Claims
- 1. A polypeptide comprising a modified human interleukin-3 (hIL-3) amino acid sequence, wherein said modified sequence differs from the sequence of native (1-133) hIL-3 by the replacement of from 4 to about 44 of the residues corresponding to positions 17-118 of native (1-133) hIL-3 by other amino acids, provided that the residues corresponding to positions 101 or 116 are not Ala or Val, respectively;
- wherein said modified sequence optionally further differs from the sequence of native (1-133) hIL-3 by the deletion of from 1 to 14 residues from the N-terminus of native (1-133) hIL-3, the deletion of from 1 to 15 residues from the C-terminus of native (1-133) hIL-3, or both; and
- wherein said 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 Methycellulose assay.
- 2. The polypeptide of claim 1 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 3. The polypeptide of claim 1 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 4. A polypeptide comprising a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:15;
- wherein;
- Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;
- Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;
- Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;
- Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;
- Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;
- Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;
- Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;
- Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;
- Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;
- Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;
- Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;
- Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;
- Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;
- Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;
- Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;
- Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;
- Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu;
- Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;
- Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;
- Xaa at position 36 is Asp, Leu, or Val;
- Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;
- Xaa at position 38 is Asn, or Ala;
- Xaa at position 40 is Leu, Trp, or Arg;
- Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;
- Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;
- Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;
- Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;
- Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;
- Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;
- Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;
- Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;
- Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;
- Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;
- Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;
- Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;
- Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;
- Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;
- Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;
- Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;
- Xaa at position 57 is Asn or Gly;
- Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;
- Xaa at position 59 is Glu, Tyr, His, Leu, Pro, or Arg;
- Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;
- Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;
- Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;
- Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;
- Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;
- Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;
- Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;
- Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;
- Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;
- Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;
- Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;
- Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;
- Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;
- Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;
- Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;
- Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;
- Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;
- Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;
- Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;
- Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;
- Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;
- Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;
- Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;
- Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;
- Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;
- Xaa at position 85 is Leu, Asn, Val, or Gln;
- Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;
- Xaa at position 87 is Leu, Ser, Trp, or Gly;
- Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;
- Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;
- Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;
- Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;
- Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;
- Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;
- Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, 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, Arg, Tyr or Pro;
- Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;
- Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;
- Xaa at position 101 is Asp, 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 can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of SEQ ID NO: 15, and wherein from 6 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 5. The polypeptide of claim 4 wherein 1-14 amino acids are deleted from the N-terminus and 1-15 amino acids are deleted from the C-terminus of said polypeptide.
- 6. The polypeptide of claim 4 wherein;
- Xaa at position 42 is Gly, Asp, Ser, Ile, Leu, Met, Tyr, or Ala;
- Xaa at position 45 is Gln, Val, Met or Asn;
- Xaa at position 46 is Asp, Ser, Gln, His or Val;
- Xaa at position 50 is Glu or Asp;
- Xaa at position 51 is Asn, Pro or Thr;
- Xaa at position 62 is Asn or Pro;
- Xaa at position 76 is Ser or Pro;
- Xaa at position 82 is Leu, Trp, Asp, Asn, Glu, His, Phe, Ser or Tyr;
- Xaa at position 95 is His, Arg, Thr, Asn or Ser;
- Xaa at position 98 is His, Ile, Leu, Ala, Gln, Lys, Met, Ser, Tyr or Val;
- Xaa at position 100 is Lys or Arg;
- Xaa at position 101 is Asp, Pro, His, Asn, Ile or Leu;
- Xaa at position 105 is Asn, or Pro;
- Xaa at position 108 is Arg, Ala, or Ser;
- Xaa at position 116 is Lys, Val, Trp, Ala, His, Phe, or Tyr;
- Xaa at position 121 is Ala, or Ile;
- Xaa at position 122 is Gln, or Ile; and
- Xaa at position 123 is Ala, Met or Glu.
- 7. The polypeptide according to claim 4 comprising a sequence selected from the group consisting of:
- (a) a sequence selected from the group consisting of:
- (18I, 25H, 29R, 32A, 37P, 42A and 45V)-(15-125)human interleukin-3 (SEQ ID NO:66);
- (18I, 25H, 29R, 32N, 37P, 42S and 45M)-(15-125)human interleukin-3 (SEQ ID NO:67);
- (18I, 25H, 29V, 32A, 37S, 42S and 45M)-(15-125)human interleukin-3 (SEQ ID NO:68);
- (51R, 55L, 59L, 62V, 67N and 69E)-(15-125)human interleukin-3 (SEQ ID NO:69);
- (51R, 55L, 60S, 62V, 67N and 69E)-(15-125)human interleukin-3 (SEQ ID NO:70);
- (51R, 55T, 59L, 62V, 67H and 69E)-(15-125)human interleukin-3 (SEQ ID NO:71 );
- (73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A and 105Q)-(15-125)human interleukin-3 (SEQ ID NO:72);
- (73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A and 105Q)-(15-125)human interleukin-3 (SEQ ID NO:73);
- (73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ. ID. NO:76);
- (73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ ID NO:77);
- (73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H and 123E)-(15-125)human interleukin-3 (SEQ ID NO:78);
- (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N and 69E)-(15-125)human interleukin-3 (SEQ ID NO:79);
- (18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H and 69E)-(15-125)human interleukin-3 (SEQ ID NO:80);
- (18I, 25H, 29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N and 69E)-(15-125)human interleukin-3 (SEQ ID NO:81);
- (42D, 45M, 46S, 50D, 95R, 98I, 100R, 116W)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:263);
- (42D, 45M, 46S, 98I, 100R, 116W)-(15-125)human interleukin-3 (SEQ ID NO:406);
- (18I, 19A, 25H, 29R, 32N, 37P, 42S, 45V, 49D, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:276);
- (18I, 19I, 25H, 29R, 32N, 37P, 42S, 45V, 49D, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:277);
- (18I, 23A, 25H, 29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:278);
- (18I, 23A, 25H, 29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ ID NO:279);
- (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,)-(15-119)human interleukin-3 (residues 3-113 of SEQ ID NO:287);
- (18I, 23A, 25H, 29R, 32A, 37P, 42D, 45V, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116V)-(15-119)human interleukin-3 (residues 3-113 of SEQ ID NO:289);
- (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116V)-(15-119)human interleukin-3 (residues 3-113 of SEQ ID NO:307);
- (18I, 20P, 23A, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:317);
- (18I, 23L, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:318);
- (18I, 23A, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, SR, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:319);
- (18I, 19I, 20L, 23A, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:320);
- (18I, 25H, 29R, 32N, 37P, 42S, 45M, 50D, 51S, 55T, 59L, 62P, 63H, 67Q, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:321);
- (18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109L, 112Q, 116S, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:322); and
- (18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109L, 112Q, 116S)-(15-118)human interleukin-3 (residues 3-113 of SEQ ID NO:323); and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 8. A polypeptide comprising a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:19;
- wherein
- Xaa 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, Lys, 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, Arg, 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, or 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 6 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 9. The polypeptide of claim 8 wherein;
- Xaa at position 28 is Gly, Asp, Ser, Ile, Leu, Met, Tyr, or Ala;
- Xaa at position 31 is Gln, Val, Met or Asn;
- Xaa at position 32 is Asp, Ser, Ala, Gln, His or Val;
- Xaa at position 36 is Glu or Asp;
- Xaa at position 37 is Asn, Pro or Thr;
- Xaa at position 48 is Asn or Pro;
- Xaa at position 62 is Ser, or Pro;
- Xaa at position 68 is Leu, Trp, Asp, Asn, Glu, His, Phe, Ser or Tyr;
- Xaa at position 81 is His, Arg, Thr, Asn or Ser;
- Xaa at position 84 is His, Ile, Leu, Ala, Arg, Gln, Lys, Met, Ser, Tyr or Val;
- Xaa at position 86 is Lys or Arg;
- Xaa at position 87 is Asp, Pro, His, Asn, Ile or Leu;
- Xaa at position 91 is Asn or Pro;
- Xaa at position 94 is Arg, Ala, or Ser;
- Xaa at position 102 is Lys, Val, Trp, Ala, His, Phe, or Tyr;
- Xaa at position 107 is Ala, or Ile;
- Xaa at position 108 is Gln, or Ile; and
- Xaa at position 109 is Ala, Met or Glu.
