US2016145340A1PendingUtilityA1

Bispecific-fc molecules

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Assignee: AMGEN INCPriority: Mar 15, 2013Filed: Mar 14, 2014Published: May 26, 2016
Est. expiryMar 15, 2033(~6.7 yrs left)· nominal 20-yr term from priority
Inventors:Luis BorgesPatrick BaeuerleWei YanMark Leo Michaels
A61P 9/10A61P 37/06A61P 33/00A61P 35/02A61P 33/02A61P 31/04A61P 35/00A61P 31/12A61P 29/00A61P 13/12A61P 11/08A61P 11/00A61P 1/16A61P 19/04C07K 2317/60C07K 2317/565C07K 2317/71C07K 16/468C07K 2319/00C07K 16/00C07K 2317/31C07K 2317/52C07K 16/2803C07K 2317/622C07K 16/32C07K 16/28C07K 16/2809C07K 2317/64C07K 2317/92A61K 2039/505C07K 2317/73C07K 2317/56C07K 16/2863C07K 16/40
62
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Claims

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.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A bispecific-Fc (Bi-Fc) comprising:
 (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 L1, L2, L3, and L4 are linkers; and wherein L2 and/or 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 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; and wherein L2 and/or 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, 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, N392D, N392E, R409D, R409E, K409D, K409E, D399K, D399R, E356R, E356K, D356R, D356K, Y349T, L351T, L368T, L398T, F405T, Y407T, and Y407R. 
     
     
         3 . (canceled) 
     
     
         4 . The Bi-Fc of  claim 1 , wherein the Fc polypeptide chain of the polypeptide chain of (a) or (b) comprises the alteration(s) L234A and/or L235A. 
     
     
         5 - 7 . (canceled) 
     
     
         8 . The Bi-Fc of  claim 1 , wherein the effector cell protein is part of the human and/or cynomolgus monkey T cell receptor (TCR)-CD3 complex. 
     
     
         9 - 12 . (canceled) 
     
     
         13 . The Bi-Fc of  claim 8 , which comprises
 a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:48;   a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:49;   a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:50;   a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:51;   a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:52; and   a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:53.   
     
     
         14 . The Bi-Fc of  claim 8 , which comprises
 a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:54;   a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:55;   a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:56;   a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:57;   a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:58; and   a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:59.   
     
     
         15 . The Bi-Fc of  claim 13 , which comprises a VH region comprising an amino acid sequence at least 95% identical to SEQ ID NO:7 and a VL region comprising an amino acid sequence at least 95% identical to SEQ ID NO:8, wherein the identity regions are at least 80 amino acids long. 
     
     
         16 . (canceled) 
     
     
         17 . The Bi-Fc of  claim 14 , which comprises a VH region comprising an amino acid sequence at least 95% identical to SEQ ID NO:29 and a VL region comprising an amino acid sequence at least 95% identical to SEQ ID NO:31, wherein the identity regions are at least 80 amino acids long. 
     
     
         18 . (canceled) 
     
     
         19 . The Bi-Fc of  claim 1 , which binds to a cell expressing human HER2. 
     
     
         20 . The Bi-Fc of  claim 19 , which comprises
 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.   
     
     
         21 . The Bi-Fc of  claim 20 , which comprises a VH region comprising an amino acid sequence at least 95% identical to SEQ ID NO:5 and a VL region comprising an amino acid sequence at least 95% identical to SEQ ID NO:6, wherein the identity regions are at least 80 amino acids long. 
     
     
         22 . (canceled) 
     
     
         23 . The Bi-Fc of  claim 1 , which binds to a cell expressing human FOLR1. 
     
     
         24 . The Bi-Fc of  claim 23 , which comprises
 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.   
     
     
         25 . The Bi-Fc of  claim 24 , which comprises a VH region comprising an amino acid sequence at least 95% identical to amino acids 1-118 of SEQ ID NO:15 and a VL region comprising an amino acid sequence at least 95% identical to amino acids 134-244 of SEQ ID NO:15, wherein the identity regions are at least 80 amino acids long. 
     
     
         26 . The Bi-Fc of  claim 25 , comprising the amino acid sequences of amino acids 1-118 and 134-244 of SEQ ID NO:15. 
     
     
         27 . The Bi-Fc of  claim 1 , which binds to a cell expressing human CD33. 
     
     
         28 . The Bi-Fc of  claim 27 , which comprises
 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.   
     
     
         29 . The Bi-Fc of  claim 28 , which comprises a VH region comprising an amino acid sequence at least 95% identical to amino acids 1-121 or 1-122 of SEQ ID NO:34 and a VL region comprising an amino acid sequence at least 95% identical to amino acids 138-251 of SEQ ID NO:34, wherein the identity regions are at least 80 amino acids long. 
     
     
         30 . (canceled) 
     
     
         31 . The Bi-Fc of  claim 1 , 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. 
     
     
         32 . The Bi-Fc of  claim 1 , wherein L2 is present and wherein L2 is not more than about 12 amino acids long. 
     
     
         33 . The Bi-Fc of  claim 1 , wherein L1 and L3 are each at least about 14 amino acids long. 
     
     
         34 - 35 . (canceled) 
     
     
         36 . A bispecific-Fc (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 L2 and/or 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 L2 and/or 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, if present, 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 CD3ε, and   wherein the Bi-Fc comprises (1) 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, or (2) a VH region comprising a CDR1, a CDR2, and a CDR3 comprising, respectively, the amino acid sequences of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56 and a VL region comprising a CDR1. a CDR2, and a CDR3 comprising, respectively, the amino acid sequence of SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59.   
     
