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.

1 - 79 . (canceled) 
     
     
         80 . 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 Fc is a human IgG Fc polypeptide chain;   wherein two of V1, V2, V3, and V4 are immunoglobulin heavy chain variable (VH) regions and the other two are immunoglobulin light chain variable (VL) regions;   wherein either V1 is a VH region and V2 is a VL region or vice versa and either V3 is a VH region and V4 is a VL region or vice versa;   wherein L1, L2, L3, and L4 are linkers, wherein L2 is present and wherein L2 is not more than 12 amino acids long;   wherein L1 and L3 are each at least 15 amino acids long; 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 Fc is a human IgG Fc polypeptide chain;   wherein either V1 is a VH region and V2 is a VL region or vice versa and either V3 is a VH region and V4 is a VL region or vice versa;   wherein two of V1, V2, V3, and V4 are VH regions and the other two are VL regions; wherein L1, L2, L3, and L4 are linkers, wherein L2 is present and wherein L2 is not more than 12 amino acids long;   wherein L1 and L3 are each at least 15 amino acids long; 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 by an immune effector cell, wherein the polypeptide chain targeting the effector cell binds to human and/or cynomolgus CD3ε;   wherein the Bi-Fc is a monomer;   wherein the Fc polypeptide chain of (a) or (b) comprises one or more the following alterations: K392D, K392E, N392D, N392E, R409D, R409E, K409D, K409E, D399K, D399R, E356R, E356K, D356R, D356K, Y349T, L351T, L368T, L398T, F405T, Y407T, and Y407R; and   wherein the Fc polypeptide chain of the Bi-Fc comprises an insertion of the amino acid sequence of any of SEQ ID NOs: 36-47 between positions 384 and 385, wherein these position numbers are assigned according to the EU numbering scheme.   
     
     
         81 . The Bi-Fc of  claim 80 , wherein the Fc polypeptide chain of (a) or (b) is an IgG1, IgG2, or IgG4 Fc polypeptide chain and comprises the alterations K392D, K409D, and Y349T. 
     
     
         82 . The Bi-Fc of claim  1 , which binds to a cell expressing human CD33, human HER2, or human FOLR1. 
     
     
         83 . A Bi-Fc, comprising:
 (a) (i) a first polypeptide chain 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 each immunoglobulin variable regions; wherein L1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent; and
 (ii) a second polypeptide chain that comprises a human IgG Fc polypeptide chain; or 
   (b) (i) a first polypeptide chain having the following formula: Fc-L4-V1 -L1 -V2-L2-V3-L3-V4; wherein Fc is a human IgG Fc polypeptide chain; wherein V1, V2, V3, and V4 are each immunoglobulin variable regions; wherein L1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent; and
 (ii) a second polypeptide chain that comprises a human IgG Fc polypeptide chain; 
   wherein the Bi-Fc binds to a target cell and an immune effector cell and/or mediates cytolysis of a target cell by an immune effector cell;   wherein L1 and L3 are at least 15 amino acids long;   wherein 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 human and/or cynomolgus CD3ε;   wherein   (1) the first polypeptide chain comprises the charge pair substitutions R409D, R409E, K409D, or K409E and N392D, N392E, K392D, or K392E, and the second polypeptide chain comprises the charge pair substitutions D399K or D399R and E356K, E356E, D356K, or D356R; or   (2) the second polypeptide chain comprises the charge pair substitutions R409D, R409E, K409D, or K409E and N392D, N392E, K392D, or K392E, and the first polypeptide chain comprises the charge pair substitutions D399K or D399R and E356K, E356E, D356K, or D356R; and   wherein the Fc polypeptide chain(s) comprise(s) an insertion of the amino acid sequence of any of SEQ ID NOs: 36-47 between positions 384 and 385 of each Fc polypeptide chain, wherein positions 384 and 385 are positions assigned according to the EU numbering scheme.   
     
     
         84 . The Bi-Fc of  claim 83 , which binds to a cell expressing human CD33, human FOLR1 or human HER2. 
     
     
         85 . The Bi-Fc of  claim 84 , which comprises:
 (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 72;   a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 73;   a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 74;   a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75;   a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76;   and   a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77;   (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 60,   a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 61,   a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 62, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 63,   a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and   a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 65; or   (c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 66,   a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 67,   a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 68,   a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69,   a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70, and   a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71.   
     
     
         86 . The Bi-Fc of  claim 83 , wherein the Fc polypeptide chains of the first and second polypeptide chains comprise one or more alteration that inhibits FcyR binding selected from the group consisting of: L234A, L235A, and any substitution at N297. 
     
     
         87 . The Bi-Fc of  claim 83 ,
 wherein V3 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 7 or 29, wherein the identity region is at least 80 amino acids long;   wherein V4 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 8 or 31, wherein the identity region is at least 80 amino acids long; and   wherein the Bi-Fc is a monomer.   
     
     
         88 . The Bi-Fc of  claim 87 , wherein V3 comprises the amino acid sequence of SEQ ID NO: 7 or 29 and V4 comprises the amino acid sequence of SEQ ID NO: 8 or 31. 
     
     
         89 . The Bi-Fc of  claim 80 , wherein the target cell is a cancer cell. 
     
     
         90 . The Bi-Fc of  claim 80 , wherein the target cell is a cell infected by a pathogen or a cell that mediates a disease. 
     
     
         91 . The Bi-Fc of  claim 90 , wherein
 (a) the pathogen is virus including human immunodeficiency virus, hepatitis virus, human papilloma virus, or cytomegalovirus, or a bacterium of the genus  Listeria ,  Mycobacterium ,  Staphylococcus , or  Streptococcus ; or   (b) the cell that mediates a disease is a fibrotic cell that mediates a fibrotic disease.   
     
     
         92 . The Bi-Fc of  claim 80 , wherein the Bi-Fc binds to an amino acid sequence within the first 27 amino acids of human or cynomolgus CD3ε as determined by alanine scanning. 
     
     
         93 . The Bi-Fc of  claim 80 , wherein the Bi-Fc comprises a VH region comprising a CDR1, a CDR2, and a CDR3 comprising, respectively, the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50 and a VL region comprising a CDR1, a CDR2, and a CDR3 comprising, respectively, the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53. 
     
     
         94 . A pharmaceutical formulation comprising the Bi-Fc of  claim 80  and a physiologically acceptable excipient. 
     
     
         95 . A nucleic acid encoding the Bi-Fc of  claim 80 . 
     
     
         96 . A vector comprising the nucleic acid of  claim 95 . 
     
     
         97 . A host cell comprising the nucleic acid of  claim 95 . 
     
     
         98 . A method of making a Bi-Fc comprising
 culturing the host cell of  claim 97  under conditions such that the nucleic acid is expressed, and   recovering the Bi-Fc from the cell mass or the culture medium.   
     
     
         99 . A composition comprising the Bi-Fc of  claim 80 .