Single-chain multivalent binding proteins with 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 .- 98 . (canceled) 
     
     
         99 . A single-chain multispecific protein comprising from amino terminus to carboxy terminus:
 (a) a first binding domain derived from an immunoglobulin-like molecule or the variable regions of an immunoglobulin;   (b) a Fc region comprising a domain derived from an immunoglobulin C H2  domain, wherein said Fc region does not comprise a domain derived from an immunoglobulin C H1  domain;   (c) a linker peptide of at least 5 amino acids; and   (d) a second binding domain derived from an immunoglobulin-like molecule or the variable regions of an immunoglobulin,   wherein the first or second binding domain binds CD3; and   wherein the first and the second binding domains recognize different molecular targets.   
     
     
         100 . The protein according to  claim 99 , wherein the first and/or second binding domain is a single-chain variable antibody fragment (scFv). 
     
     
         101 . The protein according to  claim 99 , wherein the first and/or second binding domain comprises chimeric, humanized, or human immunoglobulin variable regions. 
     
     
         102 . The protein according to  claim 99 , wherein the first and/or second binding domain comprises a light chain immunoglobulin variable region (VL1) and a heavy chain immunoglobulin variable region (VH1), wherein said variable regions are positioned in a VH1-VL1 or a VL1-VH1 orientation. 
     
     
         103 . The protein according to  claim 99 , wherein the first or second binding domain comprises variable regions derived from the G19-4 antibody. 
     
     
         104 . The protein according to  claim 103 , wherein the first or second binding domain comprises the amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 109. 
     
     
         105 . The protein according to  claim 99 , wherein the immunoglobulin-like molecule is a receptor. 
     
     
         106 . The protein according to  claim 99 , wherein the other binding domain binds to 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. 
     
     
         107 . The protein according to  claim 106 , wherein the tumor antigen is selected from the group consisting of SQUAMOUS CELL CARCINOMA ANTIGEN 1, SQUAMOUS CELL CARCINOMA ANTIGEN 2, Ovarian carcinoma antigen CA125, MUCIN 1, CTCL tumor antigen se1-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1, MAGE-B1 ANTIGEN, MAGE-B2 ANTIGEN, MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associated brain-testis-cancer antigen, Neuro-oncological ventral antigen 2, Hepatocellular carcinoma antigen gene 520, TUMOR-ASSOCIATED ANTIGEN CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1, Serologically defined breast cancer antigen NY-BR-15, Serologically defined breast cancer antigen NY-BR-16, Chromogranin A; parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195 and L6. 
     
     
         108 . The protein according to  claim 99 , wherein the Fc region further comprises an immunoglobulin hinge region. 
     
     
         109 . The protein according to  claim 108 , 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. 
     
     
         110 . The protein according to  claim 109 , wherein the hinge region is an IgG1 hinge region. 
     
     
         111 . The protein according to  claim 110 , wherein the hinge region is a human IgG1 hinge region with a mutation at one or two cysteine residues. 
     
     
         112 . The protein according to  claim 99 , wherein the Fc region further comprises a domain derived from an immunoglobulin C H3  domain. 
     
     
         113 . The protein according to  claim 99 , wherein the Fc region comprises a human immunoglobulin hinge region, C H2  domain, and C H3  domain. 
     
     
         114 . The protein according to  claim 99 , wherein the linker peptide is between 5 and 45 amino acids in length. 
     
     
         115 . The protein according to  claim 99 , wherein the linker peptide comprises an immunoglobulin core hinge region. 
     
     
         116 . The protein according to  claim 99 , wherein the linker peptide 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. 
     
     
         117 . The protein according to  claim 99 , wherein the linker peptide is derived from an interdomain region between the Ig V-like and Ig C-like region of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166, or CD244. 
     
     
         118 . The protein according to  claim 99 , wherein the protein is capable of forming dimers. 
     
     
         119 . A nucleic acid encoding the protein of  claim 99 . 
     
     
         120 . A vector comprising the nucleic acid of  claim 119 . 
     
     
         121 . A host cell comprising the nucleic acid of  claim 119 , wherein said host cell is not present in a human. 
     
     
         122 . A host cell comprising the vector of  claim 120 , wherein said host cell is not present in a human. 
     
     
         123 . A composition comprising the protein of  claim 99  and a pharmaceutically acceptable carrier.