BISPECIFIC-Fc MOLECULES

Abstract

Described herein is a bispecific molecule containing an Fc polypeptide chain and immunoglobulin variable regions. Also provided are pharmaceutical formulations comprising such molecules, nucleic acids encoding such molecules, host cells containing such nucleic acids, methods of making such molecules, and methods of using such molecules.

What is claimed is: 
     
         1 . A Bi-Fc, which comprises
 (a) a polypeptide chain comprising an amino acid sequence having the following formula: V1-L1-V2-L2-V3-L3-V4-L4-Fc; wherein two of V1, V2, V3, and V4 are heavy chain variable (VH) regions and the other two are light chain variable (VL) regions; wherein Fc is a human IgG Fc polypeptide chain; wherein L1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent; or   (b) a polypeptide chain comprising an amino acid sequence having the following formula: Fc-L4-V1-L1-V2-L2-V3-L3-V4; wherein two of V1, V2, V3, and V4 are heavy chain variable (VH) regions and the other two are light chain variable (VL) regions; wherein Fc is a human IgG Fc polypeptide chain; wherein L1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent;   wherein the Bi-Fc binds to a target cell and an immune effector cell and/or mediates cytolysis of a target cell displaying a target cell protein by an immune effector cell, and   wherein the Bi-Fc is a monomer.   
     
     
         2 . The Bi-Fc of  claim 1 , wherein Fc polypeptide chain of (a) or (b) comprises one or more the following alterations: K392D, K392E, K409D, K409E, D399K, D399R, E356R, E356K, D356R, D356K, Y349T, L351T, L368T, L398T, F405T, Y407T, and Y407R. 
     
     
         3 . The Bi-Fc of  claim 2 , wherein Fc polypeptide chain of (a) or (b) comprises K392D, K409D, and Y349T. 
     
     
         4 . The Bi-Fc of  claim 2 , wherein the Fc polypeptide chain of the polypeptide chain of (a) or (b) comprises one or more alteration that inhibits FcγR binding and/or one or more alterations that extend(s) half life. 
     
     
         5 . The Bi-Fc of  claim 1 , which is the Bi-Fc of  claim 1 (a). 
     
     
         6 . The Bi-Fc of  claim 1 , which is the Bi-Fc of  claim 1 (b). 
     
     
         7 . The Bi-Fc of  claim 1 , wherein the immune effector cell is a human T cell and/or a cynomolgus monkey T cell and the effector cell protein is part of the human and/or cynomolgus monkey T cell receptor (TCR)-CD3 complex. 
     
     
         8 . The Bi-Fc of  claim 7 , wherein the effector cell protein is human or cynomolgus monkey CD3ε. 
     
     
         9 . The Bi-Fc of  claim 8 , wherein the amino acid sequence to which the Bi-Fc binds comprises Gln-Asp-Gly-Asn-Glu (SEQ ID NO:24) as determined by alanine scanning. 
     
     
         10 . The Bi-Fc of  claim 8 , wherein the Bi-Fc comprises:
 the amino acid sequences of the CDR1, CDR2 and CDR3 of the VH region comprising the amino acid sequence of SEQ ID NO:7 or 29; and   the amino acid sequences of the CDR1, CDR2 and CDR3 of the VL region comprising the amino acid sequence of SEQ ID NO:8 or 31.   
     
     
         11 . The Bi-Fc of  claim 8 , wherein the Bi-Fc comprises:
 a VH region comprising the amino acid sequence of SEQ ID NO:7 or 29 or a variant thereof comprising no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to SEQ ID NO:7 or 29; and   a VL region comprising the amino acid sequence of SEQ ID NO:8 or 31 or a variant thereof comprising no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to SEQ ID NO:8 or 31.   
     
     
         12 . The Bi-Fc of  claim 11 , comprising the amino acid sequence of SEQ ID NO:7 or 29 and the amino acid of SEQ ID NO:8 and 31. 
     
     
         13 . The Bi-Fc of  claim 1 , wherein the target cell is a cancer cell, a cell infected by a pathogen, or a cell that mediates disease. 
     
     
         14 . The Bi-Fc of  claim 13 ,
 wherein the target cell is a cell infected by a pathogen, wherein the pathogen is selected from the group consisting of human immunodeficiency virus, hepatitis virus, human papilloma virus, cytomegalovirus, or a bacterium of the genus  Listeria, Mycobacterium, Staphylococcus , or  Streptococcus , or   wherein the target cell is a fibrotic cell that mediates a fibrotic disease.   
     
     
         15 . The Bi-Fc of  claim 13 , wherein the target cell is a cancer cell. 
     
     
         16 . A Bi-Fc, which comprises
 (i) a first polypeptide comprising an amino acid sequence having the following formula: V1-L1-V2-L2-V3-L3-V4-L4-Fc; wherein Fc is a human IgG Fc polypeptide chain; wherein V1, V2, V3 and V4 are immunoglobulin variable regions; wherein L1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent; and   (ii) a second polypeptide comprising a human IgG Fc polypeptide chain;   wherein L1 and L3 are at least 15 amino acids long and L2 is less than 12 amino acids long,   wherein either V1 is a VH region and V2 is a VL region or vice versa,   wherein either V3 is a VH region and V4 is a VL region or vice versa,   wherein the Bi-Fc binds to a target cell and an immune effector cell and/or mediates cytolysis of a target cell displaying a target cell protein by an immune effector cell,   wherein the Fc polypeptide chain in the first polypeptide chain comprises the charge pair substitutions K409E or K409E and K392D or K392E, and the Fc polypeptide chain in the second polypeptide chain comprises the charge pair substitutions D399K or D399R and D356K or D356R, or wherein the Fc polypeptide chain in the second polypeptide chain comprises the charge pair substitutions K409E or K409E and K392D or K392E, and the Fc polypeptide chain in the first polypeptide chain comprises the charge pair substitutions D399K or D399R and D356K or D356R, and   wherein the Bi-Fc comprises: a VH region comprising the amino acid sequence of SEQ ID NO:7 or 29 or a variant thereof comprising no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to SEQ ID NO:7 or 29; and a VL region comprising the amino acid sequence of SEQ ID NO:8 or 31 or a variant thereof comprising no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to SEQ ID NO:8 or 31.   
     
