Methods and compositions involving interleukin-6 receptor alpha-binding single chain variable fragments

Abstract

Disclosed are compositions comprising an isolated chimeric interleukin 6 receptor alpha (IL-6Rα) binding protein or cells expressing an isolated chimeric IL-6Rα binding protein. The isolated IL-6Rα chimeric binding protein and cells expressing the protein may be used in methods of treating cancer and reducing the risk of cytokine release syndrome.

1 . A chimeric interleukin 6 receptor alpha (IL-6Rα) binding protein comprising a heavy chain variable region comprising CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6), and CDR3 (SEQ ID NO:7) attached by a heterologous linker to a light chain variable region comprising CDR4 (SEQ ID NO:8), CDR5 (SEQ ID NO:9), and CDR6 (SEQ ID NO:10). 
     
     
         2 . The chimeric IL-6Rα binding protein, wherein the binding protein is an scFv, (scFv)2, scFvFc, Fab, Fab′, or F(ab) 2 . 
     
     
         3 . The chimeric IL-6Rα binding protein of  claim 1 , wherein the binding protein is one polypeptide. 
     
     
         4 . The chimeric IL-6Rα binding protein of  claim 3 , wherein the one polypeptide is a single chain variable fragment (scFv). 
     
     
         5 . The chimeric IL-6Rα binding protein of  claim 1 , wherein the heavy chain variable region is on the N-terminal side of the light chain variable region. 
     
     
         6 . The chimeric IL-6Rα binding protein of  claim 1 , wherein the light chain variable region is on the N-terminal side of the heavy chain variable region. 
     
     
         7 . The chimeric IL-6Rα binding protein of any of  claims 1 - 6 , wherein the linker comprises the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:11). 
     
     
         8 . The chimeric IL-6Rα binding protein of any of  claims 1 - 7 , further comprising a leader peptide. 
     
     
         9 . The chimeric IL-6Rα binding protein of any of  claims 1 - 8 , further comprising an isolation tag. 
     
     
         10 . An chimeric interleukin 6 receptor alpha (IL-6Rα) binding protein comprising a leader peptide, heavy chain variable region comprising CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6), and CDR3 (SEQ ID NO:7) attached by a heterologous linker to a light chain variable region comprising CDR4 (SEQ ID NO:8), CDR5 (SEQ ID NO:9), and CDR6 (SEQ ID NO:10), wherein the heterologous linker comprises the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:11) and the IL-6Rα binding protein is a single chain antibody fragment (scFv). 
     
     
         11 . A T cell expressing the chimeric IL-6Rα binding protein of any of  claims 1 - 10 . 
     
     
         12 . A nucleic acid molecule encoding the chimeric IL-6Rα binding protein of any of  claims 1 - 10 . 
     
     
         13 . The nucleic acid molecule of  claim 12 , further comprising a promoter controlling expression of the chimeric IL-6R binding protein. 
     
     
         14 . The nucleic acid molecule of  claim 13 , wherein the promoter is constitutive. 
     
     
         15 . The nucleic acid molecule of  claim 13 , wherein the promoter responds positively to at least one cytokine or to T-cell activation. 
     
     
         16 . The nucleic acid molecule of  claim 15 , where the at least one cytokine is IL-6, TNF-α, IFN-γ, IL-1β, IL-2, IL-8, or IL-10 or the promoter responds positively to NFAT-1 or NF-κB. 
     
     
         17 . An expression construct comprising the nucleic acid molecule of any of  claims 12 - 16 . 
     
     
         18 . The expression construct of  claim 14 , wherein the expression construct is a viral vector. 
     
     
         19 . The expression construct of  claim 14 , wherein the expression construct is a plasmid. 
     
     
         20 . A recombinant T cell comprising the nucleic acid expression construct of any of  claims 17 - 19 . 
     
     
         21 . A T cell comprising a heterologous expression construct encoding an IL-6 receptor alpha single chain variable fragment comprising a leader peptide, a heavy chain variable region comprising CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6), and CDR3 (SEQ ID NO:7) attached by a heterologous linker to a light chain variable region comprising CDR4 (SEQ ID NO:8), CDR5 (SEQ ID NO:9), and CDR6 (SEQ ID NO:10), wherein the heterologous linker comprises the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:11). 
     
     
         22 . The T cell of  claim 21 , wherein the expression construct comprises a cytokine-responsive promoter or promoter that increases expression when T cells are activated. 
     
     
         23 . The T cell of  claim 22 , wherein the promoter responds positively to one or more of the following: NFAT-1, NF-κB, IL-6, TNF-α, IFN-γ, IL-1β, IL-2, IL-8, and IL-10. 
     
     
         24 . A method for reducing the risk of cytokine release syndrome comprising administering to a patient at risk for cytokine release syndrome a composition comprising an isolated IL-6 receptor alpha (IL-6Rα) binding protein or cells comprising a heterologous nucleic acid molecule encoding the IL-6Rα binding protein. 
     
     
         25 . The method of  claim 24 , wherein the patient has cancer. 
     
     
         26 . The method of  claim 24  or  25 , wherein the patient has or will receive adoptive T-cell therapy. 
     
