 
     
     
         130 . The method of  claim 129 , wherein said disease or disorder is an infectious disease, and said epitope binding site of said diabody that is specific for said pathogenic antigen is specific for an antigen that is characteristic of said infectious disease. 
     
     
         131 . The method of  claim 129 , wherein said disease or disorder is a cancer characterized by a cancer antigen, and said epitope binding site of said diabody that is specific for said pathogenic antigen is specific for said cancer antigen. 
     
     
         132 . The use of  claim 131 , wherein said cancer antigen is Her2.