Tear lipocalin muteins binding il-4 r alpha

Abstract

The present invention relates to novel muteins derived from human tear lipocalin, which bind to IL 4 receptor alpha. The sequences of the muteins comprise particular combinations of amino acids. In particular a mutated amino acid residue is present at any one or more of the sequence positions 27, 28 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106 and 108 of the linear polypeptide sequence of the mature human tear lipocalin. A mutated amino acid residue is also present at any 2 or more of the sequence positions 26, 32, 34, 55, 56, 58 and 63 of the linear polypeptide sequence of the mature human tear lipocalin. The invention also provides a corresponding nucleic acid molecule encoding such a mutein and a method for producing such a mutein and its encoding nucleic acid molecule.

1 - 19 . (canceled) 
     
     
         20 . A mutein of human tear lipocalin, wherein the amino acid sequence of the mutein comprises:
 A SDEEI QDVSG TWYLK AMTVD SRCPR AVYNS VTPMT LTTLE GGNLE AKFTA QRKGR WQKYK LVLEK TDEPG KYTAS GGRHV AYIIR SHVKD HYIFH SEGLC PGQPV PGVWL VGRDP KNNLE ALEDF EKAAG ARGLS TESIL IPRQS ETSSP G.   
     
     
         21 . A method of generating a mutein of human tear lipocalin of  claim 20 , wherein the mutein binds to IL-4 receptor alpha, comprising:
 (a) subjecting a nucleic acid molecule encoding a human tear lipocalin to mutagenesis at the amino acid sequence positions 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106 and 108 of the linear polypeptide sequence of mature human tear lipocalin, and further, subjecting the nucleic acid molecule encoding a human tear lipocalin to mutagenesis at the amino acid sequence positions 26, 32, 34, 55, 56, 58 and 63 of the linear polypeptide sequence of mature human tear lipocalin,   (b) expressing one or more nucleic acid molecule(s) obtained in step (a) in an expression system, thereby obtaining one or more mutein(s) and   (c) enriching the one or more mutein(s) obtained in step (b) that bind to human IL-4 receptor alpha by means of selection and/or isolation.   
     
     
         22 . The method of  claim 21 , wherein step (c) comprises:
 (ci) providing IL-4 receptor alpha or an immunogenic fragment thereof,   (cii) contacting the one or more mutein(s) with the IL-4 receptor alpha or the immunogenic fragment thereof, thereby allowing the formation of a complex between the IL-4 receptor alpha or the immunogenic fragment thereof and a mutein having binding affinity for the same, and   (ciii) removing mutein(s) having no or no substantial binding affinity.   
     
     
         23 . The method of  claim 21 , wherein the selection in step (c) is carried out under competitive conditions. 
     
     
         24 . A nucleic acid molecule comprising a nucleotide sequence encoding a mutein of  claim 20 . 
     
     
         25 . A host cell containing a nucleic acid molecule of  claim 24 . 
     
     
         26 . A pharmaceutical composition comprising a mutein of human tear lipocalin as defined in  claim 20  and a pharmaceutically acceptable excipient.