Interleukin-9 Antagonist Muteins and Their Pharmacological Methods of Use

Abstract

This invention relates to IL-9 muteins that inhibit the activity of wild-type IL-9, multimers and Fc-fusion constructs of IL-9 proteins, and an efficient method to purify IL-9 proteins produced by eukaryotic cells. Related formulations, dosages and methods of administration thereof for therapeutic purposes are also provided. More particularly, these IL-9 muteins, compositions, and methods provide a treatment option for individuals afflicted with conditions where inhibiting IL-9 mediated immune responses would be beneficial, such as allergy, asthma, chronic obstructive pulmonary disease (emphysema and chronic bronchitis), pulmonary and gastro-intestinal mucus hyperplasia, inflammation, immunological disorders, leukemia, and lymphoma.

1 . An isolated and purified IL-9 mutein numbered in accordance with wild type IL-9 wherein said mutein is an IL-9 antagonist and comprises at least one amino-acid substitution. 
     
     
         2 . A isolated and purified IL-9 multimer comprising two or more IL-9 muteins as described in  claim 1  fused C-terminus to N-terminus to form the multimer, and wherein said multimer is an IL-9 antagonist. 
     
     
         3 . The isolated and purified IL-9 mutein multimer of  claim 2 , wherein said multimer is a dimer comprising a C-terminal mutein subunit having a Cys to Ser substitution at position 147 (SEQ ID NOS. 18, 20, 22, 25, 28, and 29). 
     
     
         4 . The IL-9 mutein multimer of  claim 3 , wherein said multimer is enzymatically cleaved to produce the protein shown in SEQ ID NO. 29. 
     
     
         5 . An IL-9 mutein fusion protein, wherein said protein comprises an Fc region of an immunoglobulin molecule fused to an IL-9 mutein (SEQ ID NOS. 26 and 27). 
     
     
         6 . The IL-9 mutein fusion protein of  claim 5 , wherein said protein comprises an Fc region of an immunoglobulin molecule fused to the N-terminus of an IL-9 mutein to produce the fusion protein of SEQ ID NO. 26. 
     
     
         7 . The IL-9 mutein fusion protein of  claim 5 , wherein said protein comprises an Fc region of an immunoglobulin molecule fused to the C-terminus of an IL-9 mutein to produce the fusion protein of SEQ ID NO. 27. 
     
     
         8 . A polynucleotide that encodes an IL-9 mutein selected from the group consisting of SEQ ID NOS. 17-29. 
     
     
         9 . A polynucleotide that encodes an IL-9 mutein selected from the group consisting of SEQ ID NOS. 3-14. 
     
     
         10 . An expression vector comprising the polynucleotide of  claim 9 . 
     
     
         11 . A viral vector comprising the polynucleotide of  claim 9 . 
     
     
         12 . A host cell comprising the expression vector of  claim 10 . 
     
     
         13 . A method of treating a human lung disorder selected from the group consisting of chronic obstructive pulmonary disorder, asthma, emphysema, and chronic bronchitis, comprising the steps of:
 a) providing a human having an IL-9 induced respiratory disease, and   b) administering to said human a therapeutically effective amount of the IL-9 of  claim 1 , until such time as the IL-9 receptor is antagonized and the human lung disorder attenuated.   
     
     
         14 . A method of treating human a cancer selected from the group consisting of leukemia and lymphoma comprising the steps of:
 a) providing a human having an IL-9 induced leukemia or lymphoma,   b) providing to said human a therapeutically effective amount of the IL-9 of  claim 1 , until such time as the   c) IL-9 receptor is antagonized and the human cancer attenuated.   
     
     
         15 . A method of expressing in a eukaryotic cell a chimeric protein having a high affinity amino terminal tag, the method comprising:
 a) providing a plasmid comprising a polynucleotide that encodes a high affinity amino terminal tagged protein, said tagged protein comprising a protein of interest bound at its amino terminus to a high affinity tag polypeptide which is bound at its amino terminus to a signal sequence,   b) providing a eukaryotic cell capable of expressing protein,   c) transfecting said eukaryotic cell with said plasmid,   d) expressing from said transfected cell the high affinity amino terminal tagged protein.   
     
     
         16 . The method of  claim 15 , wherein said high affinity tag polypeptide comprises a plurality of histidine amino acids. 
     
     
         17 . The method of  claim 15 , wherein said eukaryotic cells are HKB11 cells. 
     
     
         18 . The method of  claim 15 , wherein the polynucleotide that encodes said high affinity amino terminal tagged protein further comprises a nucleotide sequence encoding a protease digestible polypeptide, said protease digestible polypeptide being located between the protein of interest and the high affinity tag polypeptide. 
     
     
         19 . The method of  claim 18 , wherein said protease digestible polypeptide is digestible with TEV protease.