Interleukin-3 (IL-3) mutant polypeptides

FIELD OF THE INVENTION

[0002] The present invention relates to mutants or variants of human interleukin-3 (hIL-3) which contain one or more amino acid substitutions and which may have portions of the native hIL-3 molecule deleted. These hIL-3 single and multiple mutation polypeptides retain one or more activities of native hIL-3 and may also show improved hematopoietic cell-stimulating activity and/or an improved activity profile which may include reduction of undesirable biological activities associated with native hIL-3.

BACKGROUND OF THE INVENTION

[0003] Colony stimulating factors (CSFs) which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. CSFs in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively while GM-CSF and interleukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies.

[0004] Because of its ability to stimulate the proliferation of a number of different cell types and to support the growth and proliferation of progenitor cells, IL-3 has potential for therapeutic use in restoring hematopoietic cells to normal amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and chemotherapy.

[0005] Interleukin-3 (IL-3) is a hematopoietic growth factor which has the property of being able to promote the survival, growth and differentiation of hematopoietic cells. Among the biological properties of IL-3 are the ability (a) to support the growth and differentiation of progenitor cells committed to all, or virtually all, blood cell lineages; (b) to interact with early multipotential stem cells; (c) to sustain the growth of pluripotent precursor cells; (d) to stimulate proliferation of chronic myelogenous leukemia (CML) cells; (e) to stimulate proliferation of mast cells, eosinophils and basophils; (f) to stimulate DNA synthesis by human acute myelogenous leukemia (AML) cells; (g) to prime cells for production of leukotrienes and histamines; (h) to induce leukocyte chemotaxis; and (i) to induce cell surface molecules needed for leukocyte adhesion.

[0006] Mature human interleukin-3 (hIL-3) consists of 133 amino acids. It has one disulfide bridge and two potential glycosylation sites (Yang, et al., CELL 47:3 (1986)).

[0007] Murine IL-3 (mIL-3) was first identified by Ihle, et al., J. IMMUNOL. 126:2184 (1981) as a factor which induced expression of a T cell associated enzyme, 20-hydroxysteroid dehydrogenase. The factor was purified to homogeneity and shown to regulate the growth and differentiation of numerous subclasses of early hematopoietic and lymphoid progenitor cells.

[0008] In 1984, cDNA clones coding for murine IL-3 were isolated (Fung, et al., NATURE 307:233 (1984) and Yokota, et al., PROC. NATL. ACAD. SCI. USA 81:1070 (1984)). The murine DNA sequence coded for a polypeptide of 166 amino acids including a putative signal peptide.

[0009] The gibbon IL-3 sequence was obtained using a gibbon CDNA expression library. The gibbon IL-3 sequence was then used as a probe against a human genomic library to obtain a human IL-3 sequence.

[0010] Gibbon and human genomic DNA homologues of the murine IL-3 sequence were disclosed by Yang, et al., CELL 47:3 (1986). The human sequence reported by Yang, et al. included a serine residue at position 8 of the mature protein sequence. Following this finding, others reported isolation of Pro8 hIL-3 cDNAs having proline at position 8 of the protein sequence. Thus it appears that there may be two allelic forms of hIL-3.

[0011] Dorssers, et al., GENE 55:115 (1987), found a clone from a human cDNA library which hybridized with mIL-3. This hybridization was the result of the high degree of homology between the 3′ noncoding regions of mIL-3 and hIL-3. This cDNA coded for an hIL-3 (Pro8) sequence.

[0012] U.S. Pat. No. 4,877,729 and U.S. Pat. No. 4,959,455 disclose human IL-3 and gibbon IL-3 cDNAs and the protein sequences for which they code. The hIL-3 disclosed has serine rather than proline at position 8 in the protein sequence.

[0013] Clark-Lewis, et al., SCIENCE 231:134 (1986) performed a functional analysis of murine IL-3 analogues synthesized with an automated peptide synthesizer. The authors concluded that the stable tertiary structure of the complete molecule was required for full activity. A study on the role of the disulfide bridges showed that replacement of all four cysteines by alanine gave a molecule with {fraction (1/500)}th the activity as the native molecule. Replacement of two of the four Cys residues by Ala(Cys79, Cys140→Ala79, Ala140) resulted in an increased activity. The authors concluded that in murine IL-3 a single disulfide bridge is required between cysteines 17 and 80 to get biological activity that approximates physiological levels and that this structure probably stabilizes the tertiary structure of the protein to give a conformation that is optimal for function. (Clark-Lewis, et al., PROC. NATL. ACAD. SCI. USA 85:7897 (1988)).

[0014] International Patent Application (PCT) WO 88/00598 discloses gibbon- and human-like IL-3. The hIL-3 contains a Ser8→Pro8 replacement. Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.

[0015] EP-A-0275598 (WO 88/04691) illustrates that Ala1 can be deleted while retaining biological activity. Some mutant hIL-3 sequences are provided, e.g., two double mutants, Ala1→Asp1, Trp13→Arg13 (pGB/IL-302) and Ala1→Asp1, Met3→Thr3 (pGB/IL-304) and one triple mutant Ala1→Asp1, Leu9→Pro9, Trp13→Arg13 (pGB/IL-303).

[0016] WO 88/05469 describes how deglycosylation mutants can be obtained and suggests mutants of Arg54Arg55 and Arg108Arg109Lys110 might avoid proteolysis upon expression in Saccharomyces cerevisiae by KEX2 protease. No mutated proteins are disclosed. Glycosylation and the KEX2 protease activity are only important, in this context, upon expression in yeast.

[0017] WO 88/06161 mentions various mutants which theoretically may be conformationally and antigenically neutral. The only actually performed mutations are Met2→Ile2 and Ile131→Leu131. It is not disclosed whether the contemplated neutralities were obtained for these two mutations.

[0018] WO 91/00350 discloses nonglycosylated hIL-3 analog proteins, for example, hIL-3 (Pro8Asp15Asp70), Met3 rhul-3 (Pro8Asp15Asp70); Thr4 rhuL-3 (Pro8Asp15Asp70) and Thr6 rhuIL-3 (Pro8Asp15Asp70). It is said that these protein compositions do not exhibit certain adverse side effects associated with native hIL-3 such as urticaria resulting from infiltration of mast cells and lymphocytes into the dermis. The disclosed analog hIL-3 proteins may have N termini at Met3, Thr4, or Thr6.

[0019] WO 90/12874 discloses cysteine added variants (CAVs) of IL-3 which have at least one Cys residue substituted for a naturally occurring amino acid residue.

SUMMARY OF THE INVENTION

[0020] The present invention relates to recombinant human interleukin-3 (hIL-3) variant or mutant proteins (muteins). These hIL-3 muteins contain amino acid substitutions and may also have amino acid deletions at either/or both the N- and C-termini. Preferably, these mutant polypeptides of the present invention contain one to three amino acids which differ from the amino acids found at the corresponding positions in the native hIL-3 polypeptide. The invention also relates to pharmaceutical compositions containing the hIL-3 muteins, DNA coding for the muteins, and methods for using the muteins. Additionally, the present invention relates to recombinant expression vectors comprising nucleotide sequences encoding the hIL-3 muteins, related microbial expression systems, and processes for making the hIL-3 muteins using the microbial expression systems.

[0021] The present invention includes mutants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus and/or from 1 to 15 amino acids have been deleted from the C-terminus, and in which from one to three amino acid substitutions have been made. Preferred muteins of the present invention are those in which amino acids 1 to 14 have been deleted from the N-terminus, or amino acids 126 to 133 have been deleted from the C-terminus, and which both also contain from one to three amino acid substitutions in the polypeptide sequence. These hIL-3 multiple mutation polypeptides may have biological activities similar to or better than hIL-3 and, in some cases, may also have an improved side effect profile, i.e., some muteins may have a better therapeutic index than native hIL-3. The present invention also provides muteins which may function as IL-3 antagonists or as discrete antigenic fragments for the production of antibodies useful in immunoassay and immunotherapy protocols. In addition to the use of the hIL-3 mutant polypeptides of the present invention in vivo, it is envisioned that in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.

[0022] Antagonists of hIL-3 would be particularly useful in blocking the growth of certain cancer cells like AML, CML and certain types of B lymphoid cancers. Other conditions where antagonists would be useful include those in which certain blood cells are produced at abnormally high numbers or are being activated by endogenous ligands. Antagonists would effectively compete for ligands, presumably naturally occurring hemopoietins including and not limited to IL-3, GM-CSF and IL-5, which might trigger or augment the growth of cancer cells by virtue of their ability to bind to the IL-3 receptor complex while intrinsic activation properties of the ligand are diminished. IL-3, GM-CSF and or IL-5 also play a role in certain asthmatic responses. An antagonist of the IL-3 receptor may have utility in this disease by blocking receptor-mediated activation and recruitment of inflammatory cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]
FIG. 1 is the human IL-3 gene for E. coli expression (pMON5873), encoding the polypeptide sequence of natural (wild type) human IL-3 [SEQ ID NO:128], plus an initiator methionine, as expressed in E. coli, with the amino acids numbered from the N-terminus of the natural hIL-3.

[0024]
FIG. 2: ClaI to NsiI Replacement Fragment. FIG. 2 shows the nucleotide sequence of the replacement fragment used between the ClaI and NsiI sites of the hIL-3 gene. The codon choice used in the fragment corresponds to that found in highly expressed E. coli genes (Gouy and Gautier, 1982). Three new unique restriction sites, EcoRV, XhoI and PstI were introduced for the purpose of inserting synthetic gene fragments. The portion of the coding sequence shown encodes hIL-3 amino acids 20-70.

[0025]
FIGS. 3 and 4 shows the nucleotide and amino acid sequence of the gene in pMON5873 with the sequence extending from NcoI through HindIII. The codon choices used to encode amino acids 1-14 and 107-133 correspond to that found in highly expressed E. coli genes.

[0026]
FIG. 5 shows the construction of the plasmid vector pMON5846 which encodes [Met-(1-133) hIL-3 (Arg129)].

[0027]
FIG. 6 shows the construction of the plasmid vector pMON5847 (ATCC 68912) which encodes [Met-(1-133) hIL-3 (Arg129)].

[0028]
FIG. 7 shows the construction of plasmid vector pMON5853 which encodes [Met-(15-133) hIL-3 (Arg129)].

[0029]
FIG. 8 shows the construction of the plasmid vector pMON5854 which encodes [Met-(1-133) hIL-3 (Arg129)].

[0030]
FIG. 9 shows the DNA sequence and resulting amino acid sequence of the lamB signal peptide.

[0031]
FIG. 10 shows the construction of the plasmid vector pMON5978 which encodes Met-Ala-(15-125)hIL-3.

[0032]
FIG. 11 shows the construction of the plasmid vector pMON5988 which encodes Met-Ala(15-125)hIL-3.

[0033]
FIG. 12 shows the construction of the plasmid vector pMON5887 which encodes Met-(1-125)hIL-3.

[0034]
FIG. 13 shows the construction of pMON6457 which encodes (15-125)hIL-3; it contains the araBAD promoter and the lamB signal peptide fused to the variant hIL-3 amino acids 15-125.

[0035]
FIG. 14 shows the construction of pMON6458; it contains the araBAD promoter and the lamB signal peptide fused to the variant hIL-3 amino acids 15-125.

[0036]
FIG. 15 shows the construction of pMON6467 in which the bases encoding amino acids 35-40 of hIL-3 were deleted using site-directed PCR mutagenesis methods. pMON6467 was used as the template for the generation of single amino acid variants at positions 35-40 of hIL-3.

[0037]
FIG. 16 shows the construction of single amino acid substitutions at position 35 of hIL-3 using site-directed PCR mutagenesis methods. The mutagenesis results in 20 different single amino substitutions, which is referred to as a “library”, at position 35 of hIL-3.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention relates to muteins of human interleukin-3 (hIL-3) in which amino acid substitutions have been made at from one to three positions in the amino acid sequence of the polypeptide and to hIL-3 muteins which have substantially the same structure and substantially the same biological activity. Preferred muteins of the present invention are (15-125)hIL-3 deletion mutants which have deletions of amino acids 1 to 14 at the N-terminus and/or 126 to 133 at the C-terminus and which both also have from one to three amino acid substitutions in the polypeptide and muteins having substantially the same structure and substantially the same biological activity. As used herein human interleukin-3 corresponds to the amino acid sequence (1-133) as depicted in FIG. 1 and (15-125) hIL-3 corresponds to the 15 to 125 amino acid sequence of the hIL-3 polypeptide. Naturally occurring variants of hIL-3 polypeptide amino acids are also included in the present invention (for example, the allele in which proline rather than serine is at position 8 in the hIL-3 polypeptide sequence) as are variant hIL-3 molecules which are modified post-translationally (e.g. glycosylation).

[0039] The present invention also includes the DNA sequences which code for the mutant polypeptides, DNA sequences which are substantially similar and perform substantially the same function, and DNA sequences which differ from the DNAs encoding the muteins of the invention only due to the degeneracy of the genetic code.

[0040] Included in the present invention are novel mutant human interleukin-3 polypeptides comprising a polypeptide having the amino acid sequence of native human interleukin-3 wherein amino acids 126 to 133 have been deleted from the C-terminus of the native human interleukin-3 polypeptide and amino acids 1 to 14 have been deleted from the N-terminus of the native human interleukin-3 polypeptide and, in addition, polypeptides of the present invention also have one to three amino acid substitutions in the polypeptide sequence. The muteins of the present invention can have from one to three amino acid substitutions in the hIL-3 polypeptide chain and, in addition, can have deletions of amino acids at the N-terminus and/or the C-terminus.

[0041] Also included in the present invention are the DNA sequences coding for the muteins of the present invention; the oligonucleotide intermediates used to construct the mutant DNAs; and the polypeptides coded for by these oligonucleotides. These polypeptides may be useful as antagonists or as antigenic fragments for the production of antibodies useful in immunoassay and immunotherapy protocols.

[0042] The mutant hIL-3 polypeptides of the present invention may also have methionine, alanine, or methionine-alanine residues inserted at the N-terminus.

[0043] The present invention includes hIL-3 mutant polypeptides of the formula I:

1Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn[SEQ ID NO:15] 1 5 10 15Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 80 85 90Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 95 100 105Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 110 115 120Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe 125 130

[0044] wherein Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;

[0045] Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

[0046] Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;

[0047] Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;

[0048] Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;

[0049] Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;

[0050] Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;

[0051] Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;

[0052] Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

[0053] Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;

[0054] Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;

[0055] Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;

[0056] Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;

[0057] Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;

[0058] Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

[0059] Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;

[0060] Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu;

[0061] Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;

[0062] Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;

[0063] Xaa at position 36 is Asp, Leu, or Val;

[0064] Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;

[0065] Xaa at position 38 is Asn, or Ala;

[0066] Xaa at position 40 is Leu, Trp, or Arg;

[0067] Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;

[0068] Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;

[0069] Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;

[0070] Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;

[0071] Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;

[0072] Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;

[0073] Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;

[0074] Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;

[0075] Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;

[0076] Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;

[0077] Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;

[0078] Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;

[0079] Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;

[0080] Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;

[0081] Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;

[0082] Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;

[0083] Xaa at position 57 is Asn or Gly;

[0084] Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;

[0085] Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg;

[0086] Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;

[0087] Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;

[0088] Xaa at position 62 is Asn His, Val, Arg, Pro, Thr, Asp, or Ile;

[0089] Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;

[0090] Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;

[0091] Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;

[0092] Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

[0093] Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;

[0094] Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;

[0095] Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;

[0096] Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;

[0097] Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;

[0098] Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

[0099] Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;

[0100] Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;

[0101] Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;

[0102] Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;

[0103] Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;

[0104] Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;

[0105] Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

[0106] Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;

[0107] Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;

[0108] Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;

[0109] Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;

[0110] Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;

[0111] Xaa at position 85 is Leu, Asn, Val, or Gln;

[0112] Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;

[0113] Xaa at position 87 is Leu, Ser, Trp, or Gly;

[0114] Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;

[0115] Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;

[0116] Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;

[0117] Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;

[0118] Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;

[0119] Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;

[0120] Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro;

[0121] Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr;

[0122] Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr;

[0123] Xaa at position 97 is Ile, Val, Lys, Ala, or Asn;

[0124] Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;

[0125] Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;

[0126] Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;

[0127] Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln;

[0128] Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;

[0129] Xaa at position 103 is Asp, or Ser;

[0130] Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;

[0131] Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

[0132] Xaa at position 106 is Glu, Ser, Lys, Thr, Ile, Gly, or Pro;

[0133] Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro;

[0134] Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;

[0135] Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp;

[0136] Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;

[0137] Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;

[0138] Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;

[0139] Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;

[0140] Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;

[0141] Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;

[0142] Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;

[0143] Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;

[0144] Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;

[0145] Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln;

[0146] Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

[0147] Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

[0148] Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

[0149] and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 22 is Leu, and/or Xaa at position 34 is Gly or Glu, and/or Xaa at position 44 is Ala, and/or Xaa at position 46 is Lys or Ala, and/or Xaa at position 50 is Lys, and/or Xaa at position 59 is Pro or Arg, and/or Xaa at position 63 is Lys, and/or Xaa at position 75 is Gly or Arg, and/or Xaa at position 94 is Pro, and/or Xaa at position 98 is Arg, and/or Xaa at position 106 is Lys, and/or Xaa at position 110 is Ala or Glu, and/or Xaa at position 111 is Met, then there must be at least one additional substitution besides the ones indicated.

[0150] Included in the present invention are (1-133)hIL-3 mutant polypeptides of the Formula II:

2Ala Pro Met Ihr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn[SEQ ID NO:16] 1 5 10 15Cys Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa Xaa 35 40 45Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa 80 85 90Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa 95 100 105Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Xaa Xaa 110 115 120Xaa Xaa Xaa Gln Gln Thr Thr Lou Ser Leu Ala Ile Phe 125 130

[0151] wherein

[0152] Xaa at position 17 is Ser, Gly, Asp, Met, or Gln;

[0153] Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

[0154] Xaa at position 19 is Met, Phe, Ile, Arg, or Ala;

[0155] Xaa at position 20 is Ile or Pro;

[0156] Xaa at position 21 is Asp or Glu;

[0157] Xaa at position 23 is Ile, Val, Ala, Leu, or Gly;

[0158] Xaa at position 24 is Ile, Val, Phe, or Leu;

[0159] Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

[0160] Xaa at position 26 is His, Phe, Gly, Arg, or Ala;

[0161] Xaa at position 28 is Lys, Leu, Gln, Gly, Pro, or Val;

[0162] Xaa at position 29 is Gln, Asn, Leu, Arg, or Val;

[0163] Xaa at position 30 is Pro, His, Thr, Gly, or Gln;

[0164] Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

[0165] Xaa at position 32 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

[0166] Xaa at position 33 is Pro, Leu, Gln, Ala, or Glu;

[0167] Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

[0168] Xaa at position 35 is Leu, Ala, Asn, Pro, Gln, or Val;

[0169] Xaa at position 36 is Asp or Leu;

[0170] Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;

[0171] Xaa at position 38 is Asn or Ala;

[0172] Xaa at position 41 is Asn, Cys, Arg, His, Met, or Pro;

[0173] Xaa at position 42 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, Val or Arg;

[0174] Xaa at position 44 is Asp or Glu;

[0175] Xaa at position 45 is Gln, Val, Met, Leu, Thr, Lys, Ala, Asn, Glu, Ser, or Trp;

[0176] Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val or Gly;

[0177] Xaa at position 47 is Ile, Val, or His;

[0178] Xaa at position 49 is Met, Asn, or Asp;

[0179] Xaa at position 50 is Glu, Thr, Ala, Asn, Ser or Asp;

[0180] Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;

[0181] Xaa at position 52 is Asn or Gly;

[0182] Xaa at position 53 is Leu, Met, or Phe;

[0183] Xaa at position 54 is Arg, Ala, or Ser;

[0184] Xaa at position 55 is Arg, Thr, Val, Leu, or Gly;

[0185] Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu, Thr, Val or Lys;

[0186] Xaa at position 59 is Glu, Tyr, His, Leu, or Arg;

[0187] Xaa at position 60 is Ala, Ser, Asn, or Thr;

[0188] Xaa at position 61 is Phe or Ser;

[0189] Xaa at position 62 is Asn, Val, Pro, Thr, or Ile;

[0190] Xaa at position 63 is Arg, Tyr, Lys, Ser, His, or Val;

[0191] Xaa at position 64 is Ala or Asn;

[0192] Xaa at position 65 is Val, Thr, Leu, or Ser;

[0193] Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

[0194] Xaa at position 67 is Ser, Phe, Val, Gly, Asn, Ile, or His;

[0195] Xaa at position 68 is Leu, Val, Ile, Phe, or His;

[0196] Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

[0197] Xaa at position 70 is Asn or Pro;

[0198] Xaa at position 71 is Ala, Met, Pro, Arg, Glu, Thr, or Gln;

[0199] Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

[0200] Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro;

[0201] Xaa at position 74 is Ile or Met;

[0202] Xaa at position 75 is Glu, Gly, Asp, Ser, or Gln;

[0203] Xaa at position 76 is Ser, Val, Ala, Asn, Glu, Pro, Gly, or Asp;

[0204] Xaa at position 77 is Ile, Ser, or Leu;

[0205] Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

[0206] Xaa at position 80 is Asn, Val, Gly, Thr, Leu, Glu, or Arg;

[0207] Xaa at position 81 is Leu, or Val;

[0208] Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

[0209] Xaa at position 83 is Pro, Ala, Thr, Trp, or Met;

[0210] Xaa at position 85 is Leu or Val;

[0211] Xaa at position 87 is Leu or Ser;

[0212] Xaa at position 88 is Ala, Arg, or Trp;

[0213] Xaa at position 89 is Thr, Asp, Glu, His, Asn, or Ser;

[0214] Xaa at position 90 is Ala, Asp, or Met;

[0215] Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, or Asp;

[0216] Xaa at position 92 is Pro or Ser;

[0217] Xaa at position 93 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

[0218] Xaa at position 95 is His, Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser or Thr;

[0219] Xaa at position 96 is Pro or Tyr;

[0220] Xaa at position 97 is Ile, Val, or Ala;

[0221] Xaa at position 98 is His, Ile, Asn, Asp, Ala, Thr, Leu, Arg, Gln, Glu, lys, Met, Ser, Tyr, Val or Pro;

[0222] Xaa at position 99 is Ile, Leu, Val, or Phe;

[0223] Xaa at position 100 is Lys, Leu, His, Arg, Ile, Gln, Pro, or Ser;

[0224] Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu or Tyr;

[0225] Xaa at position 102 is Gly, Glu, Lys, or Ser;

[0226] Xaa at position 104 is Trp, Val, Tyr, Met, or Leu;

[0227] Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

[0228] Xaa at position 106 is Glu, Ser, Ala, or Gly;

[0229] Xaa at position 108 is Arg, Ala, Gln, Ser or Lys;

[0230] Xaa at position 109 is Arg, Thr, Glu, Leu, Ser, or Gly;

[0231] Xaa at position 112 is Thr, Val, Gln, Glu, His, or Ser;

[0232] Xaa at position 114 is Tyr or Trp;

[0233] Xaa at position 115 is Leu or Ala;

[0234] Xaa at position 116 is Lys, Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His, Met, Phe, Tyr or Ile;

[0235] Xaa at position 117 is Thr, Ser, or Asn;

[0236] Xaa at position 119 is Glu, Ser, Pro, Leu, Thr, or Tyr;

[0237] Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;

[0238] Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

[0239] Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

[0240] Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

[0241] and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 34 is Gly or/and Xaa or position 46 is Lys or Ala or/and Xaa at position 59 is Arg and/or Xaa at position 63 is Lys and/or Xaa at position 75 is Gly and/or Xaa at position 98 is Arg then there must be at least one additional substitution besides the ones indicated.

[0242] Included in the present invention are (1-133)hIL-3 mutant polypeptides of the Formula III:

3Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn[SEQ ID NO:17] 1 5 10 15Cys Xaa Xaa Xaa Ile Xaa Giu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa 35 40 45Xaa Ile Leu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa 50 55 60Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu 65 70 75Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala 80 85 90Xaa Pro Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa 95 100 105Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Lou Glu Xaa 110 115 120Xaa Xaa Xaa Gln Gln Thr Thr Lou Ser Lou Ala Ile Phe 125 130

[0243] wherein

[0244] Xaa at position 17 is Ser, Gly, Asp, Met, or Gln;

[0245] Xaa at position 18 is Asn, His, or Ile;

[0246] Xaa at position 19 is Met or Ile;

[0247] Xaa at position 21 is Asp or Glu;

[0248] Xaa at position 23 is Ile, Ala, Leu, or Gly;

[0249] Xaa at position 24 is Ile, Val, or Leu;

[0250] Xaa at position 25 is Thr, His, Gln, or Ala;

[0251] Xaa at position 26 is His or Ala;

[0252] Xaa at position 29 is Gln, Asn, or Val;

[0253] Xaa at position 30 is Pro, Gly, or Gln;

[0254] Xaa at position 31 is Pro, Asp, Gly, or Gln;

[0255] Xaa at position 32 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

[0256] Xaa at position 33 is Pro or Glu;

[0257] Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

[0258] Xaa at position 35 is Leu, Ala, Asn, Pro, Gln, or Val;

[0259] Xaa at position 37 is Phe, Ser, Pro, or Trp;

[0260] Xaa at position 38 is Asn or Ala;

[0261] Xaa at position 42 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr or Arg;

[0262] Xaa at position 44 is Asp or Glu;

[0263] Xaa at position 45 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser or Lys;

[0264] Xaa at position 46 is Asp, Phe, Ser, Thr, Ala, Asn Gln, Glu, His, Ile, Lys, Tyr, Val or Cys;

[0265] Xaa at position 50 is Glu, Ala, Asn, Ser or Asp;

[0266] Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;

[0267] Xaa at position 54 is Arg or Ala;

[0268] Xaa at position 54 is Arg or Ala;

[0269] Xaa at position 55 is Arg, Thr, Val, Leu, or Gly;

[0270] Xaa at position 56 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val or Lys;

[0271] Xaa at position 60 is Ala or Ser;

[0272] Xaa at position 62 is Asn, Pro, Thr, or Ile;

[0273] Xaa at position 63 is Arg or Lys;

[0274] Xaa at position 64 is Ala or Asn;

[0275] Xaa at position 65 is Val or Thr;

[0276] Xaa at position 66 is Lys or Arg;

[0277] Xaa at position 67 is Ser, Phe, or His;

[0278] Xaa at position 68 is Leu, Ile, Phe, or His;

[0279] Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

[0280] Xaa at position 71 is Ala, Pro, or Arg;

[0281] Xaa at position 72 is Ser, Glu, Arg, or Asp;

[0282] Xaa at position 73 is Ala or Leu;

[0283] Xaa at position 76 is Ser, Val, Ala, Asn, Glu, Pro, or Gly;

[0284] Xaa at position 77 is Ile or Leu;

[0285] Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

[0286] Xaa at position 80 is Asn, Gly, Glu, or Arg;

[0287] Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met, Phe, Ser, Thr, Tyr or Val;

[0288] Xaa at position 83 is Pro or Thr;

[0289] Xaa at position 85 is Leu or Val;

[0290] Xaa at position 87 is Leu or Ser;

[0291] Xaa at position 88 is Ala or Trp;

[0292] Xaa at position 91 is Ala or Pro;

[0293] Xaa at position 93 is Thr, Asp, Ser, Pro, Ala, or Arg;

[0294] Xaa at position 95 is His, Pro, Arg, Val, Gly, Asn, Phe, Ser or Thr;

[0295] Xaa at position 96 is Pro or Tyr;

[0296] Xaa at position 97 is Ile or Val;

[0297] Xaa at position 98 is His, Ile, Asn, Leu, Ala, Thr, Arg, Gln, Leu, Lys, Met, Ser, Tyr, Val or Pro;

[0298] Xaa at position 99 is Ile or Val;

[0299] Xaa at position 100 is Lys, Arg, Ile, Gln, Pro, or Ser;

[0300] Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Pro, Asn, Ile, Leu or Tyr;

[0301] Xaa at position 104 is Trp or Leu;

[0302] Xaa at position 105 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

[0303] Xaa at position 106 is Glu or Gly;

[0304] Xaa at position 108 is Arg, Ala, or Ser;

[0305] Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;

[0306] Xaa at position 112 is Thr, Val, or Gln;

[0307] Xaa at position 114 is Tyr or Trp;

[0308] Xaa at position 115 is Leu or Ala;

[0309] Xaa at position 116 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr or Ile;

[0310] Xaa at position 117 is Thr or Ser;

[0311] Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;

[0312] Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly;

[0313] Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

[0314] Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

[0315] and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and

[0316] wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 22 is Leu, and/or Xaa at position 34 is Gly or Glu, and/or Xaa at position 44 is Ala, and/or Xaa at position 46 is Lys or Ala, and/or Xaa at position 50 is Lys, and/or Xaa at position 59 is Pro or Arg, and/or Xaa at position 63 is Lys, and/or Xaa at position 75 is Gly or Arg, and/or Xaa at position 94 is Pro, and/or Xaa at position 98 is Arg, and/or Xaa at position 106 is Lys, and/or Xaa at position 110 is Ala or Glu, and/or Xaa at position 111 is Met, then there must be at least one additional substitution besides the ones indicated;

[0317] and which can additionally have Met-preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133)human interleukin-3 with the proviso that when Xaa at position 34 is Gly and/or Xaa at position 46 is Lys or Ala, and/or Xaa at position 63 is Lys, and/or Xaa at position 98 is Arg, then two or three of the amino acid designated by Xaa are different from the corresponding amino acids of the native (1-133) human interleukin-3.

[0318] Included in the present invention are (1-133)hIL-3 mutant polypeptides of the Formula IV:

4Ala Pro Met Thr Gln Thr Thr Ser Lou Lys Thr Ser Trp Val Asn[SEQ ID NO:18] 1 5 10 15Cys Xaa Xaa Met Ile Asp Glu Xaa Tie Xaa Xaa Lou Lys Xaa Xaa 20 25 30Pro Xaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa 35 40 45Xaa Ile Leu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala 50 55 60Phe Xaa Arg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu 65 70 75Xaa Xaa Leu Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala 80 85 90Xaa Pro Xaa Arg Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa 95 100 105Glu Phe Xaa Xaa Lys Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu Xaa 110 115 120Xaa Xaa Xaa Gln Gln Thr Thr Lou Ser Leu Ala Ile Phe 125 130

[0319] wherein

[0320] Xaa at position 17 is Ser, Gly, Asp, or Gln;

[0321] Xaa at position 18 is Asn, His, or Ile;

[0322] Xaa at position 23 is Ile, Ala, Leu, or Gly;

[0323] Xaa at position 25 is Thr, His, or Gln;

[0324] Xaa at position 26 is His or Ala;

[0325] Xaa at position 29 is Gln or Asn;

[0326] Xaa at position 30 is Pro or Gly;

[0327] Xaa at position 32 is Leu, Arg, Asn, or Ala;

[0328] Xaa at position 34 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met;

[0329] Xaa at position 35 is Leu, Ala, Asn, or Pro;

[0330] Xaa at position 38 is Asn or Ala;

[0331] Xaa at position 42 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;

[0332] Xaa at position 45 is Gln, Val, Met, Leu, Ala, Asn, Glu, or Lys;

[0333] Xaa at position 46 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;

[0334] Xaa at position 50 is Glu Asn, Ser or Asp;

[0335] Xaa at position 51 is Asn, Arg, Pro, Thr, or His;

[0336] Xaa at position 55 is Arg, Leu, or Gly;

[0337] Xaa at position 56 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;

[0338] Xaa at position 62 is Asn, Pro, or Thr;

[0339] Xaa at position 64 is Ala or Asn;

[0340] Xaa at position 65 is Val or Thr;

[0341] Xaa at position 67 is Ser or Phe;

[0342] Xaa at position 68 is Leu or Phe;

[0343] Xaa at position 69 is Gln, Ala, Glu, or Arg;

[0344] Xaa at position 76 is Ser, Val, Asn, Pro, or Gly;

[0345] Xaa at position 77 is Ile or Leu;

[0346] Xaa at position 79 is Lys, Asn, Met, Arg, Ile, or Gly;

[0347] Xaa at position 80 is Asn, Gly, Glu, or Arg;

[0348] Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

[0349] Xaa at position 87 is Leu or Ser;

[0350] Xaa at position 88 is Ala or Trp;

[0351] Xaa at position 91 is Ala or Pro;

[0352] Xaa at position 93 is Thr, Asp, or Ala;

[0353] Xaa at position 95 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;

[0354] Xaa at position 98 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;

[0355] Xaa at position 99 is Ile or Leu;

[0356] Xaa at position 100 is Lys or Arg;

[0357] Xaa at position 101 is Asp, Pro, Met, Lys, Thr, His, Pro, Asn, Ile, Leu or Tyr;

[0358] Xaa at position 105 is Asn, Pro, Ser, Ile or Asp;

[0359] Xaa at position 108 is Arg, Ala, or Ser;

[0360] Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;

[0361] Xaa at position 112 is Thr or Gln;

[0362] Xaa at position 116 is Lys, Val, Trp, Ala, His, Phe, Tyr or Ile;

[0363] Xaa at position 117 is Thr or Ser;

[0364] Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;

[0365] Xaa at position 121 is Ala, Ser, Ile, Pro, or Asp;

[0366] Xaa at position 122 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;

[0367] Xaa at position 123 is Ala, Met, Glu, Ser, or Leu;

[0368] and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133)human interleukin-3.

[0369] Preferred polypeptides of the present invention are (15-125)hIL-3 mutant polypeptides of the Formula V:

5Asn Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa[SEQ ID NO:19]1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75Xaa Xaa Xaa Xaa Xsa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 80 85 90Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 95 100 105Xaa Xaa Xaa Xaa Gln Gln 110

[0370] wherein

[0371] Xaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;

[0372] Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

[0373] Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;

[0374] Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;

[0375] Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;

[0376] Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly;

[0377] Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;

[0378] Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;

[0379] Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

[0380] Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp;

[0381] Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala;

[0382] Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;

[0383] Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val;

[0384] Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;

[0385] Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

[0386] Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;

[0387] Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu;

[0388] Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;

[0389] Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;

[0390] Xaa at position 22 is Asp, Leu, or Val;

[0391] Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile;

[0392] Xaa at position 24 is Asn, or Ala;

[0393] Xaa at position 26 is Leu, Trp, or Arg;

[0394] Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro;

[0395] Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met;

[0396] Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;

[0397] Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr,Met, Trp, Glu, Asn, Gln, Ala or Pro;

[0398] Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp;

[0399] Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;

[0400] Xaa at position 33 is Ile, Gly, Val, Ser, Arq, Pro, or His;

[0401] Xaa at position 34 is Leu, Ser, Cys, Arq, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;

[0402] Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;

[0403] Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;

[0404] Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;

[0405] Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr;

[0406] Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, Met, or;

[0407] Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;

[0408] Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly;

[0409] Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;

[0410] Xaa at position 43 is Asn or Gly;

[0411] Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;

[0412] Xaa at position 45 is Glu Tyr, His, Leu, Pro, or Arg;

[0413] Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr;

[0414] Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;

[0415] Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;

[0416] Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;

[0417] Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys;

[0418] Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser;

[0419] Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

[0420] Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;

[0421] Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;

[0422] Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;

[0423] Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala;

[0424] Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;

[0425] Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

[0426] Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;

[0427] Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala;

[0428] Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;

[0429] Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;

[0430] Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu;

[0431] Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;

[0432] Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp;

[0433] Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;

[0434] Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;

[0435] Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;

[0436] Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met;

[0437] Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val;

[0438] Xaa at position 71 is Leu, Asn, Val, or Gln;

[0439] Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys;

[0440] Xaa at position 73 is Leu, Ser, Trp, or Gly;

[0441] Xaa at position 74 is Ala, Lys, Arg, Val, or Trp;

[0442] Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;

[0443] Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;

[0444] Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;

[0445] Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;

[0446] Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;

[0447] Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro;

[0448] Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr;

[0449] Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr;

[0450] Xaa at position 83 is Ile, Val, Lys, Ala, or Asn;

[0451] Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;

[0452] Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;

[0453] Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, Pro;

[0454] Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu or Gln;

[0455] Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;

[0456] Xaa at position 89 is Asp, or Ser;

[0457] Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;

[0458] Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

[0459] Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;

[0460] Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro;

[0461] Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;

[0462] Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp;

[0463] Xaa at position 97 is Leu, Ile, Arg, Asp, or Met;

[0464] Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;

[0465] Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;

[0466] Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;

[0467] Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;

[0468] Xaa at position 102 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;

[0469] Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;

[0470] Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;

[0471] Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;

[0472] Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln;

[0473] Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

[0474] Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

[0475] Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

[0476] and which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

[0477] Included in the present invention are (15-125)hIL-3 mutant polypeptides of the Formula VI:

6Asn Cys Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa[SEQ ID NO:20] 1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa 20 25 30Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa 65 70 75Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa 80 85 90Xaa Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Xaa 95 100 105Xaa Xaa Xaa Xaa Gln Gln 110

[0478] wherein

[0479] Xaa at position 3 is Ser, Gly, Asp, Met, or Gln;

[0480] Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

[0481] Xaa at position 5 is Met, Phe, Ile, Arg, or Ala;

[0482] Xaa at position 6 is Ile or Pro;

[0483] Xaa at position 7 is Asp, or Glu;

[0484] Xaa at position 9 is Ile, Val, Ala, Leu, or Gly;

[0485] Xaa at position 10 is Ile, Val, Phe, or Leu;

[0486] Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

[0487] Xaa at position 12 is His, Phe, Gly, Arg, or Ala;

[0488] Xaa at position 14 is Lys, Leu, Gln, Gly, Pro, or Val;

[0489] Xaa at position 15 is Gln, Asn, Leu, Arg, or Val;

[0490] Xaa at position 16 is Pro, His, Thr, Gly, or Gln;

[0491] Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

[0492] Xaa at position 18 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

[0493] Xaa at position 19 is Pro, Leu, Gln, Ala, or Glu;

[0494] Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

[0495] Xaa at position 21 is Leu, Ala, Asn, Pro, Gln, or Val;

[0496] Xaa at position 22 is Asp or Leu;

[0497] Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile;

[0498] Xaa at position 24 is Asn or Ala;

[0499] Xaa at position 27 is Asn, Cys, Arg, His, Met, or Pro;

[0500] Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg;

[0501] Xaa at position 30 is Asp, or Glu;

[0502] Xaa at position 31 is Gln, Val, Met, Leu, Thr, Lys, Ala, Asn Glu, Ser or Trp;

[0503] Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val or Gly;

[0504] Xaa at position 33 is Ile, Val, or His;

[0505] Xaa at position 35 is Met, Asn, or Asp;

[0506] Xaa at position 36 is Glu, Thr, Ala, Asn, Ser or Asp;

[0507] Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;

[0508] Xaa at position 38 is Asn or Gly;

[0509] Xaa at position 39 is Leu, Met, or Phe;

[0510] Xaa at position 40 is Arg, Ala or Ser;

[0511] Xaa at position 41 is Arg, Thr, Val, Leu, or Gly;

[0512] Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu, Thr, Val or Lys;

[0513] Xaa at position 45 is Glu, Tyr, His, Leu, or Arg;

[0514] Xaa at position 46 is Ala, Ser, Asn, or Thr;

[0515] Xaa at position 47 is Phe or Ser;

[0516] Xaa at position 48 is Asn, Val, Pro, Thr, or Ile;

[0517] Xaa at position 49 is Arg, Tyr, Lys, Ser, His, or Val;

[0518] Xaa at position 50 is Ala or Asn;

[0519] Xaa at position 51 is Val, Thr, Leu, or Ser;

[0520] Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

[0521] Xaa at position 53 is Ser, Phe, Val, Gly, Asn, Ile, or His;

[0522] Xaa at position 54 is Leu, Val, Ile, Phe, or His;

[0523] Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

[0524] Xaa at position 56 is Asn or Pro;

[0525] Xaa at position 57 is Ala, Met, Pro, Arg, Glu, Thr, or Gln;

[0526] Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

[0527] Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro;

[0528] Xaa at position 60 is Ile or Met;

[0529] Xaa at position 61 is Glu, Gly, Asp, Ser, or Gln;