- 10. A polypeptide comprising a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:22;
- wherein
- Xaa at position 3 is Ser, Gly, Asp, or Gln;
- Xaa at position 4 is Asn, His, or Ile;
- Xaa at position 9 is Ile, Ala, Leu, or Gly;
- Xaa at position 11 is Thr, His, or Gln;
- Xaa at position 12 is His or Ala;
- Xaa at position 15 is Gln or Asn;
- Xaa at position 16 is Pro or Gly;
- Xaa at position 18 is Leu, Arg, Asn, or Ala;
- Xaa at position 20 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;
- Xaa at position 21 is Leu, Ala, Asn, or Pro;
- Xaa at position 24 is Asn or Ala;
- Xaa at position 28 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;
- Xaa at position 31 is Gln, Val, Met, Leu, Ala, Asn, Glu or Lys;
- Xaa at position 32 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;
- Xaa at position 36 is Glu, Asn, Ser or Asp;
- Xaa at position 37 is Asn, Arg, Pro, Thr, or His;
- Xaa at position 41 is Arg, Leu, or Gly;
- Xaa at position 42 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;
- Xaa at position 48 is Asn, Pro, or Thr;
- Xaa at position 50 is Ala or Asn;
- Xaa at position 51 is Val or Thr;
- Xaa at position 53 is Ser or Phe;
- Xaa at position 54 is Leu or Phe;
- Xaa at position 55 is Gln, Ala, Glu, or Arg;
- Xaa at position 62 is Ser, Val, Asn, Pro, or Gly;
- Xaa at position 63 is Ile or Leu;
- Xaa at position 65 is Lys, Asn, Met, Arg, Ile, or Gly;
- Xaa at position 66 is Asn, Gly, Glu, or Arg;
- Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;
- Xaa at position 73 is Leu or Ser;
- Xaa at position 74 is Ala or Trp;
- Xaa at position 77 is Ala or Pro;
- Xaa at position 79 is Thr, Asp, or Ala;
- Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;
- Xaa at position 84 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;
- Xaa at position 85 is Ile or Leu;
- Xaa at position 86 is Lys or Arg;
- Xaa at position 87 is Asp, Pro, Met, Lys, His, Pro, Asn, Ile, Leu or Tyr;
- Xaa at position 91 is Asn, Pro, Ser, Ile or Asp;
- Xaa at position 94 is Arg, Ala, or Ser;
- Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;
- Xaa at position 98 is Thr or Gln;
- Xaa at position 102 is Lys, Val, Trp, or Ile;
- Xaa at position 103 is Thr, Ala, His, Phe, Tyr or Ser;
- Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;
- Xaa at position 107 is Ala, Ser, Ile, Pro, or Asp;
- Xaa at position 108 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;
- Xaa at position 109 is Ala, Met, Glu, Ser, or Leu;
- wherein from 6 to 26 of the amino acids designated by Xaa are different from the amino acid sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 11. A polypeptide comprising a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:15;
- wherein Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;
- Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;
- Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;
- Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;
- Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;
- Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;
- Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;
- Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;
- Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;
- Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;
- Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;
- Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;
- Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;
- Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;
- Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;
- Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;
- Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu;
- Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;
- Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;
- Xaa at position 36 is Asp, Leu, or Val;
- Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;
- Xaa at position 38 is Asn, or Ala;
- Xaa at position 40 is Leu, Trp, or Arg;
- Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;
- Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;
- Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;
- Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;
- Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;
- Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;
- Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;
- Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;
- Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;
- Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;
- Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;
- Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;
- Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;
- Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;
- Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;
- Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;
- Xaa at position 57 is Asn or Gly;
- Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;
- Xaa at position 59 is Glu, Tyr, His, Leu, Pro, or Arg;
- Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;
- Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;
- Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;
- Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;
- Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;
- Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;
- Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;
- Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;
- Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;
- Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;
- Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;
- Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;
- Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;
- Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;
- Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;
- Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;
- Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;
- Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;
- Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;
- Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;
- Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;
- Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;
- Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;
- Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;
- Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;
- Xaa at position 85 is Leu, Asn, Val, or Gln;
- Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;
- Xaa at position 87 is Leu, Ser, Trp, or Gly;
- Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;
- Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;
- Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;
- Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;
- Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;
- Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;
- Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, 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, Arg, Tyr or Pro;
- Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;
- Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;
- Xaa at position 101 is Asp;
- Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;
- Xaa at position 103 is Asp, or Ser;
- Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;
- Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;
- Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;
- Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro;
- Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;
- Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp;
- Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;
- Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;
- Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;
- Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;
- Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;
- Xaa at position 116 is Lys;
- Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;
- Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;
- Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;
- Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln;
- Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;
- Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;
- Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;
- wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of SEQ ID NO: 15, and wherein from 4 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a);
- wherein said 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 Methycellulose assay.
- 12. The polypeptide of claim 11 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 13. The polypeptide of claim 11 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 14. The polypeptide of claim 11 wherein 1-15 amino acids are deleted from the C-terminus of said polypeptide and 1-14 amino acids are deleted from the N-terminus of said interleukin-3 polypeptide.
- 15. The polypeptide of claim 11 wherein;
- Xaa at position 42 is Gly, Asp, Ser, Ile, Leu, Met, Tyr, or Ala;
- Xaa at position 45 is Gln, Val, Met or Asn;
- Xaa at position 46 is Asp, Ser, Gln, His or Val;
- Xaa at position 50 is Glu or Asp;
- Xaa at position 51 is Asn, Pro or Thr;
- Xaa at position 62 is Asn or Pro;
- Xaa at position 76 is Ser or Pro;
- Xaa at position 82 is Leu, Trp, Asp, Asn, Glu, His, Phe, Ser or Tyr;
- Xaa at position 95 is His, Arg, Thr, Asn or Ser;
- Xaa at position 98 is His, Ile, Leu, Ala, Gln, Lys, Met, Ser, Tyr or Val;
- Xaa at position 100 is Lys or Arg;
- Xaa at position 101 is Asp;
- Xaa at position 105 is Asn, or Pro;
- Xaa at position 108 is Arg, Ala, or Ser;
- Xaa at position 116 is Lys;
- Xaa at position 121 is Ala, or Ile;
- Xaa at position 122 is Gln, or Ile; and
- Xaa at position 123 is Ala, Met or Giu.