     
         37 . The Bi-Fc of  claim 36 , comprising a VH region comprising an amino acid sequence at least 95% identical to SEQ ID NO:7 or SEQ ID NO:29 and a VL region comprising an amino acid sequence at least 95% identical to SEQ ID NO:8 or SEQ ID NO:31. 
     
     
         38 . (canceled) 
     
     
         39 . The Bi-Fc of  claim 36 , which binds to a cell expressing human CD33, human FOLR1, or human HER2. 
     
     
         40 . The Bi-Fc of  claim 39 , which comprises:
 (a) 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;   (b) 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; or   (c) 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.   
     
     
         41 . The Bi-Fc of  claim 40 , which comprises a VH region comprising an amino acid sequence at least 95% identical to SEQ ID NO:5, to amino acids 1-118 of SEQ ID NO:15, or to amino acids 1-121 of SEQ ID NO:34 and a VL region comprising an amino acid sequence at least 95% identical to SEQ ID NO:6, to amino acids 134-244 of SEQ ID NO:15, or to amino acids 138-251 of SEQ ID NO:34, wherein the identity regions are at least 80 amino acids long. 
     
     
         42 . (canceled) 
     
     
         43 . The Bi-Fc of  claim 36 , wherein the Fc polypeptide chain in the first polypeptide chain comprises a heterodimerizing alteration and wherein the Fc polypeptide chain in the second polypeptide chain comprises another heterodimerizing alteration. 
     
     
         44 . The Bi-Fc of  claim 43 , wherein the heterodimerizing alteration in the first polypeptide chain is a charge pair substitution and the heterodimerizing alteration in the second polypeptide chain is a charge pair substitution. 
     
     
         45 . The Bi-Fc of  claim 44 , wherein:
 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   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.   
     
     
         46 . The Bi-Fc of  claim 36 , wherein the Fc polypeptide chains of the first and second polypeptide chains comprise one or more alteration that inhibits FcγR binding selected from the group consisting of: L234A, L235A, and any substitution at N297. 
     
     
         47 . The Bi-Fc of  claim 36 , 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. 
     
     
         48 - 54 . (canceled) 
     
     
         55 . A bi-specific Fc (Bi-Fc) comprising:
 (i) a first polypeptide chain having 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 that have different amino acid sequences, wherein L1, L2, L3, and L4 are linkers, and wherein L2 and/or L4 can be present or absent; and   (ii) a second polypeptide chain comprising a human IgG Fc polypeptide chain;   wherein the Bi-Fc binds to a target cell and 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 and L2, if present, is less than 12 amino acids long,   wherein V1 and V3 are VH regions and V2 and V4 are VL regions,   wherein the Fc polypeptide chains of each of the first and second polypeptide chains contain a heterodimerizing alteration,   wherein the Bi-Fc comprises (1) 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 sequence of SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, or (2) a VH region comprising a CDR1, a CDR2, and a CDR3 comprising, respectively, the amino acid sequences of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56 and a VL region comprising a CDR1. a CDR2, and a CDR3 comprising, respectively, the amino acid sequence of SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59, and   wherein the first polypeptide chain comprises the charge pair substitutions K409D, K409E, R409D, or R409E and K392D, K392E, N392D, or N392E and the second polypeptide chain comprises the charge pair substitutions D399K or D399R and D356K, D356R, E356K, or E356R; or the second polypeptide chain comprises the charge pair substitutions K409D, K409E, R409D, or R409E and K392D, K392E, N392D, or N392E, and the first polypeptide chain comprises the charge pair substitutions D399K or D399R and D356K, D356R, E356K, or E356R.   
     
     
         56 . A bi-specific Fc (Bi-Fc) comprising:
 (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 V1 and V3 are VH regions and V2 and V4 are VL regions; wherein L1, L2, L3, and L4 are linkers; and wherein L2 and/or 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 V1 and V3 are VH regions and V2 and V4 are VL regions; wherein L1, L2, L3, and L4 are linkers; and wherein L2 and/or 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 V1 and V2 bind to a cancer cell antigen when the are part of an IgG and/or an scFv antibody,   wherein V3 and V4 can by to human CD3ε when they are part of an IgG and/or an scFv antibody,   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.   
     
     
         57 . The Bi-Fc of  claim 55 , 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. 
     
     
         58 - 63 . (canceled) 
     
     
         64 . A composition comprising the Bi-Fc of  claim 1  and a physiologically acceptable excipient. 
     
     
         65 . A nucleic acid encoding the Bi-Fc of  claim 1 . 
     
     
         66 . A vector comprising the nucleic acid of  claim 65 . 
     
     
         67 . A host cell comprising the nucleic acid of  claim 65 . 
     
     
         68 . A method of making a bi-specific Fc (Bi-Fc) comprising:
 culturing the host cell of  claim 67  under conditions such that the nucleic acid is expressed as a protein in the cell.   
     
     
         69 . A method for treating a cancer patient comprising administering to the patient a therapeutically effective dose of the Bi-Fc of  claim 1 . 
     
     
         70 - 71 . (canceled) 
     
     
         72 . A method for treating a patient having a fibrotic disease comprising administering to the patient a therapeutically effective dose of the Bi-Fc of  claim 1 . 
     
     
         73 . (canceled) 
     
     
         74 . A method for treating a patient having a disease mediated by a pathogen comprising administering to the patient a therapeutically effective dose of the Bi-Fc of  claim 1 . 
     
     
         75 - 79 . (canceled)

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