[0530] Xaa at position 62 is Ser, Val, Ala, Asn, Glu, Pro, Gly, or Asp;

[0531] Xaa at position 63 is Ile, Ser, or Leu;

[0532] Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp;

[0533] Xaa at position 66 is Asn, Val, Gly, Thr, Leu, Glu, or Arg;

[0534] Xaa at position 67 is Leu, or Val;

[0535] Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

[0536] Xaa at position 69 is Pro, Ala, Thr, Trp, or Met;

[0537] Xaa at position 71 is Leu or Val;

[0538] Xaa at position 73 is Leu or Ser;

[0539] Xaa at position 74 is Ala, Arg, or Trp;

[0540] Xaa at position 75 is Thr, Asp, Glu, His, Asn, or Ser;

[0541] Xaa at position 76 is Ala, Asp, or Met;

[0542] Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, or Asp;

[0543] Xaa at position 78 is Pro or Ser;

[0544] Xaa at position 79 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

[0545] Xaa at position 81 is His, Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser or Thr;

[0546] Xaa at position 82 is Pro or Tyr;

[0547] Xaa at position 83 is Ile, Val, or Ala;

[0548] Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Pro;

[0549] Xaa at position 85 is Ile, Leu, Val, or Phe;

[0550] Xaa at position 86 is Lys, Leu, His, Arg, Ile, Gln, Pro or Ser;

[0551] Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu or Tyr;

[0552] Xaa at position 88 is Gly, Glu, Lys, or Ser;

[0553] Xaa at position 90 is Trp, Val, Tyr, Met, or Leu;

[0554] Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

[0555] Xaa at position 92 is Glu, Ser, Ala, or Gly;

[0556] Xaa at position 94 is Arg, Ala, Gln, Ser or Lys;

[0557] Xaa at position 95 is Arg, Thr, Glu, Leu, Ser, or Gly;

[0558] Xaa at position 98 is Thr, Val, Gln, Glu, His, or Ser;

[0559] Xaa at position 100 is Tyr or Trp;

[0560] Xaa at position 101 is Leu or Ala;

[0561] Xaa at position 102 is Lys, Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His, Met, Phe, Tyr or Ile;

[0562] Xaa at position 103 is Thr, Ser, or Asn;

[0563] Xaa at position 105 is Glu, Ser, Pro, Leu, Thr, or Tyr;

[0564] Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;

[0565] Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

[0566] Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

[0567] Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

[0568] and which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

[0569] Included in the present invention are (15-125)hIL-3 mutant polypeptides of the Formula VII:

7Asn Cys Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa[SEQ ID NO:21] 1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa 20 25 30Xaa Xaa Ile Leu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu 35 40 45Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile 50 55 60Glu Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr 65 70 75Ala Xaa Pro Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa 80 85 90Xaa Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu 95 100 105Xaa Xaa Xaa Xaa Gln Gln 110

[0570] wherein

[0571] Xaa at position 3 is Ser, Gly, Asp, Met, or Gln;

[0572] Xaa at position 4 is Asn, His, or Ile;

[0573] Xaa at position 5 is Met or Ile;

[0574] Xaa at position 7 is Asp or Glu;

[0575] Xaa at position 9 is Ile, Ala, Leu, or Gly;

[0576] Xaa at position 10 is Ile, Val, or Leu;

[0577] Xaa at position 11 is Thr, His, Gln, or Ala;

[0578] Xaa at position 12 is His or Ala;

[0579] Xaa at position 15 is Gln, Asn, or Val;

[0580] Xaa at position 16 is Pro, Gly, or Gln;

[0581] Xaa at position 17 is Pro, Asp, Gly, or Gln;

[0582] Xaa at position 18 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

[0583] Xaa at position 19 is Pro or Glu;

[0584] Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

[0585] Xaa at position 21 is Leu, Ala, Asn, Pro, Gln, or Val;

[0586] Xaa at position 23 is Phe, Ser, Pro, or Trp;

[0587] Xaa at position 24 is Asn or Ala;

[0588] Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met Tyr or Arg;

[0589] Xaa at position 30 is Asp or Glu;

[0590] Xaa at position 31 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser or Lys;

[0591] Xaa at position 32 is Asp, Phe, Ser, Thr, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val or Cys;

[0592] Xaa at position 36 is Glu, Ala, Asn, Ser or Asp;

[0593] Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;

[0594] Xaa at position 40 is Arg or Ala;

[0595] Xaa at position 41 is Arg, Thr, Val, Leu, or Gly;

[0596] Xaa at position 42 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val or Lys;

[0597] Xaa at position 46 is Ala or Ser;

[0598] Xaa at position 48 is Asn, Pro, Thr, or Ile;

[0599] Xaa at position 49 is Arg or Lys;

[0600] Xaa at position 50 is Ala or Asn;

[0601] Xaa at position 51 is Val or Thr;

[0602] Xaa at position 52 is Lys or Arg;

[0603] Xaa at position 53 is Ser, Phe, or His;

[0604] Xaa at position 54 is Leu, Ile, Phe, or His;

[0605] Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

[0606] Xaa at position 57 is Ala, Pro, or Arg;

[0607] Xaa at position 58 is Ser, Glu, Arg, or Asp;

[0608] Xaa at position 59 is Ala or Leu;

[0609] Xaa at position 62 is Ser, Val, Ala, Asn, Glu, Pro, or Gly;

[0610] Xaa at position 63 is Ile or Leu;

[0611] Xaa at position 65 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

[0612] Xaa at position 66 is Asn, Gly, Glu, or Arg;

[0613] Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met, Phe, Ser, Thr, Tyr or Val;

[0614] Xaa at position 69 is Pro or Thr;

[0615] Xaa at position 71 is Leu or Val;

[0616] Xaa at position 73 is Leu or Ser;

[0617] Xaa at position 74 is Ala or Trp;

[0618] Xaa at position 77 is Ala or Pro;

[0619] Xaa at position 79 is Thr, Asp, Ser, Pro, Ala, or Arg;

[0620] Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Phe, Ser or Thr;

[0621] Xaa at position 82 is Pro or Tyr;

[0622] Xaa at position 83 is Ile or Val;

[0623] Xaa at position 84 is His, Ile, Asn, Leu, Ala, Thr, Arg, Gln, Lys, Met, Ser, Tyr, Val or Pro;

[0624] Xaa at position 85 is Ile, Leu, or Val;

[0625] Xaa at position 86 is Lys, Arg, Ile, Gln, Pro, or Ser;

[0626] Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Asn, Ile, Leu or Tyr;

[0627] Xaa at position 90 is Trp or Leu;

[0628] Xaa at position 91 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

[0629] Xaa at position 92 is Glu, or Gly;

[0630] Xaa at position 94 is Arg, Ala, or Ser;

[0631] Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;

[0632] Xaa at position 98 is Thr, Val, or Gln;

[0633] Xaa at position 100 is Tyr or Trp;

[0634] Xaa at position 101 is Leu or Ala;

[0635] Xaa at position 102 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr or Ile;

[0636] Xaa at position 103 is Thr or Ser;

[0637] Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;

[0638] Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly;

[0639] Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

[0640] Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

[0641] which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (15-125)human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

[0642] Included in the present invention are (15-125)hIL-3 mutant polypeptides of the Formula VIII:

8Asn Cys Xaa Xaa Met Ile Asp Gln Xaa Ile Xaa Xaa Leu Lys Xaa[SEQ ID NO:22] 1 5 10 15Xaa Pro Xaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Gln Asp 20 25 30Xaa Xaa Ile Leu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Gln 35 40 45Ala Phe Xaa Arg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile 50 55 60Glu Xaa Xaa Leu Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr 65 70 75Ala Xaa Pro Xaa Arg Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp 80 85 90Xaa Glu Phe Xaa Xaa Lys Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu 90 100 105Xaa Xaa Xaa Xaa Gln Gln 110

[0643] wherein

[0644] Xaa at position 3 is Ser, Gly, Asp, or Gln;

[0645] Xaa at position 4 is Asn, His, or Ile;

[0646] Xaa at position 9 is Ile, Ala, Leu, or Gly;

[0647] Xaa at position 11 is Thr, His, or Gln;

[0648] Xaa at position 12 is His or Ala;

[0649] Xaa at position 15 is Gln or Asn;

[0650] Xaa at position 16 is Pro or Gly;

[0651] Xaa at position 18 is Leu, Arg, Asn, or Ala;

[0652] Xaa at position 20 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

[0653] Xaa at position 21 is Leu, Ala, Asn, or Pro;

[0654] Xaa at position 24 is Asn or Ala;

[0655] Xaa at position 28 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;

[0656] Xaa at position 31 is Gln, Val, Met, Leu, Ala, Asn, Glu or Lys;

[0657] Xaa at position 32 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;

[0658] Xaa at position 36 is Glu, Asn, Ser or Asp;

[0659] Xaa at position 37 is Asn, Arg, Pro, Thr, or His;

[0660] Xaa at position 41 is Arg, Leu, or Gly;

[0661] Xaa at position 42 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;

[0662] Xaa at position 48 is Asn, Pro, or Thr;

[0663] Xaa at position 50 is Ala or Asn;

[0664] Xaa at position 51 is Val or Thr;

[0665] Xaa at position 53 is Ser or Phe;

[0666] Xaa at position 54 is Leu or Phe;

[0667] Xaa at position 55 is Gln, Ala, Glu, or Arg;

[0668] Xaa at position 62 is Ser, Val, Asn, Pro, or Gly;

[0669] Xaa at position 63 is Ile or Leu;

[0670] Xaa at position 65 is Lys, Asn, Met, Arg, Ile, or Gly;

[0671] Xaa at position 66 is Asn, Gly, Glu, or Arg;

[0672] Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

[0673] Xaa at position 73 is Leu or Ser;

[0674] Xaa at position 74 is Ala or Trp;

[0675] Xaa at position 77 is Ala or Pro;

[0676] Xaa at position 79 is Thr, Asp, or Ala;

[0677] Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;

[0678] Xaa at position 84 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;

[0679] Xaa at position 85 is Ile or Leu;

[0680] Xaa at position 86 is Lys or Arg;

[0681] Xaa at position 87 is Asp, Pro, Met, Lys, His, Pro, Asn, Ile, Leu or Tyr;

[0682] Xaa at position 91 is Asn, Pro, Ser, Ile or Asp;

[0683] Xaa at position 94 is Arg, Ala, or Ser;

[0684] Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;

[0685] Xaa at position 98 is Thr or Gln;

[0686] Xaa at position 102 is Lys, Val, Trp, or Ile;

[0687] Xaa at position 103 is Thr, Ala, His, Phe, Tyr or Ser;

[0688] Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;

[0689] Xaa at position 107 is Ala, Ser, Ile, Pro, or Asp;

[0690] Xaa at position 108 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;

[0691] Xaa at position 109 is Ala, Met, Glu, Ser, or Leu;

[0692] and which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133)human interieukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

[0693] In Formulas V, VI, VII and VIII the Asn in position 1 corresponds to the Asn in position 15 of native hIL-3 and positions 1 to 111 correspond to positions 15 to 125 in the native hIL-3 sequence shown in FIG. 1.

[0694] Also included in the present invention are polypeptides of the following formula (IX):

9 (Met)m-Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr[SEQ ID NO:129] 15 20Ser Trp Val Asn Cys Ser Xaa Met Ile Asp Glu Ile Ile25 30 35Xaa His Leu Lys Xaa Pro Pro Xaa Pro Leu Leu Asp Xaa 40 45 50Asn Asn Leu Asn Xaa Glu Asp Xaa Asp Ile Leu Met Glu 55 60Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa Arg 65 70 75Ala Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa 80 85Ile Leu Xaa Asn Leu Xaa Pro Cys Leu Pro Xaa Ala Thr90 95 100Ala Ala Pro Xaa Arg His Pro Ile Xaa Ile Lys Xaa Gly 105 110 115Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Thr Phe Tyr Leu 120 125Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln Thr Thr Leu 130Ser Leu Ala Ile Phe

[0695] wherein m is 0 or 1; Xaa at position 18 is Asn or Ile; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, or Val; Xaa at position 32 is Leu, Ala, or Asn; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 42 is Glu, Ala, or Ser; Xaa at position 45 is Gln, Val, or Met; Xaa at position 51 is Asn or Arg; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn or Val; Xaa at position 67 is Ser, Asn, or His; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser or Ala; Xaa at position 79 is Lys or Arg; Xaa at position 82 is Leu, Glu, or Val; Xaa at position 87 is Leu or Ser; Xaa at position 93 is Pro or Ser; Xaa at position 98 is His, Ile, or Thr; Xaa at position 101 is Asp or Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg or Glu; Xaa at position 116 is Lys or Val; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

[0696] Polypeptides of the present invention include those (15-125)hIL-3 muteins of the following formula (X):

10 1 5 10[SEQ ID NO:130](Metm-Alan)p-Asn Cys Ser Xaa Met Ile Asp Glu Ile Ile 15 20 Xaa His Leu Lys Xaa Pro Pro Xaa Pro Leu Leu Asp Xaa 25 30 35 Asn Asn Leu Asn Xaa Glu Asp Xaa Asp Ile Leu Met Glu 40 45 Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa Arg 50 55 60 Ala Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa 65 70 75 Ile Leu Xaa Asn Leu Xaa Pro Cys Leu Pro Xaa Ala Thr 80 85 Ala Ala Pro Xaa Arg His Pro Ile Xaa Ile Lys Xaa Gly 90 95 100 Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Thr Phe Tyr Leu 105 100 Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln

[0697] wherein m is 0 or 1; n is 0 or 1; p is 0 or 1; Xaa at position 4 is Asn or Ile; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, or Val; Xaa at position 18 is Leu, Ala, or Asn; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 28 is Glu, Ala, or Ser; Xaa at position 31 is Gln, Val, or Met; Xaa at position 37 is Asn or Arg; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn or Val; Xaa at position 53 is Ser, Asn, or His; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser or Ala; Xaa at position 65 is Lys or Arg; Xaa at position 68 is Leu, Glu, or Val; Xaa at position 73 is Leu or Ser; Xaa at position 79 is Pro or Ser; Xaa at position 84 is His, Ile, or Thr; Xaa at position 87 is Asp or Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg or Glu; Xaa at position 102 is Lys or Val; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu;

[0698] wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (15-125)human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

[0699] The present invention includes polypeptides of Formula IX and Formula X above wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3 or native (15-125) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

[0700] “Mutant amino acid sequence,” “mutant protein” or “mutant polypeptide” refers to a polypeptide having an amino acid sequence which varies from a native sequence or is encoded by a nucleotide sequence intentionally made variant from a native sequence. “Mutant protein,” “variant protein” or “mutein” means a protein comprising a mutant amino acid sequence and includes polypeptides which differ from the amino acid sequence of native hIL-3 due to amino acid deletions, substitutions, or both. “Native sequence” refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.

[0701] Human IL-3 can be characterized by its ability to stimulate colony formation by human hematopoietic progenitor cells. The colonies formed include erythroid, granulocyte, megakaryocyte, granulocytic macrophages and mixtures thereof. Human IL-3 has demonstrated an ability to restore bone marrow function and peripheral blood cell populations to therapeutically beneficial levels in studies performed initially in primates and subsequently in humans (Gillio, A. P., et al. (1990); Ganser, A., et al. (1990); Falk, S., et al. (1991). Additional activities of hIL-3 include the ability to stimulate leukocyte migration and chemotaxis; the ability to prime human leukocytes to produce high levels of inflammatory mediators like leukotrienes and histamine; the ability to induce cell surface expression of molecules needed for leukocyte adhesion; and the ability to trigger dermal inflammatory responses and fever. Many or all of these biological activities of hIL-3 involve signal transduction and high affinity receptor binding. Mutant polypeptides of the present invention may exhibit useful properties such as having similar or greater biological activity when compared to native hIL-3 or by having improved half-life or decreased adverse side effects, or a combination of these properties. They may also be useful as antagonists. hIL-3 mutant polypeptides which have little or no activity when compared to native hIL-3 may still be useful as antagonists, as antigens for the production of antibodies for use in immunology or immunotherapy, as genetic probes or as intermediates used to construct other useful hIL-3 muteins. Since hIL-3 functions by binding to its receptor(s) and triggering second messages resulting in competent signal transduction, hIL-3 muteins of this invention may be useful in helping to determine which specific amino acid sequences are responsible for these activities.

[0702] The novel hIL-3 mutant polypeptides of the present invention will preferably have at least one biological property of human IL-3 or of an IL-3-like growth factor and may have more than one IL-3-like biological property, or an improved property, or a reduction in an undesirable biological property of human IL-3. Some mutant polypeptides of the present invention may also exhibit an improved side effect profile. For example, they may exhibit a decrease in leukotriene release or histamine release when compared to native hIL-3 or (15-125) hIL-3. Such hIL-3 or hIL-3-like biological properties may include one or more of the following biological characteristics and in vivo and in vitro activities.

[0703] One such property is the support of the growth and differentiation of progenitor cells committed to erythroid, lymphoid, and myeloid lineages. For example, in a standard human bone marrow assay, an IL-3-like biological property is the stimulation of granulocytic type colonies, megakaryocytic type colonies, monocyte/macrophage type colonies, and erythroid bursts. Other IL-3-like properties are the interaction with early multipotential stem cells, the sustaining of the growth of pluripotent precursor cells, the ability to stimulate chronic myelogenous leukemia (CML) cell proliferation, the stimulation of proliferation of mast cells, the ability to support the growth of various factor-dependent cell lines, and the ability to trigger immature bone marrow cell progenitors. Other biological properties of IL-3 have been disclosed in the art. Human IL-3 also has some biological activities which may in some cases be undesirable, for example the ability to stimulate leukotriene release and the ability to stimulate increased histamine synthesis in spleen and bone marrow cultures and in vivo.

[0704] Biological activity of hIL-3 and hIL-3 mutant proteins of the present invention is determined by DNA synthesis by human acute myelogenous leukemia cells (AML). The factor-dependent cell line AML 193 was adapted for use in testing biological activity.

[0705] One object of the present invention is to provide hIL-3 muteins and hIL-3 deletion muteins with one or more amino acid substitutions in the polypeptide sequence which have similar or improved biological activity in relation to native hIL-3 or native (15-125)hIL-3.

[0706] The present invention includes mutant polypeptides comprising minimally amino acid residues 15 to 118 of hIL-3 with or without additional amino acid extensions to the N-terminus and/or C-terminus which further contain from one to three or more amino acid substitutions in the amino acid sequence of the polypeptide. It has been found that the (15-125)hIL-3 mutant is more soluble than is hIL-3 when expressed in the cytoplasm of E. coli, and the protein is secreted to the periplasm in E. coli at higher levels compared to native hIL-3.

[0707] When expressed in the E. coli cytoplasm, the above-mentioned mutant hIL-3 polypeptides of the present invention may also be constructed with Met-Ala- at the N-terminus so that upon expression the Met is cleaved off leaving Ala at the N-terminus. These mutant hIL-3 polypeptides may also be expressed in E. coli by fusing a signal peptide to the N-terminus. This signal peptide is cleaved from the polypeptide as part of the secretion process. Secretion in E. coli can be used to obtain the correct amino acid at the N-terminus (e.g., Asn15 in the (15-125) hIL-3 polypeptide) due to the precise nature of the signal peptidase. This is in contrast to the heterogeneity often observed at the N-terminus of proteins expressed in the cytoplasm in E. coli.

[0708] The hIL-3 mutant polypeptides of the present invention may have hIL-3 or hIL-3-like activity. For example, they may possess one or more of the biological activities of native hIL-3 and may be useful in stimulating the production of hematopoietic cells by human or primate progenitor cells. The hIL-3 muteins of the present invention and pharmaceutical compositions containing them may be useful in the treatment of conditions in which hematopoietic cell populations have been reduced or destroyed due to disease or to treatments such as radiation or chemotherapy.

[0709] hIL-3 muteins of the present invention may also be useful as antagonists which block the hIL-3 receptor by binding specifically to it and preventing binding of the agonist.

[0710] One potential advantage of the (15-125) hIL-3 muteins of the present invention, particularly those which retain activity similar to or better than that of native hIL-3, is that it may be possible to use a smaller amount of the biologically active mutein to produce the desired therapeutic effect. This may make it possible to reduce the number of treatments necessary to produce the desired therapeutic effect. The use of smaller amounts may also reduce the possibility of any potential antigenic effects or other possible undesirable side effects. For example, if a desired therapeutic effect can be achieved with a smaller amount of polypeptide it may be possible to reduce or eliminate side effects associated with the administration of native IL-3 such as the stimulation of leukotriene and/or histamine release. The hIL-3 muteins of the present invention may also be useful in the activation of stem cells or progenitors which have low receptor numbers. Pharmaceutical compositions containing hIL-3 muteins of the present invention can be administered parenterally, intravenously, or subcutaneously.

[0711] In variants which contain an additional cysteine the presence of the cysteine permits the labeling of the protein with ricin which permits targeting ricin and other toxins or tracers using a sulfhydryl linkage to the hIL-3 receptor.

[0712] As another aspect of the present invention, there is provided a novel method for producing the novel family of human IL-3 muteins. The method of the present invention involves culturing a suitable cell or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel hIL-3 mutant polypeptide. Suitable cells or cell lines may be bacterial cells. For example, the various strains of E-coli are well-known as host cells in the field of biotechnology. Examples of such strains include E. coli strains JM101 [Yanish-Perron, et al. (1985)] and MON105 [Obukowicz, et al. (1992)]. Various strains of B subtilis may also be employed in this method. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention.

[0713] Also suitable for use in the present invention are mammalian cells, such as Chinese hamster ovary cells (CHO). General methods for expression of foreign genes in mammalian cells are reviewed in: Kaufman, R. J. (1987) High level production of proteins in mammalian cells, in Genetic Engineering, Principles and Methods, Vol. 9, J. K. Setlow, editor, Plenum Press, New York. An expression vector is constructed in which a strong promoter capable of functioning in mammalian cells drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally fused to the coding region for the hIL-3 variant. For example, plasmids such as pcDNA I/Neo, pRc/RSV, and pRc/CMV (obtained from Invitrogen Corp., San Diego, Calif.) can be used. The eukaryotic secretion signal peptide coding region can be from the hIL-3 gene itself or it can be from another secreted mammalian protein (Bayne, M. L. et al. (1987) Proc. Natl. Acad. Sci. USA 84, 2638-2642). After construction of the vector containing the hIL-3 variant gene, the vector DNA is transfected into mammalian cells. Such cells can be, for example, the COS7, HeLa, BHK, CHO, or mouse L lines. The cells can be cultured, for example, in DMEM media (JRH Scientific). The hIL-3 variant secreted into the media can be recovered by standard biochemical approaches following transient expression 24-72 hours after transfection of the cells or after establishment of stable cell lines following selection for neomycin resistance. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al., U.S. Pat. No. 4,419,446. Another suitable mammalian cell line is the monkey COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line.

[0714] Where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein. In addition, general methods for expression of foreign genes in insect cells using Baculovirus vectors are described in: Summers, M. D. and Smith, G. E. (1987)—A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555. An expression vector is constructed comprising a Baculovirus transfer vector, in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally fused to the coding region for the hIL-3 variant polypeptide. For example, the plasmid pVL1392 (obtained from Invitrogen Corp., San Diego, Calif.) can be used. After construction of the vector carrying the hIL-3 variant gene, two micrograms of this DNA is cotransfected with one microgram of Baculovirus DNA (see Summers & Smith, 1987) into insect cells, strain SF9. Pure recombinant Baculovirus carrying the hIL-3 variant is used to infect cells cultured, for example, in Excell 401 serum-free medium (JRH Biosciences, Lenexa, Kans.). The hIL-3 variant secreted into the medium can be recovered by standard biochemical approaches.

[0715] Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel hIL-3 muteins. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Appropriate vectors which can transform microorganisms capable of expressing the hIL-3 muteins include expression vectors comprising nucleotide sequences coding for the hIL-3 muteins joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.

[0716] Vectors incorporating modified sequences as described above are included in the present invention and are useful in the production of the hIL-3 mutant polypeptides. The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.

[0717] The present invention also includes the construction and expression of (15-125)human interleukin-3 muteins having one or more amino acid substitutions in secretion vectors that optimize accumulation of correctly folded, active polypeptide. While many heterologous proteins have been secreted in E. coli there is still a great deal of unpredictability and limited success (Stader and Silhavy 1990). Full-length hIL-3 is such a protein, where attempts to secrete the protein in E. coli resulted in low levels of secretion. Secretion of the variant (15-125) hIL-3 mutant polypeptides of the present invention as a fusion with a signal peptide such as lamB results in correctly folded protein that can be removed from the periplasm of E. coli by osmotic shock fractionation. This property of the variant (15-125) hIL-3 muteins allows for the direct and rapid screening for bioactivity of the secreted material in the crude osmotic shock fraction, which is a significant advantage. Furthermore, it provides a means of using the (15-125)hIL-3 muteins to conduct structure activity relationship (SAR) studies of the hIL-3 molecule. A further advantage of secretion of (15-125) hIL-3 muteins fused to the lamB signal peptide is that the secreted polypeptide has the correct N-terminal amino acid (Asn) due to the precise nature of the cleavage of the signal peptide by signal peptidase, as part of the secretion process.

[0718] The (15-125)hIL-3 muteins of the present invention may include hIL-3 polypeptides having Met-, Ala- or Met-Ala- attached to the N-terminus. When the muteins are expressed in E. coli, polypeptides with and without Met attached to the N-terminus are obtained. The methionine can in some cases be removed by methionine aminopeptidase.

[0719] Amino terminal sequences of some of the hIL-3 muteins made in E. coli were determined using the method described by Hunkapillar et al., (1983). It was found that hIL-3 proteins made in E. coli from genes encoding Met-(15-125)hIL-3 were isolated as Met-(15-125) hIL-3. Proteins produced from genes encoding Met-Ala-(15-125) hIL-3 were produced as Ala-(15-125) hIL-3. The N-termini of proteins made in the cytoplasm of E. coli are affected by posttranslational processing by methionine aminopeptidase (Ben-Bassat et al., 1987) and possibly by other peptidases.

[0720] One method of creating the preferred hIL-3 (15-125) mutant genes is cassette mutagenesis [Wells, et al. (1985)] in which a portion of the coding sequence of hIL-3 in a plasmid is replaced with synthetic oligonucleotides that encode the desired amino acid substitutions in a portion of the gene between two restriction sites. In a similar manner amino acid substitutions could be made in the full-length hIL-3 gene, or genes encoding variants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus and/or from 1 to 15 amino acids have been deleted from the C-terminus. When properly assembled these oligonucleotides would encode hIL-3 variants with the desired amino acid substitutions and/or deletions from the N-terminus and/or C-terminus. These and other mutations could be created by those skilled in the art by other mutagenesis methods including; oligonucleotide-directed mutagenesis [Zoller and Smith (1982, 1983, 1984), Smith (1985), Kunkel (1985), Taylor, et al. (1985), Deng and Nickoloff (1992)] or polymerase chain reaction (PCR) techniques [Saiki, (1985)].

[0721] Pairs of complementary synthetic oligonucleotides encoding portions of the amino terminus of the hIL-3 gene can be made and annealed to each other. Such pairs would have protruding ends compatible with ligation to NcoI at one end. The NcoI site would include the codon for the initiator methionine. At the other end of oligonucleotide pairs, the protruding (or blunt) ends would be compatible with a restriction site that occurs within the coding sequence of the hIL-3 gene. The DNA sequence of the oligonucleotide would encode sequence for amino acids of hIL-3 with the exception of those substituted and/or deleted from the sequence.

[0722] The NcoI enzyme and the other restriction enzymes chosen should have recognition sites that occur only once in the DNA of the plasmid chosen. Plasmid DNA can be treated with the chosen restriction endonucleases then ligated to the annealed oligonucleotides. The ligated mixtures can be used to transform competent JM101 cells to resistance to an appropriate antibiotic. Single colonies can be picked and the plasmid DNA examined by restriction analysis and/or DNA sequencing to identify plasmids with mutant hIL-3 genes.

[0723] One example of a restriction enzyme which cleaves within the coding sequence of the hIL-3 gene is ClaI whose recognition site is at codons 20 and 21. The use of ClaI to cleave the sequence of hIL-3 requires that the plasmid DNA be isolated from an E. coli strain that fails to methylate adenines in the DNA at GATC recognition sites. This is because the recognition site for ClaI, ATCGAT, occurs within the sequence GATCGAT which occurs at codons 19, 20 and 21 in the hIL-3 gene. The A in the GATC sequence is methylated in most E. coli host cells. This methylation prevents ClaI from cleaving at that particular sequence. An example of a strain that does not methylate adenines is GM48.

[0724] Interpretation of Activity of Single Amino Acid Mutants in IL-3 (15-125)

[0725] As illustrated in Tables 6 and 9, there are certain positions in the IL-3 (15-125) molecule which are intolerant of substitutions, in that most or all substitutions at these positions resulted in a considerable decrease in bioactivity. There are two likely classes of such “down-mutations”: mutations that affect overall protein structure, and mutations that interfere directly with the interaction between the IL-3 molecule and its receptor. Mutations affecting the three-dimensional structure of the protein will generally lie in the interior of the protein, while mutations affecting receptor binding will generally lie on the surface of the protein. Although the three-dimensional structure of IL-3 is unknown, there are simple algorithms which can aid in the prediction of the structure. One such algorithm is the use of “helical wheels” (Kaiser, E. T. & Kezdy, F. J., Science, 223:249-255 (1984)). In this method, the presence of alpha helical protein structures can be predicted by virtue of their amphipathic nature. Helices in globular proteins commonly have an exposed hydrophilic side and a buried hydrophobic side. As a broad generalization, in globular proteins, hydrophobic residues are present in the interior of the protein, and hydrophilic residues are present on the surface. By displaying the amino acid sequence of a protein on such a “helical wheel” it is possible to derive a model for which amino acids in alpha helices are exposed and which are buried in the core of the protein. Such an analysis of the IL-3 (15-125) molecule predicts that the following helical residues are buried in the core:

[0726] M19, I20, I23, I24, L27, L58, F61, A64, L68, A71, I74, I77, L78, L81, W104, F107, L111, Y114, L115, L118.

[0727] In addition, cysteine residues at positions 16 and 84 are linked by a disulfide bond, which is important for the overall structure or “folding” of the protein. Finally, mutations which result in a major disruption of the protein structure may be expressed at low level in the secretion system used in our study, for a variety of reasons: either because the mis-folded protein is poorly recognized by the secretion machinery of the cell; because mis-folding of the protein results in aggregation, and hence the protein cannot be readily extracted from the cells; or because the mis-folded protein is more susceptible to degradation by cellular proteases. Hence, a block in secretion may indicate which positions in the IL-3 molecule which are important for maintenance of correct protein structure.

[0728] In order to retain the activity of a variant of IL-3, it is necessary to retain both the structural integrity of the protein, and retain the specific residues important for receptor contact. Hence it is possible to define specific amino acid residues in IL-3 (15-125) which must be retained in order to preserve biological activity.

[0729] Residues predicted to be important for interaction with the receptor: D21, E22, E43, D44, L48, R54, R94, D103, K110, F113.

[0730] Residues predicted to be structurally important: C16, L58, F61, A64, I74, L78, L81, C84, P86, P92, P96, F107, L111, L115, L118.

[0731] The hIL-3 muteins of the present invention may be useful in the treatment of diseases characterized by a decreased levels of either myeloid, erythroid, lymphoid, or megakaryocyte cells of the hematopoietic system or combinations thereof. In addition, they may be used to activate mature myeloid and/or lymphoid cells. Among conditions susceptible to treatment with the polypeptides of the present invention is leukopenia, a reduction in the number of circulating leukocytes (white cells) in the peripheral blood. Leukopenia may be induced by exposure to certain viruses or to radiation. It is often a side effect of various forms of cancer therapy, e.g., exposure to chemotherapeutic drugs and of infection or hemorrhage. Therapeutic treatment of leukopenia with these hIL-3 mutant polypeptides of the present invention may avoid undesirable side effects caused by treatment with presently available drugs.

[0732] The hIL-3 muteins of the present invention may be useful in the treatment of neutropenia and, for example, in the treatment of such conditions as aplastic anemia, cyclic neutropenia, idiopathic neutropenia, Chdiak-Higashi syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome and myelofibrosis.

[0733] Many drugs may cause bone marrow suppression or hematopoietic deficiencies. Examples of such drugs are AZT, DDI, alkylating agents and anti-metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, and diuretics. The hIL-3 muteins of the present invention may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies which often occur in patients treated with these drugs.

[0734] Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis. The hIL-3 muteins of the present invention may be useful in treating such hematopoietic deficiency.

[0735] The treatment of hematopoietic deficiency may include administration of the hIL-3 mutein of a pharmaceutical composition containing the hIL-3 mutein to a patient. The hIL-3 muteins of the present invention may also be useful for the activation and amplification of hematopoietic precursor cells by treating these cells in vitro with the muteins of the present invention prior to injecting the cells into a patient.

[0736] Various immunodeficiencies e.g., in T and/or B lymphocytes, or immune disorders, e.g., rheumatoid arthritis, may also be beneficially affected by treatment with the hIL-3 mutant polypeptides of the present invention. Immunodeficiencies may be the result of viral infections e.g. HTLVI, HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or the result of other medical treatment. The hIL-3 mutant polypeptides of the present invention may also be employed, alone or in combination with other hematopoietins, in the treatment of other blood cell deficiencies, including thrombocytopenia (platelet deficiency), or anemia. Other uses for these novel polypeptides are in the treatment of patients recovering from bone marrow transplants in vivo and ex vivo, and in the development of monoclonal and polyclonal antibodies generated by standard methods for diagnostic or therapeutic use.

[0737] Other aspects of the present invention are methods and therapeutic compositions for treating the conditions referred to above. Such compositions comprise a therapeutically effective amount of one or more of the hIL-3 muteins of the present invention in a mixture with a pharmaceutically acceptable carrier. This composition can be administered either parenterally, intravenously or subcutaneously. When administered, the therapeutic composition for use in this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.

[0738] The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, a daily regimen may be in the range of 0.2-150 μg/kg of non-glycosylated IL-3 protein per kilogram of body weight. This dosage regimen is referenced to a standard level of biological activity which recognizes that native IL-3 generally possesses an EC50 at or about 10 picoMolar to 100 picoMolar in the AML proliferation assay described herein. Therefore, dosages would be adjusted relative to the activity of a given mutein vs. the activity of native (reference) IL-3 and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day. In addition, there may exist specific circumstances where dosages of IL-3 mutein would be adjusted higher or lower than the range of 10-200 micrograms per kilogram of body weight. These include co-administration with other CSF or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated IL-3 mutein; and various patient-related issues mentioned earlier in this section. As indicated above, the therapeutic method and compositions may also include co-administration with other human factors. A non-exclusive list of other appropriate hematopoietins, CSFs and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, CSF-1, G-CSF, Meg-CSF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, LIF, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, or combinations thereof. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by periodic assessment of the hematological profile, e.g., differential cell count and the like.

[0739] Materials and Methods for hIL-3 Mutein Expression in E. coli

[0740] Unless noted otherwise, all specialty chemicals were obtained from Sigma Co., (St. Louis, Mo.). Restriction endonucleases, T4 poly-nucleotides kinase, E. coli DNA polymerase I large fragment (Klenow) and T4 DNA ligase were obtained from New England Biolabs (Beverly, Mass.) or Boehringer Mannheim (Indianapolis, Ind.). All chemicals and enzymes were used according to manufacturer's directions.

[0741]

Escherichia coli
Strains

[0742] Strain JM101: delta (pro lac), supE, thi, F′(traD36, proAB, lacI-Q, lacZdeltaM15) (Messing, 1979). This strain can be obtained from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, accession number 33876. MON 105 (W3110 rpoH358) (Obukowicz, et al., 1992) is a derivative of W3110 (Bachmann, 1972) and has been assigned ATCC accession number 55204. Strain GM48: dam-3, dcm-6, gal, ara, lac, thr, leu, tonA, tsx (Marinus, 1973) was used to make plasmid DNA that is not methylated at the sequence GATC.

[0743] Genes and Plasmids

[0744] The gene used for hIL-3 production in E. coli was obtained from British Biotechnology Incorporated, Cambridge, England, catalogue number BBG14. This gene is carried on a pUC based plasmid designated pP0518. The human IL-3 gene sequence is from Yang, et al. (1986).

[0745] The plasmids used for production of hIL-3 in E. coli contain genetic elements whose use has been described (Olins et al., 1988; Olins and Rangwala, 1990). The replicon used is that of pBR327 [(Bolivar et al. (1977); Soberon et al., 1980] which is maintained at a copy number of about 50 in the cell (Covarrubias, et al., (1981)). A gene encoding the beta-lactamase protein is present on the plasmids. This protein confers ampicillin resistance on the cell. This resistance serves as a selectable phenotype for the presence of the plasmid in the cell.

[0746] Intracellular Expression Plasmids

[0747] For cytoplasmic (intracellular) expression vectors the transcription promoter was derived from the recA gene of E. coli (Sancar et al., 1980). This promoter, designated precA, is contained on 72 base pairs (bp) BglII, BamHI fragment which includes the RNA polymerase binding site and the lexA repressor binding site (the operator). This segment of DNA provides high level transcription that is regulated even when the recA promoter is on a plasmid with the pBR327 origin of replication (Olins et al., 1988) incorporated herein by reference.

[0748] Secretion Expression Plasmids:

[0749] In secretion expression plasmids the transcription promoter was derived from the ara B, A, and D genes of E. coli (Greenfield et al., 1978). This promoter is designated pAraBAD and is contained on a 323 base pair SacII, BglII restriction fragment. The lamB secretion leader (Wong et al., 1988, Clement et al., 1981) was fused to the N-terminus of the hIL-3 gene at the recognition sequence for the enzyme NcoI (5′CCATGG3′). The hIL-3 genes used were engineered to have a HindIII recognition site (5′AAGCTT3′) following the coding sequence of the gene. Downstream of the gene is a 550 bp fragment containing the origin of replication of the single stranded phage f1 [Olins and Rangwala (1989)].

[0750] These hIL-3 variants were expressed as a fusion with the lamB signal peptide operatively joined to the araBAD promoter (Greenfield, 1978) and the g10-L ribosome binding site (Olins et al. 1988). The signal peptide is removed as part of the secretion process. The processed form was selectively released from the periplasm by osmotic shock as a correctly folded and fully active molecule. Secretion of (15-125) hIL-3 was further optimized by using low inducer (arabinose) concentration and by growth at 30° C. These conditions resulted in lower accumulation levels of unprocessed lamB signal peptide (15-125) hIL-3 fusion, maximal accumulation levels of processed (15-125) hIL-3 and selective release of (15-125) hIL-3 by osmotic shock fractionation. The use of a tightly regulated promoter such as araBAD from which the transcription level and hence the expression level can be modulated allowed for the optimization of secretion of (15-125) hIL-3.

[0751] The ribosome binding site (RBS) used is that from gene 10 of phage T7 (Olins et al., 1988). This is encoded in a 100 base pair (bp) fragment placed adjacent to precA. In the plasmids used herein, the recognition sequence for the enzyme NcoI (5′CCATGG3′) follows the g10-L RBS. It is at this NcoI site that the hIL-3 genes are joined to the plasmid. It is expected that the nucleotide sequence at this junction will be recognized in mRNA as a functional start site for translation (Olins et al., 1988). The hIL-3 genes used were engineered to have a HindIII recognition site (5′AAGCTT3′) following the coding sequence of the gene. Downstream of the gene is a 550 base pair fragment containing the origin of replication of the single stranded phage f1 (Dente et al., 1983; Olins, et al., 1990) both incorporated herein by reference. A plasmid containing these elements is pMON2341. Another plasmid containing these elements is pMON5847 which has been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 under the accession number ATCC 68912.