- 16. The polypeptide of claim 15 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 17. The polypeptide of claim 15 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 18. A polypeptide comprising a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:18;
- wherein
- Xaa at position 17 is Ser, Gly, Asp, or Gln;
- Xaa at position 18 is Asn, His, or Ile;
- Xaa at position 23 is Ile, Ala, Leu, or Gly;
- Xaa at position 25 is Thr, His, or Gln;
- Xaa at position 26 is His or Ala;
- Xaa at position 29 is Gln or Asn;
- Xaa at position 30 is Pro or Gly;
- Xaa at position 32 is Leu, Arg, Asn, or Ala;
- Xaa at position 34 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met;
- Xaa at position 35 is Leu, Ala, Asn, or Pro;
- Xaa at position 38 is Asn or Ala;
- Xaa at position 42 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;
- Xaa at position 45 is Gln, Val, Met, Leu, Ala, Asn, Glu, or Lys;
- Xaa at position 46 is Asp, Phe, Ser, Gln, Glu, His, Val or Thr;
- Xaa at position 50 is Glu, Asn, Ser or Asp;
- Xaa at position 51 is Asn, Arg, Pro, Thr, or His;
- Xaa at position 55 is Arg, Leu, or Gly;
- Xaa at position 56 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;
- Xaa at position 62 is Asn, Pro, or Thr;
- Xaa at position 64 is Ala or Asn;
- Xaa at position 65 is Val or Thr;
- Xaa at position 67 is Ser or Phe;
- Xaa at position 68 is Leu or Phe;
- Xaa at position 69 is Gln, Ala, Glu, or Arg;
- Xaa at position 76 is Ser, Val, Asn, Pro, or Gly;
- Xaa at position 77 is Ile or Leu;
- Xaa at position 79 is Lys, Gly, Asn, Met, Arg, Ile, or Gly;
- Xaa at position 80 is Asn, Gly, Glu, or Arg;
- Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;
- Xaa at position 87 is Leu or Ser;
- Xaa at position 88 is Ala or Trp;
- Xaa at position 91 is Ala or Pro;
- Xaa at position 93 is Thr, Asp, or Ala;
- Xaa at position 95 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;
- Xaa at position 98 is His, Ile, Asn, Ala, Thr, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;
- Xaa at position 99 is Ile or Leu;
- Xaa at position 100 is Lys or Arg;
- Xaa at position 101 is Asp;
- Xaa at position 105 is Asn, Pro, Ser, Ile or Asp;
- Xaa at position 108 is Arg, Ala, or Ser;
- Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;
- Xaa at position 112 is Thr or Gln;
- Xaa at position 116 is Lys;
- Xaa at position 117 is Thr or Ser;
- Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;
- Xaa at position 121 is Ala, Ser, Ile, Pro, or Asp;
- Xaa at position 122 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;
- Xaa at position 123 is Ala, Met, Glu, Ser, or Leu;
- wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of SEQ ID NO: 18, and wherein from 4 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a);
- wherein said 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 Methycellulose assay.
- 19. The polypeptide of claim 18 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 20. The polypeptide of claim 18 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 21. The polypeptide of claim 18;
- wherein;
- Xaa at position 28 is Gly, Asp, Ser, Ile, Leu, Met, Tyr, or Ala;
- Xaa at position 31 is Gln, Val, Met or Asn;
- Xaa at position 32 is Asp, Ser, Ala, Gln, His or Val;
- Xaa at position 36 is Glu or Asp;
- Xaa at position 37 is Asn, Pro or Thr;
- Xaa at position 48 is Asn or Pro;
- Xaa at position 62 is Ser, or Pro;
- Xaa at position 68 is Leu, Trp, Asp, Asn, Glu, His, Phe, Ser or Tyr;
- Xaa at position 81 is His, Arg, Thr, Asn or Ser;
- Xaa at position 84 is His, Ile, Leu, Ala, Arg, Gln, Lys, Met, Ser, Tyr or Val;
- Xaa at position 86 is Lys or Arg;
- Xaa at position 87 is Asp;
- Xaa at position 91 is Asn or Pro;
- Xaa at position 94 is Arg, Ala, or Ser;
- Xaa at position 102 is Lys;
- Xaa at position 107 is Ala, or Ile;
- Xaa at position 108 is Gln, or Ile; and
- Xaa at position 109 is Ala, Met or Glu.
- 22. The polypeptide of claim 21 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 23. The polypeptide of claim 21 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 24. A polypeptide comprising a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:19;
- wherein
- Xaa 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, Lys, 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, Arg, 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, or Pro;
- Xaa at position 87 is Asp;
- 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;
- 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 in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a);
- wherein said 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 Methycellulose assay.
- 25. The polypeptide of claim 24 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 26. The polypeptide of claim 24 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 27. A polypeptide according to claim 24 comprising a sequence selected from the group consisting of:
- (a) a sequence selected from the group consisting of:
- (42D, 45M, 46S, 50D)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:259);
- (42D, 45M, 46S, 116W)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:261);
- (42D, 45M, 46S, 50D, 116W)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:262); and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 28. A polypeptide comprising a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:22;
- wherein
- Xaa at position 3 is Ser, Gly, Asp, or Gln;
- Xaa at position 4 is Asn, His, or Ile;
- Xaa at position 9 is Ile, Ala, Leu, or Gly;
- Xaa at position 11 is Thr, His, or Gln;
- Xaa at position 12 is His or Ala;
- Xaa at position 15 is Gln or Asn;
- Xaa at position 16 is Pro or Gly;
- Xaa at position 18 is Leu, Arg, Asn, or Ala;
- Xaa at position 20 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;
- Xaa at position 21 is Leu, Ala, Asn, or Pro;
- Xaa at position 24 is Asn or Ala;
- Xaa at position 28 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;
- Xaa at position 31 is Gln, Val, Met, Leu, Ala, Asn, Glu or Lys;
- Xaa at position 32 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;
- Xaa at position 36 is Glu, Asn, Ser or Asp;
- Xaa at position 37 is Asn, Arg, Pro, Thr, or His;
- Xaa at position 41 is Arg, Leu, or Gly;
- Xaa at position 42 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;
- Xaa at position 48 is Asn, Pro, or Thr;
- Xaa at position 50 is Ala or Asn;
- Xaa at position 51 is Val or Thr;
- Xaa at position 53 is Ser or Phe;
- Xaa at position 54 is Leu or Phe;
- Xaa at position 55 is Gln, Ala, Glu, or Arg;
- Xaa at position 62 is Ser, Val, Asn, Pro, or Gly;
- Xaa at position 63 is Ile or Leu;
- Xaa at position 65 is Lys, Asn, Met, Arg, Ile, or Gly;
- Xaa at position 66 is Asn, Gly, Glu, or Arg;
- Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;
- Xaa at position 73 is Leu or Ser;
- Xaa at position 74 is Ala or Trp;
- Xaa at position 77 is Ala or Pro;
- Xaa at position 79 is Thr, Asp, or Ala;
- Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;
- Xaa at position 84 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;
- Xaa at position 85 is Ile or Leu;
- Xaa at position 86 is Lys or Arg;
- Xaa at position 87 is Asp;
- Xaa at position 91 is Asn, Pro, Ser, Ile or Asp;
- Xaa at position 94 is Arg, Ala, or Ser;
- Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;
- Xaa at position 98 is Thr or Gln;
- Xaa at position 102 is Lys;
- Xaa at position 103 is Thr, Ala, His, Phe, Tyr or Ser;
- Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;
- Xaa at position 107 is Ala, Ser, Ile, Pro, or Asp;
- Xaa at position 108 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;
- Xaa at position 109 is Ala, Met, Glu, Ser, or Leu;
- wherein from 4 to 26 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a);
- wherein said 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 Methycellulose assay.
