Claims
- 1. A method for ex vivo expansion of stem cells, comprising the steps of;(a) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:1 whereinXaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 18 is Asn, His, Leu, le, 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, 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, Lys, 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, 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, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln; 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, 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, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile; 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 are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus of said human interleukin-3 mutant polypeptide; and wherein from 4 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; with the proviso that no more than one of the amino acids at positions 63, 82, 87, 98, 112, and 121 are different from the corresponding amino acids in native human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; and (b) harvesting said cultured stem cells.
- 2. A method for ex vivo expansion of stem cells, comprising the steps of;(a) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, -Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:4 whereinXaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu, Tyr, His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, Pro; Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu or Gln; Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 4 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; with the proviso that no more than one of the amino acids at positions 49, 68, 73. 84, 98, and 107 are different from the corresponding amino acids in native human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; and (b) harvesting said cultured stem cells.
- 3. A method for ex vivo expansion of stem cells, comprising the steps of;(a) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:7 wherein; Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Leu; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gly; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Trp; Xaa at position 88 is Ala or Trp; Xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala or Met; Xaa at position 105 is Asn or Gln; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val, Trp or Ser; Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; wherein from four to about forty-four of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; and (b) harvesting said cultured stem cells.
- 4. A method for ex vivo expansion of stem cells, comprising the steps of;(a) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, -Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:8 wherein; Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Leu; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile, Leu or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His; Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gly; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Trp; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala or Met; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu, Leu; Xaa at position 98 Thr or Gin; Xaa at position 102 is Lys, Val, Trp or Ser; Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; wherein from four to about forty-four of the amino acids designated by Xaa are different from the corresponding amino acids of native (15-125)human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; and (b) harvesting said cultured stem cells.
- 5. The method according to claim 2 wherein R1 is selected from the group consisting of:Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:9;Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn AlaGlu Asp Val Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:10;Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:11;Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:12;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:13;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:14;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Arg Asn Leu Arg Thr ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:15;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:16;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Val Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile ThrIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn A1a Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:17;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:18;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:19;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:20;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Val Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile ThrIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:21;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Val Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile ThrIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:22;Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn AlaGlu Asp Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:23;Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:24;Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnMet Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr HisSEQ ID NO:25;Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn LeuAsn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu ArgArg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser LeuGln Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr HisSEQ ID NO:26;Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn LeuAsn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu ArgArg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser LeuGln Asn Ala Ser Gly Ile G1u Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr HisSEQ ID NO:27;Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn LeuAsn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu ArgArg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser LeuGln Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:28;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu LeuPro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg LysLeu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:29;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu LeuPro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg LysLeu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:30;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu LeuPro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg LysLeu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:31;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:32;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:33;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:34;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pr6 Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:35;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:36;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:37;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:38;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:39;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:40;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Asp Arg Asn Leu ArgLeu Ser Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ala Ile His HisSEQ ID NO:41;Leu Lys Arg Pro Pro Ala Pro Ser Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Met Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:42;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Met Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:43;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Asp Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:44;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Val Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:45;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Met Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Tyr Pro Glu Thr Asp Tyr Lys Asp Asp Asp Asp LysSEQ ID NO:46;Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysArg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn AlaGlu Asp Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnMet Ala Tyr Pro Glu Thr Asp Tyr Lys Asp Asp Asp Asp LysSEQ ID NO:47;Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysArg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnandMet Ala Asn Cys Ser Ile Met Ile Asp Glu Leu Ile His HisSEQ ID NO:48;Leu Lys Ile Pro Pro Asn Pro Ser Leu Asp Ser Ala Asn LeuAsn Ser Glu Asp Val Ser Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGln
- 6. The method of claim 1, 2, 3, 4 or 5 wherein is R2 is R1 or a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 7. The method of claim 6 wherein is R2 is selected from the group consisting of G-CSF, G-CSF Ser,17 flt3 ligand and c-mpl ligand.
- 8. The method of claim 2 wherein said chimera protein is selected from group consisting of: SEQ ID NO:121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 165, 166, 167 and 168.
- 9. The method of claim 8 wherein said chimera protein is selected from group consisting of: SEQ ID NO:124, SEQ ID NO:133, SEQ ID NO:154 and SEQ ID NO:155.