[0752] Synthesis of Oligonucleotides

[0753] Oligonucleotides were synthesized by the cyanoethyl method (Adams et al. 1983, McBride, et al. 1983, Sinba et al., 1984) on Nucleotide Synthesizer model 380A or 380B from Applied Biosystems, Inc. (Foster City, Calif.). Some oligonucleotides were purchased from Genosys Biotechnologies Inc. (The Woodlands, Tex.) or Midland Certified Reagent Co. (Midland, Tex.). The degenerate oligonucleotides were synthesized by machine mixing an equal molar ratio of the desired nucleosides in the condensation reaction at degenerate positions. Oligonucleotides were purified by polyacrylamide gel electrophoresis at concentrations from 12-20% (19:1 crosslinked) in 0.5× Tris borate (TBE) buffer (0.045 M Tris, 0.045 M boric acid, 1.25 mM EDTA) as described by Atkinson (1984). The oligonucleotides were desalted by passage through a Nensorb 20 column obtained from DuPont/New England Nuclear (Boston, Mass.) using a PREP Automated Sample Processor obtained from DuPont, Co. (Wilmington, Del.).

[0754] Quantitation of Synthetic Oligonucleotides

[0755] Synthetic oligonucleotides were resuspended in water (100 μl) and quantitated by reading the absorbance at 260 nm on a Beckman DU40 Spectrophotometer (Irvine, Calif.) using a one centimeter by one millimeter quartz cuvette (Maniatis, 1982). The concentration was determined using an extinction coefficient of 1×104 (Voet et al., 1963; Mahler and Cordes, 1966). The oligonucleotide was then diluted to the desired concentration.

[0756] Quantitation of synthetic DNA fragments can also be achieved by adding 10 to 100 picomoles of DNA to a solution containing kinase buffer (25 mM Tris pH 8.0, 10 mM MgCl2, 10 mM DTT and 2 mM spermidine). To the reaction mix is added ATP to 20 micromolar, ATP radiolabeled at the gamma phosphate (5000-10,0000 dpm/pmol) and 5 units of T4 polynucleotide kinase. Radiolabelled material is obtained from New England Nuclear (Boston, Mass.). The 10 microliter mixture is incubated at 37° C. for one hour. A 1 microliter aliquot of the mixture is chromatographed on DEAE paper (DE81 from Whatman) in 0.35 M ammonium bicarbonate. The counts that remain at the origin are used to determine the concentration of the synthetic DNA.

[0757] Recombinant DNA Methods

[0758] Isolation of plasmid DNA from E. coli cultures was performed as described (Birnboim and Doly, 1979). Some DNAs were purified by Magic™ miniprep columns, available from Promega (Madison, Wis.).

[0759] Purified plasmid DNA was treated with restriction endonucleases according to manufacturer's instructions. Analysis of the DNA fragments produced by treatment with restriction enzymes was done by agarose or polyacrylamide gel electrophoresis. Agarose (DNA grade from Fisher, Pittsburgh Pa.) was used at a concentration of 1.0% in a Tris-acetate running buffer (0.04 M Tris-acetate, 0.001M EDTA). Polyacrylamide (BioRad, Richmond Calif.) was used at a concentration of 6% (19:1 crosslinked) in 0.5× Tris-borate buffer (0.045 M Tris, 0.045 M boric acid, 1.25 mM EDTA), hereafter referred to as PAGE.

[0760] DNA polymerase I, large fragment, Klenow enzyme was used according to manufacturer's instructions to catalyze the addition of mononucleotides from 5′ to 3′ of DNA fragments which had been treated with restriction enzymes that leave protruding ends. The reactions were incubated at 65° C. for 10 minutes to heat inactivate the Klenow enzyme.

[0761] The synthetic oligonucleotides were made without 5′ or 3′ terminal phosphates. In cases where such oligonucleotides were ligated end to end, the oligonucleotides were treated at a concentration of 10 picomoles per microliter with T4 polynucleotide kinase in the following buffer: 25 mM Tris, pH 8.0, 10 mM MgCl2, 10 mM dithiothreitol, 2 mM spermidine, 1 mM rATP. After incubation for 30 minutes at 37° C., the samples were incubated at 65° C. for five minutes to heat inactivate the kinase.

[0762] Synthetic Gene Assembly

[0763] The (15-125) hIL-3 gene was divided into four regions separated by five convenient restriction sites. In each of the four regions synthetic oligonucleotides were designed so that they would anneal in complementary pairs, with protruding single stranded ends “or blunt ends” and when the pairs were properly assembled would result in a DNA sequence that encoded a portion of the hIL-3 gene. Amino acid substitutions in the hIL-3 gene were made by designing the oligonucleotides to encode the desired substitutions. The complementary oligonucleotides were annealed at concentration of 1 picomole per microliter in ligation buffer plus 50 mM NaCl. The samples were heated in a 100 ml beaker of boiling water and permitted to cool slowly to room temperature. One picomole of each of the annealed pairs of oligonucleotides were ligated with approximately 0.2 picomoles of plasmid DNA, digested with the appropriate restriction enzymes, in ligation buffer (25 mM Tris pH 8.0, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, 2 mM spermidine) with T4 DNA ligase obtained from New England Biolabs (Beverly, Mass.) in a total volume of 20 μl at room temperature overnight.

[0764] DNA fragments were isolated from agarose gels by intercepting the restriction fragments on DEAE membranes from Schleicher and Schuell (Keene, N.H.) and eluting the DNA in 10 mM Tris, 1 mM EDTA, 1 M NaCl at 55° C. for 1 hour, according to manufacturer's directions. The solutions containing the DNA fragment were concentrated and desalted by using Centricon 30 concentrators from Amicon (W. R. Grace, Beverly Mass.) according to the manufacturer's directions. Ligations were performed at 15° C. overnight, except as noted, in ligation buffer (66 mM Tris pH 7.5, 6.6 mM MgCl2, 1 mM dithiothreitol, 0.4 mM ATP) with T4 ligase obtained from New England Biolabs (Beverly, Mass.).

[0765] Polymerase Chain Reaction

[0766] Polymerase Chain Reaction (hereafter referred to as PCR) techniques (Saiki, 1985) used the reagent kit and thermal cycler from Perkin-Elmer Cetus (Norwalk, Conn.). PCR is based on a thermostable DNA polymerase from Thermus aquaticus. The PCR technique is a DNA amplification method that mimics the natural DNA replication process in that the number of DNA molecules doubles after each cycle, in a way similar to in vivo replication. The DNA polymerase mediated extension is in a 5′Ø3′ direction. The term “primer” as used herein refers to an oligonucleotide sequence that provides an end to which the DNA polymerase can add nucleotides that are complementary to a nucleotide sequence. The latter nucleotide sequence is referred to as the “template”, to which the primers are annealed. The amplified PCR product is defined as the region comprised between the 5′ ends of the extension primers. Since the primers have defined sequences, the product will have discrete ends, corresponding to the primer sequences. The primer extension reaction was carried out using 20 picomoles (pmoles) of each of the oligonucleotides and 1 picogram of template plasmid DNA for 35 cycles (1 cycle is defined as 94° C. for one minute, 50° C. for two minutes and 72° C. for three minutes). The reaction mixture was extracted with an equal volume of phenol/chloroform (50% phenol and 50% chloroform, volume to volume) to remove proteins. The aqueous phase, containing the amplified DNA, and solvent phase were separated by centrifugation for 5 minutes in a microcentrifuge (Model 5414 Eppendorf Inc, Fremont Calif.). To precipitate the amplified DNA the aqueous phase was removed and transferred to a fresh tube to which was added {fraction (1/10)} volume of 3M NaOAc (pH 5.2) and 2.5 volumes of ethanol (100% stored at minus 20° C.). The solution was mixed and placed on dry ice for 20 minutes. The DNA was pelleted by centrifugation for 10 minutes in a microcentrifuge and the solution was removed from the pellet. The DNA pellet was washed with 70% ethanol, ethanol removed and dried in a speedvac concentrator (Savant, Farmingdale, N.Y.). The pellet was resuspended in 25 microliters of TE (20 mM Tris-HCl pH 7.9, 1 mM EDTA). Alternatively the DNA was precipitated by adding equal volume of 4M NH4OAc and one volume of isopropanol [Treco, (1989)]. The solution was mixed and incubated at room temperature for 10 minutes and centrifuged. These conditions selectively precipitate DNA fragments larger than ˜20 bases and were used to remove oligonucleotide primers. One quarter of the reaction was digested with restriction enzymes [Higuchi, (1989)] and on completion heated to 70° C. to inactivate the enzymes.

[0767] Two Step Site-Directed PCR Mutagenesis

[0768] Single amino acid substitution variants were created at positions 17-123 of hIL-3 in two site-directed mutagenesis steps by PCR (Bauer et al. manuscript in preparation).

[0769] The single amino acid substitution variants at positions 94-105 of hIL-3 were created as described below. In the first mutagenesis step plasmid DNA, containing the hIL-3 gene (amino acids 15-125), was the template in the PCR reaction. The DNA sequence of one of the oligonucleotide primers was designed to replace 12 base in the hIL-3 gene (15-125) with 12 bases encoding two translation stop codons (5′TAATAA3′), followed by the recognition sequence (5′GTCGAC3′) restriction enzyme SalI. This 12 base sequence was substituted in the hIL-3 gene following the codon for amino acids 93, 97 and 101. Plasmids containing these mutagenized genes served as the templates for the second mutagenesis step.

[0770] In the second mutagenesis step, the 12 base substitution introduced in the first mutagenesis step, was replaced using a 32 fold degenerate oligonucleotide. The degenerate oligonucleotides were synthesized by machine mixing an equal molar ratio of the desired nucleosides in the condensation reaction at degenerate positions. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon. The other bases in the oliqonucleotides corresponded to the hIL-3 sequence. The degenerate oligonucleotides theoretically contain 32 different codons, encoding all 20 amino acids and one translation stop codon, at a single position. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. This pool of single amino acid substitutions at a single position is referred to as a “library”. This two step PCR site-directed mutagenesis approach was used to facilitate the identification of single amino acid substitution variants by differential DNA hybridization.

[0771] The single amino acid substitution variants at positions 17-93 and 106-123 of hIL-3 (15-125) were created as described below. In the first mutagenesis step plasmid DNA, containing the hIL-3 gene (15-125), was the template in the PCR reaction. The DNA sequence of one of the oligonucleotide primers was designed to delete 18 bases in the hIL-3 gene that encode the following amino acids; 17-22, 23-28, 29-34, 35-40, 41-46, 47-52, 53-58, 59-64, 65-70, 71-76, 77-82, 83-88, 88-93, 106-111, 112-117 and 118-123. Plasmids containing these deletion genes served as the templates for the second mutagenesis step.

[0772] In the second mutagenesis step the 18 base deletion, created in the first mutagenesis step, was restored using a 32 fold degenerate oligonucleotide. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon. The other bases in the oligonucleotides corresponded to the hIL-3 sequence. The degenerate oligonucleotides theoretically contain 32 different codons, encoding all 20 amino acids and one translation stop codon, at a single position. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. This pool of single amino acid substitutions at a single position is referred to as a “library”. This two step PCR site-directed mutagenesis approach was used to facilitate the identification of single amino acid substitution variants by differential DNA hybridization.

[0773] Recovery of Recombinant Plasmids from Ligation Mixes and Transformation of E. coli Cells with Recombinant Plasmid DNA

[0774]

E. coli
JM101 cells were made competent to take up DNA. Typically, 20 to 100 ml of cells were grown in LB medium to a density of approximately 150 Klett units and then collected by centrifugation. The cells were resuspended in one half culture volume of 50 mM CaCl2 and held at 4° C. for one hour. The cells were again collected by centrifugation and resuspended in one tenth culture volume of 50 mM CaCl2. DNA was added to a 150 microliter volume of these cells, and the samples were held at 4° C. for 30 minutes. The samples were shifted to 42° C. for one minute, one milliliter of LB was added, and the samples were shaken at 37° C. for one hour. Cells from these samples were spread on plates containing ampicillin to select for transformants. The plates were incubated overnight at 37° C. Single colonies were picked and grown in LB supplemented with ampicillin overnight at 37° C. with shaking. From these cultures DNA was isolated for restriction analysis.

[0775] Typically plasmids were constructed, using methods described herein or by references cited herein, as follows except as noted in examples included herein. DNA fragments were purified from agarose or polyacrylamide gels. Purified DNA fragments were ligated and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin containing plates. Plasmid DNA was isolated from a single colony grown in LB Broth and screened by restriction analysis for the desired construct and sequenced to determine that the DNA sequence was correct.

[0776] Culture Media

[0777] LB medium (Maniatis et al., 1982) was used for growth of cells for DNA isolation. M9 minimal medium supplemented with 1.0% casamino acids, acid hydrolyzed casein, Difco (Detroit, Mich.) was used for cultures in which recombinant hIL-3 was produced. The ingredients in the M9 medium were as follows: 3 g/liter KH2PO4, 6 g/l Na2HPO4, 0.5 g/l NaCl, 1 g/l NH4Cl, 1.2 mM MgSO4, 0.025 mM CaCl2, 0.2% glucose (0.2% glycerol with the AraBAD promoter), 1% casamino acids, 0.1 ml/l trace minerals (per liter 108 g FeCl3.6H2O, 4.0 g ZnSO4.7H2O, 7.0 CoCl2.2H2O, 7.0 g Na2MoO4.2H2O, 8.0 g CuSO4.5H2O, 2.0 g H3BO3, 5.0 g MnSO4.H2O, 100 ml concentrated HCl). Bacto agar from Difco was used for solid media and ampicillin (Polycillin-N from Bristol-Meyers, Evansville, Ind.) was added to both liquid and solid LB media at 200 micrograms per milliliter.

[0778] DNA Sequence Analysis

[0779] The nucleotide sequencing of plasmid DNA was performed using a Genesis 2000 sequencer obtained from DuPont (Wilmington, Del.) according to the methods of Prober et al. (1987) and Sanger et al. (1977). Some DNA sequences were determined using Sequenase™ polymerase according to the protocol of its supplier, U.S. Biochemicals (Cleveland, Ohio).

[0780] Production of Recombinant hIL-3 Muteins in E. coli with Vectors Employing the recA Promoter

[0781]

E. coli
strains harboring the plasmids of interest were grown at 37° C. in M9 plus casamino acids medium with shaking in a Gyrotory water bath Model G76 from New Brunswick Scientific (Edison, N.J.). Growth was monitored with a Klett Summerson meter (green 54 filter), Klett Mfg. Co. (New York, N.Y.). At a Klett value of approximately 150, an aliquot of the culture (usually one milliliter) was removed for protein analysis. To the remaining culture, nalidixic acid (10 mg/ml) in 0.1 N NaOH was added to a final concentration of 50 μg/ml. The cultures were shaken at 37° C. for three to four hours after addition of nalidixic acid. A high degree of aeration was maintained throughout the bacterial growth in order to achieve maximal production of the desired gene product. The cells were examined under a light microscope for the presence of refractile bodies (RBs). One milliliter aliquots of the culture were removed for analysis of protein content.

[0782] Production of Recombinant hIL-3 Proteins from the AraBAD Promoter in E. coli

[0783]

E. coli
strains harboring the plasmids of interest were grown at 30° C. with shaking in M9 medium plus casamino acids and glycerol. Growth was monitored with a Klett Summerson calorimeter, using a green 54 filter. At a Klett value of about 150, an aliquot of the culture (usually one milliliter) was removed for protein analysis. To the remaining culture, 20% arabinose was added to a final concentration of 0.05%. The cultures were shaken at 30° C. for three to four hours after addition of arabinose. A high degree of aeration was maintained throughout the bacterial growth in order to achieve maximal production of the desired gene product. One milliliter aliquots of the culture were removed for analysis of protein content.

[0784] Secretion and Osmotic Shock

[0785] Three hour post induction samples were fractionated by osmotic shock [Neu and Heppel (1965)]. The Klett value of the cultures was determined and 1 ml of cells were centrifuged in a Sigma microcentrifuge (West Germany) model 202MK in 1.5 mls snap top microcentrifuge tubes for 5 minutes at 10,000 rpm. The cell pellet was resuspended very gently by pipeting in a room temperature sucrose solution (20% sucrose w/v, 30 mM Tris-Hcl pH7.5, 1 mM EDTA), using 1 μl/l Klett unit. Following a 10 minute incubation at room temperature, the cells were centrifuged for 5 minutes at 10,000 rpm. The sucrose fraction was carefully removed from the cell pellet. The cell pellet was then resuspended very gently by pipeting in ice cold distilled water, using 1 μl/l Klett unit. Following a 10 minute incubation on ice, the cells were centrifuged for 5 minutes at 12,000 rpm. The water fraction was carefully removed. Equal volumes of the sucrose and water fractions were pooled and aliquoted to provide samples for ELISA and biological activity screening.

[0786] Analysis of Protein Content of E. coli Cultures Producing hIL-3 Mutant Polypeptides

[0787] Bacterial cells from cultures treated as described above were collected from the medium by centrifugation. Aliquots of these cells were resuspended in SDS loading buffer (4×: 6 g SDS, 10 ml beta-mercaptoethanol, 25 ml upper Tris gel stock (0.5 M Tris HCl pH 6.8, 0.4% SDS) brought to 50 ml with glycerol, 0.2% bromophenol blue was added) at a concentration of one microliter per Klett unit. These samples were incubated at 85° C. for five minutes and vortexed. Five or ten microliter aliquots of these samples were loaded on 15% polyacrylamide gels prepared according to the method of Laemmli (1970). Protein bands were visualized by staining the gels with a solution of acetic acid, methanol and water at 5:1:5 (volume to volume) ratio to which Coomassie blue had been added to a final concentration of 1%. After staining, the gels were washed in the same solution without the Coomassie blue and then washed with a solution of 7% acetic acid, 5% methanol. Gels were dried on a gel drier Model SE1160 obtained from Hoeffer (San Francisco, Calif.). The amount of stained protein was measured using a densitometer obtained from Joyce-Loebl (Gateshead, England). The values obtained were a measure of the amount of the stained hIL-3 protein compared to the total of the stained protein of the bacterial cells.

[0788] Western Blot Analysis of hIL-3 Muteins Made in E. coli

[0789] In some E. coli cultures producing hIL-3, the level of accumulation of the hIL-3 protein is lower than 5% of total bacterial protein. To detect hIL-3 produced at this level, Western blot analysis was used. Proteins from cultures induced with nalidixic acid or arabinose were run on polyacrylamide gels as described above except that volumes of sample loaded were adjusted to produce appropriate signals. After electrophoresis, the proteins were electroblotted to APT paper, Transa-bind, Schleicher and Schuell (Keene, N.H.) according to the method of Renart et al. (1979). Antisera used to probe these blots had been raised in rabbits, using peptides of the sequence of amino acids 20 to 41 and 94 to 118 of hIL-3 as the immunogens. The presence of bound antibody was detected with Staphylococcal protein A radiolabeled with 125I, obtained from New England Nuclear (Boston, Mass.).

[0790] Fractionation of E. coli Cells Producing hIL-3 Proteins in the Cytoplasm

[0791] Cells from E. coli cultures harboring plasmids that produce hIL-3 muteins were induced with nalidixic acid. After three hours, the hIL-3 muteins accumulated in refractile bodies. The first step in purification of the hIL-3 muteins was to sonicate cells. Aliquots of the culture were resuspended from cell pellets in sonication buffer: 10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl and 0.1 mM PMSF. These resuspended cells were subjected to several repeated sonication bursts using the microtip from a Sonicator cell disrupter, Model W-375 obtained from Heat Systems-Ultrasonics Inc. (Farmingdale, N.Y.). The extent of sonication was monitored by examining the homogenates under a light microscope. When nearly all of the cells had been broken, the homogenates were fractionated by centrifugation. The pellets, which contain most of the retractile bodies, are highly enriched for hIL-3 muteins.

[0792] Methods: Extraction, Refolding and Purification of Interleukin-3 (IL-3) Muteins Expressed as Refractile Bodies in E. coli.

[0793] Extraction of retractile bodies (RB's):

[0794] For each gram of RB's (and typically one gram is obtained from a 300 ml E. coli culture), 5 ml of a solution containing 6M guanidine hydrochloride (GnHCl), 50 mM 2-N-cyclohexylaminoethanesulfonic acid (CHES) pH 9.5 and 20 mM dithiothreitol (DTT) was added. The RB's were extracted with a Bio-Homogenizer for 15-30 seconds and gently rocked for 2 hours at 5 degrees centigrade (5° C.) to allow the protein to completely reduce and denature.

[0795] Refolding of the IL-3 Muteins

[0796] The protein solution was transferred to dialysis tubing (1000 molecular weight cut-off) and dialyzed against at least 100 volumes of 4M GnHCl-50 mM CHES pH 8.0. The dialysis was continued overnight at 5° C. while gently stirring. Subsequently dialysis was continued against at least 100 volumes of 2M GnHCl-50 mM CHES pH 8.0 and dialyzed overnight at 5° C. while gently stirring.

[0797] Purification of the IL-3 Muteins

[0798] The protein solution was removed from the dialysis tubing and acidified by the addition of 40% acetonitrile (CH3CN)-0.2% trifluoroacetic acid (TFA) to a final concentration of 20% CH3CN-0.1% TFA. This was centrifuged (16,000×g for 5 minutes) to clarify and the supernatant was loaded onto a Vydac C-18 reversed phase column (10×250 mm) available from Vydac (Hesperia, Calif.) previously equilibrated in 20% CH3CN-0.1% TFA. The column was eluted with a linear gradient (0.2% CH3CN/minute) between 40-50% CH3CN-0.1% TFA at a flow rate of 3 ml/minute while collecting 1.5 ml fractions. The fractions were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and the appropriate fractions pooled. The pooled material was dried by lyophilization or in a Speed Vac concentrator. The dry powder was reconstituted with 10 mM ammonium bicarbonate pH 7.5, centrifuged (16,000×g for 5 minutes) to clarify and assayed for protein concentration by the method of Bradford (1976) with bovine serum albumin as the standard. Such protein can be further analyzed by additional techniques such as, SDS-PAGE, electrospray mass spectrometry, reverse phase HPLC, capillary zone electrophoresis, amino acid composition analysis, and ELISA (enzyme-linked immunosorbent assay).

[0799] hIL-3 Sandwich ELISA

[0800] IL-3 protein concentrations were determined using a sandwich ELISA based on an affinity purified polyclonal goat anti-rhIL-3. Microtiter plates (Dynatech Immulon II) were coated with 150 μl goat-anti-rhIL-3 at a concentration of approximately 1 μg/ml in 100 mM NaHCO3, pH 8.2. Plates were incubated overnight at room temperature in a chamber maintaining 100% humidity. Wells were emptied and the remaining reactive sites on the plate were blocked with 200 μl of solution containing 10 mM PBS, 3% BSA and 0.05% Tween 20, pH 7.4 for 1 hour at 37° C. and 100% humidity. Wells were emptied and washed 4× with 150 mM NaCl containing 0.05% Tween 20 (wash buffer). Each well then received 150 μl of dilution buffer (10 mM PBS containing 0.1% BSA, 0.01% Tween 20, pH 7.4), containing rhIL-3 standard, control, sample or dilution buffer alone. A standard curve was prepared with concentrations ranging from 0.125 ng/ml to 5 ng/ml using a stock solution of rhIL-3 (concentration determined by amino acid composition analysis). Plates were incubated 2.5 hours at 37° C. and 100% humidity. Wells were emptied and each plate was washed 4× with wash buffer. Each well then received 150 μl of an optimal dilution (as determined in a checkerboard assay format) of goat anti-rhIL-3 conjugated to horseradish peroxidase. Plates were incubated 1.5 hours at 37° C. and 100% humidity. Wells were emptied and each plate was washed 4× with wash buffer. Each well then received 150 ul of ABTS substrate solution (Kirkegaard and Perry). Plates were incubated at room temperature until the color of the standard wells containing 5 ng/ml rhIL-3 had developed enough to yield an absorbance between 0.5-1.0 when read at a test wavelength of 410 nm and a reference wavelength of 570 nm on a Dynatech microtiter plate reader. Concentrations of immunoreactive rhIL-3 in unknown samples were calculated from the standard curve using software supplied with the plate reader.

[0801] AML Proliferation Assay for Bioactive Human Interleukin-3

[0802] The factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, Md.). This cell line, established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM/CSF supplemented medium (Lange, B., et al., (1987); Valtieri, M., et al., (1987). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al., (1987)). A cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours. The cells were then replated at 1×105 cells/well in a 24 well plate in media containing 100 U/ml IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells were maintained as AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.

[0803] AML 193 1.3 cells were washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, N.Y.) by centrifuging cell suspensions at 250×g for 10 minutes followed by decantation of supernatant. Pelleted cells were resuspended in HBSS and the procedure was repeated until six wash cycles were completed. Cells washed six times by this procedure were resuspended in tissue culture medium at a density ranging from 2×105 to 5×105 viable cells/ml. This medium was prepared by supplementing Iscove's modified Dulbecco's Medium (IMDM, Hazleton, Lenexa, Kans.) with albumin, transferrin, lipids and 2-mercaptoethanol. Bovine albumin (Boehringer-Mannheim, Indianapolis, Ind.) was added at 500 μg/ml; human transferrin (Boehringer-Mannheim, Indianapolis, Ind.) was added at 100 μg/ml; soybean lipid (Boehringer-Mannheim, Indianapolis, Ind.) was added at 50 μg/ml; and 2-mercaptoethanol (Sigma, St. Louis, Mo.) was added at 5×10−5M.

[0804] Serial dilutions of human interleukin-3 or human interleukin-3 variant protein (hIL-3 mutein) were made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 μl of medium containing interleukin-3 or interleukin-3 variant protein once serial dilutions were completed. Control wells contained tissue culture medium alone (negative control). AML 193 1.3 cell suspensions prepared as above were added to each well by pipetting 50 μl (2.5×104 cells) into each well. Tissue culture plates were incubated at 37° C. with 5% CO2 in humidified air for 3 days. On day 3, 0.5 μCi 3H-thymidine (2 Ci/mM, New England Nuclear, Boston, Mass.) was added in 50 μl of tissue culture medium. Cultures were incubated at 37° C. with 5% CO2 in humidified air for 18-24 hours. Cellular DNA was harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, Md.) using a TOMTEC cell harvester (TOMTEC, Orange, Conn.) which utilized a water wash cycle followed by a 70% ethanol wash cycle. Filter mats were allowed to air dry and then placed into sample bags to which scintillation fluid (Scintiverse II, Fisher Scientific, St. Louis, Mo. or BetaPlate Scintillation Fluid, Pharmacia LKB, Gaithersburg, Md.) was added. Beta emissions of samples from individual tissue culture wells were counted in a LKB Betaplate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, Md.) and data was expressed as counts per minute of 3H-thymidine incorporated into cells from each tissue culture well. Activity of each human interleukin-3 preparation or human interleukin-3 variant preparation was quantitated by measuring cell proliferation (3H-thymidine incorporation) induced by graded concentrations of interleukin-3 or interleukin-3 variant. Typically, concentration ranges from 0.05 pM-105 pM are quantitated in these assays. Activity is determined by measuring the dose of interleukin-3 or interleukin-3 variant which provides 50% of maximal proliferation [EC50=0.5×(maximum average counts per minute of 3H-thymidine incorporated per well among triplicate cultures of all concentrations of interleukin-3 tested-background proliferation measured by 3H-thymidine incorporation observed in triplicate cultures lacking interleukin-3]. This EC50 value is also equivalent to 1 unit of bioactivity. Every assay was performed with native interleukin-3 as a reference standard so that relative activity levels could be assigned.

[0805] Relative biological activities of some IL-3 muteins of the present invention are shown in Table 1. The Relative Biological Activity of IL-3 mutants is calculated by dividing the EC50 of (1-133) hIL-3 by the EC50 of the mutant. The Relative Biological Activity may represent the average of replicate assays.

11TABLE 1BIOLOGICAL ACTIVITY OF IL-3 MUTEINSRelativePlasmidPolypeptideBiologicalCodeStructureActivityReference(1-133) hIL-31.0pMON13286[SEQ ID NO. 69]8.0pMON13304[SEQ ID NO. 66]3.2* The Relative Biological Activity of IL-3 mutants is calculated by dividing the EC50 of (1-133) hIL-3 by the EC50 of the mutant.

[0806] The following assay is used to measure IL-3 mediated sulfidoleukotriene release from human mononuclear cells.

[0807] IL-3 Mediated Sulfidoleukotriene Release from Human Mononuclear Cells

[0808] Heparin-containing human blood was collected and layered onto an equal volume of Ficoll-Paque (Pharmacia #17-0840-02) ready to use medium (density 1.077 g/ml.). The Ficoll was warmed to room temperature prior to use and clear 50 ml polystyrene tubes were utilized. The Ficoll gradient was spun at 300×g for 30 minutes at room temperature using a H1000B rotor in a Sorvall RT6000B refrigerated centrifuge. The band containing the mononuclear cells was carefully removed, the volume adjusted to 50 mls with Dulbecco's phosphate-buffered saline (Gibco Laboratories cat. #310-4040PK), spun at 400×g for 10 minutes at 4° C. and the supernatant was carefully removed. The cell pellet was washed twice with HA Buffer [20 mM Hepes (Sigma #H-3375), 125 mM NaCl (Fisher #S271-500), 5 mM KCl (Sigma #P-9541), 0.5 mM glucose (Sigma #G-5000),0.025% Human Serum Albumin (Calbiochem #126654) and spun at 300×g, 10 min., 4° C.. The cells were resuspended in HACM Buffer (HA buffer supplemented with 1 mM CaCl2 (Fisher #C79-500) and 1 mM MgCl2 (Fisher #M -33) at a concentration of 1×106 cells/ml and 180 μl were transferred into each well of 96 well tissue culture plates. The cells were allowed to acclimate at 37° C. for 15 minutes. The cells were primed by adding 10 μls of a 20× stock of various concentrations of cytokine to each well (typically 100000, 20000, 4000, 800, 160, 32, 6.4, 1.28, 0 fM IL3). The cells were incubated for 15 minutes at 37° C. Sulfidoleukotriene release was activated by the addition of 10 μls of 20×(1000 nM) fmet-leu-phe (Calbiochem #344252) final concentration 50 nM FMLP and incubated for 10 minutes at 37° C.. The plates were spun at 350×g at 4° C. for 20 minutes. The supernatants were removed and assayed for sulfidoleukotrienes using Cayman's Leukotriene C4 EIA kit (Cat. #420211) according to manufacturers' directions. Native (15-125)hIL-3 was run as a standard control in each assay.

[0809] Native hIL-3 possesses considerable inflammatory activity and has been shown to stimulate synthesis of the arachidonic acid metabolites LTC4, LTD4, and LTE4; histamine synthesis and histamine release. Human clinical trials with native hIL-3 have documented inflammatory responses (Biesma, et al., BLOOD, 80:1141-1148 (1992) and Postmus, et al., J. CLIN. ONCOL., 10:1131-1140 (1992)). A recent study indicates that leukotrienes are involved in IL-3 actions in vivo and may contribute significantly to the biological effects of IL-3 treatment (Denzlinger, C., et al., BLOOD, 81:2466-2470 (1993))

[0810] Some muteins of the present invention may have an improved therapeutic profile as compared to native hIL-3 or (15-125)hIL-3. For example, some muteins of the present invention may have a similar or more potent growth factor activity relative to native hIL-3 or (15-125)hIL-3 without having a similar or corresponding increase in the stimulation of leukotriene or histamine. These muteins would be expected to have a more favorable therapeutic profile since the amount of polypeptide which needs to be given to achieve the desired growth factor activity (e. g. cell proliferation) would have a lesser leukotriene or histamine stimulating effect. In studies with native hIL-3, the stimulation of inflammatory factors has been an undesirable side effect of the treatment. Reduction or elimination of the stimulation of mediators of inflammation would provide an advantage over the use of native hIL-3.

[0811] Some muteins of the present invention may have antigenic profiles which differ from that of native hIL-3. For example, in a competition ELISA with an affinity purified polyclonal goat anti-hIL-3 antibody, native hIL-3 significantly blocked the binding of labeled hIL-3 to polyclonal anti-hIL-3 antibody. Some polypeptides of the present invention, particularly those with several amino acids differing from those of native hIL-3, fail to block the binding of hIL-3 to anti-hIL-3 antibody.

[0812] Table 2 lists the sequences of some oligonucleotides used in making the muteins of the present invention.

[0813] Table 3 lists the amino acid sequence of native (15-125)hIL-3 (Peptide #1) and the amino acid sequences of some mutant polypeptides of the present invention. The sequences are given with the amino acid numbering corresponding to that of native hIL-3 [FIG. 1].

[0814] Table 4 lists the DNA sequences that encode some mutated polypeptides of the present invention.