- 29. The polypeptide of claim 28 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 30. The polypeptide of claim 28 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 31. A polypeptide, comprising a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- Met-Asp-(15-119)human interleukin-3 (18I, 23A, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116V)-(15-119)human interleukin-3 (SEQ ID NO:288);
- Met-Asp-(18I, 23A, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V)-(15-119)human interleukin-3 (SEQ ID NO:290);
- Met-Asp-(18I, 23A, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15119)human interleukin-3 (SEQ DI NO:314);
- Met-Ala-Tyr-Pro-Glu-Thr-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-(18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ ID NO:315); and
- Met-Ala-Tyr-Pro-Glu-Thr-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-(18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ ID NO:316).
- 32. A polypeptide consisting of a modified human interleukin-3 (hIL-3) amino acid sequence,
- wherein said modified sequence differs from the sequence of native (1-133) hIL-3 by the replacement of from 4 to about 44 of the residues corresponding to positions 17-118 of native (1-133) hIL-3 by other amino acids, provided that the residues corresponding to positions 101 or 116 are not Ala or Val, respectively;
- wherein said modified sequence optionally further differs from the sequence of native (1-133) hIL-3 by the deletion of from 1 to 14 residues from the N-terminus of native (1-133) hIL-3, the deletion of from 1 to 15 residues from the C-terminus of native (1-133) hIL-3, or both; and
- wherein said 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 Methycellulose assay.
- 33. The polypeptide of claim 32 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 34. The polypeptide of claim 32 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 35. A polypeptide consisting of a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:15;
- wherein;
- Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;
- Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;
- Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;
- Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;
- Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;
- Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;
- Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;
- Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;
- Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;
- Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;
- Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;
- Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;
- Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;
- Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;
- Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;
- Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;
- Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu;
- Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;
- Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;
- Xaa at position 36 is Asp, Leu, or Val;
- Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;
- Xaa at position 38 is Asn, or Ala;
- Xaa at position 40 is Leu, Trp, or Arg;
- Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;
- Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;
- Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;
- Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;
- Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;
- Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;
- Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;
- Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;
- Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;
- Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;
- Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;
- Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;
- Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;
- Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;
- Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;
- Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;
- Xaa at position 57 is Asn or Gly;
- Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;
- Xaa at position 59 is Glu, Tyr, His, Leu, Pro, or Arg;
- Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;
- Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;
- Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;
- Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;
- Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;
- Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;
- Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;
- Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;
- Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;
- Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;
- Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;
- Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;
- Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;
- Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;
- Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;
- Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;
- Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;
- Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;
- Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;
- Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;
- Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;
- Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;
- Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;
- Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;
- Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;
- Xaa at position 85 is Leu, Asn, Val, or Gln;
- Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;
- Xaa at position 87 is Leu, Ser, Trp, or Gly;
- Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;
- Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;
- Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;
- Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;
- Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;
- Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;
- Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, 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, Arg, Tyr or Pro;
- Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;
- Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;
- Xaa at position 101 is Asp, 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 can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of SEQ ID NO:15, and wherein from 6 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 36. The polypeptide of claim 35 wherein 1-14 amino acids are deleted from the N-terminus and 1-15 amino acids are deleted from the C-terminus of said polypeptide.
- 37. The polypeptide of claim 35 wherein;
- Xaa at position 42 is Gly, Asp, Ser, Ile, Leu, Met, Tyr, or Ala;
- Xaa at position 45 is Gln, Val, Met or Asn;
- Xaa at position 46 is Asp, Ser, Gln, His or Val;
- Xaa at position 50 is Glu or Asp;
- Xaa at position 51 is Asn, Pro or Thr;
- Xaa at position 62 is Asn or Pro;
- Xaa at position 76 is Ser or Pro;
- Xaa at position 82 is Leu, Trp, Asp, Asn, Glu, His, Phe, Ser or Tyr;
- Xaa at position 95 is His, Arg, Thr, Asn or Ser;
- Xaa at position 98 is His, Ile, Leu, Ala, Gln, Lys, Met, Ser, Tyr or Val;
- Xaa at position 100 is Lys or Arg;
- Xaa at position 101 is Asp, Pro, His, Asn, Ile or Leu;
- Xaa at position 105 is Asn, or Pro;
- Xaa at position 108 is Arg, Ala, or Ser;
- Xaa at position 116 is Lys, Val, Trp, Ala, His, Phe, or Tyr;
- Xaa at position 121 is Ala, or Ile;
- Xaa at position 122 is Gln, or Ile; and
- Xaa at position 123 is Ala, Met or Glu.