- 10. The method of claim 1, 2, 3, 4, 5, 8 or 9 wherein said culture medium further comprises a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 11. The method of claim 6 wherein said culture medium further comprises a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 12. The method of claim 7 wherein said culture medium further comprises a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 13. The method of claim 1 wherein said mutant human interleukin-3 polypeptide has at least three times greater 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.
- 14. The method of claim 7 wherein said mutant human interleukin-3 polypeptide has at least three times greater 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.
- 15. The method of claim 1 further comprising the step of separating the stem cells from a mixed population of cells prior to culturing the stem cells.
- 16. The method of claim 15 wherein said stem cells are separated from a mixed population of cells based on the stem cells having CD34 surface antigen.
- 17. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of;(a) removing stem cells from said patient or a donor; (b) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, -Ala-R1-R2 and Ala-R2-R1; wherein R1is a human interleukin-3 mutant polypeptide of SEQ ID NO:1 whereinXaa 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, 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, Lys, 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, 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, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln; 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, 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, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile; 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 are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus of said human interleukin-3 mutant polypeptide; and wherein from 4 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; with the proviso that no more than one of the amino acids at positions 63, 82, 87, 98, 112 and 121 are different from the corresponding amino acids in native human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; (c) harvesting said cultured stem cells; and (d) transplanting said cultured stem cells into said patient.
- 18. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of;(a) removing stem cells from said patient or a donor; (b) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:4 whereinXaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu, Tyr, His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, Pro; Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu or Gln; Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 4 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; with the proviso that no more than one of the amino acids at positions 49, 68, 73, 84, 98, and 112 are different from the corresponding amino acids in native human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; (c) harvesting said cultured stem cells; and (d) transplanting said cultured stem cells into said patient.
- 19. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of;(a) removing stem cells from said patient or a donor; (b) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:7 wherein; Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Leu; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gly; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Trp; Xaa at position 88 is Ala or Trp; Xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala or Met; Xaa at position 105 is Asn or Gln; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val, Trp or Ser; Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; wherein from four to about forty-four of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3); and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; (c) harvesting said cultured stem cells; and (d) transplanting said cultured stem cells into said patient.
- 20. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of;(a) removing stem cells from said patient or a donor; (b) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1,Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:8 wherein; Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Leu; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile, Leu or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His; Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gly; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Trp; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala or Met; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu, Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val, Trp or Ser; Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; wherein from four to about forty-four of the amino acids designated by Xaa are different from the corresponding amino acids of native (15-125)human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; L is a linker capable of Linking R1 to R2; (c) harvesting said cultured stem cells; and (d) transplanting said cultured stem cells into said patient.
- 21. The method according to claim 18 wherein R1 is selected from the group consisting of:Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:9;Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn AlaGlu Asp Val Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:10;Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:11;Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:12;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:13;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:14;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Arg Asn Leu Arg Thr ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:15;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:16;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Val Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile ThrIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn A1a Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:17;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:18;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:19;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:20;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Val Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile ThrIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:21;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Val Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile ThrIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:22;Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn AlaGlu Asp Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:23;Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:24;Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnMet Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr HisSEQ ID NO:25;Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn LeuAsn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu ArgArg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser LeuGln Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr HisSEQ ID NO:26;Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn LeuAsn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu ArgArg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser LeuGln Asn Ala Ser Gly Ile G1u Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr HisSEQ ID NO:27;Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn LeuAsn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu ArgArg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser LeuGln Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:28;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu LeuPro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg LysLeu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:29;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu LeuPro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg LysLeu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:30;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu LeuPro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg LysLeu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:31;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:32;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:33;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:34;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pr6 Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:35;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:36;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:37;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:38;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:39;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:40;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Asp Arg Asn Leu ArgLeu Ser Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ala Ile His HisSEQ ID NO:41;Leu Lys Arg Pro Pro Ala Pro Ser Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Met Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:42;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Met Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:43;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Asp Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:44;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Val Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:45;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Met Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Tyr Pro Glu Thr Asp Tyr Lys Asp Asp Asp Asp LysSEQ ID NO:46;Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysArg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn AlaGlu Asp Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnMet Ala Tyr Pro Glu Thr Asp Tyr Lys Asp Asp Asp Asp LysSEQ ID NO:47;Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysArg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnandMet Ala Asn Cys Ser Ile Met Ile Asp Glu Leu Ile His HisSEQ ID NO:48.Leu Lys Ile Pro Pro Asn Pro Ser Leu Asp Ser Ala Asn LeuAsn Ser Glu Asp Val Ser Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGln.