     
         27 . The method of  claim 26 , wherein the patient has or will receive lymphodepletion. 
     
     
         28 . The method of  claim 24 , wherein the patient has an autoimmune disease. 
     
     
         29 . The method of any of  claims 24 - 28 , wherein the isolated IL-6Rα binding protein comprises a heavy chain variable region comprising CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6), and CDR3 (SEQ ID NO:7) attached by a heterologous linker to a light chain variable region comprising CDR4 (SEQ ID NO:8), CDR5 (SEQ ID NO:9), and CDR6 (SEQ ID NO:10). 
     
     
         30 . The method of  claim 29 , wherein the IL-6Rα binding protein is an scFv, (scFv)2, scFvFc, Fab, Fab′, or F(ab) 2 . 
     
     
         31 . The method of any of  claims 24 - 30 , wherein the heavy chain variable region is on the N-terminal side of the light chain variable region. 
     
     
         32 . The method of any of  claims 24 - 30 , wherein the light chain variable region is on the N-terminal side of the heavy chain variable region. 
     
     
         33 . The method of any of  claims 24 - 32 , wherein isolated IL-6Rα binding protein comprises a linker comprising the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:11). 
     
     
         34 . The method of any of  claims 24 - 33 , wherein the isolated chimeric IL-6Rα binding protein comprises a leader peptide. 
     
     
         35 . The method of any of  claims 24 - 34 , wherein the isolated chimeric IL-6Rα binding protein comprises an isolation tag. 
     
     
         36 . The method of any of  claims 24 - 35 , wherein the patient is administered T cell comprising a heterologous nucleic acid molecule encoding an IL-6Rα binding protein. 
     
     
         37 . The method of  claim 36 , wherein the T cells are autologous. 
     
     
         38 . The method of  claims 24 - 37 , wherein the heterologous nucleic acid molecule encodes a chimeric IL-6Rα binding polypeptide comprising a heavy chain variable region comprising CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6), and CDR3 (SEQ ID NO:7) attached by a heterologous linker to a light chain variable region comprising CDR4 (SEQ ID NO:8), CDR5 (SEQ ID NO:9), and CDR6 (SEQ ID NO:10). 
     
     
         39 . The method of any of  claims 24 - 38 , wherein the nucleic acid molecule comprises a promoter controlling expression of the chimeric IL-6R binding protein. 
     
     
         40 . The method of  claim 39 , wherein the promoter is constitutive. 
     
     
         41 . The method of  claim 39 , wherein the promoter responds positively to at least one cytokine or to T cell activation 
     
     
         42 . The method of  claim 41 , wherein the promoter is responsive to NFAT-1, NF-κB, IL-6, TNF-α, IFN-γ, IL-1β, IL-2, IL-8, or IL-10. 
     
     
         43 . The method of any of  claims 24 - 42 , wherein the nucleic acid molecule is an expression construct. 
     
     
         44 . The method of  claim 43 , wherein the expression construct is a viral vector. 
     
     
         45 . The method of  claim 43 , wherein the expression construct is a plasmid. 
     
     
         46 . The method of any of  claims 24 - 45 , further comprising administering to the patient an antihistamine, a corticosteroid, a steroid, acetaminophen, furosemide, and/or intravenous fluids. 
     
     
         47 . The method of any of  claims 24 - 46 , wherein the patient has one or more symptoms of cytokine release syndrome. 
     
     
         48 . A method for reducing the toxicity of adoptive cell therapy in a cancer patient comprising administering to the patient a composition comprising a composition comprising an isolated IL-6 receptor alpha (IL-6Rα) binding protein or cells comprising a heterologous nucleic acid molecule encoding the IL-6Rα binding protein. 
     
     
         49 . A method for treating an autoimmune disease in a patient comprising administering to the patient a composition comprising a composition comprising an isolated IL-6 receptor alpha (IL-6Rα) binding protein or cells comprising a heterologous nucleic acid molecule encoding the IL-6Rα binding protein. 
     
     
         50 . A method for treating a cancer patient comprising administering to the patient a composition comprising a composition comprising an isolated IL-6 receptor alpha (IL-6Rα) binding protein or cells comprising a heterologous nucleic acid molecule encoding the IL-6Rα binding protein, wherein the patient has been or will be treated with immunotherapy. 
     
     
         51 . The method of  claim 50 , wherein the immunotherapy is adoptive cell therapy. 
     
     
         52 . The method of  claim 50 , wherein the patient has symptoms of cytokine release syndrome. 
     