- 38. The polypeptide according to claim 35 comprising a sequence selected from the group consisting of:
- (a) a sequence selected from the group consisting of:
- (18I, 25H, 29R, 32A, 37P, 42A and 45V)-(15-125)human interleukin-3 (SEQ ID NO:66);
- (18I, 25H, 29R, 32N, 37P, 42S and 45M)-(15-125)human interleukin-3 (SEQ ID NO:67);
- (18I, 25H, 29V, 32A, 37S, 42S and 45M)-(15-125)human interleukin-3 (SEQ ID NO:68);
- (51R, 55L, 59L, 62V, 67N and 69E)-(15-125)human interleukin-3 (SEQ ID NO:69);
- (51R, 55L, 60S, 62V, 67N and 69E)-(15-125)human interleukin-3 (SEQ ID NO:70);
- (51R, 55T, 59L, 62V, 67H and 69E)-(15-125)human interleukin-3 (SEQ ID NO:71);
- (73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A and 105Q)-(15-125)human interleukin-3 (SEQ ID NO:72);
- (73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A and 105Q)-(15-125)human interleukin-3 (SEQ ID NO:73);
- (73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ. ID. NO:76);
- (73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ ID NO:77);
- (73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H and 123E)-(15-125)human interleukin-3 (SEQ ID NO:78);
- (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N and 69E)-(15-125)human interleukin-3 (SEQ ID NO:79)
- (18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H and 69E)-(15-125)human interleukin-3 (SEQ ID NO:80);
- (18I, 25H, 29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N and 69E)-(15-125)human interleukin-3 (SEQ ID NO:81);
- (42D, 45M, 46S, 50D, 95R, 98I, 100R, 116W)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:263);
- (42D, 45M, 46S, 98I, 100R, 116W)-(15-125)human interleukin-3 (SEQ ID NO:406);
- (18I, 19A, 25H, 29R, 32N, 37P, 42S, 45V, 49D, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:276);
- (18I, 19I, 25H, 29R, 32N, 37P, 42S, 45V, 49D, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:277);
- (18I, 23A, 25H, 29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:278);
- (18I, 23A, 25H, 29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ ID NO:279);
- (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,)-(15-119)human interleukin-3 (residues 3-113 of SEQ ID NO:287);
- (18I, 23A, 25H, 29R, 32A, 37P, 42D, 45V, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116V)-(15-119)human interleukin-3 (residues 3-113 of SEQ ID NO:289);
- (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116V)-(15-119)human interleukin-3 (residues 3-113 of SEQ ID NO:307);
- (18I, 20P, 23A, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, SiR, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:317);
- (18I, 23L, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, SiR, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:318);
- (18I, 23A, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, SiR, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human in.terleukin-3 (residues 3-113 of SEQ ID NO:319);
- (18I, 19I, 20L, 23A, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:320);
- (18I, 25H, 29R, 32N, 37P, 42S, 45M, 50D, 51S, 55T, 59L, 62P, 63H, 67Q, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:321);
- (18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109L, 112Q, 116S, 120Q and 123E)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:322); and
- (18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109L, 112Q, 116S)-(15-118)human interleukin-3 (residues 3-113 of SEQ ID NO:323); and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 39. A polypeptide consisting of a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:19;
- wherein
- Xaa 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, Lys, 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, Arg, 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, or 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 6 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 40. The polypeptide of claim 39 wherein 1-15 amino acids are deleted from the C-terminus of said polypeptide and 1-14 amino acids are deleted from the N-terminus of said interleukin-3 polypeptide.
- 41. A polypeptide consisting of a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:22;
- wherein
- Xaa at position 3 is Ser, Gly, Asp, or Gln;
- Xaa at position 4 is Asn, His, or Ile;
- Xaa at position 9 is Ile, Ala, Leu, or Gly;
- Xaa at position 11 is Thr, His, or Gln;
- Xaa at position 12 is His or Ala;
- Xaa at position 15 is Gln or Asn;
- Xaa at position 16 is Pro or Gly;
- Xaa at position 18 is Leu, Arg, Asn, or Ala;
- Xaa at position 20 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;
- Xaa at position 21 is Leu, Ala, Asn, or Pro;
- Xaa at position 24 is Asn or Ala;
- Xaa at position 28 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;
- Xaa at position 31 is Gln, Val, Met, Leu, Ala, Asn, Glu or Lys;
- Xaa at position 32 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;
- Xaa at position 36 is Glu, Asn, Ser or Asp;
- Xaa at position 37 is Asn, Arg, Pro, Thr, or His;
- Xaa at position 41 is Arg, Leu, or Gly;
- Xaa at position 42 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;
- Xaa at position 48 is Asn, Pro, or Thr;
- Xaa at position 50 is Ala or Asn;
- Xaa at position 51 is Val or Thr;
- Xaa at position 53 is Ser or Phe;
- Xaa at position 54 is Leu or Phe;
- Xaa at position 55 is Gln, Ala, Glu, or Arg;
- Xaa at position 62 is Ser, Val, Asn, Pro, or Gly;
- Xaa at position 63 is Ile or Leu;
- Xaa at position 65 is Lys, Asn, Met, Arg, Ile, or Gly;
- Xaa at position 66 is Asn, Gly, Glu, or Arg;
- Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;
- Xaa at position 73 is Leu or Ser;
- Xaa at position 74 is Ala or Trp;
- Xaa at position 77 is Ala or Pro;
- Xaa at position 79 is Thr, Asp, or Ala;
- Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;
- Xaa at position 84 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;
- Xaa at position 85 is Ile or Leu;
- Xaa at position 86 is Lys or Arg;
- Xaa at position 87 is Asp, Pro, Met, Lys, His, Pro, Asn, Ile, Leu or Tyr;
- Xaa at position 91 is Asn, Pro, Ser, Ile or Asp;
- Xaa at position 94 is Arg, Ala, or Ser;
- Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;
- Xaa at position 98 is Thr or Gln;
- Xaa at position 102 is Lys, Val, Trp, or Ile;
- Xaa at position 103 is Thr, Ala, His, Phe, Tyr or Ser;
- Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;
- Xaa at position 107 is Ala, Ser, Ile, Pro, or Asp;
- Xaa at position 108 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;
- Xaa at position 109 is Ala, Met, Glu, Ser, or Leu;
- wherein from 6 to 26 of the amino acids designated by Xaa are different from the amino acid sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 42. The polypeptide of claim 41 wherein;
- Xaa at position 28 is Gly, Asp, Ser, Ile, Leu, Met, Tyr, or Ala;
- Xaa at position 31 is Gln, Val, Met or Asn;
- Xaa at position 32 is Asp, Ser, Ala, Gln, His or Val;
- Xaa at position 36 is Glu or Asp;
- Xaa at position 37 is Asn, Pro or Thr;
- Xaa at position 48 is Asn or Pro;
- Xaa at position 62 is Ser, or Pro;
- Xaa at position 68 is Leu, Trp, Asp, Asn, Glu, His, Phe, Ser or Tyr;
- Xaa at position 81 is His, Arg, Thr, Asn or Ser;
- Xaa at position 84 is His, Ile, Leu, Ala, Arg, Gln, Lys, Met, Ser, Tyr or Val;
- Xaa at position 86 is Lys or Arg;
- Xaa at position 87 is Asp, Pro, His, Asn, Ile or Leu;
- Xaa at position 91 is Asn or Pro;
- Xaa at position 94 is Arg, Ala, or Ser;
- Xaa at position 102 is Lys, Val, Trp, Ala, His, Phe, or Tyr;
- Xaa at position 107 is Ala, or Ile;
- Xaa at position 108 is Gln, or Ile; and
- Xaa at position 109 is Ala, Met or Glu.