- 22. The method of claim 17, 18, 19, 20 or 21 wherein is R2 is R1 or a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 23. The method of claim 22 wherein is R2 is selected from the group consisting of G-CSF, G-CSF Ser,17 flt3 ligand and c-mpl ligand.
- 24. The method of claim 18 wherein said chimera protein is selected from group consisting of: SEQ ID NO:121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 165, 166, 167 and 168.
- 25. The method of claim 24 wherein said chimera protein is selected from group consisting of: SEQ ID NO:124, SEQ ID NO:133, SEQ ID NO:154 and SEQ ID NO:155.
- 26. The method of claim 17, 18, 19, 20, 21, 24 or 25 wherein said culture medium further comprises a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 27. The method of claim 22 wherein said culture medium further comprises a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 28. The method of claim 23 wherein said culture medium further comprises a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 29. The method of claim 17 wherein said mutant human interleukin-3 polypeptide has at least three times greater 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.
- 30. The method of claim 23 wherein said mutant human interleukin-3 polypeptide has at least three times greater 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.
- 31. The method of claim 17 further comprising the step of separating the stem cells from a mixed population of cells prior to culturing the stem cells.
- 32. The method of claim 31 wherein said stem cells are separated from a mixed population of cells based on the stem cells having CD34 surface antigen.
- 33. A method of enhancing the efficiency of the transduction of cultured stem cells by a heterologous gene, comprising the steps of;(a) removing stem cells from a patient or donor; (b) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:1 whereinXaa 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, 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, Lys, 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, 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, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln; 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, 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, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile; 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, le, 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 are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus of said human interleukin-3 mutant polypeptide; and wherein from 4 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; with the proviso that no more than one of the amino acids at positions 63, 82, 87, 98, 112 and 121 are different from the corresponding amino acids in native human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; (c) traducing DNA into said cultured cells; and (d) harvesting said transduced cells.
- 34. A method of enhancing the efficiency of the transduction of cultured stem cells by a heterologous gene, comprising the steps of;(a) removing stem cells from a patient or a donor; (b) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:4 whereinXaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu, Tyr, His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, Pro; Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu or Gln; Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 4 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; with the proviso that no more than one of the amino acids at positions 49, 68, 73, 84, 98 and 107 are different from the corresponding amino acids in native human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; (c) transducing DNA into said cultured cells; and (d) harvesting said transduced cells.
- 35. A method of enhancing the efficiency of the transduction of cultured stem cells by a heterologous gene, comprising the steps of;(a) removing stem cells from a patient or a donor; (b) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:7 wherein; Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Leu; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gly; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Trp; Xaa at position 88 is Ala or Trp; Xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala or Met; Xaa at position 105 is Asn or Gln; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val, Trp or Ser; Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; wherein from four to about forty-four of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; (c) transducing DNA into said cultured cells; and (d) harvesting said transduced cells.
- 36. A method of enhancing the efficiency of the transduction of cultured stem cells by a heterologous gene, comprising the steps of;(a) removing stem cells from a patient or a donor; (b) culturing said stem cells with a growth medium comprising a chimera protein having the formula selected from the group consisting of: R1-L-R2, R2-L-R1, R1-R2, R2-R1, R1-L-R1, R1-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1; wherein R1 is a human interleukin-3 mutant polypeptide of wherein R1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:8wherein; Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Leu; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile, Leu or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His; Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gly; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Trp; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala or Met; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu, Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val, Trp or Ser; Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; wherein from four to about forty-four of the amino acids designated by Xaa are different from the corresponding amino acids of native (15-125)human interleukin-3; and wherein said modified human interleukin-3 (hIL-3) amino acid sequence has increased activity, relative to 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;R2 is a hematopoietic growth factor; and L is a linker capable of Linking R1 to R2; (c) transducing DNA into said cultured cells; and (d) harvesting said transduced cells.