     
         17 . A nucleic acid encoding the Bi-Fc of  claim 1 . 
     
     
         18 . A vector comprising the nucleic acid of  claim 17 . 
     
     
         19 . A host comprising the nucleic acid of  claim 17  or the vector of  claim 18 . 
     
     
         20 . One or more nucleic acid(s) encoding the Bi-Fc of  claim 16 . 
     
     
         21 . One or more vector(s) comprising the nucleic acid(s) of  claim 20 . 
     
     
         22 . A host cell containing the nucleic acid(s) of  claim 20  or the vector(s) of  claim 21 . 
     
     
         23 . A method of making a Bi-Fc comprising
 culturing a host cell under conditions such that a nucleic acid in the host cell encoding the Bi-Fc is expressed, and   recovering the Bi-Fc from the cell mass or the culture medium,   wherein the Bi-Fc comprises:   (a)
 (i) a polypeptide chain comprising an amino acid sequence having the following formula: V1-L1-V2-L2-V3-L3-V4-L4-Fc; wherein two of V1, V2, V3, and V4 are heavy chain variable (VH) regions and the other two are light chain variable (VL) regions; wherein Fc is a human IgG Fc polypeptide chain; wherein L1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent; or 
 (ii) a polypeptide chain comprising an amino acid sequence having the following formula: Fc-L4-V1-L1-V2-L2-V3-L3-V4; wherein two of V1, V2, V3, and V4 are heavy chain variable (VH) regions and the other two are light chain variable (VL) regions; wherein Fc is a human IgG Fc polypeptide chain; wherein L1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent; 
 wherein the Bi-Fc binds to a target cell and an immune effector cell and/or mediates cytolysis of a target cell displaying a target cell protein by an immune effector cell, and 
 wherein the Bi-Fc is a monomer; or 
   (b)
 (i) a first polypeptide comprising an amino acid sequence having the following formula: V1-L1-V2-L2-V3-L3-V4-L4-Fc; wherein Fc is a human IgG Fc polypeptide chain; wherein V1, V2, V3 and V4 are immunoglobulin variable regions; wherein L1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent; and 
 (ii) a second polypeptide comprising a human IgG Fc polypeptide chain; 
 wherein L1 and L3 are at least 15 amino acids long and L2 is less than 12 amino acids long, 
 wherein either V1 is a VH region and V2 is a VL region or vice versa, 
 wherein either V3 is a VH region and V4 is a VL region or vice versa, 
 wherein the Bi-Fc binds to a target cell and an immune effector cell and/or mediates cytolysis of a target cell displaying a target cell protein by an immune effector cell, 
 wherein the Fc polypeptide chain in the first polypeptide chain comprises the charge pair substitutions K409E or K409E and K392D or K392E, and the Fc polypeptide chain in the second polypeptide chain comprises the charge pair substitutions D399K or D399R and D356K or D356R, or wherein the Fc polypeptide chain in the second polypeptide chain comprises the charge pair substitutions K409E or K409E and K392D or K392E, and the Fc polypeptide chain in the first polypeptide chain comprises the charge pair substitutions D399K or D399R and D356K or D356R, and 
 wherein the Bi-Fc comprises: a VH region comprising the amino acid sequence of SEQ ID NO:7 or 29 or a variant thereof comprising no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to SEQ ID NO:7 or 29, and a VL region comprising the amino acid sequence of SEQ ID NO:8 or 31 or a variant thereof comprising no more than 10 amino acid substitutions, insertions, and/or deletions of a single amino acid per 100 amino acids relative to SEQ ID NO:8 or 31. 
   
     
     
         24 . A method for treating a patient having cancer, a fibrotic disease, or a disease mediated by a pathogen comprising administering to the patient a therapeutically effective dose of the Bi-Fc of  claim 1 . 
     
     
         25 . The method of  claim 24 , wherein the patient has cancer and the method further comprises administering radiation, a chemotherapeutic agent, or a non-chemotherapeutic, anti-neoplastic agent before, after, or concurrently with the administration of the Bi-Fc. 
     
     
         26 . A method for treating a patient having cancer, a fibrotic disease, or a disease mediated by a pathogen comprising administering to the patient a therapeutically effective dose of the Bi-Fc of  claim 16 . 
     
     
         27 . The method of  claim 26 , wherein the patient has cancer and the method further comprises administering radiation, a chemotherapeutic agent, or a non-chemotherapeutic, anti-neoplastic agent before, after, or concurrently with the administration of the Bi-Fc. 
     
     
         28 . A pharmaceutical composition comprising a therapeutically effective dose of the Bi-Fc of  claim 1  and a physiologically acceptable carrier, excipient, and/or diluent. 
     
     
         29 . A pharmaceutical composition comprising a therapeutically effective dose of the Bi-Fc of  claim 16  and a physiologically acceptable carrier, excipient, and/or diluent.