Binding domain-immunoglobulin fusion proteins

Abstract

The invention relates to novel binding domain-immunoglobulin fusion proteins that feature a binding domain for a cognate structure such as an antigen, a counterreceptor or the like, a wild-type IgG1, IGA or IgE hinge region polypeptide or a mutant IgG1 hinge region polypeptide having either zero, one or two cysteine residues, and immunoglobulin CH2 and CH3 domains, and that are capable of ADCC and/or CDC while occurring predominantly as polypeptides that are compromised in their ability to form disulfide-linked multimers. The fusion proteins can be recombinantly produced at high express levels. Also provided are related compositions and methods, including cell surface forms of the fusion proteins and immunotherapeutic applications of the fusion proteins and of polynucleotides encoding such fusion proteins.

1 . A recombinant CTLA-4 Ig fusion protein comprising, from N-terminus to C-terminus, a CTLA-4 domain, an immunoglobulin hinge, a CH2 immunoglobulin heavy chain constant region, and a CH3 immunoglobulin heavy chain constant region. 
     
     
         2 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein said CTLA-4 domain binds a ligand selected from the group consisting of CD80 and CD86. 
     
     
         3 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CTLA-4 domain is a CTLA-4 ectodomain polypeptide or extracellular domain polypeptide. 
     
     
         4 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CTLA-4 domain is an scFv comprising a variable heavy chain and a variable light chain joined by a linker and the CTLA-4 domain binds CD80 or CD86. 
     
     
         5 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CTLA-4 domain comprises an amino acid sequence with at least 90% identity to amino acid sequence of SEQ ID NO:309 or 314. 
     
     
         6 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CTLA-4 domain comprises an amino acid sequence with at least 95% identity to amino acid sequence of SEQ ID NO:309 or 314. 
     
     
         7 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CTLA-4 domain is encoded by a nucleic acid comprising a nucleic acid sequence with at least 95% identity to the nucleic acid sequence of SEQ ID NOS:308 or 313. 
     
     
         8 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the recombinant CTLA-4 Ig fusion protein does not contain a CH1 domain. 
     
     
         9 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the hinge is an IgG1 hinge. 
     
     
         10 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the IgG1 hinge is a wild-type IgG1 hinge or comprises a mutation at one, two or three cysteine residues. 
     
     
         11 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CH2 immunoglobulin heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 583 
     
     
         12 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the hinge is an IgA hinge. 
     
     
         13 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the IgA hinge is a wild-type IgA hinge or comprises a mutation at one or two cysteine residues. 
     
     
         14 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the hinge is an IgE hinge. 
     
     
         15 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CH2 immunoglobulin heavy chain constant region is an IgG1 CH2 immunoglobulin heavy chain constant region and the CH3 immunoglobulin heavy chain constant region is an IgG1 CH3 immunoglobulin heavy chain constant region. 
     
     
         16 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CH2 immunoglobulin heavy chain constant region is an IgA immunoglobulin heavy chain constant region and the CH3 immunoglobulin heavy chain constant region is an IgA immunoglobulin heavy chain constant region. 
     
     
         17 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CH2 immunoglobulin heavy chain constant region is an IgE immunoglobulin heavy chain constant region and the CH3 immunoglobulin heavy chain constant region is an IgE immunoglobulin heavy chain constant region. 
     
     
         18 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the hinge, the CH2 immunoglobulin heavy chain constant region, and the CH3 immunoglobulin constant region are encoded by a nucleic acid comprising a nucleic acid sequence with at least 90% identity to the nucleic acid sequence of SEQ ID NO: 287 or 315. 
     
     
         19 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the immunoglobulin hinge is an IgG1 hinge, the CH2 immunoglobulin heavy chain constant region is an IgG1 CH2 constant region, and the CH3 immunoglobulin heavy chain constant region is an IgG1 CH2 constant region. 
     
     
         20 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the immunoglobulin hinge is an IgA hinge, the CH2 immunoglobulin heavy chain constant region is an IgA CH2 constant region, and the CH3 immunoglobulin heavy chain constant region is an IgA CH2 constant region. 
     
     
         21 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the CH3 immunoglobulin heavy chain constant region is an IgE CH3 domain with a mutation that prevents association with a J chain. 
     
     
         22 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the immunoglobulin hinge is an IgE hinge, the CH2 immunoglobulin heavy chain constant region is an IgE CH2 constant region, and the CH3 immunoglobulin heavy chain constant region is an IgE CH2 constant region. 
     
     
         23 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the recombinant CTLA-4 Ig fusion protein comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:307, 316 or 530. 
     
     
         24 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the recombinant CTLA-4 Ig fusion protein mediates antibody dependent cell-mediated cytoxicity of Reh CD80.10 cells in vitro. 
     
     
         25 . The recombinant CTLA-4 Ig fusion protein of  claim 1 , wherein the recombinant CTLA-4 Ig fusion protein is capable of at least one immunological activity selected from the group consisting of antibody dependent cell-mediated cytoxicity and complement fixation. 
     
     
         26 . An isolated polynucleotide encoding the recombinant CTLA-4 Ig fusion protein of  claim 1 . 
     
     
         27 . A recombinant expression construct comprising the polynucleotide of  claim 26 . 
     
     
         28 . A host cell transformed or transfected with the recombinant expression construct of  claim 27 . 
     
     
         29 . A CHO or COS cell capable of expressing the recombinant CTLA-4 Ig fusion protein of  claim 1 . 
     
     
         30 . A pharmaceutical composition comprising the recombinant CTLA-4 Ig fusion protein of  claim 1  and a physiologically acceptable carrier. 
     
     
         31 . A method of treating a subject having or suspected of having a malignant condition or a B-cell disorder, comprising administering to the subject a therapeutically effective amount of a recombinant CTLA-4 Ig fusion protein of  claim 1 . 
     
     
         32 . The method of  claim 31 , wherein the malignant condition or B-cell disorder is selected from the group consisting of rheumatoid arthritis, myasthenia gravis, Grave's disease, type I diabetes mellitus, multiple sclerosis and an autoimmune disease.