- 37. The method according to claim 33 wherein R1 is selected from the group consisting of:Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:9;Arg Pro Pro AIa Pro Leu Leu Asp Pro Asn Asn Leu Asn AlaGlu Asp Val Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala A1a Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:10;Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:11;Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:12;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:13;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:14;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Arg Asn Leu Arg Thr ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:15;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:16;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Val Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile ThrIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:17;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:18;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:19;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:20;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Val Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile ThrIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnAsn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu LysSEQ ID NO:21;Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn GlyGlu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg ProAsn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Val Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile ThrIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:22;Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn AlaGlu Asp Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:23;Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnAsn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysSEQ ID NO:24;Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile HisIle Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu ThrPhe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln GlnMet Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr HisSEQ ID NO:25;Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn LeuAsn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu ArgArg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser LeuGln Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His PtoIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr HisSEQ ID NO:26;Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn LeuAsn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu ArgArg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser LeuGln Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr HisSEQ ID NO:27;Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn LeuAsn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu ArgArg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser LeuGln Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:28;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu LeuPro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg LysLeu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:29;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu LeuPro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg LysLeu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:30;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu LeuPro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg LysLeu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:31;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:32;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:33;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys A1a Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:34;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:35;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:36;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:37;Leu Lys Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:38;Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn LeuAsn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:39;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu ValPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:40;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Asp Arg Asn Leu ArgLeu Ser Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ala Ile His HisSEQ ID NO:41;Leu Lys Arg Pro Pro Ala Pro Ser Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Met Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:42;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Met Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:43;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Ala Glu Asp Val Asp Ile Leu Met Asp Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:44;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Val Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His HisSEQ ID NO:45;Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn LeuAsn Asp Glu Asp Met Ser Ile Leu Met Glu Arg Asn Leu ArgLeu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGlnMet Ala Tyr Pro Glu Thr Asp Tyr Lys Asp Asp Asp Asp LysSEQ ID NO:46;Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysArg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn AlaGlu Asp Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu ProAsn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnMet Ala Tyr Pro Glu Thr Asp Tyr Lys Asp Asp Asp Asp LysSEQ ID NO:47;Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu LysArg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn SerGlu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr ProAsn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu AsnAla Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro CysLeu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile IleIle Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu ThrPhe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln GlnandMet Ala Asn Cys Ser Ile Met Ile Asp Glu Leu Ile His HisSEQ ID NO:48.Leu Lys Ile Pro Pro Asn Pro Ser Leu Asp Ser Ala Asn LeuAsn Ser Glu Asp Val Ser Ile Leu Met Glu Arg Asn Leu ArgThr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His LeuGlu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu GlnPro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His ProIle Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu LysLeu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu GlnGln
- 38. The method of claim 33, 34, 35, 36 or 37 wherein is R2 is R1 or a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 39. The method of claim 38 wherein is R2 is selected from the group consisting of G-CSF, G-CSF Ser,17 flt3 ligand or c-mpl ligand.
- 40. The method of claim 34 wherein said chimera protein is selected from group consisting of: SEQ ID NO:121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 165, 166, 167 and 168.
- 41. The method of claim 40 wherein said chimera protein is selected from group consisting of: SEQ ID NO:124, SEQ ID NO:133, SEQ ID NO:154 and SEQ ID NO:155.
- 42. The method of claim 33, 34, 35, 36 or 37 wherein said culture medium further comprises a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 43. The method of claim 38 wherein said culture medium further comprises a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 44. The method of claim 39 wherein said culture medium further comprises a hematopoietic growth factor selected from the group consisting of: GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
- 45. The method of claim 33 wherein said mutant human interleukin-3 polypeptide has at least three times greater 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.
- 46. The method of claim 38 wherein said mutant human interleukin-3 polypeptide has at least three times greater 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.
- 47. The method of claim 33 further comprising the step of separating the stem cells from a mixed population of cells prior to culturing the stem cells.
- 48. The method of claim 47 wherein said stem cells are separated from a mixed population of cells based on the stem cells having CD34 surface antigen.
Parent Case Info
This is a continuation-in-part of U.S. Ser. No. 08/446,872, filed Jun. 6, 1995, pending, which was filed under 35 USC §371 from PCT/US95/01185, filed Feb. 4, 1995; which is a continuation-in-part of U.S. Ser. No. 08/192,325, filed Feb. 4, 1994, now U.S. Patent No. 6,057,133; which is a continuation-in-part of U.S. Ser. No. 08/411,795, filed Apr. 6, 1995, now U.S. Pat. No. 5,604,116; said Ser. No. 08/411,795 was filed under 35 USC §371 from PCT/US93/11197, filed Nov. 22, 1993; which is a continuation-in-part of U.S. Ser. No. 07/981,044, filed Nov. 24, 1992, now abandoned. The noted applications are incorporated herein by reference.
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Continuation in Parts (4)
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