12TABLE 2OLIGONUCLEOTIDESOligo #1AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAGT AATA[SEQ ID NO:8]Oligo #2AGCTTATTAC TGTTGAGCCT GCGCGTTCTC CAAGGTTTTC AGATAGAAGG TCAGTTTACG ACGG[SEQ ID NO:9]Oligo #3CTAGCCACGG CCGCACCCAC GCGACATCCA ATCCATATCA AGGACGGTGA CTGGAATG[SEQ ID NO:24]Oligo #4TTAACATTCC AGTCACCGTC CTTGATATGG ATTGGATGTC GCGTGGGTGC GGCCGTGG[SEQ ID NO:25]Oligo #5CATGGCTAAC TGCTCTAACA TGAT[SEQ ID NO:151]Oligo #6CGATCAT GTTAGAGCAGTTAGC[SEQ ID NO:152]Oligo #7 IL3MUTNCOTGTCTGCTCA GGCCATGGCT[SEQ ID NO:26]Oligo #8 IL3T93GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGTCGAC TTATTACGTG GGTGCGGCCG TGGCTAG[SEQ ID NO:27]Oligo #9 IL3T97GCGCGAATTC ATTCCAGTCA CCGTCGACTT ATTAGATTGG ATGTCGCGTG GGTGG[SEQ ID NO:28]Oligo #10 IL3T101GCGCGAATTC GTCGACTTAT TAGTCCTTGA TATGGATTGG ATG[SEQ ID NO:31]Oligo #11 IL3R94GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG ATGSNNCGTG GGTGCGGCCG TGGCTAG[SEQ ID NO:32]Oligo #12 IL3R95GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG SNNTCGCGTG GGTGCGGCCG TGGC[SEQ ID NO:33]Oligo #13 IL3R96GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATSNN ATGTCGCGTG GGTGCGGCCG T[SEQ ID NO:34]Oligo #14 IL3R97GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGSNNTGG ATGTCGCGTG GGTGCGGC[SEQ ID NO:35]Oligo #15 IL3P9497GATATGGATT GGATGTCGCG TGGG[SEQ ID NO:36]Oligo #16 IL3R98GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TSNNGATTGG ATGTCGCGTG GGTGC[SEQ ID NO:37]Oligo #17 IL3R99GCGCGAATTC ATTCCAGTCA CCGTCCTTSN NATGGATTGG ATGTCGCGTG GG[SEQ ID NO:38]Oligo #18 IL3R100GCGCGAATTC ATTCCAGTCA CCGTCSNNGA TATGGATTGG ATGTCGCGT[SEQ ID NO:39]Oligo #19 IL3R101GCGCGAATTC ATTCCAGTCA CCSNNCTTGA TATGGATTGG ATGTCG[SEQ ID NO:40]Oligo #20 IL3P98100GTCACCGTCC TTGATATGGA TTGG[SEQ ID NO:41]Oligo #21 IL3R102GCGCGAATTC ATTCCAGTCS NNGTCCTTGA TATGGATTGG ATG[SEQ ID NO:42]Oligo #22 IL3R103GCGCGAATTC ATTCCASNNA CCGTCCTTGA TATGGATTGG[SEQ ID NO:43]Oligo #23 IL3R104GCGCGAATTC ATTSNNGTCA CCGTCCTTGA TATGGAT[SEQ ID NO:44]Oligo #24 IL3R105GCGCGAATTC SNNCCAGTCA CCGTCCTTGA TATG[SEQ ID NO:45]Oligo #25 IL3P102105GAATTCATTC CAGTCACCGT TCCTT[SEQ ID NO:46]Oligo #26 IL3MDTR1CGCGCGGAAT TCATTCCAGT CACCGT[SEQ ID NO:47]Oligo #27 DEL1722CGCGCGCCAT GGCTAACTGC ATTATAACAC ACACTTAAAG CA[SEQ ID NO:48]Oligo #28 DEL2328CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAACA GCCACCTTTG CCTTTGCT[SEQ ID NO:49]Oligo #29 DEL2934CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCTGG[SEQ ID NO:50]ACTTCAACAA CCTCAAOligo #30 DEL3540GCGCGCGATA TCTTGGTCTT CTTCACCATT CAGCGGCAGC GGTGGCTGCT[SEQ ID NO:51]Oligo #31 DEL4146GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATGAGGT TGTTGAAGTC CAGCA[SEQ ID NO:52]Oligo #32 DEL4752GCGCGCCTCG AGGTTTGGAC GACGAAGATC TTGGTCTTCA CCATTGA[SEQ ID NO:53]Oligo #33 DEL5358GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGT TATTTTCCAT CAGGATAT[SEQ ID NO:54]Oligo #34 DEL5964GCGCGCTGAT GCATTCTGCA GAGACTTGAC GAGGTTTGGA CGACGAAGGT[SEQ ID NO:55]Oligo #35 DEL6570GCGCGCCTCG AGGCATTCAA CCGTGCTGCA TCAGCAATTG AGAGCAT[SEQ ID NO:56]Oligo #36 DEL7176GCGCGCCTGC AGAATATTCT TAAAAATCTC CTGCC[SEQ ID NO:57]Oligo #37 DEL7782GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCCCATGTC TGCCGCTAGC GAG[SEQ ID NO:58]Oligo #38 DEL8388GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GACGGCCGCA[SEQ ID NO:59]CCCACGCGAC AOligo #39 DEL8893CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCA GGGCAGACAT GGCAGGA[SEQ ID NO:60]Oligo #40 DEL106111CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTAT[SEQ ID NO:61]TCCAGTCACC GTCCTTGAOligo #41 DEL112117CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA CAGTTTACGA CGCAATTCAT[SEQ ID NO:62]Oligo #42 DEL118123CGCGCGAAGC TTATTACTGT TGGGTTTTCA GATAGAAGGT CA[SEQ ID NO:63]Oligo #43 R17IL3 Length: 000058CGCGCGCCAT GGCTAACTGC NNSAACATGA TCGATGAAAT TATAACACAC TTAAAGCA[SEQ ID NO:64]Oligo #44 R18IL3 Length: 000058CGCGCGCCAT GGCTAACTGC TCTNNSATGA TCGATCAAAT TATAACACAC TTAAAGCA[SEQ ID NO:222]Oligo #45 R19IL3 Length: 000058CGCGCGCCAT GGCTAACTGC TCTAACNNSA TCGATGAAAT TATAACACAC TTAAAGCA[SEQ ID NO:223]Oligo #46 R20IL3 Length: 000058CGCGCGCCAT GGCTAACTGC TCTAACATGN NSGATGAAAT TATAACACAC TTAAAGCA[SEQ ID NO:224]Oligo #47 R21IL3 Length: 000050CGCGCGCCAT GGCTAACTGC TCTAACATGA TCNNSGAAAT TATAACACAC TTAAAGCA[SEQ ID NO:225]Oligo #48 R22IL3 Length: 000058CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATNNSAT TATAACACAC TTAAAGCA[SEQ ID NO:226]Oligo #49 R23IL3 Length: 000076CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAANN SATAACACAC TTAAAGCAGC[SEQ ID NO:227]CACCTTTGCC TTTGCTOligo #50 R24IL3 Length: 000076CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TNNSACACAC TTAAAGCAGC[SEQ ID NO:228]CACCTTTGCC TTTGCTOligo #51 R25IL3 Length: 000076CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATANNSCAC TTAAAGCAGC[SEQ ID NO:229]CACCTTTGCC TTTGCTOligo #52 R26IL3 Length: 000076CGCGCGCCAT GGCTAACTGC TCTAACATGA TCOATGAAAT TATAACANNS TTAAAGCAGC[SEQ ID NO:74]CACCTTTGCC TTTGCTOligo #53 R27IL3 Length: 000076CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC NNSAAGCAGC[SEQ ID NO:75]CACCTTTGCC TTTGCTOligo #54 R28IL3 Length: 000076CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTANNSCAGC[SEQ ID NO:76]CACCTTTGCC TTTGCTOligo #55 R29IL3 Length: 000094CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGNNSC[SEQ ID NO:77]CACCTTTGCC TTTGCTGGAC TTCAACAACC TCAAOligo #56 R30IL3 Length: 000094CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGN[SEQ ID NO:78]NSCCTTTGCC TTTGCTGGAC TTCAACAACC TCAAOligo #57 R31IL3 Length: 000094CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC[SEQ ID NO:79]CANNSTTGCC TTTGCTGGAC TTCAACAACC TCAAOligo #58 R32IL3 Length: 000094CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC[SEQ ID NO:80]CACCTNNSCC TTTGCTGGAC TTCAACAACC TCAAOligo #59 R33IL3 Length: 000094CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC[SEQ ID NO:81]CACCTTTGNN STTGCTGGAC TTCAACAACC TCAAOligo #60 R34IL3 Length: 000094CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC[SEQ ID NO:82]CACCTTTGCC TNNSCTGGAC TTCAACAACC TCAAOligo #61 R35IL3 Length: 000065GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAG TCSNNCAGCG GCAGCGGTGG CTGCT[SEQ ID NO:83]Oligo #62 R36IL3 Length: 000065GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAS NNCAGCAGCG GCAGCG GTGGCTGCT[SEQ ID NO:84]Oligo #63 R37IL3 Length: 000065GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTSNNG TCCAGCAGCG GCAGCGGTGG CTGCT[SEQ ID NO:85]Oligo #64 R38IL3 Length: 000065GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTSNNGAAG TCCAGCAGCG GCAGCGGTGG CTGCT[SEQ ID NO:86]Oligo #65 R39IL3 Length: 000065GCGCGCGATA TCTTGGTCTT CACCATTGAG SNNGTTGAAG TCCAGCAGCG GCAGCGGTGG CTGCT[SEQ ID NO:87]Oligo #66 R40IL3 Length: 000065GCGCGCGATA TCTTGGTCTT CACCATTSNN GTTGTTGAAG TCCAGCAGCG GCAGCGGTGC CTGCT[SEQ ID NO:88]Oligo #67 R41IL3 Length: 000083GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCACC[SEQ ID NO:89]SNNGAGGTTG TTGAAGTCCA GCAOligo #68 R42IL3 Length: 000083GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCSNN[SEQ ID NO:90]ATTGAGGTTG TTGAAGTCCA GCAOligo #69 R43IL3 Length: 000083GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCSNNACC[SEQ ID NO:91]ATTGAGGTTG TTGAAGTCCA GCAOligo #70 R44IL3 Length: 000083GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GSNNTTCACC[SEQ ID NO:92]ATTGAGGTTG TTGAAGTCCA GCAOligo #71 R45IL3 Length: 000083GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCSN NGTCTTCACC[SEQ ID NO:93]ATTGAGGTTG TTGAAGTCCA GCAOligo #72 R46IL3 Length: 000.083GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATSNNTT GGTCTTCACC[SEQ ID NO:94]ATTGAGGTTG TTGAAGTCCA GCAOligo #73 R47IL3 Length: 000065GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGSNNATCTT GGTCTTCACC ATTGA[SEQ ID NO:95]Oligo #74 R48IL3 Length: 000065GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATS NNGATATCTT GGTCTTCACC ATTGA[SEQ ID NO:96]Oligo #75 R49IL3 Length: 000065GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCSNNC AGGATATCTT GGTCTTCACC ATTGA[SEQ ID NO:97]Oligo #76 R50IL3 Length: 000065GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTSNNCATC AGGATATCTT GGTCTTCACC ATTGA[SEQ ID NO:98]Oligo #77 R51IL3 Length: 000065GCGCGCCTCG AGGTTTGGAC GACGAAGGTT SNNTTCCATC AGGATATCTT GGTCTTCACC ATTGA[SEQ ID NO:99]Oligo #78 R52IL3 Length: 000065GCGCGCCTCG AGGTTTGGAC GACGAAGSNN ATTTTCCATC AGGATATCTT GGTCTTCACC ATTGA[SEQ ID NO:100]Oligo #79 R53IL3 Length: 000086GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG[SEQ ID NO:101]ACGSNNGTTA TTTTCCATCA GGATATOligo #80 R54IL3 Length: 000086GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG[SEQ ID NO:102]SNNAAGGTTA TTTTCCATCA GGATATOligo #81 R55IL3 Length: 000086GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGSNN[SEQ ID NO:103]ACGAAGGTTA TTTTCCATCA GGATATOligo #82 R56IL3 Length: 000086GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTSNNACG[SEQ ID NO:104]ACGAAGGTTA TTTTCCATCA GGATATOligo #83 R57IL3 Length: 000086GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GSNNTGGACG[SEQ ID NO:105]ACGAAGGTTA TTTTCCATCA GGATATOligo #84 R58IL3 Length: 000086GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCSN NGTTTGGACG[SEQ ID NO:106]ACGAAGGTTA TTTTCCATCA GGATATOligo #85 R59IL3 Length: 000068GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCSNNGA GGTTTGGACG ACGAAGGT[SEQ ID NO:107]Oligo #86 R60IL3 Length: 000068GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AASNNCTCGA GGTTTGGACG ACGAAGGT[SEQ ID NO:108]Oligo #87 R61IL3 Length: 000068GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTS NNTGCCTCGA GGTTTGGACG ACGAAGGT[SEQ ID NO:109]Oligo #88 R62IL3 Length: 000068GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGSNNG AATGCCTCGA GGTTTGGACG ACGAAGGT[SEQ ID NO:110]Oligo #89 R63IL3 Length: 000068GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCSNNGTTG AATGCCTCGA GGTTTGGACG ACGAAGGT[SEQ ID NO:111]Oligo #90 R64IL3 Length: 000068GCGCGCTGAT GCATTCTGCA GAGACTTGAC SNNACGGTTG AATGCCTCGA GGTTTGGACG ACGAAGGT[SEQ ID NO:112]Oligo #91 R65IL3 Length: 000065GCGCGCCTCG AGGCATTCAA CCGTGCTNNS AAGTCTCTGC AGAATGCATC AGCAATTGAG AGCAT[SEQ ID NO:113]Oligo #92 R66IL3 Length: 000065GCGCGCCTCG AGGCATTCAA CCGTGCTGTC NNSTCTCTGC AGAATGCATC AGCAATTGAG AGCAT[SEQ ID NO:114]Oligo #93 R67IL3 Length: 000065GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGNNSCTGC AGAATGCATC AGCAATTGAG AGCAT[SEQ ID NO:115]Oligo #94 R60IL3 Length: 000065GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTNNSC AGAATGCATC AGCAATTGAG AGCAT[SEQ ID NO:116]Oligo #95 R69IL3 Length: 000065GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGN NSAATGCATC AGCAATTGAG AGCAT[SEQ ID NO:117]Oligo #96 R70IL3 Length: 000065GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGC AGNNSGCATC AGCAATTGAG AGCAT[SEQ ID NO:118]Oligo #97 R71IL3 Length: 000053GCGCGCCTGC AGAATNNSTC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCC[SEQ ID NO:119]Oligo #98 R72IL3 Length: 000053GCGCGCCTGC AGAATGCANN SGCAATTGAG AGCATTCTTA AAAATCTCCT GCC[SEQ ID NO:120]Oligo #99 R73IL3 Length: 000053GCGCGCCTGC AGAATGCATC ANNSATTGAG AGCATTCTTA AAAATCTCCT GCC[SEQ ID NO:121]Oligo #100 R74IL3 Length: 000053GCGCGCCTGC AGAATGCATC AGCANNSGAG AGCATTCTTA AAAATCTCCT GCC[SEQ ID NO:122]Oligo #101 R75IL3 Length: 000053GCGCGCCTGC AGAATGCATC AGCAATTNNS AGCATTCTTA AAAATCTCCT GCC[SEQ ID NO:123]Oligo #102 R76IL3 Length: 000053GCGCGCCTGC AGAATGCATC AGCAATTGAG NNSATTCTTA AAAATCTCCT GCC[SEQ ID NO:124]Oligo #103 R77IL3 Length: 000071GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCNNSCTTA AAAATCTCCT GCCATCTCTG[SEQ ID NO:125]CCGCTACCCA COligo #104 R78IL3 Length: 000071GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTNNSA AAAATCTCCT GCCATGTCTG[SEQ ID NO:126]CCGCTAGCCA COligo #105 R79IL3 Length: 000071GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTN NSAATCTCCT GCCATGTCTG[SEQ ID NO:127]CCGCTAGCCA COligo #106 R80IL3 Length: 000071GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AANNSCTCCT GCCATGTCTG[SEQ ID NO:138]CCGCTAGCCA COligo #107 R81IL3 Length: 000071GCCCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATNNSCT GCCATGTCTG[SEQ ID NO:139]CCGCTAGCCA COligo #108 R82IL3 Length: 000071GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCNN SCCATGTCTG[SEQ ID NO:140]CCGCTAGCCA COligo #109 R83IL3 Length: 000089GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GNNSTGTCTG[SEQ ID NO:141]CCGCTAGCCA CGGCCGCACC CACGCGACAOligo #110 R84IL3 Length: 000089GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCANNSCTG[SEQ ID NO:142]CCGCTAGCCA CGGCCGCACC CACGCGACAOligo #111 R85IL3 Length: 000089GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTNNS[SEQ ID NO:143]CCGCTAGCCA CGGCCGCACC CACGCGACAOligo #112 R86IL3 Length: 000089GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG[SEQ ID NO:157]NNSCTAGCCA CGGCCGCACC CACGCGACAOligo #113 R87IL3 Length: 000009GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG[SEQ ID NO:158]CCGNNSGCCA CGGCCGCACC CACGCGACAOligo #114 R88IL3 Length: 000009GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG[SEQ ID NO:159]CCGCTANNSA CGGCCGCACC CACGCGACAOligo #115 R89IL3 Length: 000086CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCGGC[SEQ ID NO:160]SNNGGCCAGG GGCAGACATG GCAGGAOligo #116 R90IL3 Length: 000086CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCSNN[SEQ ID NO:161]CGTGGCCAGG GGCAGACATG GCAGGAOligo #117 R91IL3 Length: 000086CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGSNNGGC[SEQ ID NO:162]CGTGGCCAGG GGCAGACATG GCAGGAOligo #118 R92IL3 Length: 000086CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TSNNTGCGGC[SEQ ID NO:163]CGTGGCCAGG GGCAGACATG GCAGGAOligo #119 R93IL3 Length: 000086CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGSN NGGGTGCGGC[SEQ ID NO:164]CGTGGCCAGG GGCAGACATG GCAGGAOligo #120 3PR106 Length: 000048TTTCAGATAG AAGGTCAGTT TACGACGGAA SNNATTCCAG TCACCGTC[SEQ ID NO:165]Oligo #121 3PR107 Length: 000048TTTCAGATAG AAGGTCAGTT TACGACGSNN TTCATTCCAG TCACCGTC[SEQ ID NO:166]Oligo #122 3PR108 Length: 000048TTTCAGATAG AAGGTCAGTT TACGSNNGAA TTCATTCCAG TCACCGTC[SEQ ID NO:167]Oligo #123 3PR109 Length: 000048TTTCAGATAG AAGGTCAGTT TSNNACGGAA TTCATTCCAG TCACCGTC[SEQ ID NO:168]Oligo #124 3PR110 Length: 000048TTTCAGATAG AAGGTCAGSN NACGACGGAA TTCATTCCAG TCACCGTC[SEQ ID NO:169]Oligo #125 3PR111 Length: 000048TTTCAGATAG AAGGTSNNTT TACGACGGAA TTCATTCCAG TCACCGTC[SEQ ID NO:170]Oligo #126 IL3MUTD3 Length: 000023CGCGCGAAGC TTATTACTGT TGA[SEQ ID NO:171]Oligo #127 R112IL3 Length: 000078CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAASNNCA[SEQ ID NO:172]GTTTACGACG GAATTCATOligo #128 R113IL3 Length: 000078CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TASNNGGTCA[SEQ ID NO:173]GTTTACGACG GAATTCATOligo #129 R114IL3 Length: 000078CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGS NNGAAGGTCA[SEQ ID NO:174]GTTTACGACG GAATTCATOligo #130 R115IL3 Length: 000078CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTSNNA TAGAAGGTCA[SEQ ID NO:175]GTTTACGACG GAATTCATOligo #131 R116IL3 Length: 000078CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTSNNCAGA TAGAAGGTCA[SEQ ID NO:176]GTTTACGACG GAATTCATOligo #132 R117IL3 Length: 000078CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA SNNTTTCAGA TAGAAGGTCA[SEQ ID NO:177]GTTTACGACG GAATTCATOligo #133 R118IL3 Length: 000060CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCSNN GGTTTTCAGA TAGAAGGTCA[SEQ ID NO:178]Oligo #134 R119IL3 Length: 000060CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTSNNCAA GGTTTTCAGA TAGAAGGTCA[SEQ ID NO:179]Oligo #135 R120IL3 Length: 000060CGCGCGAAGC TTATTACTGT TGAGCCTGCG CSNNCTCCAA GGTTTTCAGA TAGAAGGTCA[SEQ ID NO:180]Oligo #136 R121IL3 Length: 000060CGCGCGAAGC TTATTACTGT TGAGCCTGSN NGTTCTCCAA GGTTTTCAGA TAGAAGGTCA[SEQ ID NO:181]Oligo #137 R122IL3 Length: 000060CGCGCGAAGC TTATTACTGT TGAGCSNNCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA[SEQ ID NO:182]Oligo #138 R123IL3 Length: 000060CGCGCGAAGC TTATTACTGT TGSNNCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA[SEQ ID NO:183]Oligo #139 P1722IL3 Length: 000024TGCTCTAACA TGATCGATGA AATT[SEQ ID NO:184]Oligo #140 P2328IL3 Length: 000024GAAATTATAA CACACTTAAA GCAG[SEQ ID NO:185]Oligo #141 P2934IL3 Length: 000024AAGCAGCCAC CTTTGCCTTT GCTG[SEQ ID NO:186]Oligo #142 P3540IL3 Length: 000024AAGCAGCCAC CGCTGCCGCT GCTG[SEQ ID NO:187]Oligo #143 PRB41-46 Length: 000024CTCAATGGTG AAGACCAAGA TATC[SEQ ID NO:188]Oligo #144 PRB47-52 Length: 000024GATATCCTGA TGGAAAATAA CCTT[SEQ ID NO:189]Oligo #145 PRB53-58 Length: 000024AACCTTCGTC GTCCAAACCT CGAG[SEQ ID NO:190]Oligo #146 PRB59-64 Length: 000024CTCGAGGCAT TCAACCGTGC TGTC[SEQ ID NO:191]Oligo #147 PRB65-70 Length: 000024GCTGTCAAGT CTCTGCAGAA TGCA[SEQ ID NO:192]Oligo #148 P7176IL3 Length: 000024AATGCATCAG CAATTGAGAG CATT[SEQ ID NO:193]Oligo #149 P7782IL3 Length: 000024AGCATTCTTA AAAATCTCCT GCCA[SEQ ID NO:194]Oligo #150 P8388IL3 Length: 000024CTGCCATGTC TGCCCCTGGC CACG[SEQ ID NO:195]Oligo #151 P8893IL3 Length: 000024CTGGCCACGG CCGCACCCAC GCGA[SEQ ID NO:196]Oligo #152 P106111 Length: 000024AATGAATTCC GTCGTAAACT GACC[SEQ ID NO:197]Oligo #153 P112117 Length: 000024CTGACCTTCT ATCTGAAAAC CTTG[SEQ ID NO:198]Oligo #154 P118123 Length: 000024ACCTTGGAGA ACGCGCAGGC TCAA[SEQ ID NO:199]Oligo #155 PSTECRI1.REQ Length: 000022GAATGCATCA GCAATTGAGA GC[SEQ ID NO:200]Oligo #156 PSTECRI5.REQ Length: 000020AATTGCTGAT GCATTCTGCA[SEQ ID NO:201]Oligo #157 PSTECRI2.REQ Length: 000024ATTCTTAAAA ATCTCCTGCC ATGT[SEQ ID NO:202]Oligo #158 PSTECRI6.REQ Length: 000024CAGGAGATTT TTAAGAATGC TCTC[SEQ ID NO:203]Oligo #159 PSTECRI3.REQ Length: 000030CTGCCCCTGG CCACGGCCGC ACCCACGCGA[SEQ ID NO:204]Oligo #160 PSTECRI7.REQ Length: 000030GGGTGCGGCC GTGGCCAGGG GCAGACATGG[SEQ ID NO:205]Oligo #161 98I100R4.REQ Length: 000034CATCCAATCA TCATCCGTGA CGGTGACTGG AATG[SEQ ID NO:206]Oligo #162 98I100R8.REQ Length: 000044AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGATGTC GCGT[SEQ ID NO:207]Oligo #163 95R8I0R4.REQ Length: 000034CGCCCAATCA TCATCCGTGA CGGTGACTGG AATG[SEQ ID NO:208]Oligo #164 95R810R8.REQ Length: 000044AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGGCGTC GCGT[SEQ ID NO:209]Oligo #165 NCOECRV1.REQ Length: 000040CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA[SEQ ID NO:210]Oligo #166 NCOECRV4.REQ Length: 000045CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC[SEQ ID NO:211]Oligo #167 NCOECRV2.REQ Length: 000036CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC[SEQ ID NO:212]Oligo #168 NCOECRV5.REQ Length: 000036GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG[SEQ ID NO:213]Oligo #169 2D5M6SUP.REQ Length: 000027AACAACCTCA ATGACGAAGA CATGTCT[SEQ ID NO:214]Oligo #170 2D5M65LO.REQ Length: 000010AGACATGTCT TCGTCATT[SEQ ID NO:215]Oligo #15(A) Length: 000016 TGAACCATAT GTCAGG[SEQ ID NO:29]Oligo #16(A) Length: 000024 AATTCCTGAC ATATGGTTCA TGCA[SEQ ID NO:30]Oligo #51(A) Length: 000034GCCGATACCGCGGCATACTCCCACCATTCAGAGA[SEQ ID NO:155]Oligo #52(A) Length: 000033GCCGATAAGATCTAAAACGGGTATGGAGAAACA[SEQ ID NO:156]Oligo #171 Length: 000040CATGGCTAAC TGCTCTAACA TGATCAACGA AATTATAACA[SEQ. ID NO:69]Oligo #172 Length: 000045CTTTAAGTGT GTTATAATTT CGTTGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:70]Oligo #173 Length: 000040CATGGCTAAC TGCTCTAACA TGATCCAAGA AATTATAACA[SEQ. ID NO:71]Oligo #174 Length: 000045CTTTAAGTGT GTTATAATTT CTTGGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:72]Oligo #175 Length: 000040CATGGCTAAC TGCTCTAACA TGATCGAAGA AATTATAACA[SEQ. ID NO:73]Oligo #176 Length: 000045CTTTAAGTGT GTTATAATTT CTTCGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:219]Oligo #177 Length: 000040CATGGCTAAC TGCTCTAACA TGATCAGCGA AATTATAACA[SEQ. ID NO:230]Oligo #178 Length: 000045CTTTAAGTGT GTTATAATTT CGCTGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:231]Oligo #179 Length: 000040CATGGCTAAC TGCTCTAAACA TGATCACCGA AATTATAACA[SEQ. ID NO:232]Oligo #100 Length: 000045CTTTAAGTGT GTTATAATTT CCGTGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:233]Oligo #181 Length: 000040CATGGCTAAC TGCTCTAACA TGATCGATAA CATTATAACA[SEQ. ID NO:234]Oligo #182 Length: 000045CTTTAAGTGT GTTATAATGT TATCGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:235]Oligo #183 Length: 000040CATGGCTAAC TGCTCTAACA TGATCGATGA CATTATAACA[SEQ. ID NO:236]Oligo #184 Length: 000045CTTTAAGTGT GTTATAATGT CATCGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:237]Oligo #185 Length: 000040CATGGCTAAC TGCTCTAACA TGATCGATCA GATTATAACA[SEQ. ID NO:238]Oligo #186 Length: 000045CTTTAAGTGT GTTATAATCT GATCGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:239]Oligo #187 Length: 000040CATGGCTAAC TGCTCTAACA TGATCGATCT GATTATAACA[SEQ. ID NO:240]Oligo #188 Length: 000045CTTTAAGTGT GTTATAATCA GATCGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:241]Oligo #189 Length: 000040CATGGCTAAC TGCTCTAACA TGATCGATGT TATTATAACA[SEQ. ID NO:242]Oligo #190 Length: 000045CTTTAAGTGT GTTATAATAA CATCGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:243]Oligo #191 Length: 000036CACTTAAAGC AGCCACCTTT GCCTGCTCTG GACTTC[SEQ. ID NO:244]Oligo #192 Length: 000036GAGGTTGTTG AAGTCCAGAG CAGGCAAAGG TGGCTG[SEQ. ID NO:245]Oligo #193 Length: 000036CACTTAAAGC AGCCACCTTT GCCTCGTCTG GACTTC[SEQ. ID NO:246]Oligo #194 Length: 000036GAGGTTGTTG AAGTCCAGAC GAGGCAAAGG TGGCTG[SEQ. ID NO:247]Oligo #195 Length: 000036CACTTAAAGC AGCCACCTTT GCCTCAGCTG GACTTC[SEQ. ID NO:248]Oligo #196 Length: 000036GAGGTTGTTG AAGTCCAGCT GAGGCAAAGG TGGCTG[SEQ. ID NO:249]Oligo #197 Length: 000036CACTTAAAGC AGCCACCTTT GCCTGAACTG GACTTC[SEQ. ID NO:250]Oligo #198 Length: 000036GAGGTTGTTG AAGTCCAGCT CAGGCAAAGG TGGCTG[SEQ. ID NO:251]Oligo #199 Length: 000036CACTTAAAGC AGCCACCTTT GCCTATCCTG GACTTC[SEQ. ID NO:252]Oligo #200 Length: 000036GAGGTTGTTG AAGTCCAGGA TAGGCAAAGG TGGCTG[SEQ. ID NO:253]Oligo #201 Length: 000036CACTTAAAGC AGCCACCTTT CCCTTTCCTG GACTTC[SEQ. ID NO:254]Oligo #202 Length: 000036GAGGTTGTTG AAGTCCAGGA AAGGCAAAGG TGGCTG[SEQ. ID NO:255]Oligo #203 Length: 000036CACTTAAAGC AGCCACCTTT GCCTACCCTG GACTTC[SEQ. ID NO:256]Oligo #204 Length: 000036GAGGTTGTTG AAGTCCAGGG TAGGCAAAGG TGGCTG[SEQ. ID NO:257]Oligo #205 Length: 000027AACAACCTCA ATCGTGAAGA CCAAGAT[SEQ. ID NO:258]Oligo #206 Length: 000018ATCTTGGTCT TCACGATT[SEQ. ID NO:259]Oligo #207 Length: 000027AACAACCTCA ATAACGAAGA CCAAGAT[SEQ. ID NO:260]Oligo #208 Length: 000018ATCTTGGTCT TCGTTATT[SEQ. ID NO:261]Oligo #209 Length: 000027AACAACCTCA ATGAAGAAGA CCAAGAT[SEQ. ID NO:262]Oligo #210 Length: 000018ATCTTGGTCT TCTTCATT[SEQ. ID NO:263]Oligo #211 Length: 000027AACAACCTCA ATATCGAAGA CCAAGAT[SEQ. ID NO:264]Oligo #212 Length: 000018ATCTTGGTCT TCGATATT[SEQ. ID NO:265]Oligo #213 Length: 000027AACAACCTCA ATCTGGAAGA CCAAGAT[SEQ. ID NO:266]Oligo #214 Length: 000018ATCTTGGTCT TCCAGATT[SEQ. ID NO:267]Oligo #215 Length: 000027AACAACCTCA ATAAAGAAGA CCAAGAT[SEQ. ID NO:268]Oligo #216 Length: 000018ATCTTGGTCT TCTTTATT[SEQ. ID NO:269]Oligo #217 Length: 000027AACAACCTCA ATATGGAAGA CCAAGAT[SEQ. ID NO:270]Oligo #218 Length: 000018ATCTTGGTCT TCCATATT[SEQ. ID NO:271]Oligo #219 Length: 000027AACAACCTCA ATTTCGAAGA CCAAGAT[SEQ. ID NO:272]Oligo #220 Length: 000018ATCTTGGTCT TCGAAATT[SEQ. ID NO:273]Oligo #221 Length: 000027AACAACCTCA ATACCGAAGA CCAAGAT[SEQ. ID NO:274]Oligo #222 Length: 000018ATCTTGGTCT TCGGTATT[SEQ. ID NO:275]Oligo #223 Length: 000027AACAACCTCA ATTACGAAGA CCAAGAT[SEQ. ID NO:276]Oligo #224 Length: 000018ATCTTGGTCT TCGTAATT[SEQ. ID NO:277]Oligo #225 Length: 000027AACAACCTCA ATGTTGAAGA CCAAGAT[SEQ. ID NO:278]Oligo #226 Length: 000010ATCTTGGTCT TCAACATT[SEQ. ID NO:279]Oligo #227 Length: 000027AACAACCTCA ATGGGCGTGA CCAAGAT[SEQ. ID NO:280]Oligo #228 Length: 000018ATCTTGGTCT CGCCCATT[SEQ. ID NO:281]Oligo #229 Length: 000027AACAACCTCA ATGGGCAGGA CCAAGAT[SEQ. ID NO:282]Oligo #230 Length: 000010ATCTTGGTCC TGCCCATT[SEQ. ID NO:283]Oligo #231 Length: 000027AACAACCTCA ATGGGGGTGA CCAAGAT[SEQ. ID NO:284]Oligo #232 Length: 000018ATCTTGGTCA CCCCCATT[SEQ. ID NO:285]Oligo #233 Length: 000027AACAACCTCA ATGGGACCGA CCAAGAT[SEQ. ID NO:286]Oligo #234 Length: 000018ATCTTGGTCG GTCCCATT[SEQ. ID NO:287]Oligo #235 Length: 000027AACAACCTCA ATGGGGAAGC TCAAGAT[SEQ. ID NO:288]Oligo #236 Length: 000018ATCTTGAGCT TCCCCATT[SEQ. ID NO:289]Oligo #237 Length: 000027AACAACCTCA ATGGGGAAAA CCAAGAT[SEQ. ID NO:290]Oligo #238 Length: 000018ATCTTGGTTT TCCCCATT[SEQ. ID NO:291]Oligo #239 Length: 000027AACAACCTCA ATGGGGAACA GCAAGAT[SEQ. ID NO:292]Oligo #240 Length: 000018ATCTTGCTGT TCCCCATT[SEQ. ID NO:293]Oligo #241 Length: 000027AACAACCTCA ATGGGGAAGA ACAAGAT[SEQ. ID NO:294]Oligo #242 Length: 000018ATCTTGTTCT TCCCCATT[SEQ. ID NO:295]Oligo #243 Length: 000027AACAACCTCA ATGGGGAAGA CGCTGAT[SEQ. ID NO:296]Oligo #244 Length: 000010ATCAGCGTCT TCCCCATT[SEQ. ID NO:297]Oligo #245 Length: 000027AACAACCTCA ATGGGGAAGA CCGTGAT[SEQ. ID NO:298]Oligo #246 Length: 000018ATCACGGTCT TCCCCATT[SEQ. ID NO:299]Oligo #247 Length: 000027AACAACCTCA ATGGGGAAGA CAACGAT[SEQ. ID NO:300]Oligo #248 Length: 000018ATCGTTGTCT TCCCCATT[SEQ. ID NO:301]Oligo #249 Length: 000027AACAACCTCA ATGGGGAAGA CGACGAT[SEQ. ID NO:302]Oligo #250 Length: 000018ATCGTCGTCT TCCCCATT[SEQ. ID NO:303]Oligo #251 Length: 000027AACAACCTCA ATGGTGAAGA CGAAGAT[SEQ. ID NO:304]Oligo #252 Length: 000018ATCTTCGTCT TCCCCATT[SEQ. ID NO:305]Oligo #253 Length: 000027AACAACCTCA ATGGTGAAGA CCACGAT[SEQ. ID NO:306]Oligo #254 Length: 000018ATCGTGGTCT TCCCCATT[SEQ. ID NO:307]Oligo #255 Length: 000027AACAACCTCA ATGGGGAAGA CATCGAT[SEQ. ID NO:308]Oligo #256 Length: 000018ATCGATGTCT TCCCCATT[SEQ. ID NO:309]Oligo #257 Length: 000027AACAACCTCA ATGGGGAAGA CTCCGAT[SEQ. ID NO:310]Oligo #258 Length: 000018ATCGGAGTCT TCCCCATT[SEQ. ID NO:311]Oligo #259 Length: 000027AACAACCTCA ATGGGGAAGA CCAAGCT[SEQ. ID NO:312]Oligo #260 Length: 000018AGCTTGGTCT TCCCCATT[SEQ. ID NO:313]Oligo #261 Length: 000027AACAACCTCA ATGGGGAAGA CCAAAAC[SEQ. ID NO:314]Oligo #262 Length: 000018GTTTTGGTCT TCCCCATT[SEQ. ID NO:315]Oligo #263 Length: 000027AACAACCTCA ATGGGGAAGA CCAACAG[SEQ. ID NO:316]Oligo #264 Length: 000018CTGTTGGTCT TCCCCATT[SEQ. ID NO:317]Oligo #265 Length: 000027AACAACCTCA ATGGGGAAGA CCAAGAA[SEQ. ID NO:318]Oligo #266 Length: 000018TTCTTGGTCT TCCCCATT[SEQ. ID NO:319]Oligo #267 Length: 000027AACAACCTCA ATGGGGAAGA CCAACAC[SEQ. ID NO:320]Oligo #260 Length: 000018GTGTTGGTCT TCCCCATT[SEQ. ID NO:321]Oligo #269 Length: 000027AACAACCTCA ATGGGGAAGA CCAAATC[SEQ. ID NO:322]Oligo #270 Length: 000018GATTTGGTCT TCCCCATT[SEQ. ID NO:323]Oligo #271 Length: 000027AACAACCTCA ATGGGGAAGA CCAACTG[SEQ. ID NO:324]Oligo #272 Length: 000018CAGTTGGTCT TCCCCATT[SEQ. ID NO:325]Oligo #273 Length: 000027AACAACCTCA ATGGGGAAGA CCAAAAA[SEQ. ID NO:326]Oligo #274 Length: 000018TTTTTGGTCT TCCCCATT[SEQ. ID NO:327]Olign #275 Length: 000027AACAACCTCA ATGGGGAAGA CCAATAC[SEQ. ID NO:328]Oligo #276 Length: 000018GTATTGGTCT TCCCCATT[SEQ. ID NO:329]Oligo #277 Length: 000027AACAACCTCA ATGGGGAAGA CCAAGTT[SEQ. ID NO:330]Oligo #278 Length: 000018AACTTGGTCT TCCCCATT[SEQ. ID NO:331]Oligo #279 Length: 000036ATCGCTATGG AAAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:332]Oligo #200 Length: 000027CCTTCGAAGG TTATTTTCCA TAGCGAT[SEQ. ID NO:333]Oligo #281 Length: 000036ATCGAAATGG AAAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:334]Oligo #282 Length: 000027CCTTCGAAGG TTATTTTCCA TTTCGAT[SEQ. ID NO:335]Oligo #283 Length: 000036ATCAAAATGG AAAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:336]Oligo #284 Length: 000027CCTTCGAAGG TTATTTTCCA TTTTGAT[SEQ. ID NO:337]Oligo #285 Length: 000036ATCATGATGG AAAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:338]Oligo #286 Length: 000027CCTTCGAAGG TTATTTTCCA TCATGAT[SEQ. ID NO:339]Oligo #287 Length: 000036ATCACCATGG AAAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:340]Oligo #288 Length: 000027CCTTCGAAGG TTATTTTCCA TGGTGAT[SEQ. ID NO:341]Oligo #289 Length: 000036ATCGTTATGG AAAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:342]Oligo #290 Length: 000027CCTTCGAAGG TTATTTTCCA TAACGAT[SEQ. ID NO:343]Oligo #291 Length: 000036ATCCTGATGC ACAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:344]Oligo #292 Length: 000027CCTTCGAAGG TTATTGTGCA TCAGGAT[SEQ. ID NO:345]Oligo #293 Length: 000036ATCCTGATGA TGAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:346]Oligo #294 Length: 000027CCTTCGAAGG TTATTCATCA TCAGGAT[SEQ. ID NO:347]Oligo #295 Length: 000036ATCCTGATGT TCAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:348]Oligo #296 Length: 000027CCTTCGAAGG TTATTGAACA TCAGGAT[SEQ. ID NO:349]Oligo #297 Length: 000036ATCCTGATGG CTAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:350]Oligo #298 Length: 000027CCTTCGAAGG TTATTAGCCA TCAGGAT[SEQ. ID NO:351]Oligo #299 Length: 000036ATCCTGATGA ACAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:352]Oligo #300 Length: 000027CCTTCGAAGG TTATTGTTCA TCAGGAT[SEQ. ID NO:353]Oligo #301 Length: 000036ATCCTGATGA TCAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:354]Oligo #302 Length: 000027CCTTCGAAGG TTATTGATCA TCAGGAT[SEQ. ID NO:355]Oligo #303 Length: 000036ATCCTGATGA AAAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:356]Oligo #304 Length: 000027CCTTCGAAGG TTATTTTTCA TCAGGAT[SEQ. ID NO:357]Oligo #305 Length: 000036ATCCTGATGT CCAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:358]Oligo #306 Length: 000027CCTTCGAAGG TTATTGGACA TCAGGAT[SEQ. ID NO:359]Oligo #307 Length: 000036ATCCTGATGG TTAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:360]Oligo #308 Length: 000027CCTTCGAAGG TTATTAACCA TCAGGAT[SEQ. ID NO:361]Oligo #309 Length: 000036ATCCTGATGG AAAATAACCT TGCTAGGCCA AACCTG[SEQ. ID NO:362]Oligo #310 Length: 000027CCTAGCAAGG TTATTTTCCA TCAGGAT[SEQ. ID NO:363]Oligo #311 Length: 000036ATCCTGATGG AAAATAACCT TAACAGGCCA AACCTG[SEQ. ID NO:364]Oligo #312 Length: 000027CCTGTTAAGG TTATTTTCCA TCAGGAT[SEQ. ID NO:365]Oligo #313 Length: 000036ATCCTGATGG AAAATAACCT TCACAGGCCA AACCTG[SEQ. ID NO:366]Oligo #314 Length: 000027CCTGTGAAGG TTATTTTCCA TCAGGAT[SEQ. ID NO:367]Oligo #315 Length: 000036ATCCTGATGG AAAATAACCT TAAAAGGCCA AACCTG[SEQ. ID NO:368]Oligo #316 Length: 000027CCTTTTAAGG TTATTTTCCA TCAGGAT[SEQ. ID NO:369]Oligo #317 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGGCT AACCTG[SEQ. ID NO:370]Oligo #318 Length: 000024CCTGTTGAAT GCCTCCAGGT TAGC[SEQ. ID NO:371]Oligo #319 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGCCGT AACCTG[SEQ. ID NO:372]Oligo #320 Length: 000024CCTGTTGAAT GCCTCCAGGT TACG[SEQ. ID NO:373]Oligo #321 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGAAC AACCTG[SEQ. ID NO:374]Oligo #322 Length: 000024CCTGTTGAAT GCCTCCAGGT TGTT[SEQ. ID NO:375]Oligo #323 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGGAA AACCTG[SEQ. ID NO:376]Oligo #324 Length: 000024CCTGTTGAAT GCCTCCAGGT TTTC[SEQ. ID NO:377]Oligo #325 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGCAC AACCTG[SEQ. ID NO:378]Oligo #326 Length: 000024CCTGTTGAAT GCCTCCAGGT TGTG[SEQ. ID NO:379]Oligo #327 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGCTG AACCTG[SEQ. ID NO:380]Oligo #328 Length: 000024CCTGTTGAAT GCCTCCAGGT TCAG[SEQ. ID NO:381]Oligo #329 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGTTC AACCTG[SEQ. ID NO:382]Oligo #330 Length: 000024CCTGTTGAAT GCCTCCAGGT TGAA[SEQ. ID NO:383]Oligo #331 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGACC AACCTG[SEQ. ID NO:384]Oligo #332 Length: 000024CCTGTTGAAT GCCTCCAGGT TGGT[SEQ. ID NO:385]Oligo #333 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGTAC AACCTG[SEQ. ID NO:386]Oligo #334 Length: 000024CCTGTTGAAT GCCTCCAGGT TGTA[SEQ. ID NO:387]Oligo #335 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGGTT AACCTG[SEQ. ID NO:388]Oligo #336 Length: 000024CCTGTTGAAT GCCTCCAGGT TAAC[SEQ. ID NO:389]Oligo #337 Length: 000018AAAAATCTCG CTCCATGT[SEQ. ID NO:390]Oligo #338 Length: 000018AGCGAGATTT TTAAGAAT[SEQ. ID NO:391]Oligo #339 Length: 000018AAAAATCTCA ACCCATGT[SEQ. ID NO:392]Oligo #340 Length: 000018GTTGAGATTT TTAAGAAT[SEQ. ID NO:393]Oligo #341 Length: 000018AAAAATCTCG AACCATGT[SEQ. ID NO:394]Oligo #342 Length: 000018TTCGAGATTT TTAAGAAT[SEQ. ID NO:395]Oligo #343 Length: 000018AAAAATCTCC ACCCATGT[SEQ. ID NO:396]Oligo #344 Length: 000018GTGGAGATTT TTAAGAAT[SEQ. ID NO:397]Oligo #345 Length: 000018AAAAATCTCA TCCCATGT[SEQ. ID NO:398]Oligo #346 Length: 000018GATGAGATTT TTAAGAAT[SEQ. ID NO:399]Oligo #347 Length: 000018AAAAATCTCA TGCCATGT[SEQ. ID NO:400]Oligo #348 Length: 000018CATGAGATTT TTAAGAAT[SEQ. ID NO:401]Oligo #349 Length: 000018AAAAATCTCT TCCCATGT[SEQ. ID NO:402]Oligo #350 Length: 000018GAAGAGATTT TTAAGAAT[SEQ. ID NO:403]Oligo #351 Length: 000018AAAAATCTCT CCCCATGT[SEQ. ID NO:404]Oligo #352 Length: 000018GGAGAGATTT TTAAGAAT[SEQ. ID NO:405]Oligo #353 Length: 000018AAAAATCTCA CCCCATGT[SEQ. ID NO:406]Oligo #354 Length: 000018GGTGAGATTT TTAAGAAT[SEQ. ID NO:407]Oligo #355 Length: 000018AAAAATCTCT ACCCATGT[SEQ. ID NO:408]Oligo #356 Length: 000018GTAGAGATTT TTAAGAAT[SEQ. ID NO:409]Oligo #357 Length: 000027CTGCCCCTGG CCACGGCCGC AGCTACG[SEQ. ID NO:410]Oligo #358 Length: 000024ATGGATTGGA TGTCGCGTAG CTGC[SEQ. ID NO:411]Oligo #359 Length: 000027CTGCCCCTGG CCACGGCCGC AGGTACG[SEQ. ID NO:412]Oligo #360 Length: 000024ATGGATTGGA TGTCGCGTAC CTGC[SEQ. ID NO:413]Oligo #361 Length: 000027CTGCCCCTGG CCACGGCCGC AATCACG[SEQ. ID NO:414]Oligo #362 Length: 000024ATGGATTGGA TGTCGCGTGA TTGC[SEQ. ID NO:415]Oligo #363 Length: 000021GCTCATCCAA TCCATATCAA G[SEQ. ID NO:416]Oligo #364 Length: 000024ATGGATTGGA TGAGCCGTGG GTGC[SEQ. ID NO:417]Oligo #365 Length: 000021CAGCATCCAA TCCATATCAA G[SEQ. ID NO:418]Oligo #366 Length: 000024ATGGATTGGA TGCTGCGTGG GTGC[SEQ. ID NO:419]Oligo #367 Length: 000021CACCATCCAA TCCATATCAA G[SEQ. ID NO:420]Oligo #368 Length: 000024ATGGATTGGA TGGTGCGTGG GTGC[SEQ. ID NO:421]Oligo #369 Length: 000021AAACATCCAA TCCATATCAA G[SEQ. ID NO:422]Oligo #370 Length: 000024ATGGATTGGA TGTTTCGTGG GTGC[SEQ. ID NO:423]Oligo #371 Length: 000021CGAGCTCCAA TCCATATCAA G[SEQ. ID NO:424]Oligo #372 Length: 000024ATGGATTGGA GCTCGCGTGG GTGC[SEQ. ID NO:425]Oligo #373 Length: 000021CGAAACCCAA TCCATATCAA G[SEQ. ID NO:426]Oligo #374 Length: 000024ATGGATTGGG TTTCGCGTGG GTGC[SEQ. ID NO:427]Oligo #375 Length: 000021CGAGACCCAA TCCATATCAA G[SEQ. ID NO:428]Oligo #376 Length: 000024ATGGATTGGG TCTCGCGTGG GTGC[SEQ. ID NO:429]Oligo #377 Length: 000021CGAATCCCAA TCCATATCAA G[SEQ. ID NO:430]Oligo #378 Length: 000024ATGGATTGGG ATTCGCGTGG GTGC[SEQ. ID NO:431]Oligo #379 Length: 000021CGAAAACCAA TCCATATCAA G[SEQ. ID NO:432]Oligo #380 Length: 000024ATGGATTGGT TTTCGCGTGG GTGC[SEQ. ID NO:433]Oligo #381 Length: 000021CGAATGCCAA TCCATATCAA G[SEQ. ID NO:434]Oligo #382 Length: 000024ATGGATTGGC ATTCGCGTGG GTGC[SEQ. ID NO:435]Oligo #383 Length: 000021CGATTCCCAA TCCATATCAA G[SEQ. ID NO:436]Oligo #384 Length: 000024ATGGATTCGG AATCCCCTGG GTGC[SEQ. ID NO:437]Oligo #385 Length: 000021CGATCCCCAA TCCATATCAA G[SEQ. ID NO:438]Oligo #386 Length: 000024ATGGATTGGG GATCGCGTGG CTGC[SEQ. ID NO:439]Oligo #387 Length: 000021CGATGGCCAA TCCATATCAA G[SEQ. ID NO:440]Oligo #388 Length: 000024ATGGATTGGC CATCGCGTGG GTGC[SEQ. ID NO:441]Oligo #389 Length: 000021CGATACCCAA TCCATATCAA G[SEQ. ID NO:442]Oligo #390 Length: 000024ATGGATTGGG TATCGCGTGG GTGC[SEQ. ID NO:443]Oligo #391 Length: 000034CATCCAATCC AAATCAAGGA CGGTGACTGG AATG[SEQ. ID NO:444]Oligo #392 Length: 000044AATTCATTCC AGTCACCGTC CTTGATTTGG ATTGGATGTC GCGT[SEQ. ID NO:445]Oligo #393 Length: 000034CATCCAATCG AAATCAAGGA CGGTGACTGG AATG[SEQ. ID NO:446]Oligo #394 Length: 000044AATTCATTCC AGTCACCGTC CTTGATTTCG ATTGGATGTC GCGT[SEQ. ID NO:447]Oligo #395 Length: 000034CATCCAATCA TGATCAAGGA CGGTGACTGG AATG[SEQ. ID NO:448]Oligo #396 Length: 000044AATTCATTCC AGTCACCGTC CTTGATCATG ATTGGATGTC GCGT[SEQ. ID NO:449]Oligo #397 Length: 000034CATCCAATCT TCATCAAGGA CGGTGACTGG AATG[SEQ. ID NO:450]Oligo #398 Length: 000044AATTCATTCC AGTCACCGTC CTTGATGAAG ATTGGATGTC GCGT[SEQ. ID NO:451]Oligo #399 Length: 000034CATCCAATCT CCATCAAGGA CGGTGACTGG AATG[SEQ. ID NO:452]Oligo #400 Length: 000044AATTCATTCC AGTCACCGTC CTTGATGGAG ATTGGATGTC GCGT[SEQ. ID NO:453]Oligo #401 Length: 000034CATCCAATCg taATCAAGGA CGGTGACTGG AATG[SEQ. ID NO:454]Oligo #402 Length: 000044AATTCATTCC AGTCACCGTC CTTGATTACG ATTGGATGTC GCGT[SEQ. ID NO:455]Oligo #403 Length: 000021CGACATCCAA TCCGTATCAA G[SEQ. ID NO:456]Oligo #404 Length: 000024ACGGATTGGA TGTCGCGTGG GTGC[SEQ. ID NO:457]Oligo #405 Length: 000021CGACATCCAA TCAAAATCAA G[SEQ. ID NO:458]Oligo #406 Length: 000024TTTGATTGGA TGTCGCGTGG GTGC[SEQ. ID NO:459]Oligo #407 Length: 000021CGACATCCAA TCTACATCAA G[SEQ. ID NO:460]Oligo #408 Length: 000024GTAGATTGGA TGTCGCGTGG GTGC[SEQ. ID NO:461]Oligo #409 Length: 000016GCTGGTGACT GGAATG[SEQ. ID NO:462]Oligo #410 Length: 000026[SEQ. ID NO:463]AATTCATTCC AGTCACCAGC CTTGATOligo #411 Length: 000016AACGGTGACT GGAATG[SEQ. ID NO:464]Oligo #412 Length: 000026AATTCATTCC AGTCACCGTT CTTGAT[SEQ. ID NO:465]Oligo #413 Length: 000016GAAGGTGACT GGAATG[SEQ. ID NO:466]Oligo #414 Length: 000026AATTCATTCC AGTCACCTTC CTTGAT[SEQ. ID NO:467]Oligo #415 Length: 000016GGTGGTGACT GGAATG[SEQ. ID NO:468]Oligo #416 Length: 000026AATTCATTCC AGTCACCACC CTTGAT[SEQ. ID NO:469]Oligo #417 Length: 000016ATCGGTGACT GGAATG[SEQ. ID NO:470]Oligo #418 Length: 000026AATTCATTCC AGTCACCGAT CTTGAT[SEQ. ID NO:471]Oligo #419 Length: 000016CTGGGTGACT GGAATG[SEQ. ID NO:472]Oligo #420 Length: 000026AATTCATTCC AGTCACCCAG CTTGAT[SEQ. ID NO:473]Oligo #421 Length: 000016TTCGGTGACT GGAATG[SEQ. ID NO:474]Oligo #422 Length: 000026AATTCATTCC AGTCACCGAA CTTGAT[SEQ. ID NO:475]Oligo #423 Length: 000016TCCGGTGACT GGAATG[SEQ. ID NO:476]Oligo #424 Length: 000026AATTCATTCC AGTCACCGGA CTTGAT[SEQ. ID NO:477]Oligo #425 Length: 000032AATTCGCTAG GAAACTGACG TTCTATCTGA AA[SEQ. ID NO:478]Oligo #426 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTAGCG[SEQ. ID NO:479]Oligo #427 Length: 000032AATTCCAGAG GAAACTGACG TTCTATCTGA AA[SEQ. ID NO:480]Oligo #428 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCTGG[SEQ. ID NO:481]Oligo #429 Length: 000032AATTCCACAG GAAACTGACG TTCTATCTGA AA[SEQ. ID NO:482]Oligo #430 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGTGG[SEQ. ID NO:483]Oligo #431 Length: 000032AATTCTCCAG GAAACTGACG TTCTATCTGA AA[SEQ. ID NO:484]Oligo #432 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGGAG[SEQ. ID NO:485]Oligo #433 Length: 000032AATTCCGGAG GCGTCTGACG TTCTATCTGA AA[SEQ. ID NO:486]Oligo #434 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGACG CCTCCGG[SEQ. ID NO:487]Oligo #435 Length: 000032AATTCCGGAG GGAACTGACG TTCTATCTGA AA[SEQ. ID NO:488]Oligo #436 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGTTC CCTCCGG[SEQ. ID NO:489]Oligo #437 Length: 000032AATTCCGGAG GCACCTGACG TTCTATCTGA AA[SEQ. ID NO:490]Oligo #438 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGGTG CCTCCGG[SEQ. ID NO:491]Oligo #439 Length: 000032AATTCCGGAG GATCCTGACG TTCTATCTGA AA[SEQ. ID NO:492]Oligo #440 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGGAT CCTCCGG[SEQ. ID NO:493]Oligo #441 Length: 000032AATTCCGGAG GTCCCTGACG TTCTATCTGA AA[SEQ. ID NO:494]Oligo #442 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGGGA CCTCCGG[SEQ. ID NO:495]Oligo #443 Length: 000032AATTCCGGAG GAAACTGACG GACTATCTGA AA[SEQ. ID NO:496]Oligo #444 Length: 000037CTCAAGGGTT TTCAGATAGT CCGTCAGTTT CCTCCGG[SEQ. ID NO:497]Oligo #445 Length: 000032AATTCCGGAG GAAACTGACG ATCTATCTGA AA[SEQ. ID NO:498]Oligo #446 Length: 000037CTCAACGGTT TTCACATAGA TCCTCAGTTT CCTCCGG[SEQ. ID NO:499]Oligo #447 Length: 000032AATTCCGGAG GAAACTGACG CTGTATCTGA AA[SEQ. ID NO:500]Oligo #448 Length: 000037CTCAAGGGTT TTCAGATACA GCGTCAGTTT CCTCCGG[SEQ. ID NO:501]Oligo #449 Length: 000032AATTCCGGAG GAAACTCACG AAATATCTCA AA[SEQ. ID NO:502]Oligo #450 Length: 000037CTCAAGGGTT TTCAGATATT TCGTCAGTTT CCTCCGG[SEQ. ID NO:503]Oligo #451 Length: 000032AATTCCGGAG GAAACTGACG GTTTATCTGA AA[SEQ. ID NO:504]Oligo #452 Length: 000037CTCAAGGGTT TTCAGATAAA CCGTCACTTT CCTCCCG[SEQ. ID NO:505]Oligo #453 Length: 000032AATTCCGCAG GAAACTGACG TTCTATCTGG CT[SEQ. ID NO:506]Oligo #454 Length: 000037CTCAAGGGTA GCCAGATAGA ACCTCAGTTT CCTCCGG[SEQ. ID NO:507]Oligo #455 Length: 000032AATTCCGGAG GAAACTGACG TTCTATCTGC GT[SEQ. ID NO:508]Oligo #456 Length: 000037CTCAAGGGTA CGCAGATAGA ACGTCAGTTT CCTCCGG[SEQ. ID NO:509]Oligo #457 Length: 000032AATTCCGGAG GAAACTGACG TTCTATCTGA AC[SEQ. ID NO:510]Oligo #458 Length: 000037CTCAAGGGTG TTCAGATAGA ACGTCAGTTT CCTCCGG[SEQ. ID NO:511]Oligo #459 Length: 000032AATTCCGGAG GAAACTGACG TTCTATCTGC AG[SEQ. ID NO:512]Oligo #460 Length: 000037CTCAAGGGTC TGCAGATAGA ACGTCAGTTT CCTCCGG[SEQ. ID NO:513]Oligo #461 Length: 000032AATTCCGGAG GAAACTGACG TTCTATCTGC AC[SEQ. ID NO:514]Oligo #462 Length: 000037CTCAAGGGTG TGCAGATAGA ACGTCAGTTT CCTCCGG[SEQ. ID NO:515]Oligo #463 Length: 000032AATTCCGGAG GAAACTGACG TTCTATCTGA TG[SEQ. ID NO:516]Oligo #464 Length: 000037CTCAAGGGTC ATCAGATAGA ACGTCAGTTT CCTCCGG[SEQ. ID NO:517]Oligo #465 Length: 000032AATTCCGGAG GAAACTGACG TTCTATCTGT TC[SEQ. ID NO:518]Oligo #466 Length: 000037CTCAAGGGTG AACAGATAGA ACGTCAGTTT CCTCCGG[SEQ. ID NO:519]Oligo #467 Length: 000032AATTCCGGAG GAAACTGACG TTCTATCTGT AC[SEQ. ID NO:520]Oligo #468 Length: 000037CTCAAGGGTG TACAGATAGA ACGTCAGTTT CCTCCGG[SEQ. ID NO:521]Oligo #469 Length: 000040CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA[SEQ. ID NO:522]Oligo #470 Length: 000036CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC[SEQ. ID NO:523]Oligo #471 Length: 000027AACAACCTCA ATGGGGAAGA CCAAGAT[SEQ. ID NO:524]Oligo #472 Length: 000045CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC[SEQ. ID NO:525]Oligo #473 Length: 000036GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG[SEQ. ID NO:526]Oligo #474 Length: 000018ATCTTGGTCT TCCCCATT[SEQ. ID NO:527]Oligo #475 Length: 000036ATCCTGATGG AAAATAACCT TCGAAGGCCA AACCTG[SEQ. ID NO:528]Oligo #476 Length: 000024GAGGCATTCA ACAGGGCTGT CAAG[SEQ. ID NO:529]Oligo #477 Length: 000015AGTTTACAGA ATGCA[SEQ. ID NO:530]Oligo #478 Length: 000027CCTTCGAAGG TTATTTTCCA TCAGGAT[SEQ. ID NO:531]Oligo #479 Length: 000024CCTGTTGAAT GCCTCCAGGT TTGG[SEQ. ID NO:532]Oligo #480 Length: 000020TTCTGTAAAC TCTTGACAGC[SEQ. ID NO:533]Oligo #481 Length: 000021TCAGCAATTG AGAGCATTCT T[SEQ. ID NO:534]Oligo #482 Length: 000018AAAAATCTCC TGCCATGT[SEQ. ID NO:535]Oligo #483 Length: 000040CTGCCCCTGG CCACGGCCGC ACCCACGCGA CATCCAATCC ATATCAAG[SEQ. ID NO:536]Oligo #484 Length: 000027CTGCCCCTGG CCACGGCCGC ACCCACG[SEQ. ID NO:537]Oligo #485 Length: 000021CGACATCCAA TCCATATCAA G[SEQ. ID NO:538]Oligo #486 Length: 000016GACGGTGACT GGAATG[SEQ. ID NO:539]Oligo #487 Length: 000019GCTCTCAATT GCTGATGCA[SEQ. ID NO:540]Oligo #488 Length: 000018CAGGAGATTT TTAAGAAT[SEQ. ID NO:541]Oligo #489 Length: 000040ATGGATTGGA TGTCGCGTGG GTGCGGCCGT GGCCAGGGGC AGACATGG[SEQ. ID NO:542]Oligo #490 Length: 000024GGCCGTGGCC AGGGGCAGAC ATGG[SEQ. ID NO:543]Oligo #491 Length: 000024ATGGATTGGA TGTCGCGTGG GTGC[SEQ. ID NO:544]Oligo #492 Length: 000026AATTCATTCC AGTCACCGTC CTTGAT[SEQ. ID NO:545]Oligo #493 Length: 000032AATTCCGGAG GAAACTGACG TTCTATCTGA AA[SEQ. ID NO:546]Oligo #494 Length: 000032ACCCTTGAGA ATGCGCAGGC TCAACAGTAA TA[SEQ. ID NO:547]Oligo #495 Length: 000037CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCCGG[SEQ. ID NO:548]Oligo #496 Length: 000027AGCTTATTAC TGTTGAGCCT GCGCATT[SEQ. ID NO:549]

