Single-chain multivalent binding proteins effector function

Abstract

Multivalent binding peptides, including bi-specific binding peptides, having immunoglobulin effector function are provided, along with encoding nucleic acids, vectors and host cells as well as methods for making such peptides and methods for using such peptides to treat or prevent a variety of diseases, disorders or conditions, as well as to ameliorate at least one symptom associated with such a disease, disorder or condition.

1 . A polypeptide comprising, from amino to carboxy terminus:
 (a) a first binding domain comprising variable regions from an immunoglobulin;   (b) a constant sub-region comprising an immunoglobulin hinge region, C H2  domain and a C H3  domain;   (c) a linker peptide, wherein the linker peptide does not comprise a Gly 4 Ser sequence; and   (d) a second binding domain comprising variable regions from an immunoglobulin,   wherein the variable regions of the first and second binding domains do not comprise a mutated complementarity determining region (CDR) derived from anti-CD20 2H7 antibody.   
     
     
         2 . The polypeptide of  claim 1 , wherein the variable regions of the first and second binding domains do not comprise the sequence of SEQ ID NO: 332, SEQ ID NO: 335, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 344, SEQ ID NO: 341, or SEQ ID NO: 345. 
     
     
         3 . The polypeptide of  claim 1 , wherein the variable regions of the first and second binding domains do not comprise a mutated CDR1 or CDR3 derived from the light chain of the anti-CD20 2H7 antibody. 
     
     
         4 . The polypeptide of  claim 1 , wherein the variable regions of the first and second binding domains do not comprise a mutated CDR2 or CDR3 derived from the heavy chain of the anti-CD20 2H7 antibody. 
     
     
         5 . The polypeptide of  claim 1 , wherein at least one of the first binding domain and second binding domain is a single-chain variable antibody fragment (scFv). 
     
     
         6 . The polypeptide of  claim 1 , wherein at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of 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 and an angiogenesis target. 
     
     
         7 . The polypeptide of  claim 1 , wherein said constant sub-region does not comprise a C H1  domain. 
     
     
         8 . The polypeptide of  claim 1 , wherein the hinge region is a hinge region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgE, IgA2, synthetic hinge and the hinge-like C H2  domain of IgM. 
     
     
         9 . The polypeptide of  claim 8 , wherein the hinge region is an IgG1 hinge region. 
     
     
         10 . The polypeptide of  claim 9 , wherein the hinge region is a human IgG1 hinge region with a mutation at one or two cysteine residues. 
     
     
         11 . The polypeptide of  claim 1 , wherein the linker is at least 5 amino acids in length. 
     
     
         12 . The polypeptide of  claim 11 , wherein the linker is between 5 and 45 amino acids in length. 
     
     
         13 . The polypeptide of  claim 1 , wherein the linker comprises an immunoglobulin core hinge region. 
     
     
         14 . The polypeptide of  claim 1 , wherein the linker is derived from a stalk region of a Type II Membrane Protein C-type lectin. 
     
     
         15 . The polypeptide of  claim 14 , wherein the Type II Membrane Protein C-type lectin is selected from the group consisting of CD69, CD72, CD94, NKG2A, and NKG2D. 
     
     
         16 . The polypeptide of  claim 1 , wherein the 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, or 377. 
     
     
         17 . The polypeptide of  claim 1 , wherein the first and/or second binding domains comprise chimeric, humanized, or human immunoglobulin variable domains. 
     
     
         18 . The polypeptide of  claim 1 , wherein the constant sub-region comprises IgG1 immunoglobulin C H2  and C H3  domains. 
     
     
         19 . The polypeptide of  claim 1 , wherein the constant sub-region comprises C H2  and C H3  domains from a human immunoglobulin. 
     
     
         20 . The polypeptide of  claim 1 , wherein the immunoglobulin constant sub-region provides an effector function selected from the group consisting of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, complement fixation, antibody-dependent cellular phagocytosis, binding to Fc receptors, and protein A binding. 
     
     
         21 . The polypeptide of  claim 1 , wherein the polypeptide is capable of forming dimers. 
     
     
         22 . A nucleic acid encoding the polypeptide of  claim 1 . 
     
     
         23 . A vector comprising the nucleic acid of  claim 22 . 
     
     
         24 . A host cell comprising the nucleic acid of  claim 22 . 
     
     
         25 . A composition comprising the polypeptide of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         26 . A host cell comprising the vector of  claim 23 .