- 43. A polypeptide consisting of a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:15;
- wherein
- Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;
- Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;
- Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;
- Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;
- Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;
- Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;
- Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;
- Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;
- Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;
- Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;
- Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;
- Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;
- Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;
- Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;
- Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;
- Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;
- Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu;
- Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;
- Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;
- Xaa at position 36 is Asp, Leu, or Val;
- Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;
- Xaa at position 38 is Asn, or Ala;
- Xaa at position 40 is Leu, Trp, or Arg;
- Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;
- Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;
- Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;
- Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;
- Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;
- Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;
- Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;
- Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;
- Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;
- Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;
- Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;
- Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;
- Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;
- Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;
- Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;
- Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;
- Xaa at position 57 is Asn or Gly;
- Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;
- Xaa at position 59 is Glu, Tyr, His, Leu, Pro, or Arg;
- Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;
- Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;
- Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;
- Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;
- Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;
- Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;
- Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;
- Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;
- Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;
- Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;
- Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;
- Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;
- Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;
- Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;
- Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;
- Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;
- Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;
- Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;
- Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;
- Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;
- Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;
- Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;
- Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;
- Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;
- Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;
- Xaa at position 85 is Leu, Asn, Val, or Gln;
- Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;
- Xaa at position 87 is Leu, Ser, Trp, or Gly;
- Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;
- Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;
- Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;
- Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;
- Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;
- Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;
- Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, 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, Arg, Tyr or Pro;
- Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;
- Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;
- Xaa at position 101 is Asp;
- Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;
- Xaa at position 103 is Asp, or Ser;
- Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;
- Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;
- Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;
- Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro;
- Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;
- Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp;
- Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;
- Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;
- Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;
- Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;
- Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;
- Xaa at position 116 is Lys;
- Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;
- Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;
- Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;
- Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln;
- Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;
- Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;
- Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;
- wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of SEQ ID NO:15, and wherein from 4 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a);
- wherein said 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 Methycellulose assay.
- 44. The polypeptide of claim 43 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 45. The polypeptide of claim 43 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 46. The polypeptide of claim 43 wherein;
- Xaa at position 42 is Gly, Asp, Ser, Ile, Leu, Met, Tyr, or Ala;
- Xaa at position 45 is Gln, Val, Met or Asn;
- Xaa at position 46 is Asp, Ser, Gln, His or Val;
- Xaa at position 50 is Glu or Asp;
- Xaa at position 51 is Asn, Pro or Thr;
- Xaa at position 62 is Asn or Pro;
- Xaa at position 76 is Ser or Pro;
- Xaa at position 82 is Leu, Trp, Asp, Asn, Glu, His, Phe, Ser or Tyr;
- Xaa at position 95 is His, Arg, Thr, Asn or Ser;
- Xaa at position 98 is His, Ile, Leu, Ala, Gln, Lys, Met, Ser, Tyr or Val;
- Xaa at position 100 is Lys or Arg;
- Xaa at position 101 is Asp;
- Xaa at position 105 is Asn, or Pro;
- Xaa at position 108 is Arg, Ala, or Ser;
- Xaa at position 116 is Lys;
- Xaa at position 121 is Ala, or Ile;
- Xaa at position 122 is Gln, or Ile; and
- Xaa at position 123 is Ala, Met or Glu.
- 47. The polypeptide of claim 46 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 48. The polypeptide of claim 46 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 49. A polypeptide consisting of a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:18;
- wherein
- Xaa at position 17 is Ser, Gly, Asp, or Gln;
- Xaa at position 18 is Asn, His, or Ile;
- Xaa at position 23 is Ile, Ala, Leu, or Gly;
- Xaa at position 25 is Thr, His, or Gln;
- Xaa at position 26 is His or Ala;
- Xaa at position 29 is Gln or Asn;
- Xaa at position 30 is Pro or Gly;
- Xaa at position 32 is Leu, Arg, Asn, or Ala;
- Xaa at position 34 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met;
- Xaa at position 35 is Leu, Ala, Asn, or Pro;
- Xaa at position 38 is Asn or Ala;
- Xaa at position 42 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;
- Xaa at position 45 is Gln, Val, Met, Leu, Ala, Asn, Glu, or Lys;
- Xaa at position 46 is Asp, Phe, Ser, Gln, Glu, His, Val or Thr;
- Xaa at position 50 is Glu, Asn, Ser or Asp;
- Xaa at position 51 is Asn, Arg, Pro, Thr, or His;
- Xaa at position 55 is Arg, Leu, or Gly;
- Xaa at position 56 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;
- Xaa at position 62 is Asn, Pro, or Thr;
- Xaa at position 64 is Ala or Asn;
- Xaa at position 65 is Val or Thr;
- Xaa at position 67 is Ser or Phe;
- Xaa at position 68 is Leu or Phe;
- Xaa at position 69 is Gln, Ala, Glu, or Arg;
- Xaa at position 76 is Ser, Val, Asn, Pro, or Gly;
- Xaa at position 77 is Ile or Leu;
- Xaa at position 79 is Lys, Gly, Asn, Met, Arg, Ile, or Gly;
- Xaa at position 80 is Asn, Gly, Glu, or Arg;
- Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;
- Xaa at position 87 is Leu or Ser;
- Xaa at position 88 is Ala or Trp;
- Xaa at position 91 is Ala or Pro;
- Xaa at position 93 is Thr, Asp, or Ala;
- Xaa at position 95 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;
- Xaa at position 98 is His, Ile, Asn, Ala, Thr, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;
- Xaa at position 99 is Ile or Leu;
- Xaa at position 100 is Lys or Arg;
- Xaa at position 101 is Asp;
- Xaa at position 105 is Asn, Pro, Ser, Ile or Asp;
- Xaa at position 108 is Arg, Ala, or Ser;
- Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;
- Xaa at position 112 is Thr or Gln;
- Xaa at position 116 is Lys;
- Xaa at position 117 is Thr or Ser;
- Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;
- Xaa at position 121 is Ala, Ser, Ile, Pro, or Asp;
- Xaa at position 122 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;
- Xaa at position 123 is Ala, Met, Glu, Ser, or Leu;
- wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of SEQ ID NO:18, and wherein from 4 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a);
- wherein said 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 Methycellulose assay.
- 50. The polypeptide of claim 49 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 51. A polypeptide according to claim 50 comprising a sequence selected from the group consisting of:
- (a) a sequence selected from the group consisting of:
- (42D, 45M, 46S, 50D)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:259);
- (42D, 45M, 46S, 116W)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:261); and
- (42D, 45M, 46S, 50D, 116W)-(15-125)human interleukin-3 (residues 3-113 of SEQ ID NO:262); and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a).
- 52. The polypeptide of claim 49 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 53. The polypeptide of claim 49;
- wherein;
- Xaa at position 28 is Gly, Asp, Ser, Ile, Leu, Met, Tyr, or Ala;
- Xaa at position 31 is Gln, Val, Met or Asn;
- Xaa at position 32 is Asp, Ser, Ala, Gln, His or Val;
- Xaa at position 36 is Glu or Asp;
- Xaa at position 37 is Asn, Pro or Thr;
- Xaa at position 48 is Asn or Pro;
- Xaa at position 62 is Ser, or Pro;
- Xaa at position 68 is Leu, Trp, Asp, Asn, Glu, His, Phe, Ser or Tyr;
- Xaa at position 81 is His, Arg, Thr, Asn or Ser;
- Xaa at position 84 is His, Ile, Leu, Ala, Arg, Gln, Lys, Met, Ser, Tyr or Val;
- Xaa at position 86 is Lys or Arg;
- Xaa at position 87 is Asp;
- Xaa at position 91 is Asn or Pro;
- Xaa at position 94 is Arg, Ala, or Ser;
- Xaa at position 102 is Lys;
- Xaa at position 107 is Ala, or Ile;
- Xaa at position 108 is Gln, or Ile; and
- Xaa at position 109 is Ala, Met or Glu.