[0815]

13

TABLE 3

POLYPEPTIDES

The numbering of the mutated amino acid positions which

describe the following polypeptides corresponds to the

sequence of the full-length, native (1-133)hIL-3.

PEPTIDE #1: pMON5988 (Example 43), (15-125)hIL-3 SEQ ID NO: 65;

PEPTIDE #2: pMON13344 (Example 8), (15-125)hIL-3 (18I, 25H, 29R, 32A,

37P, 42A and 45V) SEQ ID NO: 66;

PEPTIDE #3: pMON13345 (Example 9), (15-125)hIL-3 (18I, 25H, 29R, 32N,

37P, 42S and 45M) SEQ ID NO: 67;

PEPTIDE #4: pMON13346 (Example 10), (15-125)hIL-3 (18I, 25H, 29V, 32A,

37S, 42S and 45M) SEQ ID NO: 68;

PEPTIDE #5: pMON13347 (Example 12), (15-125)hIL-3 (51R, 55L, 59L, 62V,

67N and 69E) SEQ ID NO: 69;

PEPTIDE #6: pMON13348 (Example 13), (15-125)hIL-3 (51R, 55L, 60S, 62V,

67N and 69E) SEQ ID NO: 70;

PEPTIDE #7: pMON13349 (Example 14), (15-125)hIL-3 (51R, 55T, 59L, 62V,

67H and 69E) SEQ ID NO: 71;

PEPTIDE #8: pMON13350 (Example 16), (15-125)hIL-3 (73G, 76A, 79R, 82Q,

87S, 93S, 98I, 101A and 105Q) SEQ ID NO: 72;

PEPTIDE #9: pMON13355 (Example 17), (15-125)hIL-3 (73G, 76A, 79R, 82V,

87S, 93S, 98T, 101A and 105Q) SEQ ID NO: 73;

PEPTIDE #10: pMON13352 (Example 19), (15-125)hIL-3 (109E, 116V, 120Q

and 123E) SEQ ID NO: 74;

PEPTIDE #11: pMON13354 (Example 20), (15-125)hIL-3 (109E, 116V, 117S, 120H and

123E) SEQ ID NO: 75;

PEPTIDE #12: pMON13360 (Example 21), (15-125)hIL-3 (73G, 76A, 79R,

82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ. ID. NO: 76;

PEPTIDE #13: pMON13361 (Example 22), (15-125)hIL-3 (73G, 76A, 79R,

82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and 123E),

SEQ ID NO: 77;

PEPTIDE #14: pMON13362 (Example 23), (15-125)hIL-3 (73G, 76A, 79R,

82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H and 123E)

SEQ ID NO: 78;

PEPTIDE #15: pMON13363 (Example 24), (15-125)hIL-3 (18I, 25H, 29R,

32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N and 69E)

SEQ ID NO: 79;

PEPTIDE #16: pMON13364 (Example 25), (15-125)hIL-3 (18I, 25H, 29R,

32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H and 69E)

SEQ ID NO: 80;

PEPTIDE #17: pMON13365 (Example 26), (15-125)hIL-3 (18I, 25H, 29V,

32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N and 69E)

SEQ ID NO: 81;

PEPTIDE #18: pMON13298 (Example 27), Met-Ala-(15-125)hIL-3 (73G, 76A,

79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E) SEQ ID

NO: 82;

PEPTIDE #19: pMON13299 (Example 28), Met-Ala-(15-125)hIL-3 (73G, 76A,

79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and 123E); SEQ

ID NO: 83;

PEPTIDE #20: pMON13300 (Example 29), Met-Ala-(15-125)hIL-3 (73G, 76A,

79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H and 123E) SEQ ID NO:

84;

PEPTIDE #21: pMON13301 (Example 30), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N and 69E)

SEQ ID NO: 85;

PEPTIDE #22: pMON13302 (Example 31), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H and 69E)

SEQ ID NO: 86;

PEPTIDE #23: pMON13303 (Example 32), Met-Ala-(15-125)hIL-3 (18I, 25H,

29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N and 69E)

SEQ ID NO: 87;

PEPTIDE #24: pMON13287 (Example 33), Met-Ala-(15-125)hIL-3 (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) SEQ ID

NO: 88;

PEPTIDE #25: pMON13288 (Example 34), Met-Ala-(15-125)hIL-3 (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) SEQ ID

NO: 89;

PEPTIDE #26: pMON13289 (Example 35), Met-Ala-(15-125)hIL-3 (18I, 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) SEQ ID

NO: 90;

PEPTIDE #27: pMON13290 (Example 36), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R,

82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and 123E) SEQ ID

NO: 91;

PEPTIDE #28: pMON13292 (Example 37), Met-Ala-(15-125)hIL-3 (18I, 25H,

29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N, 69E, 73G, 76A, 79R,

82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and 123E) SEQ ID

NO: 92;

PEPTIDE #29: pMON13294 (Example 38), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R,

82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H and 123E) SEQ

ID NO: 93;

PEPTIDE #30: pMON13295 (Example 39), Met-Ala-(15-125)hIL-3 (18I, 25H,

29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N, 69E, 73G, 76A, 79R,

82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H and 123E) SEQ

ID NO: 94;

PEPTIDE #31: pMON13312 (Example 40), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E, 73G, 76A, 79R,

82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and 123E) SEQ ID

NO: 95;

PEPTIDE #32: pMON13313 (Example 41), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R,

82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H and 123E) SEQ

ID NO: 96;

PEPTIDE #A3: pMON13285 (Example 60), Met-Ala-(15-125)hIL-3 (42D, 45M,

46S, 50D) SEQ ID NO: 259;

PEPTIDE #A4: pMON13286 (Example 61), Met-Ala-(15-125)hIL-3 (42D, 45M,

46S) SEQ ID NO: 260;

PEPTIDE #A5: pMON13325 (Example 62), Met-Ala-(15-125)hIL-3 (42D, 45M,

46S, 116W) SEQ ID NO: 261;

PEPTIDE #A6: pMON13326 (Example 63), Met-Ala-(15-125)hIL-3 (42D, 45M,

46S, 50D, 116W) SEQ ID NO: 262;

PEPTIDE #A7: pMON13330 (Example 65), Met-Ala-(15-125)hIL-3 (42D, 45M,

46S, 50D, 95R, 98I, 100R, 116W) SEQ ID NO: 263;

PEPTIDE #A8: pMON13329 (Example 66), Met-Ala-(15-125)hIL-3, (42D, 45M,

46S, 98I, 100R, 116W) SEQ ID NO: 406;

PEPTIDE #B1: pMON13406 (Example 69), Met-Ala-(15-125)hIL-3 (18I, 19A,

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) SEQ ID

NO: 264;

PEPTIDE #B2: pMON13414 (Example 70), Met-Ala-(15-125)hIL-3

(18I, 19I, 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) SEQ ID NO: 265;

PEPTIDE #B3: pMON13407 (Example 71), Met-Ala-(15-125)hIL-3

(18I, 25H, 29R, 32N, 37P, 42S, 45V, 51R, 55T, 59L, 62V, 67H, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 266;

PEPTIDE #B4: pMON13405 (Example 72), Met-Ala-(15-125)hIL-3

(18I, 19A, 25H, 29R, 32N, 37P, 42S, 45V, 51R, 55T, 59L, 62V, 67H, 69E,

73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 267;

PEPTIDE #B5: pMON13415 (Example 73), Met-Ala-(15-125)hIL-3

(18I, 19I, 25H, 29R, 32N, 37P, 42S, 45V, 51R, 55T, 59L, 62V, 67H, 69E,

73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 268;

PEPTIDE #B6: pMON13408 (Example 74), Met-Ala-(15-125)hIL-3

(18I, 25H, 29R, 32N, 37P, 42S, 45M, 49I, 51R, 55T, 59L, 62V, 67H, 69E,

73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 269;

PEPTIDE #B7: pMON13409 (Example 75), Met-Ala-(15-125)hIL-3

(18I, 25H, 29R, 32N, 37P, 42S, 45M, 49L, 51R, 55T, 59L, 62V, 67H, 69E,

73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 270;

PEPTIDE #B8: pMON13410 (Example 76), Met-Ala-(15-125)hIL-3

(18I, 25H, 29R, 32N, 37P, 42S, 45M, 49D, 51R, 55T, 59L, 62V, 67H, 69E,

73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 271;

PEPTIDE #B9: pMON13422 (Example 77), Met-Ala-(15-125)hIL-3

(18I, 19A, 25H, 29R, 32N, 37P, 42S, 45V, 49I, 51R, 55T, 59L, 62V, 67H,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 272;

PEPTIDE #B10: pMON13423 (Example 78), Met-Ala-(15-125)hIL-3

(18I, 19I, 25H, 29R, 32N, 37P, 42S, 45V, 49I, 51R, 55T, 59L, 62V, 67H,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 273;

PEPTIDE #B11: pMON13424 (Example 79), Met-Ala-(15-125)hIL-3

(18I, 19A, 25H, 29R, 32N, 37P, 42S, 45V, 49L, 51R, 55T, 59L, 62V, 67H,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 274;

PEPTIDE #B12: pMON13425 (Example 80), Met-Ala-(15-125)hIL-3

(18I, 19I, 25H, 29R, 32N, 37P, 42S, 45V, 49L, 51R, 55T, 59L, 62V, 67H,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 275;

PEPTIDE #B13: pMON13426 (Example 81), Met-Ala-(15-125)hIL-3 (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)

SEQ ID NO: 276;

PEPTIDE #B14: pMON13429 (Example 82), Met-Ala-(15-125)hIL-3

(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) SEQ ID NO: 277;

PEPTIDE #B15: pMON13368 (Example 83), Met-Ala-(15-125)hIL-3 (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) SEQ ID

NO: 278;

PEPTIDE #B16: pMON13380 (Example 84), Met-Ala-(15-125)hIL-3 (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)

SEQ ID NO: 279;

PEPTIDE #B17: pMON13475 (Example 86), Met-Ala-(15-125)hIL-3 (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, 116V, 120Q and

123E) SEQ ID NO: 280;

PEPTIDE #B18: pMON13366 (Example 87), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42N, 45V, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 281;

PEPTIDE #B19: pMON13367 (Example 88), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42A, 45V, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 282;

PEPTIDE #B20: pMON13369 (Example 89), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42D, 45V, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 283;

PEPTIDE #B21: pMON13370 (Example 90), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42A, 45M, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 284;

PEPTIDE #B22: pMON13373 (Example 91), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42D, 45M, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 285;

PEPTIDE #B23: pMON13374 (Example 92), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42S, 45M, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 286;

PEPTIDE #B24: pMON13375 (Example 93), Met-Ala-(15-119)hIL-3 (18I, 25H,

29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R,

82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,) SEQ ID NO: 287;

PEPTIDE #B25: pMON13376 (Example 94), Met-Asp-(15-119)hIL-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) SEQ ID NO: 288;

PEPTIDE #B26: pMON13377 (Example 95), Met-Ala-(15-119)hIL-3 (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) SEQ

ID NO: 289;

PEPTIDE #B27: pMON13378 (Example 96), Met-Asp-(15-119)hIL-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, 116V) SEQ ID NO: 290;

PEPTIDE #B28: pMON13379 (Example 97), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42A, 45V, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116V, 120Q and

123E) SEQ ID NO: 291;

PEPTIDE #B29: pMON13385 (Example 98), Met-Ala-(15-125)hIL-3 (18I, 25H,

29V, 32R, 34S, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A,

79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E) SEQ ID

NO: 292;

PEPTIDE #B30: pMON13381 (Example 99), Met-Ala-(15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R,

82W, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E) SEQ ID

NO: 293;

PEPTIDE #B31: pMON13383 (Example 100), Met-Ala-(15-125)hIL-3 (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,

120Q and 123E) SEQ ID NO: 294;

PEPTIDE #B32: pMON13384 (Example 101), Met-Ala-(15-125)hIL-3 (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, 120Q and 123E)

SEQ ID NO: 295;

PEPTIDE #B33: pMON13388 (Example 102), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42A, 45V, 50D, 51R, 55L, 56S, 60S, 62V, 67N, 69E,

73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 296;

PEPTIDE #B34: pMON13389 (Example 103), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42D, 45M, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A,

79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E) SEQ ID

NO: 297;

PEPTIDE #B35: pMON13391 (Example 104), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 34S, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 298;

PEPTIDE #B36: pMON13392 (Example 105), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42D, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A,

79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E) SEQ ID

NO: 299;

PEPTIDE #B37: pMON13393 (Example 106), Met-Ala-(15-125)hIL-3 (18I,

23A, 25H, 29R, 32A, 34S, 37P, 42D, 45M, 46S, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 300;

PEPTIDE #B38: pMON13394 (Example 107), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42D, 45M, 46S, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 301;

PEPTIDE #B39: pMON13395 (Example 108), Met-Ala-(15-125)hIL-3 (18I,

23A, 25H, 29V, 32R, 34S, 37P, 42D, 45V, 46S, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 302;

PEPTIDE #B40: pMON13396 (Example 109), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A,

79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 100R, 101M, 116V, 120Q and

123E) SEQ ID NO: 303;

PEPTIDE #B41: pMON13397 (Example 110), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A,

79R, 82W, 87S, 93S, 98I, 101A, 105Q, 109E, 100R, 101M, 116V, 120Q and

123E) SEQ ID NO: 304;

PEPTIDE #B42: pMON13398 (Example 111), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42D, 45V, 46S, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 305;

PEPTIDE #B43: pMON13399 (Example 112), Met-Ala-(15-125)hIL-3 (18I,

23A, 25H, 29V, 32R, 34S, 37P, 42D, 45V, 46S, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 306;

PEPTIDE #B44: pMON13404 (Example 113), Met-Ala-(15-119)hIL-3 (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) SEQ ID NO: 307;

PEPTIDE #B45: pMON13387 (Example 114), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42A, 45V, 50D, 51R, 55L, 60S, 62V, 67N, 69E, 73G,

76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 308;

PEPTIDE #B46: pMON13416 (Example 115), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42D, 45V, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E,

73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 309;

PEPTIDE #B47: pMON13417 (Example 116), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42D, 45M, 46S, 50D, 51R, 55L, 60S, 62V, 67N, 69E,

73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 310;

PEPTIDE #B48: pMON13420 (Example 117), Met-Ala-(15-125)hIL-3 (18I,

23A, 25H, 29R, 32A, 34S, 37P, 42D, 45V, 46S, 50D, 51R, 55L, 56S, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 311;

PEPTIDE #B49: pMON13421 (Example 118), Met-Ala-(15-125)hIL-3 (18I,

23A, 25H, 29R, 32A, 34S, 37P, 42D, 45M, 46S, 50D, 51R, 55L, 56S, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 331;

PEPTIDE #B50: pMON13432 (Example 119), Met-Ala-(15-125)hIL-3 (18I,

23A, 25H, 29R, 32A, 34S, 37P, 42D, 45M, 46S, 50D, 51R, 55L, 60S, 62V,

67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 312;

PEPTIDE #B51: pMON13382 (Example 120), Met-Ala-(15-125)hIL-3 (18I,

25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E, 73G, 76A,

79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116W, 120Q and 123E)

SEQ ID NO: 313;

PEPTIDE #B52: pMON13476 (Example 85), Met-Asp-(15-125)hIL-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, 116V, 120Q and 123E) SEQ DI

NO: 314;

PEPTIDE #B53: pMON13446 (Example 121), Met-Ala-Tyr-Pro-Glu-Thr-Asp-

Tyr-Lys-Asp-Asp-Asp-Asp-Lys-(15-125)hIL-3 (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) SEQ ID NO: 315;

PEPTIDE #B54: pMON13390 (Example 122), Met-Ala-Tyr-Pro-Glu-Thr-Asp-

Tyr-Lys-Asp-Asp-Asp-Asp-Lys-(15-125)hIL-3 (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) SEQ ID NO: 316;

PEPTIDE #C-2: pMON13400 (Example 124), Met-Ala-(15-125)hIL-3 (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) SEQ ID NO: 317;

PEPTIDE #C-3: pMON13402 (Example 125), Met-Ala-(15-125)hIL-3 (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) SEQ ID NO: 318;

PEPTIDE #C-10: pMON13440 (Example 131), Met-Ala-(15-125)hIL-3 (18I,

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) SEQ ID NO: 319;

PEPTIDE #C-11: pMON13451 (Example 132), Met-Ala-(15-125)hIL-3 (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) SEQ ID NO: 320;

PEPTIDE #C-4: pMON13403 (Example 126), Met-Ala-(15-125)hIL-3 (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) SEQ ID NO: 321;

PEPTIDE #C-5: pMON13411 (Example 127), Met-Ala-(15-125)hIL-3 (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)

SEQ ID NO: 322;

PEPTIDE #C-6: pMON13412 (Example 128), Met-Ala-(15-118)hIL-3 (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) SEQ ID NO: 323.

[0816]

14

TABLE 4

DNA SEQUENCES

The numbering of the mutated amino acid positions in the

modified hIL-3 proteins corresponds to the sequence of the

full-length, native (1-133)hIL-3.

DNA Sequence #1: pMON13287 (Example 33), encoding Met-Ala-(15-125)hIL-3

(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) SEQ ID NO: 97;

DNA Sequence #2: pMON13290 (Example 36), encoding Met-Ala-(15-125)hIL-3

(18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E,

73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 98;

DNA Sequence #3: pMON13313 (Example 41), encoding Met-Ala-(15-125)hIL-3

(18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N, 69E,

73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H

and 123E) SEQ ID NO: 98;

DNA Sequence #4: pMON13288 (Example 34), encoding Met-Ala-(15-125)hIL-3

(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) SEQ ID NO: 100;

DNA Sequence #5: pMON13312 (Example 40), encoding Met-Ala-(15-125)hIL-3

(18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E,

73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and

123E) SEQ ID NO: 101;

DNA Sequence #6: pMON13294 (Example 38), encoding Met-Ala-(15-125)hIL-3

(18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H, 69E,

73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H

and 123E) SEQ ID NO: 102;

DNA Sequence #7: pMON13289 (Example 35), encoding Met-Ala-(15-125)hIL-3

(18I, 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) SEQ ID

NO: 103;

DNA Sequence #8: pMON13292 (Example 37), encoding Met-Ala-(15-125)hIL-3

(18I, 25H, 29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N, 69E,

73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and 123E)

SEQ ID NO: 104;

DNA Sequence #9: pMON13295 (Example 39), encoding Met-Ala-(15-125)hIL-3

(18I, 25H, 29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N, 69E,

73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H

and 123E) SEQ ID NO: 105;

DNA Sequence #10: pMON13344 (Example 8), (15-125)hIL-3 (18I, 25H, 29R,

32A, 37P, 42A and 45V) SEQ ID NO: 106;

DNA Sequence #11: pMON13345 (Example 9), (15-125)hIL-3 (18I, 25H, 29R,

32N, 37P, 42S and 45M) SEQ ID NO: 107;

DNA Sequence #12: pMON13346 (Example 10), (15-125)hIL-3 (18I, 25H,

29V, 32A, 37S, 42S and 45M) SEQ ID NO: 108;

DNA Sequence #13: pMON13347 (Example 12), (15-125)hIL-3 (51R, 55L,

59L, 62V, 67N and 69E) SEQ ID NO: 109;

DNA Sequence #14: pMON13348 (Example 13), (15-125)hIL-3 (51R, 55L,

60S, 62V, 67N and 69E) SEQ ID NO: 110;

DNA Sequence #15: pMON13349 (Example 14), (15-125)hIL-3 (51R, 55T,

59L, 62V, 67H and 69E) SEQ ID NO: 111;

DNA Sequence #16: pMON13350 (Example 16), (15-125)hIL-3 (73G, 76A,

79R, 82Q, 87S, 93S, 98I, 101A and 105Q) SEQ ID NO: 112;

DNA Sequence #17: pMON13355 (Example 17), (15-125)hIL-3 (73G, 76A,

79R, 82V, 87S, 93S, 98T, 101A and 105Q) SEQ ID NO: 113;

DNA Sequence #18: pMON13352 (Example 19), (15-125)hIL-3 (109E, 116V,

120Q and 123E) SEQ ID NO: 114;

DNA Sequence #19: pMON13354 (Example 20), (15-125)hIL-3 (109E, 116V,

117S, 120H and 123E) SEQ ID NO: 115;

DNA Sequence #20: pMON13363 (Example 24), (15-125)hIL-3 (18I, 25H,

29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N and 69E)

SEQ ID NO: 116;

DNA Sequence #21: pMON13364 (Example 25), (15-125)hIL-3 (18I, 25H,

29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H and 69E)

SEQ ID NO: 117;

DNA Sequence #22: pMON13365 (Example 26), (15-125)hIL-3 (18I, 25H,

29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N and 69E)

SEQ ID NO: 118;

DNA Sequence #23: pMON13360 (Example 21), (15-125)hIL-3 (73G, 76A,

79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q and 123E),

SEQ ID NO: 119;

DNA Sequence #24: pMON13361 (Example 22), (15-125)hIL-3 (73G, 76A,

79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 120Q and 123E),

SEQ ID NO: 120;

DNA Sequence #25: pMON13362 (Example 23), (15-125)hIL-3 (73G, 76A,

79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V, 117S, 120H and 123E)

SEQ ID NO: 121;

DNA Sequence #26: pMON13301 (Example 30), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N

and 69E) SEQ ID NO: 122;

DNA Sequence #27: pMON13302 (Example 31), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H

and 69E) SEQ ID NO: 123;

DNA Sequence #28: pMON13303 (Example 32), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29V, 32A, 37S, 42S, 45M, 51R, 55L, 59L, 62V, 67N

and 69E) SEQ ID NO: 124;

DNA Sequence #29: pMON13298 (Example 27), encoding Met-Ala-(15-125)

hIL-3 (73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 125;

DNA Sequence #30: pMON13299 (Example 28), encoding Met-Ala-(15-125)

hIL-3 (73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V,

120Q and 123E); SEQ ID NO: 126;

DNA Sequence #31: pMON13300 (Example 29), encoding Met-Ala-(15-125)

hIL-3 (73G, 76A, 79R, 82V, 87S, 93S, 98T, 101A, 105Q, 109E, 116V,

117S, 120H and 123E) SEQ ID NO: 127;

DNA Sequence #33: pMON13438 (Example 59), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 161;

DNA Sequence #A3: pMON13285 (Example 60), encoding Met-Ala-(15-125)

hIL-3 (42D, 45M, 46S, 50D) SEQ ID NO: 398;

DNA Sequence #A4: pMON13286 (Example 61), encoding Met-Ala-(15-125)

hIL-3 (42D, 45M, 46S) SEQ ID NO: 399;

DNA Sequence #B1: pMON13406 (Example 69), encoding Met-Ala-(15-125)

hIL-3 (18I, 19A, 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) SEQ ID NO: 332;

DNA Sequence #B2: pMON13414 (Example 70), encoding Met-Ala-(15-125)

hIL-3 (18I, 19I, 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) SEQ ID NO: 333;

DNA Sequence #B3: pMON13407 (Example 71), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32N, 37P, 42S, 45V, 51R, 55T, 59L, 62V, 67H,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 334;

DNA Sequence #B4: pMON13405 (Example 72), encoding Met-Ala-(15-125)

hIL-3 (18I, 19A, 25H, 29R, 32N, 37P, 42S, 45V, 51R, 55T, 59L, 62V,

67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 335;

DNA Sequence #B5: pMON13415 (Example 73), encoding Met-Ala-(15-125)

hIL-3 (18I, 19I, 25H, 29R, 32N, 37P, 42S, 45V, 51R, 55T, 59L, 62V,

67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 336;

