Interleukin-3 (IL-3) multiple mutation polypeptides

Abstract

The present invention relates to recombinant human interleukin-3 (hIL-3) variant or mutant proteins (muteins). These hIL-3 muteins contain amino acid substitutions and may also have amino acid deletions at both the N- and C-termini. The invention also relates to pharmaceutical compositions containing the hIL-3 muteins and methods for using them. Additionally, the present invention relates to recombinant expression vectors comprising nucleotide sequences encoding the hIL-3 muteins, related microbial expression systems, and processes for making the hIL-3 muteins using the microbial expression systems. Included in the present invention are deletion mutants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus, and from 1 to 15 amino acids 119 to 133 have been deleted from the C-terminus, and which also contain amino acid substitutions in the polypeptide. These hIL-3 multiple mutation polypeptides may have biological activities similar to or better than hIL-3 and, in some cases, may also have an improved side effect profile.

1 - 65 . (canceled)  
     
     
         66 . A method of producing a human interleukin-3 mutant polypeptide in a bacterial cell wherein the human interleukin-3 mutant polypeptide comprises a modified hIL-3 amino acid sequence of SEQ ID NO: 15 wherein: 
 Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;    Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;    Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;    Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;    Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;    Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;    Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;    Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;    Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;    Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;    Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;    Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;    Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;    Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;    Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;    Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;    Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu;    Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;    Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;    Xaa at position 36 is Asp, Leu, or Val;    Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;    Xaa at position 38 is Asn, or Ala;    Xaa at position 40 is Leu, Trp, or Arg;    Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;    Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;    Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;    Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;    Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;    Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;    Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;    Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;    Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;    Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;    Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;    Xaa at position 52 is Asn, His, Arg, Leu; Gly, Ser, or Thr;    Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;    Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;    Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;    Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;    Xaa at position 57 is Asn or Gly;    Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;    Xaa at position 59 is Glu, Tyr, His, Leu, Pro, or Arg;    Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;    Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser:    Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;    Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;    Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;    Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;    Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;    Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;    Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;    Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;    Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;    Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;    Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;    Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;    Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;    Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;    Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;    Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;    Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;    Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;    Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;    Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;    Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;    Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;    Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;    Xaa at position 85 is Leu, Asn, Val, or Gln;    Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;    Xaa at position 87 is Leu, Ser, Trp, or Gly;    Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;    Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;    Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;    Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;    Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;    Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;    Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lye, His, Ala, or Pro;    Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr;    Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr;    Xaa at position 97 is Ile, Val, Lys, Ala, or Asn;    Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;    Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;    Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;    Xaa at position 101 is Asp, Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;    Xaa at position 103 is Asp, or Ser;    Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;    Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His:    Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;    Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro;    Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;    Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, Ala, or Trp;    Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;    Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;    Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;    Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;    Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;    Xaa at position 116 is Lys, Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;    Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;    Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;    Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln;    Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;    Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;    Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;    wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus; and wherein from 4 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and said mutant human interleukin-3 polypeptide has at least three times greater cell proliferative activity than native human interleukin-3 in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay.    
     
     
         67 . The method of  claim 66  wherein the bacterial cell is  E. coli.    
     
     
         68 . The method of  claim 67  wherein the human interleukin-3 mutant polypeptide is expressed in the cytoplasm of  E. coli.    
     
     
         69 . The method of  claim 67  wherein  E. coli  is JM101 having accession number 33876.  
     
     
         70 . The method of  claim 67  wherein  E. coli  is MON 105 having accession number 55204.  
     
     
         71 . The method of  claim 66  wherein the human interleukin-3 mutant polypeptide is expressed as a fusion with a signal peptide.  
     
     
         72 . The method of  claim 71  wherein the signal peptide is LamB.