Binding constructs and methods for use thereof

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-acting region, i.e., IgE CH2, 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 expression 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 fusion protein comprising from amino-terminus to carboxy-terminus: (i) an immunoglobulin binding domain polypeptide capable of binding a target molecule, wherein the binding domain polypeptide comprises a heavy chain variable region having a mutated amino acid residue; (ii) an altered immunoglobulin hinge polypeptide comprising a cysteine and a proline substituted for a different amino acid; and (iii) an amino-terminally truncated immunoglobulin heavy chain constant region polypeptide. 
     
     
         2 . The fusion protein of  claim 1  wherein the binding domain polypeptide comprises an immunoglobulin light chain variable region polypeptide and an immunoglobulin heavy chain variable region polypeptide. 
     
     
         3 . The fusion protein of  claim 1  wherein the heavy chain variable region has an amino acid substitution at position 11 that is serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, or histidine. 
     
     
         4 . The fusion protein of  claim 1  wherein the binding domain is a single chain Fv polypeptide. 
     
     
         5 . The fusion protein of  claim 1  wherein the immunoglobulin binding domain polypeptide comprises a human or humanized immunoglobulin variable region. 
     
     
         6 . The fusion protein of  claim 1  wherein said binding domain polypeptide binds to an antigen on an immune effector cell. 
     
     
         7 . The fusion protein of  claim 1  wherein the immunoglobulin binding domain polypeptide binds to a CD2, CD3, CD4, CD19, CD20, CD28, CD8α, CD37, CD40, CD69, CD137, CD152, CD154, or PD-L1. 
     
     
         8 . The fusion protein of  claim 4  wherein the single chain Fv is from HD37, 2H7, G28-1, 5B9, 10A8, 2e12, G19-4, G28-5, or 1D8. 
     
     
         9 . The fusion protein of  claim 1  wherein the immunoglobulin binding domain polypeptide comprises a light chain variable region attached to the heavy chain variable region by a linker peptide comprising a peptide sequence of Gly-Gly-Gly-Gly-Ser (SEQ ID NO:516). 
     
     
         10 . The fusion protein of  claim 1  wherein the amino-terminally truncated immunoglobulin heavy chain constant region polypeptide comprises a human CH2 constant region polypeptide attached to a human CH3 constant region polypeptide. 
     
     
         11 . The fusion protein of  claim 10  wherein the CH2 and CH3 constant region polypeptides are an IgA, IgD, IgG1, IgG2, IgG3, or IgG4 constant region polypeptides. 
     
     
         12 . The fusion protein of  claim 11  wherein CH2 and CH3 constant region polypeptides are IgG 1  constant region polypeptides, wherein the IgG 1  CH2 constant region polypeptide comprises one or more amino acid mutations at positions 238, 255, 256, 257, 258, 290, 322, 331, and 339. 
     
     
         13 . The fusion protein of  claim 12  wherein the IgG 1  CH3 constant region polypeptide comprises one or more amino acid mutations at positions 405 and 407. 
     
     
         14 . The fusion protein of  claim 1  wherein the amino-terminally truncated immunoglobulin heavy chain constant region polypeptide comprises an IgE CH3 constant region polypeptide attached to an IgE CH4 constant region polypeptide. 
     
     
         15 . The fusion protein of  claim 1  wherein the altered immunoglobulin hinge polypeptide comprises an altered human IgG, IgA, or IgE hinge region. 
     
     
         16 . The fusion protein of  claim 1  wherein the altered immunoglobulin hinge polypeptide comprises an altered human IgG1, IgG2, IgG3, or IgG4 hinge region. 
     
     
         17 . The fusion protein of  claim 1  wherein the hinge region comprises no more than one cysteine residue. 
     
     
         18 . The fusion protein of  claim 1  wherein the altered immunoglobulin hinge polypeptide comprises an altered wild type IgG1 immunoglobulin hinge region, wherein the wild type IgG1 hinge region comprises first, second, and third cysteine residues, and a proline, wherein the first cysteine reside is N-terminal to the second cysteine, the second cysteine is N-terminal to the third cysteine, and the third cysteine is N-terminal to the proline residue, and wherein the altered immunoglobulin hinge polypeptide has only the third cysteine or has only the first and third cysteines of a wild type IgG1 hinge region. 
     
     
         19 . The fusion protein of  claim 18 , wherein the binding domain is 5B9 scFv or HD37 scFv. 
     
     
         20 . The fusion protein of  claim 1  wherein the altered immunoglobulin hinge polypeptide comprises an altered wild type IgG1 immunoglobulin hinge region, wherein (i) the wild type IgG1 hinge region comprises first, second, and third cysteine residues, and a proline, wherein the first cysteine reside is N-terminal to the second cysteine, the second cysteine is N-terminal to the third cysteine, and the third cysteine is N-terminal to the proline residue, (ii) the proline N-terminal to the third cysteine in the hinge is substituted, and (iii) (a) the second cysteine is substituted; (b) the third cysteine is substituted; (c) the first and second cysteines are substituted; (d) the first and third cysteines are substituted; or (e) the second and third cysteines are substituted. 
     
     
         21 . The fusion protein of  claim 20  wherein the substitution is with a serine, alanine, threonine, glycine, aspartate, or asparagine. 
     
     
         22 . A pharmaceutical composition comprising a binding domain-immunoglobulin fusion protein according to  claim 1  in combination with a physiologically acceptable carrier. 
     
     
         23 . A method of treating a malignant condition, comprising administering to a patient a therapeutically effective amount of a fusion protein according to  claim 1 . 
     
     
         24 . The method of  claim 23  wherein the malignant condition is cancer. 
     
     
         25 . The method of  claim 23  wherein the malignant condition is a melanoma, carcinoma, or sarcoma.