DNA Sequence #B6: pMON13408 (Example 74), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32N, 37P, 42S, 45M, 49I, 51R, 55T, 59L, 62V,

67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 337;

DNA Sequence #B7: pMON13409 (Example 75), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32N, 37P, 42S, 45M, 49L, 51R, 55T, 59L, 62V,

67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 338;

DNA Sequence #B8: pMON13410 (Example 76), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32N, 37P, 42S, 45M, 49D, 51R, 55T, 59L, 62V,

67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 339;

DNA Sequence #B9: pMON13422 (Example 77), encoding Met-Ala-(15-125)

hIL-3 (18I, 19A, 25H, 29R, 32N, 37P, 42S, 45V, 49I, 51R, 55T, 59L,

62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 340;

DNA Sequence #B10: pMON13423 (Example 78), encoding Met-Ala-(15-125)

hIL-3 (18I, 19I, 25H, 29R, 32N, 37P, 42S, 45V, 49I, 51R, 55T, 59L,

62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 341;

DNA Sequence #B11: pMON13424 (Example 79), encoding Met-Ala-(15-125)

hIL-3 (18I, 19A, 25H, 29R, 32N, 37P, 42S, 45V, 49L, 51R, 55T, 59L,

62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 342;

DNA Sequence #B12: pMON13425 (Example 80), encoding Met-Ala-(15-125)

hIL-3 (18I, 19I, 25H, 29R, 32N, 37P, 42S, 45V, 49L, 51R, 55T, 59L,

62V, 67H, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 343;

DNA Sequence #B13: pMON13426 (Example 81), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 344;

DNA Sequence #B14: pMON13429 (Example 82), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 345;

DNA Sequence #B15: pMON13368 (Example 83), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 346;

DNA Sequence #B16: pMON13380 (Example 84), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 347;

DNA Sequence #B17: pMON13475 (Example 86), encoding Met-Ala-(15-125)

hIL-3 (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, 116V, 120Q and 123E) SEQ ID NO: 348;

DNA Sequence #B18: pMON13366 (Example 87), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42N, 45V, 46S, 50D, 51R, 55L, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 349;

DNA Sequence #B19: pMON13367 (Example 88), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 46S, 50D, 51R, 55L, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 350;

DNA Sequence #B20: pMON13369 (Example 89), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42D, 45V, 46S, 50D, 51R, 55L, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 351;

DNA Sequence #B21: pMON13370 (Example 90), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45M, 46S, 50D, 51R, 55L, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 352;

DNA Sequence #B22: pMON13373 (Example 91), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42D, 45M, 46S, 50D, 51R, 55L, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 353;

DNA Sequence #B23: pMON13374 (Example 92), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42S, 45M, 46S, 50D, 51R, 55L, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 354;

DNA Sequence #B24: pMON13375 (Example 93), encoding Met-Ala-(15-119)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,) SEQ

ID NO: 355;

DNA Sequence #B25: pMON13376 (Example 94), Met-Asp-(15-119)hIL-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) SEQ ID

NO: 356;

DNA Sequence #B26: pMON13377 (Example 95), encoding Met-Ala-(15-119)

hIL-3 (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) SEQ ID NO: 357;

DNA Sequence #B27: pMON13378 (Example 96), Met-Asp-(15-119)hIL-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, 116V) SEQ ID NO: 358;

DNA Sequence #B28: pMON13379 (Example 97), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 46S, 50D, 51R, 55L, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

112Q, 116V, 120Q and 123E) SEQ ID NO: 359;

DNA Sequence #B29: pMON13385 (Example 98), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29V, 32R, 34S, 37P, 42A, 45V, 51R, 55L, 60S, 62V,

67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 360;

DNA Sequence #B30: pMON13381 (Example 99), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82W, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 361;

DNA Sequence #B31: pMON13383 (Example 100), encoding Met-Ala-(15-125)

hIL-3 (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, 120Q and 123E) SEQ ID NO: 362;

DNA Sequence #B32: pMON13384 (Example 101), encoding Met-Ala-(15-125)

hIL-3 (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,

120Q and 123E) SEQ ID NO: 363;

DNA Sequence #B33: pMON13388 (Example 102), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 50D, 51R, 55L, 56S, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 346;

DNA Sequence #B34: pMON13389 (Example 103), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42D, 45M, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 365;

DNA Sequence #B35: pMON13391 (Example 104), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 34S, 37P, 42A, 45V, 51R, 55L, 60S, 62V,

67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 366;

DNA Sequence #B36: pMON13392 (Example 105), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42D, 45V, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V, 120Q

and 123E) SEQ ID NO: 367;

DNA Sequence #B37: pMON13393 (Example 106), encoding Met-Ala-(15-125)

hIL-3 (18I, 23A, 25H, 29R, 32A, 34S, 37P, 42D, 45M, 46S, 51R, 55L,

60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q,

109E, 116V, 120Q and 123E) SEQ ID NO: 368;

DNA Sequence #B38: pMON13394 (Example 107), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42D, 45M, 46S, 51R, 55L, 60S, 62V,

67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 369;

DNA Sequence #B39: pMON13395 (Example 108), encoding Met-Ala-(15-125)

hIL-3 (18I, 23A, 25H, 29V, 32R, 34S, 37P, 42D, 45V, 46S, 51R, 55L,

60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q,

109E, 116V, 120Q and 123E) SEQ ID NO: 370;

DNA Sequence #B40: pMON13396 (Example 109), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 100R, 101M,

116V, 120Q and 123E) SEQ ID NO: 371;

DNA Sequence #B41: pMON13397 (Example 110), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82W, 87S, 93S, 98I, 101A, 105Q, 109E, 100R, 101M,

116V, 120Q and 123E) SEQ ID NO: 372;

DNA Sequence #B42: pMON13398 (Example 111), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42D, 45V, 46S, 51R, 55L, 60S, 62V,

67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 373;

DNA Sequence #B43: pMON13399 (Example 112), encoding Met-Ala-(15-125)

hIL-3 (18I, 23A, 25H, 29V, 32R, 34S, 37P, 42D, 45V, 46S, 51R, 55L,

60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q,

109E, 116V, 120Q and 123E) SEQ ID NO: 374;

DNA Sequence #B44: pMON13404 (Example 113), encoding Met-Ala-(15-119)

hIL-3 (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)

SEQ ID NO: 375;

DNA Sequence #B45: pMON13387 (Example 114), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 50D, 51R, 55L, 60S, 62V,

67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 116V,

120Q and 123E) SEQ ID NO: 376;

DNA Sequence #B46: pMON13416 (Example 115), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42D, 45V, 46S, 50D, 51R, 55L, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 377;

DNA Sequence #B47: pMON13417 (Example 116), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42D, 45M, 46S, 50D, 51R, 55L, 60S,

62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E,

116V, 120Q and 123E) SEQ ID NO: 378;

DNA Sequence #B48: pMON13420 (Example 117), encoding Met-Ala-(15-125)

hIL-3 (18I, 23A, 25H, 29R, 32A, 34S, 37P, 42D, 45V, 46S, 50D, 51R,

55L, 56S, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A,

105Q, 109E, 116V, 120Q and 123E) SEQ ID NO: 379;

DNA Sequence #B49: pMON13421 (Example 118), encoding Met-Ala-(15-125)

hIL-3 (18I, 23A, 25H, 29R, 32A, 34S, 37P, 42D, 45M, 46S, 50D, 51R,

55L, 56S, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A,

105Q, 109E, 116V, 120Q and 123E) SEQ ID NO: 380;

DNA Sequence #B50: pMON13432 (Example 119), encoding Met-Ala-(15-125)

hIL-3 (18I, 23A, 25H, 29R, 32A, 34S, 37P, 42D, 45M, 46S, 50D, 51R,

55L, 60S, 62V, 67N, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A,

105Q, 109E, 116V, 120Q and 123E) SEQ ID NO: 381;

DNA Sequence #B51: pMON13382 (Example 120), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32A, 37P, 42A, 45V, 51R, 55L, 60S, 62V, 67N,

69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q, 109E, 112Q, 116W,

120Q and 123E) SEQ ID NO: 382;

DNA Sequence #B52: pMON13476 (Example 85), Met-Asp-(15-125)hIL-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, 116V, 120Q and 123E)

SEQ DI NO: 383;

DNA Sequence #B53: pMON13446 (Example 121), encoding Met-Ala-Tyr-Pro-

Glu-Thr-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-(15-125)hIL-3 (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) SEQ ID NO: 404;

DNA Sequence #B54: pMON13390 (Example 122), encoding Met-Ala-Tyr-Pro-

Glu-Thr-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-(15-125)hIL-3 (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) SEQ ID NO: 405;

DNA Sequence #C-1: pMON13418 (Example 123), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H,

69E, 73G, 76P, 793, 82Q, 85V, 87Y, 88W, 91P, 93S, 95T, 98T, 101A,

105Q, 109E, 116V, 120Q and 123E) SEQ ID NO: 393;

DNA Sequence #C-2: pMON13400 (Example 124), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 384;

DNA Sequence #C-3: pMON13402 (Example 125), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 385;

DNA Sequence #C-4: pMON13403 (Example 126), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 388;

DNA Sequence #C-5: pMON13411 (Example 127), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 390;

DNA Sequence #C-6: pMON13412 (Example 128), encoding Met-Ala-(15-118)

hIL-3 (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)

SEQ ID NO: 391;

DNA Sequence #C-7: pMON13413 (Example 129), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 392;

DNA Sequence #C-8: pMON13419 (Example 126), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32N, 37P, 42S, 45M, 50D, 51S, 55T, 59L, 62P,

63H, 65S, 67Q, 69E, 73G, 76A, 79R, 82Q, 87S, 93S, 98I, 101A, 105Q,

109E, 116V, 120Q and 123E) SEQ ID NO: 389;

DNA Sequence #C-9: pMON13428 (Example 133), encoding Met-Ala-(15-125)

hIL-3 (18I, 25H, 29R, 32N, 37P, 42S, 45M, 51R, 55T, 59L, 62V, 67H,

69E, 73G, 76P, 79S, 82Q, 85V, 87Y, 91P, 93S, 95T, 98T, 101A, 105Q,

109L, 112Q, 116S, 120Q and 123E) SEQ ID NO: 394;

DNA Sequence #C-10: pMON13440 (Example 131), encoding Met-Ala-(15-125)

hIL-3 (18I, 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) SEQ ID NO: 386;

DNA Sequence #C-11: pMON13451 (Example 132), encoding Met-Ala-(15-125)

hIL-3 (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) SEQ ID NO: 387;

DNA Sequence #C-12: pMON13459 (Example 134), encoding Met-Ala-(15-125)

hIL-3 (18I, 23L, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, 51R,

55T, 59L, 62V, 67H, 69E, 73G, 76P, 79S, 82Q, 85V, 87Y, 91P, 93S, 95T,

98T, 101A, 105Q, 109L, 112Q, 116S, 120Q and 123E) SEQ ID NO: 395;

DNA Sequence #C-13: pMON13467 (Example 135), encoding Met-Ala-(15-125)

hIL-3 (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, 109L, 112Q, 116S, 120Q and 123E) SEQ ID NO: 396;

DNA Sequence #C-14: pMON13492 (Example 136), encoding Met-Ala-(15-125)

hIL-3 (18I, 23L, 25H, 29I, 32N, 34S, 37S, 38A, 42S, 45V, 46S, 51R,

55T, 59L, 62V, 67H, 69E, 73G, 76P, 79S, 82Q, 85V, 87Y, 91P, 93S, 95T,

98T, 101A, 105Q, 109E, 116V, 120Q and 123E) SEQ ID NO: 397.

[0817] Polypeptides corresponding to SEQ ID NOS. 15, 16, 17, 18 and 129 comprising (1-133)hIL-3 containing one or more amino acid substitutions can be made using the procedures described above and in the following examples by starting with the appropriate oligonuctiotides and then constructing the DNA encoding the polypeptide and expressing it in an appropriate host cell. In a similar manner polypeptides which correspond to SEQ ID NOS. 19, 20, 21, 22 and 130 and contain one or more amino acid substitutions and wherein from 1 to 14 amino acids have been sequentially deleted from the N-terminus, or from 1 to 15 amino acids have been deleted from the C-terminus or deletions of amino acids have been made from both the N-terminus and the C-terminus can also be made by following the procedures described above and in the following examples, beginning with the appropriate starting materials.

[0818] Additional details may be found in U.S. patent application Ser. No. 07/981,044 filed Nov. 24, 1992, which is hereby incorporated by reference in its entirety.

[0819] Additional details may be found in co filed U.S. patent application Attorney docket number 2713/2, which is hereby incorporated by reference in its entirety.

[0820] All references, patents or applications cited herein are incorporated by reference in their entirety.

[0821] Further details known to those skilled in the art may be found in T. Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1982) and references cited therein, incorporated herein by reference in its entirety; and in J. Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (1989) and references cited therein, incorporated herein by reference in its entirety.

[0822] The following examples will illustrate the invention in greater detail although it will be understood that the invention is not limited to these specific examples.

[0823] Amino acids are shown herein by standard one letter or three letter abbreviations as follows:

15AbbreviatedDesignationAmino AcidAAlaAlanineCCysCysteineDAspAspartic acidEGluGlutamic acidFPhePhenylalanineGGlyGlycineHHisHistidineIIleIsoleucineKLysLysineLLeuLeucineMMetMethionineNAsnAsparaginePProProlineQGlnGlutamineRArgArginineSSerSerineTThrThreonineVValValineWTrpTryptophanYTyrTyrosine

[0824] Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.

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[0876] Treco, D. A., in Current protocols in Molecular Biology, Seidman et al., eds. J Wiley N.Y., unit 2.1. (1989)

[0877] Valtieri, M., D. Santoli, D. Caracciolo, B. L. Kreider, S. W. Altmann, D. J. Tweardy, I. Gemperlein, F. Mavilio, B. J. Lange and G. Rovera. Establishment and characterization of an undifferentiated human T leukemia cell line which requires granulocyte-macrophage colony stimulating factor for growth. J. Immunol. 138:4042 (1987).

[0878] Voet, D., W. B. Gatzer, R. A. Cox, P. Doty. Absorption spectra of the common bases. Biopolymers 1: 193 (1963).

[0879] Wells, J. A., Vasser, M., and Powers, D. B. Cassette mutagenesis: an effective method for generation of multiple mutants at defined sites. Gene, 34:315-323 (1985).

[0880] Wong, E. Y., R. Seetharam, C. Kotts, R. A. Heeren, B. K. Klein, S. B. Braford, K. J. Mathis, B. F. Bishop, N. R. Siegel, C. E. Smith and W. C. Tacon. Expression of secreted IGF-1 in Escherichia coli. Gene, 68: 193-203 (1988).

[0881] Yang, Y., A. B. Clarletta, P. A. Temple, M. P. Chung, S. Koviacic, J. S. Witek-Giannotti, A. C. Leary, R. Kriz, R. E. Donahue, G. G. Wong and S. C. Clark. Human IL-3 (Multi-CSF): Identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell 47: 3-10 (1985).

[0882] Yanisch-Perron, C., J. Viera and J. Messing. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103-119 (1985).

[0883] Zoller, M. J. and Smith, M. Oligonucleotide-directed muta-genesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any frag-ment of DNA. Nucleic Acid Research, 10: 6487-6500 (1982).

[0884] Zoller, M. J. and Smith, M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods in Enzymology, 100:468-500 (1983).

[0885] Zoller, M. J. and Smith, M. Oligonucleotide-directed Mutagenesis: A simple method using two oligonucleotide primers and a single-stranded DNA template. DNA, 3: 479 (1984).

EXAMPLE 1

[0886] Construction of pMON 5846 (FIG. 4) which Encodes [Met-(1-133)hIL-3 (Arg129)]

[0887] A plasmid containing the gene for the cDNA of hIL-3 cloned into pUC18 on an EcoRI to HindIII fragment was obtained from British Biotechnology Limited (Cambridge, England). This plasmid was designated pPO518. The purified plasmid DNA was cleaved by the restriction endonucleases NheI and BamHI. Approximately 0.5 micrograms of cleaved plasmid DNA was ligated to 1.0 picomoles of a pair of annealed oligonucleotides with the following sequence:

165′-CTAGCGATCTTTTAATAAGCTTG-3′[SEQ ID NO:1]3′-GCTAGAAAATTATTCGAACCTAG-5′[SEQ ID NO:2]

[0888] The ligation mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked, and plasmid DNA was purified and subjected to restriction enzyme analysis. An isolate was identified in which the above oligonucleotide sequence had replaced the portion of the gene that encodes the extreme C-terminus. Within the new sequence was a new stop codon, TAA, and a recognition site for the enzyme HindIII. The new plasmid was designated pMON5846.

EXAMPLE 2

[0889] (a) Construction of Expression Vector Plasmid pMON2341

[0890] The plasmid pMON2341 was used to supply the particular replicon and expression elements used for construction of many of the plasmids used to produce hIL-3 and hIL-3 muteins in E. coli. These expression elements are described in the materials and methods section. pMON2341 is derived from pMON5515 (Olins et al., 1988) and from pMON2429. pMON2429 consists of the phage mp18 (Yanisch-Perron et al., 1985) with a BclI fragment carrying the chloramphenicol acetyl transferase (cat) gene from pBR328 (Covarrubias et al., 1981) inserted into the BamHI site. The cat gene in pMON2429 has been altered from that in pBR328 by site directed mutagenesis (Kunkel, 1985). The recognition sites for NcoI and EcoRI which occur in the native gene were altered so that these two restriction enzymes no longer recognize these sites. The changes did not alter the protein specified by the gene. Also, an NcoI site was introduced at the N-terminus of the coding sequence so that it overlaps the codon for initiator methionine.

[0891] The steps involved in construction of pMON2341 are listed below:

[0892] (1) The DNAs of pMON5515 and pMON2429 were treated with NcoI and HindIII. The fragments were ligated and used to transform competent E. coli to ampicillin resistance. From these colonies, some were identified that were chloramphenicol resistant. From one of these colonies, plasmid DNA was isolated in which the rat atriopeptigen gene of pMON5515 had been replaced by the NcoI to HindIII fragment containing the cat gene from pMON2429. This fragment contains the recognition sites for several restriction enzymes in the portion derived from the multilinker region of mp18. The new plasmid was designated pMON2412.

[0893] (2) pMON2412 was treated with the enzyme ClaI which cleaves at one location in the pBR327 derived portion of the DNA. The protruding ends were rendered blunt by treatment with Klenow in the presence of nucleotide precursors. This DNA was mixed with an isolated 514 bp RsaI fragment derived from pEMBL8 (Dente et al., 1983). This RsaI fragment contains the origin of replication of phage f1. This ligation mixture was used to transform competent E. coli cells to ampicillin resistance. Among the plasmid DNAs isolated from these cells was pMON5578. This plasmid has the structure of pMON2412 with the f1 origin region inserted into the ClaI site. This is illustrated in the Figures and in Olins and Rangwala (1990).

[0894] (3) The DNA of pMON5578 was treated with restriction enzymes HindIII and MstII. The DNA was then treated with Klenow enzyme in the presence of nucleotide precursors to render the ends blunt. This treated DNA was ligated and used to transform competent E. coli to ampicillin resistance. From the ampicillin resistant colonies, one plasmid was recovered from which the portion between HindIII and MstII was absent. This deletion resulted in the removal of sequences from the plasmid which are recognized by a number of restriction endonuclease sites. The new plasmid was designated pMON5582.

[0895] (4) The DNA of pMON5582 was treated with SstII and BclI and ligated in the presence of annealed oligonucleotides with the sequences shown below.

17[SEQ ID NO:3]5′- GGCAACAATTTCTACAAAACACTTGATACTGTATGAGCAT-[SEQ ID NO:4]3′-CGCCGTTGTTAAAGATGTTTTGTGAACTATGACATACTCGTA-ACAGTATAATTGCTTCAACAGAACAGATC-3′TGTCATATTAACGAACTTGTCTTGT-5′

[0896] This sequence encodes the essential elements of the recA promoter of E. coli including the transcription start site and the lexA repressor binding site (the operator) (Sancar et al., 1980). The plasmid recovered from the ligation mixes contained this recA promoter in place of the one in pMON5582 (and in pMON5515). The functionality of the recA promoter was illustrated by Olins and Rangwala (1990). The new plasmid was designated pMON5594.

[0897] (5) To eliminate the single EcoRI site in pMON5594, the DNA was treated with EcoRI, then with Klenow in the presence of nucleotide precursors to render the ends blunt and then the DNA was ligated. From this ligation mix a plasmid was recovered whose DNA was not cleaved with EcoRI. This plasmid was designated pMON5630.

[0898] (6) To alter the single recognition site for PstI, plasmid pMON5630 was subjected to site directed mutagenesis (Kunkel, 1985). The oligonucleotide used in this procedure has the sequence shown below.

[0899] 5′-CCATTGCTGCCGGCATCGTGGTC-3′ [SEQ ID NO:5]

[0900] The result of the procedure was to construct pMON2341 which differs from pMON5630 in that the PstI site in the beta-lactamase gene was altered so that PstI no longer recognizes the site. The single nucleotide change does not alter the amino acid sequence of the beta-lactamase protein.

[0901] (b) Construction of pMON5847 (FIG. 5) which Encodes [Met-(1-133)hIL-3(Arg129)]

[0902] Plasmid pMON2341 was used to supply the replicon, promoter, ribosome binding site, transcription terminator and antibiotic resistance marker for the plasmids used to produce hIL-3 in E. coli from cDNA derived hIL-3 genes.

[0903] Plasmid pMON2341 was treated with restriction enzymes NcoI and HindIII. The restriction fragment containing the replication origin was purified. The DNA of plasmid pMON5846 was treated with NcoI and HindIII. The restriction fragment containing the hTL-3 gene was gel purified. These purified restriction fragments were mixed and ligated. The ligation mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked, and plasmid DNA was purified and analyzed using restriction enzymes. pMON5847 was identified as a plasmid with the replicon of pMON2341 and the hIL-3 gene in place of the chloramphenicol acetyl transferase gene. JM101 cells harboring this plasmid were cultured in M9 medium and treated with nalidixic acid as described above. Samples of the culture were examined for protein content. It was found that this hIL-3 mutein was produced at about 6% of total cell protein as measured on Coomassie stained polyacrylamide gels.

EXAMPLE 3

[0904] Construction of pMON5854 (FIG. 7) which Encodes [Met-(1-133)hIL-3(Arg129)]

[0905] To increase the accumulation of hIL-3 in E. coli, the coding sequence of the amino terminal portion of the protein was altered to more closely reflect the codon bias found in E. coli genes that produce high levels of proteins (Gouy and Gautier, 1982). To change the coding sequence for the amino terminal portion of the gene, a pair of synthetic oligonucleotides were inserted between the NcoI and HpaI sites within the coding sequence. About 0.5 micrograms of DNA of the plasmid pMON5847 (Example 2) was treated with NcoI and HpaI. This DNA was mixed with an annealed pair of oligonucleotides with the following sequence:

18[SEQ ID NO:6]5′-CATGGCTCCAATGACTCAGACTACTTCTCTTAAGACT-[SEQ ID NO:7] 3′-CGAGGTTACTGAGTCTGATGAAGAGAATTCTGA-TCTTGGGTT-3′AGAACCCAA-5′

[0906] The fragments were ligated. The ligation mixture was used to transform competent JM101 to ampicillin resistance. Colonies were picked into broth. From the cultures plasmid DNA was made and examined for the presence of a DdeI site (CTNAG) which occurs in the synthetic sequence but not between the NcoI and HpaI sites in the sequence of pMON5847. The new recombinant plasmid was designated pMON5854. The nucleotide sequence of the DNA in the coding sequence of the amino terminal portion of the hIL-3 gene in pMON5854 was determined by DNA sequencing and found to be the same as that of the synthetic oligonucleotide used in ligation. Cultures of JM101 cells harboring this plasmid were grown and treated with nalidixic acid to induce production of the hIL-3 mutant protein. Analysis of the proteins on Coomassie gels showed that the accumulation of hIL-3 mutein was about 25% of total cell protein in cultures harboring pMON5854, significantly higher than it was in cultures harboring pMON5847.

EXAMPLE 4

[0907] Construction of pMON5887 (FIG. 12) which Encodes [Met-(1-125)hIL-3]

[0908] The plasmid DNA of pMON5854 (Example 3) was treated with EcoRI and HindIII and the larger fragment was gel purified. About 0.5 microgram of this DNA was ligated to 1 picomole of an annealed pair of oligonucleotides which encode amino acids 107 through 125 of hIL-3. The sequences of these oligonucleotides are shown below. EcoRI to HindIII

19[SEQ ID NO:8]5′-AATTCCGTCGTAAACTGACCTTCTATCTCAAAA-[SEQ ID NO:9] 3′-GGCAGCATTTGACTGGAAGATAGACTTTT-CCTTCGAGAACGCGCAGGCTCAACAGTAATA-3′GGAACCTCTTGCGCGTCCGAGTTGTCATTATTCGA-5′

[0909] After ligation, the DNA was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked into broth and plasmid DNA was isolated from each culture. Restriction analysis of the plasmid DNA showed the presence of an EcoRI to HindIII fragment smaller than that of pMON5854. The nucleotide sequence of the portion of the coding sequence between the EcoRI and HindIII sites was determined to confirm the accuracy of the replaced sequence. The new plasmid was designated pMON5887 encoding Met-(1-125)hIL-3 which has the following amino acid sequence:

20[SEQ ID NO:10]Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys ThrSer Trp Val Asn Cys Ser Asn Met Ile Asp Glu IleIle Thr His Leu Lys Gln Pro Pro Leu Pro Leu LeuAsp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp IleLeu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu GluAla Phe Asn Arg Ala Val Lys Ser Leu Gln Asn AlaSer Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu ProGys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg HisPro Ile His Ile Lys Asp Gly Asp Trp Asn Glu PheArg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu GluAsn Ala Gln Ala Gln Gln

EXAMPLE 5

[0910] Construction of pMON5967 which Encodes [Met-Ala-(15-125)hIL-3]

[0911] Plasmid DNA of pMON5887 isolated from E. coli GM48 (dam-) was cleaved with NcoI and ClaI and ligated to 1 picomole of an annealed pair of oligonucleotides, Nco I and ClaI, encoding amino acids [Met Ala (15-20)hIL-3]. The sequence of these oligonucleotides is shown below.

215′-CATGGCTAACTGCTCTAACATGAT-3′[SEQ ID NO:11] 3′-CGATTGACGAGATTGTACTAGC-5′[SEQ ID NO:12]

[0912] The resulting ligation mix was used to transform competent E. coli JM101 cells to ampicillin resistant colonies. Plasmid DNA was isolated from these cells and the size of the inserted fragment was determined to be smaller than that of pMON5887 by restriction analysis using NcoI and NsiI. The nucleotide sequence of the region between NcoI and ClaI was determined and found to be that of the synthetic oligonucleotides. The new plasmid was designated pMON5967 and cells containing it were induced for protein production. Sonicated cell pellets and supernatants were used for protein purification and bio-assay.

EXAMPLE 6

[0913] Construction of pMON5978 which Encodes [Met-Ala-(15-125)hIL-3]

[0914] Plasmid DNA of pMON5967 isolated from E. coli GM48(dam-) was cleaved with ClaI and NsiI and ligated to 1 picomole of an annealed assembly of six oligonucleotides encoding hIL-3 amino acids 20-70 (FIG. 2). This synthetic fragment encodes three unique restriction sites, EcoRV, XhoI and PstI. The sequence of these oligonucleotides is shown in FIG. 2.

[0915] The resulting ligation mix was used to transform competent E. coli JM101 cells to ampicillin resistant colonies. Plasmid DNA was isolated and screened with XbaI and EcoRV for the presence of the new restriction site EcoRV. The DNA sequence of the region between ClaI and NsiI was determined and found to be the same as that of the synthetic oligonucleotides. The new plasmid was designated pMON5978, and cells containing it were induced for protein production. Sonicated cell pellets and supernatants were used for protein purification and bio-assay.

[0916] Plasmid pMON5978 encodes [Met-Ala-(15-125)hIL-3] which has the following amino acid sequence:

22[SEQ ID NO:13]Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile IleThr His Leu Lys Gln Pro Pro Leu Pro Leu Leu AspPhe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile LeuMet Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu AlaPhe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala SerAla Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro CysLeu Pro Leu Ala Thr Ala Ala Pro Thr Arg His ProIle His Ile Lys Asp Gly Asp Trp Asn Glu Phe ArgArg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu AsnAla Gln Ala Gln Gln

EXAMPLE 7

[0917] Construction of pMON5898

[0918] Plasmid pMON5851 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3695 base pair NcoI,HindIII fragment. The genetic elements derived from pMON5851 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, AraBAD promoter, g10L ribosome binding site and the lamB secretion leader. The AraBAD promoter is identical to that described in plasmid pMON6235 and the lamB signal peptide sequence used is that shown in FIG. 8 fused to hIL-3 at the NcoI recognition site. Plasmid pMON5873 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 408 base pair NcoI,HindIII fragment. The genetic element derived from pMON5873 is the hIL-3 gene (1-133). Clones containing the hIL-3 (1-133) gene contained a 408 base pair NcoI, HindIII restriction fragment. This construct was designated pMON5898.

EXAMPLE 8

[0919] Construction of pMON5987

[0920] Plasmid pMON6458 DNA was digested with restriction enzymes NcoI and HindIII, resulting in a 3940 base pair NcoI,HindIII fragment. The genetic elements derived from pMON6458 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, AraBAD promoter, g10L ribosome binding site and lamB secretion leader. Plasmid pMON5978 DNA was digested with NcoI and NsiI. The resulting 170 base pair NcoI, NsiI fragment encodes amino acids 15-71 of (15-125)hIL-3. Plasmid pMON5976 DNA was digested with NsiI and HindIII. The resulting 175 base pair NsiI,HindIII fragment encodes amino acids 72-125 of (15-125)hIL-3. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and screened for the restriction sites EcoRV and NheI and DNA sequenced to confirm the correct insert.

EXAMPLE 9

[0921] Construction of pMON5988

[0922] The plasmid DNA of pMON5987 was digested with NheI and EcoRI, resulting in a 3903 base pair NheI, EcoRI fragment. The 3903 base pair NheI, EcoRI fragment was ligated to 1.0 picomoles of the following annealed oligonucleotides (Oligo #3 and Oligo #4):

23[SEQ ID NO:131]5′-CTAGCCACGGCCGCACCCACGCGACATCCAATCCATATCAA-[SEQ ID NO:132] 3′-GGTGCCGGCGTGGGTGCGCTGTAGGTTAGGTATAGTT-GGACGGTGACTGGAATG-3′CCTGCCACTGACCTTACAATT-5′

[0923] The ligation reaction mixture was used to transform E. coli K-12 strain JM101 and transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm positive clones. This plasmid was constructed to change alanine 101 to aspartic acid in the hIL-3 gene (15-125). The Ala101 to Asp101 change was confirmed by DNA sequencing. This plasmid was designated pMON5988 and encodes Peptide #1 [SEQ ID NO:65].

EXAMPLE 10

[0924] Construction of pMON5873 which Encodes [Met-(1-133)hIL-3]

[0925] The gene obtained from British Biotechnology, Ltd. specified arginine at codon position 129. The amino acid specified in the native hIL-3 cDNA is serine. To produce a protein with the native sequence at this position, the portion of the coding sequence between the EcoRI site at codons 106 and 107 and the NheI site at codons 129 and 130 was replaced. Plasmid DNA of pMON5854 (Example 3) and pMON5853 (Example 64) were treated with EcoRI and NheI. The larger fragments of each were gel purified. These were ligated to a pair of an annealed oligonucleotides with the following sequences:

24[SEQ ID NO:136]5′-AATTCCGTCGTAAACTGACCTTCTATCTGAAAACC-[SEQ ID NO:137] 3′-GGCAGCATTTGACTGGAAGATAGACTTTTGG-TTGGAGAACGCGCAGGCTCAACAGACCACTCTGTCG-3′AACCTCTTGCGCGTCCGAGTTGTCTGGTGAGACAGCGATC-5′

[0926] The ligation reaction mixtures were used to transform competent JM101 cells to ampicillin resistance. Colonies were picked into broth and grown. Plasmid DNA was isolated and screened for the presence of a new StyI recognition site present in the synthetic DNA and not in pMON5854 and pMON5853. The nucleotide sequence of the gene in the region between EcoRI and NheI was determined and found to be that of the synthetic oligonucleotides. The new plasmids were designated pMON5873 encoding [Met-(1-133)hIL-3] and pMON5872 encoding [Met-(15-133)hIL-3].

[0927] The plasmid, pMON5873, encodes Met-(1-133)hIL-3 which has the following amino acid sequence:

25[SEQ ID NO:128]Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys ThrSer Trp Val Asn Cys Ser Asn Met Ile Asp Glu IleIle Thr His Leu Lys Gln Pro Pro Leu Pro Leu LeuAsp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp IleLeu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu GluAla Phe Asn Arg Ala Val Lys Ser Leu Gln Asn AlaSer Ala Ile Gln Ser Ile Leu Lys Asn Leu Leu ProCys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg HisPro Ile His Ile Lys Asp Gly Asp Trp Asn Glu PheArg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu GluAsn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu AlaIle Phe

EXAMPLE 11

[0928] Construction of pMON6458

[0929] Plasmid pMON6525 DNA was digested with restriction enzymes HindIII and SalI and the resulting 3172 base pair fragment was isolated from a 1% agarose gel by interception onto DEAE membrane. The genetic elements derived from pMON6525 are the beta-lactamase gene (AMP), pBR327 origin of replication, and phage f1 origin of replication as the transcription terminator. (The genetic elements derived from plasmid pMON6525 are identical to those in plasmid pMON2341 which could also be used to construct pMON6458.) Plasmid pMON6457 was digested with restriction enzymes HindIII and SalI and the resulting 1117 base pair fragment was isolated by PAGE and crush and soak elution. The genetic elements derived from pMON6457 are the pAraBAD promoter, g10L ribosome binding site, lamB secretion leader and the (15-125) hIL-3 gene. The restriction fragments were ligated and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Clones containing the hIL-3 gene (encoding amino acids 15-125) contained a 345 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6458. This plasmid was constructed to eliminate an EcoRI restriction site outside the hIL-3 gene coding region in plasmid pMON6457.

EXAMPLE 12

[0930] Construction of pMON6455

[0931] Plasmid pMON5905 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3936 base pair fragment. The genetic elements derived from pMON5905 are the beta-lactamase gene (AMP), pBR327 origin of replication, pAraBAD promoter, g10L ribosome binding site, lamB secretion leader and phage f1 origin of replication as the transcription terminator. The following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, g10L ribosome binding site and phage f1 origin of replication as the transcription terminator, derived from plasmid pMON5905 are identical to those in plasmid pMON5594 which could also be used to construct pMON6455. The AraBAD promoter is identical to that described in pMON6235. The lamB signal peptide sequence used in pMON6455 is that shown in FIG. 8 fused to hIL-3 (15-125) at the NcoI site. Plasmid pMON5887 DNA was digested with restriction enzymes HindIII and NcoI, resulting in a 384 base pair NcoI, HindIII fragment. The restriction fragments were ligated, and the ligation reaction mixture was used to transform into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Positive clones containing the hIL-3 gene (encoding amino acids 1-125) contained a 384 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6455.

EXAMPLE 13

[0932] Construction of pMON6456

[0933] Plasmid pMON5905 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3936 base pair fragment. The genetic elements derived from pMON5905 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, pAraBAD promoter, g10L ribosome binding site and the lamB secretion leader. Plasmid pMON5871 was digested with restriction enzymes HindIII and NcoI, resulting in a 330 base pair NcoI, HindIII fragment. The genetic element derived from pMON5871 encompassed the bases encoding the (1-107) hIL-3 gene. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Clones containing the hIL-3 gene (encoding amino acids 1-107) contained a 330 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6456.

EXAMPLE 14

[0934] Construction of pMON6457

[0935] Plasmid pMON6455 DNA grown in E. coli strain GM48 (dam-)was digested with restriction enzymes NcoI and ClaI, resulting in a 4263 base pair NcoI, ClaI fragment. The restriction fragment was ligated to 1.0 picomoles of annealed oligonucleotides (Oligo #5 and Oligo #6) with the following sequence coding for Met Ala 14-20 hIL-3:

265′-CATGGCTAACTGCTCTAACATGAT-3′[SEQ ID NO:151]3′-CGATTGACGAGATTGTACTAGC-5′[SEQ ID NO:152]

[0936] The resulting DNA was transformed into E. coli K-12 strain JM101 and transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes XbaI and EcoRI in double digest. Positive clones containing the hIL-3 gene (encoding aa 15-125 of hIL-3) contained a 433 base pair XbaI, EcoRI restriction fragment and were DNA sequenced. This construct was designated pMON6457. This plasmid was constructed to delete the first 14 amino acids of hIL-3. The coding sequence of the resulting gene begins as follows:

275′ ATG GCT AAC TGC . . . 3′[SEQ ID NO:153] Met Ala Asn Cys . . .[SEQ ID NO:154] 15

[0937] The first two amino acids (Methionine, Alanine) create an NcoI restriction site and a signal peptidase cleavage site between the lamB signal peptide and (15-125) hIL-3. Plasmid pMON6457 encodes (15-125) hIL-3 which has the amino acid sequence designated SEQ ID NO:65.

EXAMPLE 15

[0938] Construction of pMON6235

[0939] One of the DNA fragments used to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotides, Oligo #51(A) [SEQ ID NO:155] and Oligo #52(A) [SEQ ID NO:156], were used as primers in this procedure. The template for the PCR reaction was E. coli strain W3110 chromosomal DNA, prepared as described in Maniatis (1982). The oligonucleotide primers were designed to amplify the AraBAD promoter (Greenfield et al., 1978). The resulting DNA product was digested with the restriction enzymes SacII and BglII. The reaction mixture was purified as described previously. Plasmid, pMON5594, DNA was digested with SacII and BglII, resulting in a 4416 base pair SacII,BglII restriction fragment which contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, G10L ribosome binding site, phage f1 origin of replication as the transcription terminator and the chloramphenicol acetyl transferase (cat) gene. The 4416 base pair SacII,BglII restriction fragment from pMON5594 was ligated to the PCR-generated SacII, BglII DNA fragment. The ligation mixture was used to transform E. coli K-12 strain JM101. Positive clones contained a 323 base pair SacII,BglII fragment and were DNA sequenced to confirm that the SacII,BglII fragment was the AraBAD promoter. This construct was designated pMON6235.

EXAMPLE 16

[0940] Construction of pMON6460

[0941] One of the DNA fragments to construct this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the oligonucleotides, Oligo #7 [SEQ ID NO: 26] and Oligo #8 [SEQ ID NO: 27] as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. Upon completion, the digest was heated at 70□ C. for 15 minutes to inactivate the enzymes. The restriction fragment was purified by phenol/chloroform extraction and precipitation with equal volume isopropanol in the presence of 2M NH4OAc. The oligonucleotide, Oligo #8, introduces two stop codons (TAA) after amino acid 93 of hIL-3 and creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones containing the above mentioned changes released a 1023 base pair SalI fragment. This construct was designated pMON6460. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 94, 95, 96 and 97 of hIL-3.

EXAMPLE 17

[0942] Construction of pMON6461

[0943] One of the DNA fragments to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotide, Oligo #7 [SEQ. ID NO: 26] and Oligo #9 [SEQ. ID NO: 28], as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. The oligonucleotide, Oligo #9, introduces two stop codons (TAA) after amino acid 97 of hIL-3 and creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON5458 was ligated to the PCR-generated NcoI, EcoRI DNA fragment. Positive clones containing the above mentioned changes released a 1035 base pair SalI fragment. This construct was designated pMON6461. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 98, 99, 100 and 101 of hIL-3.