- 54. The polypeptide of claim 53 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 55. The polypeptide of claim 53 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 56. A polypeptide consisting of a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:19;
- wherein
- Xaa 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, Lys, 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, Arg, 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, or Pro;
- Xaa at position 87 is Asp;
- 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;
- 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 in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a);
- wherein said 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 Methycellulose assay.
- 57. The polypeptide of claim 56 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 58. The polypeptide of claim 56 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 59. A polypeptide consisting of a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- (a) a sequence of SEQ ID NO:22;
- wherein
- Xaa at position 3 is Ser, Gly, Asp, or Gln;
- Xaa at position 4 is Asn, His, or Ile;
- Xaa at position 9 is Ile, Ala, Leu, or Gly;
- Xaa at position 11 is Thr, His, or Gln;
- Xaa at position 12 is His or Ala;
- Xaa at position 15 is Gln or Asn;
- Xaa at position 16 is Pro or Gly;
- Xaa at position 18 is Leu, Arg, Asn, or Ala;
- Xaa at position 20 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;
- Xaa at position 21 is Leu, Ala, Asn, or Pro;
- Xaa at position 24 is Asn or Ala;.
- Xaa at position 28 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;
- Xaa at position 31 is Gln, Val, Met, Leu, Ala, Asn, Glu or Lys;
- Xaa at position 32 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;
- Xaa at position 36 is Glu, Asn, Ser or Asp;
- Xaa at position 37 is Asn, Arg, Pro, Thr, or His;
- Xaa at position 41 is Arg, Leu, or Gly;
- Xaa at position 42 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;
- Xaa at position 48 is Asn, Pro, or Thr;
- Xaa at position 50 is Ala or Asn;
- Xaa at position 51 is Val or Thr;
- Xaa at position 53 is Ser or Phe;
- Xaa at position 54 is Leu or Phe;
- Xaa at position 55 is Gln, Ala, Glu, or Arg;
- Xaa at position 62 is Ser, Val, Asn, Pro, or Gly;
- Xaa at position 63 is Ile or Leu;
- Xaa at position 65 is Lys, Asn, Met, Arg, Ile, or Gly;
- Xaa at position 66 is Asn, Gly, Glu, or Arg;
- Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;
- Xaa at position 73 is Leu or Ser;
- Xaa at position 74 is Ala or Trp;
- Xaa at position 77 is Ala or Pro;
- Xaa at position 79 is Thr, Asp, or Ala;
- Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;
- Xaa at position 84 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;
- Xaa at position 85 is Ile or Leu;
- Xaa at position 86 is Lys or Arg;
- Xaa at position 87 is Asp;
- Xaa at position 91 is Asn, Pro, Ser, Ile or Asp;
- Xaa at position 94 is Arg, Ala, or Ser;
- Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;
- Xaa at position 98 is Thr or Gln;
- Xaa at position 102 is Lys;
- Xaa at position 103 is Thr, Ala, His, Phe, Tyr or Ser;
- Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;
- Xaa at position 107 is Ala, Ser, Ile, Pro, or Asp;
- Xaa at position 108 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;
- Xaa at position 109 is Ala, Met, Glu, Ser, or Leu;
- wherein from 4 to 26 of the amino acids designated by Xaa are different from the corresponding amino acids in the sequence of native (1-133) human interleukin-3; and
- (b) a sequence having an N-terminal methionine residue, alanine residue, or methionine-alanine dipeptide immediately preceding a sequence according to (a);
- wherein said 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 Methycellulose assay.
- 60. The polypeptide of claim 59 wherein said polypeptide has at least five times greater activity than native human interleukin-3.
- 61. The polypeptide of claim 59 wherein said polypeptide has at least ten times greater activity than native human interleukin-3.
- 62. A polypeptide, consisting of a modified human interleukin-3 amino acid sequence selected from the group consisting of:
- Met-Asp-(15-119)human interleukin-3 (18I, 23A, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116V)-(15-119)human interleukin-3 (SEQ ID NO:288);
- Met-Asp-(18I, 23A, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V)-(15-119)human interleukin-3 (SEQ ID NO:290);
- Met-Asp-(18I, 23A, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-119)human interleukin-3 (SEQ DI NO:314);
- Met-Ala-Tyr-Pro-Glu-Thr-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-(18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ ID NO:315); and
- Met-Ala-Tyr-Pro-Glu-Thr-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-(18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)-(15-125)human interleukin-3 (SEQ ID NO:316).
- 63. A pharmaceutical composition comprising a polypeptide of any one of claims 1-31, pharmaceutically acceptable carrier.
- 64. A pharmaceutical composition comprising a polypeptide of any one of claims 32-62 and a pharmaceutically acceptable carrier.
- 65. A nucleic acid molecule encoding a polypeptide of claim 1.
- 66. A nucleic acid molecule according to claim 65 comprising a nucleotide sequence, selected from the group consisting of:
- SEQ ID NO:106;
- SEQ ID NO:107;
- SEQ ID NO:108;
- SEQ ID NO:109;
- SEQ ID NO:110;
- SEQ ID NO:111;
- SEQ ID NO:112;
- SEQ ID NO:113;
- SEQ ID NO:114;
- SEQ ID NO:115;
- SEQ ID NO:116;
- SEQ ID NO:117;
- SEQ ID NO:118;
- SEQ ID NO:119;
- SEQ ID NO:120;
- SEQ ID NO:121;
- SEQ ID NO:122;
- SEQ ID NO:123;
- SEQ ID NO:124;
- SEQ ID NO:125;
- SEQ ID NO:126;
- SEQ ID NO:127;
- SEQ ID NO:160;
- SEQ ID NO:161;
- SEQ ID NO:398;
- SEQ ID NO:399;
- SEQ ID NO:346;
- SEQ ID NO:347;
- SEQ ID NO:303;
- SEQ ID NO:404;
- SEQ ID NO:405;
- SEQ ID NO:344;
- SEQ ID NO:345;
- SEQ ID NO:355;
- SEQ ID NO:356;
- SEQ ID NO:357;
- SEQ ID NO:358;
- SEQ ID NO:375;
- SEQ ID NO:384;
- SEQ ID NO:385;
- SEQ ID NO:386;
- SEQ ID NO:387;
- SEQ ID NO:388;
- SEQ ID NO:389;
- SEQ ID NO:390;
- SEQ ID NO:391; and
- SEQ ID NO:392.