     
         53 . A method for reducing the toxicity of one or more cytokines comprising administering to a patient with an autoimmune disease or condition or a patient being treated with immunotherapy an effective amount of a composition comprising a single chain variable fragment comprising an isolated IL-6Rα binding protein comprises a heavy chain variable region comprising CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6), and CDR3 (SEQ ID NO:7) attached by a heterologous linker to a light chain variable region comprising CDR4 (SEQ ID NO:8), CDR5 (SEQ ID NO:9), and CDR6 (SEQ ID NO:10). AMENDMENTS This listing of claims will replace all prior versions, and listings of claims in the application. CLAIMS:  1 . (Original) A chimeric interleukin  6  receptor alpha (IL- 6 Ra) binding protein comprising a heavy chain variable region comprising CDR 1  (SEQ ID NO: 5 ), CDR 2  (SEQ ID NO: 6 ), and CDR 3  (SEQ ID NO: 7 ) attached by a heterologous linker to a light chain variable region comprising CDR 4  (SEQ ID NO: 8 ), CDR 5  (SEQ ID NO: 9 ), and CDR 6  (SEQ ID NO: 10 ).  2 - 10 . (Canceled)  11 . (Currently Amended) A T cell expressing the chimeric IL- 6 Ra binding protein of any of elaims  1 - 10   claim 1 .  12 . (Currently Amended) A nucleic acid molecule encoding the chimeric IL- 6 Ra binding protein of any of  claims 1 - 10   claim 1 .  13 - 19 . (Canceled)  20 . (Currently Amended) A recombinant T cell comprising the nucleic acid expression construct of any of  claims 17 - 19 of  claim 12 .  21 - 23 . (Canceled)  24 . (Original) A method for reducing the risk of cytokine release syndrome comprising administering to a patient at risk for cytokine release syndrome a composition comprising an isolated IL- 6  receptor alpha (IL- 6 Ra) binding protein or cells comprising a heterologous nucleic acid molecule encoding the IL- 6 Ra binding protein.  25 . (Original) The method of  claim 24 , wherein the patient has cancer.  26 . (Currently Amended) The method of  claim 24  er  25 , wherein the patient has or will receive adoptive T-cell therapy.  27 . (Original) The method of  claim 26 , wherein the patient has or will receive lymphodepletion.  28 . (Original) The method of  claim 24 , wherein the patient has an autoimmune disease.  29 . (Currently Amended) The method of any of elaims  24   28   claim 24 , wherein the isolated IL- 6 Ra binding protein comprises a heavy chain variable region comprising CDR 1  (SEQ ID NO: 5 ), CDR 2  (SEQ ID NO: 6 ), and CDR 3  (SEQ ID NO: 7 ) attached by a heterologous linker to a light chain variable region comprising CDR 4  (SEQ ID NO: 8 ), CDR 5  (SEQ ID NO: 9 ), and CDR 6  (SEQ ID NO: 10 ).  30 . (Original) The method of  claim 29 , wherein the IL- 6 Ra binding protein is an scFv, (scFv) 2 , scFvFc, Fab, Fab', or F(ab) 2 .  31 - 32 . (Canceled)  33 . (Currently Amended) The method of any of elaims  24 - 32   claim 24 , wherein isolated IL-  6 Ra binding protein comprises a linker comprising the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 11 ).  34 . (Currently Amended) The method of any of elaims  24   33   claim 24 , wherein the isolated chimeric IL- 6 Ra binding protein comprises a leader peptide and/or an isolation tag.  35 . (Canceled)  36 . (Currently Amended) The method of any of elaims  24   35   claim 24 , wherein the patient is administered T cell comprising a heterologous nucleic acid molecule encoding an IL- 6 Ra binding protein.  37 . (Original) The method of  claim 36 , wherein the T cells are autologous.  38 . (Currently Amended) The method of elaims  24   37   claim 24 , wherein the heterologous nucleic acid molecule encodes a chimeric IL- 6 Ra binding polypeptide comprising a heavy chain variable region comprising CDR 1  (SEQ ID NO: 5 ), CDR 2  (SEQ ID NO: 6 ), and CDR 3  (SEQ ID NO: 7 ) attached by a heterologous linker to a light chain variable region comprising CDR 4  (SEQ ID NO: 8 ), CDR 5  (SEQ ID NO: 9 ), and CDR 6  (SEQ ID NO: 10 ).  39 - 45 . (Canceled)  46 . (Currently Amended) The method of any of elaims  24   45   claim 24 , further comprising administering to the patient an antihistamine, a corticosteroid, a steroid, acetaminophen, furosemide, and/or intravenous fluids.  47 . (Currently Amended) The method of any of elaims  24 - 46   claim 24 , wherein the patient has one or more symptoms of cytokine release syndrome.  48 . (Canceled)  49 . (Original) A method for treating an autoimmune disease in a patient comprising administering to the patient a composition comprising a composition comprising an isolated IL- 6  receptor alpha (IL- 6 Ra) binding protein or cells comprising a heterologous nucleic acid molecule encoding the IL- 6 Ra binding protein.  50 - 52 . (Canceled)  53 . (Original) A method for reducing the toxicity of one or more cytokines comprising administering to a patient with an autoimmune disease or condition or a patient being treated with immunotherapy an effective amount of a composition comprising a single chain variable fragment comprising an isolated IL- 6 Ra binding protein comprises a heavy chain variable region comprising CDR 1  (SEQ ID NO: 5 ), CDR 2  (SEQ ID NO: 6 ), and CDR 3  (SEQ ID NO: 7 ) attached b y a heterologous linker to a light chain variable region comprising CDR 4  (SEQ ID NO: 8 ), CDR 5  (SEQ ID NO: 9 ), and CDR 6  (SEQ ID NO: 10 ).