EXAMPLE 18

[0944] Construction of pMON6462

[0945] One of the DNA fragments to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotide, Oligo #7 [SEQ. ID NO: 26] and Oligo #10 [SEQ. ID NO: 31], as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. The oligonucleotide, Oligo #10 [SEQ. ID NO: 31] introduces two stop codons (TAA) after amino acid 101 of hIL-3 and creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON5458 was ligated to the PCR-generated NcoI, EcoRI DNA fragment. Positive clones containing the above mentioned changes released a 1047 base pair SalI fragment. This construct was designated pMON6462. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 102, 103, 104 and 105 of hIL-3.

EXAMPLE 19

[0946] Construction of Single Amino Acid Substitution Libraries at Positions 94, 95, 96 and 97

[0947] One of the DNA fragments used to construct the plasmids containing single amino acid substitution at positions 94, 95, 96 and 97 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction plasmid pMON6460 DNA was the template and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus. The degenerate oligonucleotides, Oligo #11 [SEQ. ID NO: 32], Oligo #12 [SEQ. ID NO: 33], Oligo #13 [SEQ. ID NO: 34] and Oligo #14 [SEQ. ID NO: 35], were the primers at the C-terminus. These oligonucleotides are 32-fold degenerate, with G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 94, 95, 96 and 97 of hIL-3 respectively. These degenerate oligonucleotide primers theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at a single position. The degenerate oligonucleotides (Oligo #11 [SEQ. ID NO: 32], Oligo #12 [SEQ. ID NO: 33], Oligo #13 [SEQ. ID NO: 34] and Oligo #14 [SEQ. ID NO: 35]) replace the twelve bases introduced into pMON6460, that encode the two stop codons (TAA) after amino acid 93 of hIL-3 and the SalI recognition sequence. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. The resulting PCR-generated DNA products were digested with the restriction enzymes NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6460 was ligated to the PCR-generated NcoI, EcoRI DNA fragments. Plasmid DNA from individual colonies was isolated as described previously and screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #15 [SEQ. ID NO: 36], as the probe which had been labeled with p32. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 20

[0948] Construction of Single Amino Acid Substitution Libraries at Positions 98, 99, 100 and 101

[0949] Single amino acid substitutions variants were constructed at position 98, 99, 100 and 101 as described previously, with the following changes. In the PCR reaction the template was plasmid pMON6461 DNA and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus. The degenerate oligonucleotides, Oligo #16 [SEQ. ID NO: 37], Oligo #17 [SEQ. ID NO: 38], Oligo #18 [SEQ. ID NO: 39] and Oligo #19 [SEQ. ID NO: 40], were used as primers at the C-terminus. The resulting PCR-generated DNA products were purified and digested with restriction enzymes NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6461 was ligated to the PCR-generated DNA NcoI, EcoRI restriction fragment. Single colonies were screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #20 [SEQ. ID NO: 41], as the probe. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 21

[0950] Construction of Single Amino Acid Substitution Libraries at Positions 102, 103, 104 and 105

[0951] Single amino acid substitutions variants were constructed at position 102, 103, 104 and 105 as described previously, with the following changes. The template was pMON6462 and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus. The degenerate oligonucleotides, Oligo #21 [SEQ. ID NO: 42], Oligo #22 [SEQ. ID NO: 43], Oligo #23 [SEQ. ID NO: 44] and Oligo #24 [SEQ. ID NO: 45] were used as primers at the C-terminus. The resulting PCR-generated DNA products were purified and digested with restriction enzymes, NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6462 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Single colonies were screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #25 [SEQ. ID NO: 46], as the probe. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 22

[0952] Construction of Plasmid pMON6464

[0953] Amino acids 17-22 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the PCR reaction using the oligonucleotides, Oligo #26 and Oligo #27 as primers. The resulting PCR-generated DNA products were purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 17-22 of hIL-3 have been deleted. pMON6464 was made to serve as the template for the creation of single amino acid substitution variants at positions 17, 18, 19, 20, 21 and 22 of hIL-3.

EXAMPLE 23

[0954] Construction of Plasmid pMON6465

[0955] Amino acids 23-28 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #28, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 23-28 of hIL-3 have been deleted. pMON6465 was made to serve as the template for the creation of single amino acid substitution variants at positions 23, 24, 25, 26, 27 and 28 of hIL-3.

EXAMPLE 24

[0956] Construction of Plasmid pMON6466

[0957] Amino acids 29-34 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #29 as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 29-34 of hIL-3 have been deleted. pMON6466 was made to serve as the template for the creation of single amino acid substitution variants at positions 29, 30, 31, 32, 33 and 34 of hIL-3.

EXAMPLE 25

[0958] Construction of Plasmid pMON6467

[0959] Amino acids 35-40 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #30, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRV. The NcoI, EcoRV restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, EcoRV restriction fragment. Positive clones contained a 81 base pair NcoI, EcoRV restriction fragment in which the bases encoding amino acids 35-40 of hIL-3 have been deleted. pMON6467 was made to serve as the template for the creation of single amino acid substitution variants at positions 35, 36, 37, 38, 39 and 40 of hIL-3.

EXAMPLE 26

[0960] Construction of Plasmid pMON6468

[0961] Amino acids 41-46 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #31, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and XhoI. The NcoI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, XhoI restriction fragment. Positive clones contained a 119 base pair NcoI, XhoI restriction fragment in which the bases encoding amino acids 41-46 of hIL-3 have been deleted. pMON6468 was made to serve as the template for the creation of single amino acid substitution variants at positions 41, 42, 43, 44, 45 and 46 of hIL-3.

EXAMPLE 27

[0962] Construction of Plasmid pMON6469

[0963] Amino acids 47-52 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #32, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and XhoI. The NcoI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, XhoI restriction fragment. Positive clones contained a 119 base pair NcoI, XhoI restriction fragment in which the bases encoding amino acids 47-52 of hIL-3 have been deleted. pMON6469 was made to serve as the template for the creation of single amino acid substitution variants at positions 47, 48, 49, 50, 51 and 52 of hIL-3.

EXAMPLE 28

[0964] Construction of Plasmid pMON6470

[0965] Amino acids 53-58 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid, pMON5988, DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #33, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and NsiI. The NcoI, NsiI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, NsiI restriction fragment. Positive clones contained a 152 base pair NcoI, NsiI restriction fragment in which the bases encoding amino acids 53-58 of hIL-3 have been deleted. pMON6470 was made to serve as the template for the creation of single amino acid substitution variants at positions 53, 54, 55, 56, 57 and 58 of hIL-3.

EXAMPLE 29

[0966] Construction of Plasmid pMON6471

[0967] Amino acids 59-64 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #34, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and NsiI. The NcoI, NsiI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, NsiI restriction fragment. Positive clones contained a 152 base pair NcoI, NsiI restriction fragment in which the bases encoding amino acids 59-64 of hIL-3 have been deleted. pMON6471 was made to serve as the template for the creation of single amino acid substitution variants at positions 59, 60, 61, 62, 63 and 64 of hIL-3.

EXAMPLE 30

[0968] Construction of Plasmid pMON6472

[0969] Amino acids 65-70 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #35, as primers. The resulting PCR-generated DNA product was purified and digested with EcoRI and XhoI. The EcoRI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated EcoRI, XhoI restriction fragment. Positive clones contained a 126 base pair EcoRI, XhoI restriction fragment in which the bases encoding amino acids 65-70 of hIL-3 have been deleted. pMON6472 was made to serve as the template for the creation of single amino acid substitution variants at positions 65, 66, 67, 68, 69 and 70 of hIL-3.

EXAMPLE 31

[0970] Construction of Plasmid pMON6473

[0971] Amino acids 71-76 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid, pMON5988, DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #36, as primers. The resulting PCR-generated DNA product was and digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment, in which the bases encoding amino acids 71-76 of hIL-3 have been deleted, and lost the NsiI restriction site. pMON6473 was made to serve as the template for the creation of single amino acid substitution variants at positions 71, 72, 73, 74, 75 and 76 of hIL-3.

EXAMPLE 32

[0972] Construction of Plasmid pMON6474

[0973] Amino acids 77-82 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #37, as primers. The resulting PCR-generated DNA product was purified and digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was liqated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 170 base pair NcoI, NsiI restriction fragment and a 93 base pair NsiI, EcoRI restriction fragment in which the bases encoding amino acids 77-82 of hIL-3 have been deleted. pMON6474 was made to serve as the template for the creation of single amino acid substitution variants at positions 77, 78, 79, 80, 81 and 82 of hIL-3.

EXAMPLE 33

[0974] Construction of Plasmid pMON6475

[0975] Amino acids 83-88 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #38, as primers. The resulting PCR-generated DNA product was digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 170 base pair NcoI, NsiI restriction fragment and a 93 base pair NsiI, EcoRI restriction fragment in which the bases encoding amino acids 83-88 of hIL-3 have been deleted. pMON6475 was made to serve as the template for the creation of single amino acid substitution variants at positions 83, 84, 85, 86, 87 and 88 of hIL-3.

EXAMPLE 34

[0976] Construction of Plasmid pMON6476

[0977] Amino acids 88-93 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #39, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 88-93 of hIL-3 have been deleted. pMON6476 was made to serve as the template for the creation of single amino acid substitution variants at positions 88, 89, 90, 91, 92 and 93 of hIL-3.

EXAMPLE 35

[0978] Construction of Plasmid pMON6477

[0979] Amino acids 106-111 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #40, as primers. The resulting PCR-generated DNA fragment was purified and digested with NcoI and HindIII. The NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 106-111 of hIL-3 have been deleted. pMON6477 was made to serve as the template for the creation of single amino acid substitution variants at positions 106, 107, 108, 109, 110 and 111 of hIL-3.

EXAMPLE 36

[0980] Construction of Plasmid pMON6478

[0981] Amino acids 112-117 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #41, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and HindIII. The 4008 bp NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 112-117 of hTL-3 have been deleted. pMON6478 was made to serve as the template for the creation of single amino acid substitution variants at positions 112, 113, 114, 115, 116 and 117 of hIL-3.

EXAMPLE 37

[0982] Construction of Plasmid pMON6479

[0983] Amino acids 118-123 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #42, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and HindIII. The NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 118-123 of hIL-3 have been deleted. pMON6479 was made to serve as the template for the creation of single amino acid substitution variants at positions 118, 119, 120, 121, 122 and 123 of hIL-3.

EXAMPLE 38

[0984] Construction of Single Amino Acid Substitution Libraries at Positions 17, 18, 19, 20, 21 and 22

[0985] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 17, 18, 19, 20, 21 and 22 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6464 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #43, Oligo #44, Oligo #45, Oligo #46, Oligo #47 and Oligo #48 were the primers at the C-terminus. The oligonucleotide, Oligo #26, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6464. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 17, 18, 19, 20, 21 and 22 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA product was digested with NcoI and EcoRV. Plasmid pMON6464 DNA was digested with restriction enzymes NcoI and EcoRV resulting in a 4190 base pair fragment which was ligated to the PCR-generated NcoI, EcoRV restriction fragments. Plasmid DNA was isolated and screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #139, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 39

[0986] Construction of Single Amino Acid Substitution Libraries at Positions 23, 24, 25, 26, 27 and 28

[0987] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 23, 24, 25, 26, 27 and 28 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6465 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #49, Oligo #50, Oligo #51, Oligo #52, Oligo #53 and Oligo #54 were the primers at the C-terminus. The oligonucleotide, Oligo #26, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6465. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 23, 24, 25, 26, 27 and 28 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with restriction enzymes NcoI and EcoRV. Plasmid pMON6465 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #140, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 40

[0988] Construction of Single Amino Acid Substitution Libraries at Positions 29, 30, 31, 32, 33 and 34

[0989] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 29, 30, 31, 32, 33 and 34 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6466 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #55, Oligo #56, Oligo #57, Oligo #58, Oligo #59 and Oligo #60 were the primers at the C-terminus. The oligonucleotide Oligo #26 was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6466. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 29, 30, 31, 32, 33 and 34 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and EcoRV. Plasmid pMON6466 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #141, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 41

[0990] Construction of Single Amino Acid Substitution Libraries at Positions 35, 36, 37, 38, 39 and 40

[0991] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 35, 36, 37, 38, 39 and 40 of hIL-3 were generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6467 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #61, Oligo #62, Oligo #63, Oligo #64, Oligo #65 and Oligo #66 were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6467. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 35, 36, 37, 38, 39 and 40 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored and at the other position, 32 different codons substitutions were created at positions independently. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and EcoRV. Plasmid pMON6467 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #142, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 42

[0992] Construction of Single Amino Acid Substitution Libraries at Positions 41, 42, 43, 44, 45 and 46

[0993] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 41, 42, 43, 44, 45 and 46 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6468 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #67, Oligo #68, Oligo #69, Oligo #70, Oligo #71 and Oligo #72 were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6468. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 41, 42, 43, 44, 45 and 46 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and XhoI. Plasmid pMON6468 DNA was digested with restriction enzymes NcoI and XhoI and the resulting 4152 base pair fragment was ligated to the PCR-generated NcoI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #143, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 43

[0994] Construction of Single Amino Acid Substitution Libraries at Positions 47, 48, 49, 50, 51 and 52

[0995] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 47, 48, 49, 50, 51 and 52 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6469 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #73, Oligo #74, Oligo #75, Oligo #76, Oligo #77 and Oligo #78, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6469. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 47, 48, 49, 50, 51 and 52 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and XhoI. Plasmid pMON6469 DNA was digested with restriction enzymes NcoI and XhoI and the resulting 4152 base pair fragment was ligated to the PCR-generated NcoI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #144, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 44

[0996] Construction of Single Amino Acid Substitution Libraries at Positions 53, 54, 55, 56, 57 and 58

[0997] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 53, 54, 55, 56, 57 and 58 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6470 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #79, Oligo #80, Oligo #81, Oligo #82, Oligo #83 and Oligo #84, were the primers at the C-terminus. The oligonucleotide, oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6470. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 53, 54, 55, 56, 57 and 58 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and NsiI. Plasmid pMON6470 DNA was digested with restriction enzymes NcoI and NsiI and the resulting 4119 base pair fragment was ligated to the PCR-generated NcoI, NsiI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #145, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 45

[0998] Construction of Single Amino Acid Substitution Libraries at Positions 59, 60, 61, 62, 63 and 64

[0999] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 59, 60, 61, 62, 63 and 64 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6471 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #85, Oligo #86, Oligo #87, Oligo #88, Oligo #89 and Oligo #90, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6471. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 59, 60, 61, 62, 63 and 64 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and NsiI. Plasmid pMON6471 DNA was digested with restriction enzymes NcoI and NsiI and the resulting 4119 base pair fragment was ligated to the PCR-generated NcoI, NsiI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #146, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 46

[1000] Construction of Single Amino Acid Substitution Libraries at Positions 65, 66, 67, 68, 69 and 70

[1001] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 65, 66, 67, 68, 69 and 70 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6472 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #91, Oligo #92, Oligo #93, Oligo #94, Oligo #95 and Oligo #96, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6472. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 65, 66, 67, 68, 69 and 70 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and XhoI. Plasmid pMON6472 DNA was digested with restriction enzymes EcoRI and XhoI and the resulting 4145 base pair fragment was ligated to the PCR-generated EcoRI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #147, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 47

[1002] Construction of Single Amino Acid Substitution Libraries at Positions 71, 72, 73, 74, 75 and 76

[1003] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 71, 72, 73, 74, 75 and 76 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6473 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #97, Oligo #98, Oligo #99, Oligo #100, Oligo #101 and Oligo #102, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6473. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 71, 72, 73, 74, 75 and 76 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA fragments were purified and digested with the restriction enzymes EcoRI and PstI. Plasmid pMON6473 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #148, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 48

[1004] Construction of Single Amino Acid Substitution Libraries at Positions 77, 78, 79, 80, 81 and 82

[1005] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 77, 78, 79, 80, 81 and 82 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the reaction the plasmid pMON6474 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #103, Oligo #104, Oligo #105, Oligo #106, Oligo #107 and Oligo #108, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6474. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 77, 78, 79, 80, 81 and 82 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and PstI as described previosly. Plasmid pMON6474 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #149, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 49

[1006] Construction of Single Amino Acid Substitution Libraries at Positions 83, 84, 85, 86, 87 and 88

[1007] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 83, 84, 85, 86, 87 and 88 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6475 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #109, Oligo #110, Oligo #111, Oligo #112, Oligo #113 and Oligo #114, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6475. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 83, 84, 85, 86, 87 and 88 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA producs were purified and digested with the restriction enzymes EcoRI and PstI. Plasmid pMON6475 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #150, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 50

[1008] Construction of Single Amino Acid Substitution Libraries at Positions 88, 89, 90, 91, 92 and 93

[1009] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 88, 89, 90, 91, 92 and 93 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6476 DNA was the template and the following degenerate oligonucleotides, Oligo #114, Oligo #115, Oligo #116, Oligo #117, Oligo #118 and Oligo #119, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6476. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 88, 89, 90, 91, 92 and 93 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and NcoI. Plasmid pMON6476 DNA was digested with restriction enzymes EcoRI and NcoI and the resulting 4008 base pair fragment was ligated to the PCR-generated EcoRI, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #151, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 51

[1010] Construction of Single Amino Acid Substitution Libraries at Positions 106, 107, 108, 109, 110 and 111

[1011] One of the DNA fragments used to construct the plasmids containing the single amino acid substitutions at positions 106, 107, 108, 109, 110 and 111 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously in two sequential PCR reactions. In the first PCR reaction, plasmid pMON6477 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #120, Oligo #121, Oligo #122, Oligo #123, Oligo #124 and Oligo #125 were the primers at the C-terminus. The oligonucleotide, Oligo #7 was the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6477. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 106, 107, 108, 109, 110 and 111 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The DNA generated in this PCR reaction was purified by phenol/chloroform extraction and precipitation with equal volume isopropanol in the presence of 2M NH4OAc to remove any primer that was not extended. This DNA was then used as a primer in the second PCR reaction.

[1012] In the second PCR reaction plasmid pMON6477 DNA was the template, the DNA product generated in the first PCR reaction (described above) was the primer at the N-terminus and the oligonucleotide, Oligo #126 (DNA sequence shown in Table 1), was the primer at the C-terminus. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6477 was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #152, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 52

[1013] Construction of Single Amino Acid Substitution Libraries at Positions 112, 113, 114, 115, 116 and 117

[1014] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 112, 113, 114, 115, 116 and 117 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6478 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #127, Oligo #128, Oligo #129, Oligo #130, Oligo #131 and Oligo #132, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6478. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 112, 113, 114, 115, 116 and 117 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6478 was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #153, which had been labeled with P32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 53

[1015] Construction of Single Amino Acid Substitution Libraries at Positions 118, 119, 120, 121, 122 and 123

[1016] One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 118, 119, 120, 121, 122 and 123 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6479 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #133, Oligo #134, Oligo #135, Oligo #136, Oligo #137 and Oligo #138, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6479. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 118, 119, 120, 121, 122 and 123 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6479 DNA was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #154, which had been labeled with p32 Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 54

[1017] Construction of pMON13358

[1018] Plasmid pMON5978 DNA (Example 6) was digested with restriction enzymes NsiI and EcoRI and the resulting 3853 base pair NsiI,EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-71 and 106-125 of (15-125) hIL-3. The 3853 base pair NsiI,EcoRI restriction fragment from pMON5978 was ligated to the following annealed complementary oligonucleotides.

[1019] Oligo #15(A) [SEQ ID NO: 29]

[1020] Oligo #16(A) [SEQ ID NO: 30]

[1021] In the resulting plasmid the 111 bases between the NsiI and EcoRI restriction sites in the (15-125) hIL-3 gene are replaced with 24 bases from the above mentioned oligonucleotides. This linker also creates a NdeI recognition sequence.

EXAMPLE 55

[1022] Construction of pMON13304

[1023] Plasmid pMON13358 DNA is digested with restriction enzymes PstI and EcoRI and the resulting 3846 base pair PstI,EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-69 and 106-125 of (15-125) hIL-3. The 3846 base pair NsiI,EcoRI restriction fragment from pMON13358 is ligated to the following annealed complementary oligonucleotides.

28Oligo #155[SEQ ID NO:200]Oligo #156[SEQ ID NO:201]Oligo #157[SEQ ID NO:202]Oligo #158[SEQ ID NO:203]Oligo #159[SEQ ID NO:204]Oligo #160[SEQ ID NO:205]Oligo #161[SEQ ID NO:206]Oligo #162[SEQ ID NO:207]

[1024] When assembled, the oligonucleotides create PstI and EcoRI restriction ends and the DNA sequence that encodes amino acids 70-105 of (15-125) hIL-3 with the following amino acid substitutions; 98I and 100R. The codons encoding amino acids 70-105 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13304, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

[1025] Peptide #A1 [SEQ ID NO:66]

EXAMPLE 56

[1026] Construction of pMON13305

[1027] Plasmid pMON13358 DNA is digested with restriction enzymes PstI and EcoRI and the resulting 3846 base pair PstI,EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-69 and 106-125 of (15-125) hIL-3. The 3846 base pair NsiI,EcoRI restriction fragment from pMON13358 is ligated to the following annealed complementary oligonucleotides.

29Oligo #155[SEQ ID NO:200]Oligo #156[SEQ ID NO:201]Oligo #157[SEQ ID NO:202]Oligo #158[SEQ ID NO:203]Oligo #159[SEQ ID NO:204]Oligo #160[SEQ ID NO:205]Oligo #163[SEQ ID NO:208]Oligo #164[SEQ ID NO:209]

[1028] When assembled, the oligonucleotides create PstI and EcoRI restriction ends and the DNA sequence that encodes amino acids 70-105 of (15-125) hIL-3 with the following amino acid substitutions; 95R, 98I and 100R. The codons encoding amino acids 70-105 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13305, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

[1029] Peptide #A2 [SEQ ID NO:67]

EXAMPLE 57

[1030] Construction of pMON13286

[1031] Plasmid pMON5978 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 3865 base pair NcoI,EcoRV fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, precA promoter, g10L ribosome binding site and the bases encoding amino acids 47-125 of (15-125) hIL-3. The 3865 base pair NcoI,EcoRV restriction fragment from pMON5978 was ligated to the following annealed complementary oligonucleotides.

30Oligo #165[SEQ ID NO:210]Oligo #166[SEQ ID NO:211]Oligo #167[SEQ ID NO:212]Oligo #168[SEQ ID NO:213]Oligo #169[SEQ ID NO:214]Oligo #170[SEQ ID NO:215]

[1032] When assembled, the oligonucleotides create NcoI and EcoRV restriction ends and the DNA sequence that encodes amino acids 15-46 of (15-125) hIL-3 with the following amino acid substitutions; 42D, 45M and 46S. The codons encoding amino acids 15-46 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13286, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

[1033] Peptide #A3 [SEQ ID NO:69]

[1034] DNA sequence #A4 pMON13286 42D, 45M, 46S

31ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA[SEQ ID NO:68]GCCACCGCTG CCGCTGCTGG ACTTCAACAA CCTCAATGAC GAAGACATGTCTATCCTGAT GGAAAATAAC CTTCGTCGTC CAAACCTCGA GGCATTCAACCGTGCTGTCA AGTCTCTGCA GAATGCATCA GCAATTGAGA GCATTCTTAAAAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC ACGCGACATCCAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACCTTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG

EXAMPLE 58

[1035] Construction of pMON5853 (FIG. 6) which Encodes [Met-(15-133)hIL-3(Arg129)]

[1036] Plasmid DNA of pMON5847 (Example 2) was treated with NcoI. The restriction enzyme was inactivated by heat treatment (65° C. for 10 minutes). The DNA was then treated with large fragment of DNA polymerase I (Klenow) in the presence of all four nucleotide precursors. This produces DNA termini with non-overlapping ends. After 5 minutes at 37° C., the polymerase was inactivated by heat treatment at 65° C. for 10 minutes. The DNA was then treated with HpaI, an enzyme which produces non-overlapping termini. The DNA was ethanol precipitated and ligated. The ligation reaction mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked and plasmid DNA was analyzed by restriction analysis. A plasmid designated pMON5853 was identified as one containing a deletion of the amino terminal 14 codons of the hIL-3 gene. The DNA sequence for the junction of the ribosome binding site to the (15-133) hIL-3 gene was determined to be the following:

32[SEQ ID NO:133]5′-AAGGAGATATATCCATGAACTGCTCTAAC-3′[SEQ ID NO:134] M N C S N

[1037] The lower line contains the one-letter code for the amino acids specified by the coding sequence of the amino terminus of the 15-133 hIL-3 gene. These are methionine, asparagine, cysteine, serine and asparagine.

[1038] When cultures of JM101 cells harboring this plasmid were induced with nalidixic acid, it was found that hIL-3 (15-133) accumulated at levels higher than hIL-3 (pMON5847).

[1039] The plasmid, pMON5853, encodes Met-(15-133) hIL-3 (Arg129) which has the following amino acid sequence:

33 Met Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr[SEQ ID NO:135]His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe AsnAsn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu AsnAsn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg AlaVal Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser IleLeu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr AlaAla Pro Thr Arg His Pro Ile His Ile Lys Asp Gly AspTrp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu LysThr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu ArgLeu Ala Ile Phe

[1040] Formula XI shown below is a representation of a [(15-125)hIL-3 mutein] with numbers in bold type added above the amino acids to represent the position at which the amino acid below the bolded number appears in native (1-133)hIL-3 [e. g. the amino acid at position 1 of Formula XI corresponds to the Asn which appears at position 15 in native (1-133)hIL-3]. The number shown in bold indicates the amino acids that correspond to the native IL-3(1-133). The non-bold members below the amino acids sequences are for Seq Id reference numbers. When the muteins are expressed the initial amino acid may be preceded by Met- or Met-Ala-.

34 15 20 25Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln[SEQ ID NO:23] 1 5 10 15 30 35 40Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp 20 25 30 45 50 55Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu 35 40 45 60 65 70Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile 50 55 60 75 80 85Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr 65 70 75 90 95 100Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp 80 85 90105 110 115Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu 95 100 105120 125Asn Ala Gln Ala Gln Gln 110

[1041] Table 9 shows (15-125)hIL-3 muteins of the present invention which have one (and in some cases two) amino acid substitutions in the (15-125)hIL-3 polypeptide and which were constructed as described in the Examples. The mutants in Table 9 were secreted into the periplasmic space in E.coli. The periplasmic content was released by osmotic shock and the material in the crude osmotic shock fraction was screened for growth promoting activity. Biological activity is the growth promoting activity of AML cells relative to (15-125) hIL-3 (pMON6458 or pMMON5988). The numbers in parentheses indicate the number of repeat assays. When a variant was assayed more than once the standard deviation is indicated. An “−” indicates that the hIL3 variant protein level was less than 1.0 μg/ml and was not screened for growth promoting activity.