- 67. A nucleic acid molecule according to claim 65 comprising a nuleotide sequence selected from the group consisting of:
- SEQ ID NO:106;
- SEQ ID NO:107;
- SEQ ID NO:108;
- SEQ ID NO:109;
- SEQ ID NO:110;
- SEQ ID NO:111;
- SEQ ID NO:112;
- SEQ ID NO:113;
- SEQ ID NO:114;
- SEQ ID NO:115;
- SEQ ID NO:116;
- SEQ ID NO:117;
- SEQ ID NO:118;
- SEQ ID NO:119;
- SEQ ID NO:120;
- SEQ ID NO:121;
- SEQ ID NO:122;
- SEQ ID NO:123;
- SEQ ID NO:124;
- SEQ ID NO:125;
- SEQ ID NO:126;
- SEQ ID NO:127;
- SEQ ID NO:160;
- SEQ ID NO:161;
- SEQ ID NO:398;
- SEQ ID NO:399;
- SEQ ID NO:346;
- SEQ ID NO:347;
- SEQ ID NO:303;
- SEQ ID NO:404;
- SEQ ID NO:405;
- SEQ ID NQ:344;
- SEQ ID NO:345;
- SEQ ID NO:355;
- SEQ ID NO:356;
- SEQ ID NO:357;
- SEQ ID NO:358;
- SEQ ID NO:375;
- SEQ ID NO:384;
- SEQ ID NO:385;
- SEQ ID NO:386;
- SEQ ID NO:387;
- SEQ ID NO:388;
- SEQ ID NO:389;
- SEQ ID NO:390;
- SEQ ID NO:391; and
- SEQ ID NO:392.
- 68. A nucleic acid molecule encoding a polypeptide of claim 2.
- 69. A nucleic acid molecule encoding a polypeptide of claim 3.
- 70. A nucleic acid molecule encoding a polypeptide of claim 4.
- 71. A nucleic acid molecule encoding a polypeptide of claim 5.
- 72. A nucleic acid molecule encoding a polypeptide of claim 6.
- 73. A nucleic acid molecule encoding a polypeptide of claim 7.
- 74. A nucleic acid molecule encoding a polypeptide of claim 8.
- 75. A nucleic acid molecule encoding a polypeptide of claim 9.
- 76. A nucleic acid molecule encoding a polypeptide of claim 10.
- 77. A nucleic acid molecule encoding a polypeptide of claim 11.
- 78. A nucleic acid molecule encoding a polypeptide of claim 12.
- 79. A nucleic acid molecule encoding a polypeptide of claim 13.
- 80. A nucleic acid molecule encoding a polypeptide of claim 14.
- 81. A nucleic acid molecule encoding a polypeptide of claim 15.
- 82. A nucleic acid molecule encoding a polypeptide of claim 16.
- 83. A nucleic acid molecule encoding a polypeptide of claim 17.
- 84. A nucleic acid molecule encoding a polypeptide of claim 18.
- 85. A nucleic acid molecule encoding a polypeptide of claim 19.
- 86. A nucleic acid molecule encoding a polypeptide of claim 20.
- 87. A nucleic acid molecule encoding a polypeptide of claim 21.
- 88. A nucleic acid molecule encoding a polypeptide of claim 22.
- 89. A nucleic acid molecule encoding a polypeptide of claim 23.
- 90. A nucleic acid molecule encoding a polypeptide of claim 24.
- 91. A nucleic acid molecule encoding a polypeptide of claim 25.
- 92. A nucleic acid molecule encoding a polypeptide of claim 26.
- 93. A nucleic acid molecule encoding a polypeptide of claim 27.
- 94. A nucleic acid molecule encoding a polypeptide of claim 28.
- 95. A nucleic acid molecule encoding a polypeptide of claim 29.
- 96. A nucleic acid molecule encoding a polypeptide of claim 30.
- 97. A nucleic acid molecule encoding a polypeptide of claim 31.
- 98. A nucleic acid molecule encoding a polypeptide of claim 32.
- 99. A nucleic acid molecule encoding a polypeptide of claim 33.
- 100. A nucleic acid molecule encoding a polypeptide of claim 34.
- 101. A nucleic acid molecule encoding a polypeptide of claim 35.
- 102. A nucleic acid molecule encoding a polypeptide of claim 36.
- 103. A nucleic acid molecule encoding a polypeptide of claim 37.
- 104. A nucleic acid molecule encoding a polypeptide of claim 38.
- 105. A nucleic acid molecule encoding a polypeptide of claim 39.
- 106. A nucleic acid molecule encoding a polypeptide of claim 40.
- 107. A nucleic acid molecule encoding a polypeptide of claim 41.
- 108. A nucleic acid molecule encoding a polypeptide of claim 42.
- 109. A nucleic acid molecule encoding a polypeptide of claim 43.
- 110. A nucleic acid molecule encoding a polypeptide of claim 44.
- 111. A nucleic acid molecule encoding a polypeptide of claim 45.
- 112. A nucleic acid molecule encoding a polypeptide of claim 46.
- 113. A nucleic acid molecule encoding a polypeptide of claim 47.
- 114. A nucleic acid molecule encoding a polypeptide of claim 48.
- 115. A nucleic acid molecule encoding a polypeptide of claim 49.
- 116. A nucleic acid molecule encoding a polypeptide of claim 50.
- 117. A nucleic acid molecule encoding a polypeptide of claim 51.
- 118. A nucleic acid molecule encoding a polypeptide of claim 52.
- 119. A nucleic acid molecule encoding a polypeptide of claim 53.
- 120. A nucleic acid molecule encoding a polypeptide of claim 54.
- 121. A nucleic acid molecule encoding a polypeptide of claim 55.
- 122. A nucleic acid molecule encoding a polypeptide of claim 56.
- 123. A nucleic acid molecule encoding a polypeptide of claim 57.
- 124. A nucleic acid molecule encoding a polypeptide of claim 58.
- 125. A nucleic acid molecule encoding a polypeptide of claim 59.
- 126. A nucleic acid molecule encoding a polypeptide of claim 60.
- 127. A nucleic acid molecule encoding a polypeptide of claim 61.
- 128. A nucleic acid molecule encoding a polypeptide of claim 62.
- 129. A method of producing a polypeptide comprising: growing under suitable nutrient conditions, a host cell transformed or transfected with a replicable vector comprising the nucleic acid molecule of any one of claims 65, 66, or 68-97 in a manner allowing expression of said polypeptide and recovering said polypeptide.
- 130. A method of producing a polypeptide comprising: growing under suitable nutrient conditions, a host cell transformed or transfected with a replicable vector comprising the nucleic acid molecule of any one of claims 67 or 98-128 in a manner allowing expression of said polypeptide and recovering said polypeptide.
Parent Case Info
This is a divisional of application Ser. No. 08/411,795, now U.S. Pat. No. 5,604,116, which was filed as International Application serial number PCT/US93/11197 on Nov. 22, 1993, and as a national stage (35 U.S.C. .sctn. 371) application on Apr. 6, 1995. Application Ser. No. PCT/US93/11197 is in turn is a continuation-in-part of U.S. application Ser. No. 07/981,044, filed Nov. 24, 1992, and now abandoned. The noted applications are incorporated herein by reference.
US Referenced Citations (6)
Foreign Referenced Citations (1)
Number |
Date |
Country |
275598 |
Jul 1988 |
EPX |
Divisions (1)
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Number |
Date |
Country |
Parent |
411795 |
Apr 1995 |
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Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
981044 |
Nov 1992 |
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