35TABLE 6(15-125) HUMAN INTERLEUKIN-3 MUTANTShIL-3 aaPARENTAL(15-125) hIL-3 MUTANTPOSITION1aaCODONaaSEQ ID NO:CODONBIOL ACTIVITY 17/311SERTCTLYS19AAG<0.018 (1)17/3 SERTCTGLY19GGG 1.2 ± 1.1 (3)17/3 SERTCTASP19GAC 1.0 ± 0.7 (3)17/3 SERTCTMET19ATG 0.50 (1)17/3 SERTCTGLN19CAG 1.2 ± 0.7 (3)17/3 SERTCTARG19AGG<0.070 (1)18/4 ASNAACHIS19CAC 1.2 ± 0.3 (3)18/4 ASNAACLEU19CTC 0.45 ± 0.42 (4)18/4 ASNAACILE19ATC 1.5 ± 0.2 (2)18/4 ASNAACPHE19TTC 0.19 ± 0.26 (2)18/4 ASNAACARG19CGG 0.10 (1)18/4 ASNAACGLN19CAA 0.37 (1)19/5 METATGPHE19TTC 0.25 (1)19/5 METATGILE19ATC 0.77 ± 0.70 (9)19/5 METATGARG19AGG 0.17 (1)19/5 METATGGLY19GGA 0.06 (1)19/5 METATGALA19GCG 0.19 (1)19/5 METATGCYS19TGC-20/6 ILEATCCYS19TGC-20/6 ILEATCGLN19CAG-20/6 ILEATCGLU19GAG<0.025 (1)20/6 ILEATCARG19CGC<0.025 (1)20/6 ILEATCPRO19CCG 0.29 ± 0.16 (3)20/6 ILEATCALA19GCG 0.18 (1)21/7 ASPGATPHE19TTC<0.016 (1)21/7 ASPGATLYS19AAG 0.027 ± 0.027 (2)21/7 ASPGATARG19AGG<0.008 (1)21/7 ASPGATALA19GCG 0.07 ± 0.06 (3)21/7 ASPGATGLY19GGG 0.032 (1)21/7 ASPGATVAL19GTG<0.008 (1)22/8 GLUGAATRP19TGG-22/8 GLUGAAPRO19CCG<0.015 (1)22/8 GLUGAASER19TCG<0.015 (1)22/8 GLUGAAALA19GCC<0.015 (1)22/8 GLUGAAHIS19CAC<0.015 (1)22/8 GLUGAAGLY19GGC<0.008 (1)23/9 ILEATTVAL19GTG 0.18 (1)23/9 ILEATTALA219GCG 1.16 ± 0.16 (3)23/9 ILEATTLEU19TTG 1.3 (1)23/9 ILEATTGLY219GGG 0.06 (1)23/9 ILEATTTRP19TGG-23/9 ILEATTLYS219AAG-23/9 ILEATTPHE19TTC-23/9 ILEATTLEU219TTG 3.0 ± 1.1 (3)23/9 ILEATTSER219AGC<0.005 (1)23/9 ILEATTARG219CGC-24/10ILEATAGLY19GGG<0.004 (1)24/10ILEATAVAL19GTC 0.89 ± 0.23 (4)24/10ILEATAARG319CGG-24/10ILEATASER19AGC<0.003 (1)24/10ILEATAPHE19TTC 0.29 ± 0.24 (2)24/10ILEATALEU19CTG 0.52 ± 0.12 (3)25/11THRACAHIS19CAC 1.11 ± 0.2 (3)25/11THRACAGLY19GGC 0.48 ± 0.27 (4)25/11THRACAGLN19CAG 1.0 ± 0.8 (4)25/11THRACAARG19CGG 0.26 ± 0.17 (2)25/11THRACAPRO19CCG 0.36 (1)31/17PROCCTGLY19GGG 0.79 ± 0.61 (2)31/17PROCCTALA19GCC 0.49 (1)31/17PROCCTARG19CGC 0.25 ± 0.20 (2)31/17PROCCTLEU19CTG 0.22 (1)31/17PROCCTGLN19CAG 0.62 ± 0.04 (2)31/17PROCCTLEU419CTG 0.30 ± 0.20 (3)32/18LEUTTGVAL19GTG 0.01 (1)32/18LEUTTGARG19CGC 1.5 ± 1.0 (4)32/18LEUTTGGLN19CAG 0.93 ± 0.18 (3)32/18LEUTTGASN19AAC 1.2 ± 0.5 (5)32/18LEUTTGGLY519GGC 0.84 ± 1.0 (3)32/18LEUTTGALA19GCG 1.4 ± 0.7 (5)32/18LEUTTGGLU19GAG 0.88 ± 0.37 (2)33/19PROCC(T/C)LEU19CTG 0.13 (1)33/19PROCC(T/C)GLN19CAG 0.22 ± 0.20 (2)33/19PROCC(T/C)ALA19GCG 0.30 ± 0.14 (2)33/19PROCC(T/C)THR19ACC<0.018 (1)33/19PROCC(T/C)GLU19GAG 0.54 ± 0.43 (2)34/20LEUTTGVAL19GTG 1.2 ± 0.6 (3)34/20LEUTTGGLY19GGG 0.64 ± 0.74 (2)34/20LEUTTGSER19TCG 1.5 ± 0.7 (4)34/20LEUTTGLYS19AAG 0.97 ± 0.28 (2)34/20LEUTTGMET19ATG 1.7 ± 0.5 (3)35/21LEUCTGALA19GCC 1.6 ± 0.5 (3)35/21LEUCTGGLY19GGC<0.006 ± 0.002 (3)35/21LEUCTGASN19AAC 1.1 ± 1.7 (5)35/21LEUCTGPRO19CCC 1.8 ± 2.0 (5)35/21LEUCTGGLN19CAA 0.98 ± 1.1 (5)35/21LEUCTGVAL19GTG 0.76 ± 0.86 (5)36/22ASPGACLEU19CTC 0.20 (1)25/11THRACAALA19GCC 10.86 ± 0.27 (3)26/12HISCACTHR19ACG 0.010 (1)26/12HISCACPHE19TTC 0.26 (1)26/12HISCACGLY19GGG 0.19 (1)26/12HISCACARG19CGG 0.21 (1)26/12HISCACALA19GCC 0.56 ± 0.03 (2)26/12HISCACTRP19TGG-27/13LEUTTAGLY19GGG-27/13LEUTTAARG19AGG-27/13LEUTTATHR19ATC 0.084 (1)27/13LEUTTASER19TCC-27/13LEUTTAALA19GCG 0.01 (1)28/14LYSAAGARG19CGG 0.42 ± 0.07 (2)28/14LYSAAGLEU19TTG-28/14LYSAAGTRP19TGG-28/14LYSAAGGLN19CAG 0.27 (1)28/14LYSAAGGLY19GGC 0.36 ± 0.07 (2)28/14LYSAAGPRO19CCC 0.10 ± 0.04 (2)28/14LYSAAGVAL19GTG 0.19 ± 0.12 (2)29/15GLNCAGASN19AAC 1.62 ± 1.7 (3)29/15GLNCAGLEU19CTG 0.28429/15GLNCAGPRO19CCG-29/15ARGCAGARG19AGG 0.44 ± 0.16 (4)29/15GLNCAGVAL19GTG 0.62 ± 0.40 (4)30/16PROCCAHIS19CAC 0.26 (1)30/16PROCCATHR19ACG 0.36 (1)30/16PROCCAGLY19GGG 1.2 ± 0.8 (3)30/16PROCCAASP19GAC-30/16PROCCAGLN19CAG 0.61 ± 0.37 (3)30/16PROCCASER19TCG-30/16PROCCALEU19TTC-30/16PROCCALYS19AAG-31/17PROCCTASP19GAC 0.66 ± 0.71(3)36/22ASPGACVAL19GTG-37/23PHETTCSER19AGC 0.62 ± 0.40 (4)37/23PHETTCPRO19CCG 0.65 ± 0.39 (4)37/23PHETTCTRP19TGG-37/23PHETTCILE19ATC 0.1 (1)38/24ASNAACALA19GCN 1.9 (1)40/26LEUCTCTRP19TGG-40/26LEUCTCARG19CGC-41/27ASNAATCYS19TGC 0.18 (1)41/27ASNAATARG19CGC 0.13 ± 0.13 (2)41/27ASNAATLEU19CTG 0.09 ± 0.07 (2)41/27ASNAATHIS19CAC 0.49 ± 0.26 (4)41/27ASNAATMET19ATG 0.30 ± 0.38 (4)41/27ASNAATPRO19CCG 0.12 (1)42/28GLYGGGASP19GAC 5.7 ± 5.7 (6)42/28GLYGGGSER19AGC 4.3 ± 4.8 (7)42/28GLYGGGCYS19TGC 0.53 (1)42/28GLYGGGALA19GCC 5.9 ± 4.1 (7)43/29GLUGAAASN19AAC 0.050 (1)43/29GLUGAATYR19TAC 0.010 (1)43/29GLUGAALEU19CTC<0.009 (1)43/29GLUGAAPHE19TTC<0.009 (1)43/29GLUGAAASP19GAC 0.044 (1)43/29GLUGAAALA19GCC<0.009 (1)43/29GLUGAACYS19TGC<0.009 (1)43/29GLUGAASER19AGC<0.009 (1)44/30ASPGACSER19TCA 0.007 (1)44/30ASPGACLEU19CTG<0.007 (1)44/30ASPGACARG19AGG<0.007 (1)44/30ASPGACLYS19AAG<0.007 (1)44/30ASPGACTHR19ACG-44/30ASPGACMET19ATG<0.007 (1)44/30ASPGACTRP19TGG<0.007 (1)44/30ASPGACPRO19CCC<0.007 (1)45/31GLNCAAPRO19CCC-45/31GLNCAAPHE19TTC 0.007 (1)45/31GLNCAAVAL19GTC 6.7 ± 6.1 (5)45/31GLNCAAMET19ATG 3.4 ± 1.8 (5)45/31GLNCAALEU19TTG 1.1 ± 1.3 (2)45/31GLNCAATHR19ACG 0.96 ± 1.5 (3)45/31GLNCAALYS19AAG 1.6 ± 2.2 (5)45/31GLNCAATRP19TGG 0.10 (1)46/32ASPGACPHE19TTC 1.2 ± 0.5 (3)46/32ASPGACSER19TCC 7.9 ± 6.4 (4)46/32ASPGACTHR19ACC 1.8 ± 0.2 (2)46/32ASPGACCYS19TGC 0.80 (1)46/32ASPGACGLY19GGC 0.25 (1)47/33ILEATTGLY19GGC<0.015 (1)47/33ILEATTVAL19GTG 0.38 (1)47/33ILEATTHIS19CAC 0.10 (1)47/33ILEATTSER19TCC 0.03 (1)47/33ILEATTARG19AGG 0.09 (1)47/33ILEATTPRO19CCG<0.015 (1)48/34LEUCTGSER19AGC<0.009 (1)48/34LEUCTGCYS19TCG-48/34LEUCTGARG19CGC<0.009 (1)48/34LEUCTGILE19ATC 0.036 (1)48/34LEUCTGHIS19CAC<0.009 (1)48/34LEUCTGPHE19TTC<0.009 (1)48/34LEUCTGASN19AAC<0.009 (1)49/35METATGARG19CGC 0.007 (1)49/35METATGALA19GCC 0.091 (1)49/35METATGGLY19GGC 0.036 (1)49/35METATGPRO19CCC<0.009 (1)49/35METATGASN19AAC 0.23 (1)49/35METATGHIS19CAC<0.009 (1)49/35METATGASP19GAC 0.28 ± 0.48 (3)50/36GLUGAALEU19CTC 0.01 (1)50/36GLUGAATHR19ACC 0.20 (1)50/36GLUGAAASP19GAC-50/36GLUGAATYR19TAC 0.09 (1)50/36GLUGAAGLN19CTG 0.02 (1)51/37ASNAATARG19CGC 2.0 ± 0.8 (3)51/37ASNAATMET19ATG 0.75 ± 0.50 (2)51/37ASNAATPRO19CCG 2.77 ± 1.6 (3)51/37ASNAATSER19TCC 0.87 ± 0.44 (3)51/37ASNAATTHR19ACG 2.3 ± 1.6 (3)51/37ASNAATHIS19CAC 1.3 ± 0.9 (5)52/38ASNAACHIS19CAC 0.004 (1)52/38ASNAACARG19CGC 0.004 (1)52/38ASNAACLEU19TGG 0.003 (1)52/38ASNAACGLY19GGC 0.22 (1)52/38ASNAACSER19AGC 0.07 (1)52/38ASNAACTHR19ACG 0.44 ± 0.30 (3)53/39LEUCTTTHR19ACC<0.005 (1)53/39LEUCTTALA19GCG-53/39LEUCTTGLY19GGC<0.005 (1)53/39LEUCTTGLU19GAG<0.005 (1)53/39LEUCTTPRO19CCG<0.005 (1)53/39LEUCTTLYS19AAG<0.005 (1)53/39LEUCTTSER19AGC 0.008 (1)53/39LEUCTTMET19ATG 0.31 (1)54/40ARGCGAASP19GAC<0.005 (1)54/40ARGCGAILE19ATC 0.05 (1)54/40ARGCGASER19TCC 0.10 (1)54/40ARGCGAVAL19GTG<0.005 (1)54/40ARGCGATHR19ACC 0.015 (1)54/40ARGCGAGLN19CAG 0.04 (1)54/40ARGCGALEU19TTG 0.03 (1)55/41ARGAGGTHR19ACC 0.65 ± 1.1 (4)55/41ARGAGGVAL19GTC 0.96 ± 0.36 (3)55/41ARGAGGSER19TCG 0.065 (1)55/41ARGAGGLEU19CTG 1.1 ± 1.2 (4)55/41ARGAGGGLY19GGC 1.0 ± 0.6 (4)56/42PROCCAGLY19GGC 1.1 ± 0.8 (3)56/42PROCCACYS19TGC 0.21 (1)56/42PROCCASER19AGC 1.4 ± 0.4 (2)56/42PROCCAGLN19CAG 1.8 (1)56/42PROCCALYS19AAG 0.60 (1)57/43ASNAACGLY619GGC-58/44LEUCTGSER19AGC<0.041 (1)58/44LEUCTGASP19GAC<0.041 (1)58/44LEUCTGARG19CGG<0.041 (1)58/44LEUCTGGLN19CAG<0.041 (1)58/44LEUCTGVAL19GTC<0.041 (1)58/44LEUCTGCYS19TGC-59/45GLUGAGTYR19TAC 0.41 ± 0.37 (5)59/45GLUGAGHIS19CAC 0.38 ± 0.31 (2)59/45GLUGAGLEU19CTC 0.46 ± 0.36 (6)59/45GLUGAGPRO19CCC-59/45GLUGAGARG19CGC 0.15 (1)60/46ALAGCASER19AGC 0.91 ± 0.55 (4)60/46ALAGCAPRO19CCC-60/46ALAGCATYR19TAC<0.008 (1)60/46ALAGCAASN19AAC 0.38 (1)60/46ALAGCATHR19ACG 0.21 (1)61/47PHETTCASN19AAC-61/47PHETTCGLU19GAG<0.010 (1)61/47PHETTCPRO19CCC-61/47PHETTCLYS19AAG<0.010 (1)61/47PHETTCARG19CGC 0.006 (1)61/47PHETTCSER19TCG 0.17 (1)62/48ASNAACHIS19CAC-62/48ASNAACVAL19GTG 0.37 ± 0.25 (4)62/48ASNAACARG19AGG-62/48ASNAACPRO719CCG 1.6 ± 0.4 (3)62/48ASNAACPRO19CCG 2.0 ± 0.3 (3)62/48ASNAACTHR819ACG 2.3 ± 1.1 (3)62/48ASNAACASP19GAC-62/48ASNAACILE19ATC 0.56 ± 0.24 (4)63/49ARGA(G/A)GTYR19TAC 0.47 (1)63/49ARGA(G/A)GTRP19TGG 0.09 (1)63/49ARGA(G/A)GLYS19AGG 0.52 (1)63/49ARGA(G/A)GSER919TCC 0.13 (1)63/49ARGA(G/A)GHIS19CAC 0.42 ± 0.25 (7)63/49ARGA(G/A)GPRO19CCG<0.014 ± 0.013 (2)63/49ARGA(G/A)GVAL19GTG 0.39 ± 0.34 (3)64/50ALAGCTASN19AAC 1.5 ± 2.9 (4)64/50ALAGCTPRO19CCG<0.023 (1)64/50ALAGCTSER19AGC<0.023 (1)64/50ALAGCTLYS19AAG<0.047 (1)65/51VALGTCTHR19ACC 0.71 ± 0.64 (3)65/51VALGTCPRO19CCG<0.014 (1)65/51VALGTCHIS19CAC<0.014 (1)65/51VALGTCLEU19CTC 0.42 (1)65/51VALGTCPHE19TTC 0.061 (1)65/51VALGTCSER19TCC 0.34 (1)66/52LYSAAGILE1019ATC 0.42 (1)66/52LYSAAGARG19AGG 0.79 ± 0.18 (2)66/52LYSAAGVAL19GTC 0.38 ± 0.17 (2)66/52LYSAAGASN19AAC 0.32 (1)66/52LYSAAGGLU19GAG 0.14 (1)66/52LYSAAGSER19TCG 0.31 (1)66/52LYSAAGVAL1119GTG 0.055 (1)67/53SERAGTALA19GCG<0.014 (1)67/53SERAGTPHE19TTC 1.2 ± 0.2 (2)67/53SERAGTVAL19GTG 0.24 (1)67/53SERAGTGLY19GGG 0.50 ± 0.29 (4)67/53SERAGTASN19AAC 0.52 ± 0.28 (7)67/53SERAGTILE19ATC 0.29 (1)67/53SERAGTPRO19CCG 0.055 (1)67/53SERAGTHIS19CAC 0.99 ± 0.62 (6)68/54LEUTTAVAL19GTC 0.14 (1)68/54LEUTTATRP19TGG 0.07 (1)68/54LEUTTASER19AGC<0.003 (1)68/54LEUTTAILE19ATC 0.84 ± 0.47 (3)68/54LEUTTAPHE19TTC 1.7 ± 0.3 (3)68/54LEUTTATHR19ACG 0.011 (1)68/54LEUTTAHIS19CAC 0.82 ± 0.45 (2)69/55GLNCAGALA19GCG 1.2 ± 0.8 (3)69/55GLNCAGPRO19CCA0.74 0.45 (4)69/55GLNCAGTHR19ACG 0.97 ± 0.46 (4)69/55GLNCAGTRP19TGG-69/55GLNCAGGLU19GAG 1.4 ± 0.7 (3)69/55GLNCAGARG19CGG 1.4 ± 1.1 (3)69/55GLNCAGGLY19GGG 0.68 ± 0.02 (2)69/55GLNCAGLEU19CTC-70/56ASNAA(C/T)LEU19TTG 0.032 (1)70/56ASNAA(C/T)VAL19GTG-70/56ASNAA(C/T)TRP19TGG-70/56ASNAA(C/T)PRO1219CCG 0.43 ± 0.29 (2)70/56ASNAA(C/T)ALA1319GCC 0.03 (1)71/57ALAGCAMET19ATG 0.23 (1)71/57ALAGCALEU19CTG<0.005 (1)71/57ALAGCAPRO19CCC 0.58 (1)71/57ALAGCAARG19AGG 0.66 (1)71/57ALAGCAGLU19GAG 0.46 ± 0.2771/57ALAGCATHR19ACC 0.34 ± 0.41 (3)71/57ALAGCAGLN19GGC 0.42 ± 0.32 (3)71/57ALAGCATRP19TGG-71/57ALAGCAASN19AAC 0.09 (1)72/58SERTCAGLU19GAG 0.62 ± 0.27 (3)72/58SERTCAMET19ATG 0.45 ± 0.55 (3)72/58SERTCAALA19GCC 0.48 ± 0.33 (3)72/58SERTCAHIS19CAC 0.10 (1)72/58SERTCAASN19AAC 0.38 ± 0.44 (3)72/58SERTCAARG19CGG 0.81 ± 0.43 (4)72/58SERTCAASP19GAC 0.58 ± 0.39 (3)73/59ALAGCAGLU19GAG 0.49 ± 0.32 (3)73/59ALAGCAASP19GAC 0.27 (1)73/59ALAGCALEU19CTG 0.55 ± 0.45 (4)73/59ALAGCASER19AGC 0.37 ± 0.36 (2)73/59ALAGCAGLY19GGG 0.38 ± 0.32 (3)73/59ALAGCATHR19ACC 0.31 (1)73/59ALAGCAARG19AGG 0.40 ± 0.18 (3)74/60ILEAT(T/C)MET19ATG<0.16 (1)74/60ILEAT(T/C)THR19ACG-74/60ILEAT(T/C)PRO19CCG-74/60ILEAT(T/C)ARG19AGG-74/60ILEAT(T/C)GLY19GGG 0.006 (1)74/60ILEAT(T/C)ALA19GCG-75/61GLUGAGLYS19AAG 0.07 ± 0.07 (2)75/61GLUGAGGLY19GGG 0.27 ± 0.20 (2)75/61GLUGAGASP19GAC 0.18 (1)75/61GLUGAGPRO19CCG-75/61GLUGAGTRP19TGG-75/61GLUGAGARG19CGG-75/61GLUGAGSER19TCG 0.27 ± 0.22 (3)75/61GLUGAGGLN19CAG 0.40 ± 0.38 (3)75/61GLUGAGLEU19TTG-76/62SERAGCVAL19GTG 1.0 ± 0.2 (2)76/62SERAGCALA19GCG 0.94 ± 0.46 (2)76/62SERAGCASN19AAC 1.2 (1)76/62SERAGCTRP19TGG-76/62SERAGCGLU19GAG 0.90 ± 0.19 (2)76/62SERAGCPRO19CCG 2.1 ± 0.8 (4)76/62SERAGCGLY19GGC 1.3 ± 1.0 (4)76/62SERAGCASP19GAC0.29 (1)77/63ILEATTSER19AGC 0.48 ± 0.38 (4)77/63ILEATTARG19CGC 0.09 ± 0.04 (2)77/63ILEATTTHR19ACG<0.008 (1)77/63ILEATTLEU19TTG 2.0 ± 0.1 (3)78/64LEUCTTALA19GCG-78/64LEUCTTSER19TCC-78/64LEUCTTGLU19GAG<0.006 (1)78/64LEUCTTPHE19TTC-78/64LEUCTTGLY19GGG-78/64LEUCTTARG19AGG-79/65LYSAA(A/G)THR19ACA 0.77 ± 0.91 (6)79/65LYSAA(A/G)GLY19GGG 11.1 ± 0.9 (6)79/65LYSAA(A/G)ASN19AAC 1.0 ± 0.6 (6)79/65LYSAA(A/G)MET19ATG 1.6 ± 0.7 (6)79/65LYSAA(A/G)ARG19CGC 1.04 ± 0.7 (7)79/65LYSAA(A/G)ILE19ATC 1.0 ± 0.6 (6)79/65LYSAA(A/G)GLY19GGG 1.2 ± 0.4 (6)79/65LYSAA(A/G)ASP19GAC 0.72 ± 0.38 (7)80/66ASNAATTRP19TGG-80/66ASNAATVAL19GTC 0.32 (1)80/66ASNAATGLY19GGC 1.5 ± 1.4 (4)80/66ASNAATTHR19ACG 0.13 (1)80/66ASNAATLEU19CTG 0.33 ± 0.14 (2)80/66ASNAATGLU19GAG 1.1 ± 0.8 (4)80/66ASNAATARG19AGG 1.0 ± 0.8 (4)81/67LEUCTCGLN19CAA-81/67LEUCTCGLY19GGC<0.023 (1)81/67LEUCTCALA19GCG<0.047 (1)81/67LEUCTCTRP19TGG<0.005 (1)81/67LEUCTCARG19CGG-81/67LEUCTCVAL19GTG 0.16 ± 0.18 (2)81/67LEUCTCLYS19AAG-82/68LEUC(TG/CC)GLN19CAG 1.8 ± 0.3 (3)82/68LEUC(TG/CC)LYS19AAG 0.05 (1)82/68LEUC(TG/CC)TRP19TGG 2.7 ± 1.3 (4)82/68LEUC(TG/CC)ARG19AGC 1.1 ± 0.2 (3)82/68LEUC(TG/CC)ASP19GAC 2.7 ± 1.3 (4)82/68LEUC(TG/CC)VAL19GTG 1.5 ± 1.1 (5)83/69PROCCAALA19GCA 0.41 (1)83/69PROCCATHR19ACC 0.66 ± 0.12 (3)83/69PROCCAARG19CGG-83/69PROCCATRP19TGG 0.29 (1)83/69PROCCAMET19ATG 0.43 ± 0.28 (3)84/70CYSTG(T/C)GLU19GAG<0.014 (1)84/70CYSTG(T/C)GLY19GGG<0.006 (1)84/70CYSTG(T/C)ARG19AGG-84/70CYSTG(T/C)MET19ATG-84/70CYSTG(T/C)VAL19GTG-85/71LEUCTGASN19AAC-85/71LEUCTGVAL19GTG 0.52 ± 0.21 (5)85/71LEUCTGGLN19CAG-86/72PROCCCCYS19TGC-86/72PROCCCARG19AGG-86/72PROCCCALA19GCG-86/72PROCCCLYS19AAG-87/73LEU(C/A)TGSER19AGC 1.5 ± 0.4 (3)87/73LEU(C/A)TGTRP19TGG-87/73LEU(C/A)TGGLY19GGG-88/74ALAGCCLYS19AAG-88/74ALAGCCARG19AGG 0.11 ± 0.10 (2)88/74ALAGCCVAL19GTG 0.09 ± 0.02 (2)88/74ALAGCCTRP19TGG 1.8 ± 0.2 (2)89/75THRAC(G/A)ASP19GAC 0.24 ± 0.10 (2)89/75THRAC(G/A)CYS19TGC-89/75THRAC(G/A)LEU19CTC 0.01 (1)89/75THRAC(G/A)VAL19GTG 0.08 (1)89/75THRAC(G/A)GLU19GAG 0.11 (1)89/75THRAC(G/A)HIS19CAC 0.16 ± 0.06 (2)89/75THRAC(G/A)ASN19AAC 0.21 ± 0.04 (2)89/75THRAC(G/A)SER19TCG 0.25 ± 0.07 (2)90/76ALAGCCPRO19CCC 0.03 (1)90/76ALAGCCSER19TCG-90/76ALAGCCTHR19ACC 0.48 (1)90/76ALAGCCGLY19GGC<0.006 (1)90/76ALAGCCASP19GAC 0.44 ± 0.29 (4)90/76ALAGCCILE19ATC-90/76ALAGCCMET19ATG 0.25 ± 0.13 (2)91/77ALAGCAPRO19CCC 1.9 ± 1.2 (3)91/77ALAGCASER19TCC 0.12 ± 0.07 (2)91/77ALAGCATHR19ACC 0.48 ± 0.16 (2)91/77ALAGCAPHE19TTC 0.44 ± 0.50 (3)91/77ALAGCALEU19CTC 0.43 ± 0.27 (5)91/77ALAGCAASP19GAC 0.55 ± 0.09 (2)91/77ALAGCAHIS19CAC-92/78PROCCCPHE19TTC-92/78PROCCCARG19CGG-92/78PROCCCSER19AGC 0.26 (1)92/78PROCCCLYS19AAG-92/78PROCCCHIS19CAC-92/78PROCCCLEU19CTG-93/79THRACGASP19GAC 1.3 ± 0.7 (4)93/79THRACGSER19TCG 0.70 ± 0.56 (4)93/79THRACGASN19AAC-93/79THRACGPRO19CCC 0.53 ± 0.36 (4)93/79THRACGALA19GCG 1.13 ± 0.2 (3)93/79THRACGLEU19CTG 0.69 ± 0.4293/79THRACGARG19CGC 0.93 ± 0.96 (4)94/80ARGCGAILE19ATC<0.020 (1)94/80ARGCGASER19TCC<0.100 (1)94/80ARGCGAGLU19GAG<0.020 (1)94/80ARGCGALEU19CTG<0.020 (1)94/80ARGCGAVAL19GTG<0.024 (1)94/80ARGCGAPRO19CCC<0.024 (1)95/81HISCATGLN19CAG<0.010 (1)95/81HISCATPRO19CCG 1.6 ± 0.8 (3)95/81HISCATARG19CGC 4.7 ± 5.9 (2)95/81HISCATVAL19GTG 1.2 ± 1.7 (2)95/81HISCATLEU19CTC 0.7 (1)95/81HISCATGLY19GGC 1.7 ± 2.4 (5)95/81HISCATTHR19ACC 2.9 ± 4.5 (4)95/81HISCATTYR19TAC 0.07 (1)96/82PROCCALYS19AAG<0.010 ± 0.001 (2)96/82PROCCATYR19TAC 0.69 (1)96/82PROCCAGLY19GGG<0.040 (1)96/82PROCCAILE19ATC<0.040 (1)96/82PROCCATHR19ACC<0.040 (1)97/83ILEATCVAL19GTC 0.91 ± 1.2 (8)97/83ILEATCLYS19AAG<0.02497/83ILEATCALA19GCG 0.15 (1)97/83ILEATCASN19AAT<0.02 (1)98/84HISCATILE19ATC 5.0 ± 4.9 (12)98/84HISCATASN19AAC 1.4 ± 0.4 (2)98/84HISCATLEU19CTC 2.4 ± 1.0 (2)98/84HISCATASP19GAC 0.38 ± 0.49 (5)98/84HISCATALA19GCC 2.0 ± 1.0 (3)98/84HISCATTHR19ACG 1.6 ± 0.3 (2)98/84HISCATLEU19TTG 1.5 (1)98/84HISCATPRO19CCG 0.55 (1)99/85ILEATCLEU19CTG 1.4 ± 1.4 (7)99/85ILEATCARG19CGC<0.025 (1)99/85ILEATCASP19GAC<0.025 (1)99/85ILEATCVAL19GTC 0.51 ± 0.59 (3)99/85ILEATCPRO19CCG<0.025 (1)99/85ILEATCGLN19CAG<0.018 ± 0.010 (2)99/85ILEATCGLY19GGG<0.018 ± 0.10 (2)99/85ILEATCSER19TCG<0.025 (1)99/85ILEATCPHE19TTC 0.45 (1)99/85ILEATCHIS19CAC<0.025 (1)100/86 LYSAAGTYR19TAC 0.03 (1)100/86 LYSAAGLEU19TTG 0.33 ± 0.31 (3)100/86 LYSAAGHIS19CAC 0.36 ± 0.22 (9)100/86 LYSAAGARG19AGC 4.7 ± 5.9 (4)100/86 LYSAAGILE19ATC 0.95 (1)100/86 LYSAAGSER19AGC 0.95 (1)100/86 LYSAAGGLN19CAG 0.78 ± 0.80 (7)100/86 LYSAAGPRO19CCG 0.70 (1)101/87 ASPGACPRO19CCC 2.3 ± 3.1 (4)101/87 ASPGACMET19ATG 1.8 ± 2.5 (6)101/87 ASPGACLYS19AAG 1.2 ± 1.7 (3)101/87 ASPGACHIS19CAC 2.5 (1)101/87 ASPGACTHR19ACG 0.90 ± 0.77 (3)101/87 ASPGACTYR19TAC 0.59 (1)101/87 ASPGACVAL19GTC 0.42 (1)101/87 ASPGACTYR19TAC 1.0 ± 0.02 (2)101/87 ASPGACGLN19CAG 0.07 (1)102/88 GLYGGTLEU19CTC<0.015 ± 0.007 (2)102/88 GLYGGTGLU19GAG 0.40 ± 0.07 (3)102/88 GLYGGTLYS19AGG 0.16 ± 0.14 (2)102/88 GLYGGTSER19TCC 0.29 (1)102/88 GLYGGTTYR19TAC 0.04 (1)102/88 GLYGGTPRO19CCC<0.011 (1)103/89 ASPGACSER19TCC 0.02 (1)104/90 TRPTGGVAL19GTG 0.11 ± 0.06 (5)104/90 TRPTGGCYS19AGC 0.07 ± 0.03 (5)104/90 TRPTGGTYR19TAC 0.34 ± 0.42 (5)104/90 TRPTGGTHR19ACC 0.04 ± 0.02 (2)104/90 TRPTGGMET19ATG 0.14 (1)104/90 TRPTGGPRO19CCC 0.02 ± 0.02 (2)104/90 TRPTGGLEU19TTG 0.65 ± 1.0 (3)104/90 TRPTGGGLN19CAG 0.008 (1)104/90 TRPTGGLYS19AAG-104/90 TRPTGGGLY19GAG-104/90 TRPTGGALA19GCC-104/90 TRPTGGPHE19TTC-104/90 TRPTGGGLY19GGC-105/91 ASNAATPRO19CCG 14.8 ± 8.5 (5)105/91 ASNAATALA19GCC 0.65 ± 0.30 (3)105/91 ASNAATPHE19TTC 0.13 (1)105/91 ASNAATSER19TCC 1.9 ± 2.7 (5)105/91 ASNAATTRP19TGG 0.95 (1)105/91 ASNAATGLN19CAA 0.57 ± 0.52 (3)105/91 ASNAATTYR19TAC 0.66 ± 0.53 (4)105/91 ASNAATLEU19CTC 0.87 ± 0.79 (2)105/91 ASNAATLYS19AAG 0.70 (1)105/91 ASNAATILE19ATC 1.0 (1)105/91 ASNAATASP19GAC 1.0 ± 0.9 (4)105/91 ASNAATHIS19CAC 0.71 ± 0.48 (2)106/92 GLUGAASER19TCC 0.17 ± 0.21 (2)106/92 GLUGAAALA19GCG 0.235 ± 0.26 (2)106/92 GLUGAALYS19AAG-106/92 GLUGAATHR19ACC-106/92 GLUGAAILE19ATC-106/92 GLUGAAGLY19GGC 0.70 ± 0.76 (4)106/92 GLUGAAPRO19CCC-108/94 ARGCGGLYS19AAG 0.11 ± 0.03 (2)108/94 ARGCGGASP19GAC-108/94 ARGCGGLEU19TTG 0.01 (1)108/94 ARGCGGTHR19ACG 0.08 (1)108/94 ARGCGGILE19ATC<0.01 (1)108/94 ARGCGGPRO19CCC-109/95 ARGAGGTHR19ACC 1.1 ± 0.2 (3)109/95 ARGAGGPRO19CCC-109/95 ARGAGGGLU19GAG 1.1 ± 0.1 (3)109/95 ARGAGGTYR19TAC<0.006 (1)109/95 ARGAGGLEU19CTC 1.2 ± 0.9 (4)109/95 ARGAGGSER19TCG 1.7 ± 0.8 (4)109/95 ARGAGGGLY19GGG 0.17 (1)110/96 LYSAAAALA19GCC<0.08 (1)110/96 LYSAAAASN19AAC-110/96 LYSAAATHR19ACG-110/96 LYSAAALEU19CTC-110/96 LYSAAAARG19CGG-110/96 LYSAAAGLN19CAG-110/96 LYSAAATRP19TGG-111/97 LEUCTGILE19ATC-111/97 LEUCTGARG19CGG-111/97 LEUCTGASP19GAC-111/97 LEUCTGMET19ATG-112/98 THRACGVAL19GTG 0.55 ± 0.44 (3)112/98 THRACGGLN19CAG 1.7 ± 1.0 (3)112/98 THRACGTYR19TAC<0.018 (1)112/98 THRACGGLU19GAG 0.12 (1)112/98 THRACGHIS19CAC 0.25 ± 0.40 (3)112/98 THRACGSER19TCC 0.17 ± 0.15 (2)112/98 THRACGPHE19TTC-113/99 PHETTCSER19AGC-113/99 PHETTCCYS19TGC-113/99 PHETTCHIS19CAC<0.009 (1)113/99 PHETTCGLY19GGC-113/99 PHETTCTRP19TGG-113/99 PHETTCTYR19TAC 0.07 (1)113/99 PHETTCASN19AAC-114/100TYRTATCYS19TGC-114/100TYRTATHIS19CAC-114/100TYRTATSER19AGC-114/100TYRTATTRP19TGG 0.88 (1)114/100TYRTATARG19AGG-114/100TYRTATLEU19CTC<0.018 (1)115/101LEUCTGASN19AAC<0.004 (1)115/101LEUCTGVAL19GTG-115/101LEUCTGPRO19CCC<0.004 (1)115/101LEUCTGARG19AGG<0.004 (1)115/101LEUCTGALA19GCG 0.50 (1)115/101LEUCTGHIS19CAC-115/101LEUCTGTHR19ACC-115/101LEUCTGTRP19TGG-115/101LEUCTGMET19ATG<0.008 (1)116/102LYSAAALEU1419TTG-116/102LYSAAAPRO1419CCG<0.004 (1)116/102LYSAAATHR1419ACC 0.50 (1)116/102LYSAAAMET1419ATG 0.13 (1)116/102LYSAAAASP1419GAC<0.018 (1)116/102LYSAAAVAL19GTG 2.3 ± 1.2 (5)116/102LYSAAAGLU19GAG 0.06 (1)116/102LYSAAAARG19CGC 0.06 (1)116/102LYSAAATRP19TGG 2.3 ± 1.0 (4)116/102LYSAAASER19TCG 0.69 ± 0.51 (5)116/102LYSAAALEU19CTC 0.14 ± 0.02 (2)116/102LYSAAAILE19ATC 1.3 ± 0.3 (3)116/102LYSAAATHR19ACG 0.84 ± 0.30 (4)117/103THRACCSER19AGC 1.1 ± 0.2 (3)117/103THRACCASN19AAC 0.31 ± 0.39 (3)117/103THRACCILE19ATC-117/103THRACCTRP19TGG 0.02 (1)117/103THRACCLYS19AAG<0.005 (1)117/103THRACCPRO19CCG-118/104LEUCTTSER19TCA-118/104LEUCTTPRO19CCC-118/104LEUCTTALA19GCC-118/104LEUCTTGLU19GAG-118/104LEUCTTCYS19TGC-118/104LEUCTTASP19GAC-118/104LEUCTTTYR19TAC-119/105GLUGAGSER19TCC 0.26 ± 0.19 (2)119/105GLUGAGLYS19AAG 0.04 (1)119/105GLUGAGPRO19CCG 0.31 ± 0.27 (3)119/105GLUGAGLEU19CTG 0.35 ± 0.35 (3)119/105GLUGAGTHR19ACC 0.25 ± 0.27 (3)119/105GLUGAGTYR19TAC 0.30 ± 0.32 (3)119/105GLUGAGARG19CGC 0.06 (1)120/106ASNAATALA19GCC<0.009 (1)120/106ASNAATPRO19CCC 1.7 ± 0.7 (3)120/106ASNAATLEU19TTG 1.2 ± 0.3 (3)120/106ASNAATHIS19CAC 1.0 ± 0.3 (2)120/106ASNAATVAL19GTG 1.7 ± 0.3 (3)120/106ASNAATGLN19CAG 0.85 ± 0.16 (2)121/107ALAGCGSER19AGC 1.2 ± 0.2 (3)121/107ALAGCGILE19ATC 2.8 ± 2.5 (2)121/107ALAGCGASN19AAC 0.91 ± 0.77 (5)121/107ALAGCGPRO19CCG 1.3 (1)121/107ALAGCGLYS19AAG 0.26 ± 0.24 (2)121/107ALAGCGASP19GAC 1.8* ± 0.9 (3)121/107ALAGCGGLY19GGC 0.69 (1)122/108GLNGCGSER19AGC 0.96 ± 0.41 (3)122/108GLNCA(G/A)MET19ATG 1.7 ± 0.5 (3)122/108GLNCA(G/A)TRP19TGG 1.4 (1)122/108GLNCA(G/A)ARG19AGG 0.78 (1)122/108GLNCA(G/A)PHE19TTC 2.3 ± 1.1 (3)122/108GLNCA(G/A)PRO19CCG 1.0 (1)122/108GLNCA(G/A)HIS19CAC 1.4 (1)122/108GLNCA(G/A)ILE19ATC 2.7 ± 0.8 (3)122/108GLNCA(G/A)TYR19TAC 1.7 ± 0.3 (2)122/108GLNCA(G/A)CYS19TGC 0.58 (1)123/109ALAGCTMET19ATG 2.0 ± 0.2 (3)123/109ALAGCTGLU19GAG 2.1 ± 1.0 (3)123/109ALAGCTHIS19CAC 0.98 ± 0.72 (3)123/109ALAGCTSER19AGC 1.4 ± 0.8 (3)123/109ALAGCTPRO19CCC 0.64 ± 0.16 (2)123/109ALAGCTTYR19TAC 0.51 ± 0.25 (2)123/109ALAGCTLEU19CTG 1.2 ± 0.1 (2)1The first position number representes the amino acid position in (1-133) hIL-3 and the second number represents the position in (15-125) hIL-3 in which the Asn at position 15 of native hIL-3 is position 1 in (15-125) hIL-3 (See the numbering for Formula XI) 2Double mutant; has PRO at position 35. 3Double mutant; has THR at position 49. 4Double mutant; has Gly at position 32. 5Double mutant; has Leu at position 31. 6Double mutant; has Gly at position 46. 7Double mutant; Arg at position 42. 8Double mutant; Phe at position 53. 9Double mutant; has Val at position 49. 10Double mutant; has Pro at position 73. 11Double mutant; has Thr at position 64. 12Double mutant; has Pro at position 73. 13Double mutant; has Met at position 74. 14Double mutant; has Ser at position 105.

EXAMPLE 64

[1042] Construction of pMON13215

[1043] Plasmid, pMON2341, DNA was digested with restriction enzymes NcoI and HindIII resulting in a 3619 base pair NcoI/HindIII fragment. The genetic elements derived from pMON2341 are the beta-lactamase gene (AMP), pBR327 origin of replication, F1 phage origin of replication as the transcription terminator, precA, g10L ribosome binding site. The plasmid encoding the hIL-3 (15-125) Trp(116) variant, from Table 9 was digested with NcoI and HindIII resulting in a 345 base pair NcoI/HindIII fragment. The 345 Base pair NcoI/HindIII fragment was ligated with the 3619 base pair fragment from pMON2341 and the ligation reaction mixture was used to transform E.coli K-12 strain JM101. Plasmid DNA was isolated and screened by restriction anaylsis using NcoI and HindIII. Positive clones contained a 345 base pair NcoI/HindIII. This construct was designated PMON13215. The plasmid, pMON13215, encodes the (15-125)hIL-3 variant with the following amino acid sequence:

[1044] PEPTIDE A9; (15-125)HIL-3 TRP(116) PMON13215

36Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly[SEQ ID NO:217] 30 35 40Gln Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn 45 50 55Leu Gln Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser 60 65 70Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu 75 80 85Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly 90 95 100Asp Trp Asn Gln Phe Arg Arg Lys Leu Thr Phe Tyr Leu Trp Thr 105 110 115Leu Gln Asp Ala Gln Ala Gln Gln 120 125

[1045] DNA sequence #A9 pMON13215 116w

37ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTCAAGCA[SEQ ID NO:220]GCCACCGCTG CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAGATATCCTGAT GGAAAATAAC CTTCGTCGTC CAAACCTCGA GGCATTCAACCGTGCTGTCA ACTCTCTGCA GAATGCATCA GCAATTGAGA GCATTCTTAAAAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC ACGCGACATCCAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACCTTCTATCTGT GGACCTTGGA GAACGCGCAG GCTCAACAG

EXAMPLE 65

[1046] Construction of pMON13252

[1047] Plasmid, pMON2341, DNA was digested with restriction enzymes NcoI and HindIII resulting in a 3619 base pair NcoI/HindIII fragment. The genetic elements derived from pMON2341 are the beta-lactamase gene (AMP), pBR327 origin of replication F1 phage origin of replication as the transcription terminator, precA, g10L ribosome binding site. The plasmid encoding the hIL-3 (15-125) Asp(50) variant, from Table 9, was digested with NcoI and HindIII resulting in a 345 base pair NcoI/HindIII fragment. This 345 Base pair NcoI/HindIII fragment was ligated with the 3619 base pair fragment from pMON2341 and the ligation reaction mixture was used to transform E.coli K-12 strain JM101. Plasmid DNA was isolated and screened by restriction analysis using NcoI and HindIII. Positive clones contained a 345 base pair NcoI/HindIII. This construct was designated pMON13252. The plasmid, pMON13252, encodes the (15-125)hIL-3 variant with the following amino acid sequence:

[1048] PEPTIDE A10; (15-125)HIL-3 ASP(50) pMON13252

38 Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu[SEQ ID NO:218] 15 20 25Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly 30 35 40Gln Asp Gln Asp Ile Leu Met Asp Asn Asn Leu Arg Arg Pro Asn 45 50 55Leu Gln Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser 60 65 70Ala Ile Gln Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu 75 80 85Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly 90 95 100Asp Trp Asn Gln Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr 105 110 115Leu Glu Asn Ala Gln Ala Gln Gln 120 125

[1049] DNA sequence #A10 pMON13252 50D

39ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA[SEQ ID NO:216]GCCACCGCTG CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAACACCAAGATATCCTGAT GGAAAATAAC CTTCGTCGTC CAAACCTCGA GGCATTCAACCGTGCTGTCA ACTCTCTGCA GAATCCATCA GCAATTGAGA GCATTCTTAAAAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC ACGCGACATCCAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACCTTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG

[1050]

40

TABLE 7

NATIVE
SUBSTITUTION

RELATIVE

pMON number

amino acid
amino acid
SEQ ID NO:
POTENCY

position/mutant

(15-125)

hIL-3

pMON13201
45/31M
Gln
Met
19
6.3

pMON13202
51/37R
Asn
Arg
19
1.58

pMON13203
51/37P
Asn
Pro
19
2.5

pMON13204
51/37T
Asn
Thr
19
3.16

pMON13205
56/425
Pro
Ser
19
6.3

pMON13206
98/84I
His
Ile
19
6.3

pMON13207
45/31V
Gln
Val
19
4

pMON13208
42/28D
Gly
Asp
19
6.3

pMON13209
42/28S
Gly
Ser
19
12.6

pMON13210
42/28A
Gly
Ala
19
2.5

pMON13211
46/32S
Asp
Ser
19
16

pMON13212
82/68W
Leu
Trp
19
5

pMON13213
82/68D
Leu
Asp
19
4

pMON13214
100/86R
Lys
Arg
19
4

pMON13215
116/102W
Lys
Trp
19
31

pMON13216
23/9L
Ile
Leu
19
4

pMON13217
32/18R
Leu
Arg
19
7.9

pMON13218
32/18N
Leu
Asn
19
2

pMON13219
32/1BA
Leu
Ala
19
1.58

pMON13220
34/205
Leu
Ser
19
6.3

pMON13221
34/20M
Leu
Met
19
6.3

pMON13222
50/36D
Glu
Asp
19
7.9

pMON13223
62/48I
Asn
Ile
19
*

pMON13224
66/52R
Lys
Arg
19
4

pMON13225
76/62P
Ser
Pro
19
1.25

pMON13226
77/63L
Ile
Leu
19
1.58

pMON13227
22/8G
Glu
Gly
19
0.008

pMON13228
115/101M
Leu
Met
19
0.04

pMON13229
122/108I
Gln
Ile
19
1

pMON13231
51/37H
Asn
His
19
1.25

pMON13232
59/45L
Glu
Leu
19
1.99

pMON13233
63/49H
Arg
His
19
*

pMON13234
64/50N
Ala
Asn
19
0.03

DM0N13235
65/51T
Val
Thr
19
1.58

pMON13236
76/62V
Ser
Val
19
2.5

pMON13237
76/62A
Ser
Ala
19
5

pM0N13238
91/77P
Ala
Pro
19
*

pM0N13240
100/860
Lys
Gln
19
2.5

pMON13241
101/87M
Asp
Met
19
6.3

pMON13242
105/91N
Asn
Asn
19
*

pMON13243
161/102V
Lys
Val
19
7.9

pMON13244
122/108F
Gln
Phe
19
6.3

pMON13245
123/109E
Ala
Glu
19
1.58

position/mutant

(15-125)

pMON13246
42D
Gly
Asp
15
20

pMON13247
42S
Gly
Ser
15
*

pMON13248
42A
Gly
Ala
15
16

pMON13249
45V
Gln
Val
15
5

pMON13250
45M
Gln
Met
15
*

pMON13251
46S
Asp
Ser
15
5

pMON13252
50D
Glu
Asp
15
5

pMON13253
981
His
Ile
15
*

pMON13264
97V
Ile
Val
15
4

pMON13266
75K
Glu
Lys
15
0.25

pMON13267
89N
Thr
Asn
15
2.5

[1051]

41

TABLE 8

position/
substi-

oligo
oligo
parental
restriction

native a.a.
tution
codon
oligo pair
oligo pair
oligo pair
pair
pair
plasmid
digest

21 asp
glu
GAA
21glu1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 73
SEQ ID NO: 523
SEQ ID NO: 524

21glu4
NcoRV5
NcoRV6

SEQ ID NO: 219
SEQ ID NO: 526
SEQ ID NO: 527

21 asp
gln
CAA
21gln1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 71
SEQ ID NO: 523
SEQ ID NO: 524

21gln4
NcoRV5
NcoRV6

SEQ ID NO: 72
SEQ ID NO: 526
SEQ ID NO: 527

21 asp
asn
AAC
21asn1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 68
SEQ ID NO: 523
SEQ ID NO: 524

21asn4
NcoRV5
NcoRV6

SEQ ID NO: 70
SEQ ID NO: 526
SEQ ID NO: 527

21 asp
thr
ACC
21thr1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 232
SEQ ID NO: 523
SEQ ID NO: 524

21thr4
NcoRV5
NcoRV6

SEQ ID NO: 233
SEQ ID NO: 526
SEQ ID NO: 527

21 asp
ser
AGC
21ser1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 230
SEQ ID NO: 523
SEQ ID NO: 524

21 ser4
NcoRV5
NcoRV6

SEQ ID NO: 231
SEQ ID NO: 526
SEQ ID NO: 527

22 glu
asp
GAC
22asp1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 236
SEQ ID NO: 523
SEQ ID NO: 524

22asp4
NcoRV5
NcoRV6

SEQ ID NO: 237
SEQ ID NO: 526
SEQ ID NO: 527

22 glu
asn
AAC
22asn1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 234
SEQ ID NO: 523
SEQ ID NO: 524

22asn4
NcoRV5
NcoRV6

SEQ ID NO: 235
SEQ ID NO: 526
SEQ ID NO: 527

22 glu
gln
CAG
22gln1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 238
SEQ ID NO: 523
SEQ ID NO: 524

22gln4
NcoRV5
NcoRV6

SEQ ID NO: 239
SEQ ID NO: 526
SEQ ID NO: 527

22 glu
leu
CTG
22leu1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 240
SEQ ID NO: 523
SEQ ID NO: 524

22leu4
NcoRV5
NcoRV6

SEQ ID NO: 241
SEQ ID NO: 526
SEQ ID NO: 527

22 glu
val
GTT
22val1
NcoRv2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 242
SEQ ID NO: 523
SEQ ID NO: 524

22val4
NcoRV5
NcoRV6

SEQ ID NO: 243
SEQ ID NO: 526
SEQ ID NO: 527

34 leu
glu
GAA
NcoRV1
34Glu2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 251
SEQ ID NO: 524

NcoRV4
34Glu5
NcoRV6

SEQ ID NO: 525
SEQ ID NO: 252
SEQ ID NO: 527

34 leu
gln
CAG
NcoRV1
34gln2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 248
SEQ ID NO: 524

NcoRV4
34gln5
NcoRV6

SEQ ID NO: 525
SEQ ID NO: 249
SEQ ID NO: 527

34 leu
thr
ACC
NcoRV1
34thr2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 256
SEQ ID NO: 524

NcoRV4
34thr5
NcoRV6

SEQ ID NO: 525
SEQ ID NO: 257
SEQ ID NO: 527

34 leu
arg
CGT
NcoRV1
34arg2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 246
SEQ ID NO: 524

NcoRV4
34arg5
NcoRV6

SEQ ID NO: 525
SEQ ID NO: 247
SEQ ID NO: 527

34 leu
ala
GCT
NcoRV1
34ala2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 244
SEQ ID NO: 524

NcoRV4
34ala5
NcoRV6

SEQ ID NO: 525
SEQ ID NO: 245
SEQ ID NO: 527

34 leu
phe
TTC
NcoRV1
34phe2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 254
SEQ ID NO: 524

NcoRV4
34phe5
NcoRV6

SEQ ID NO: 525
SEQ ID NO: 255
SEQ ID NO: 527

34 leu
ile
ATC
NcoRV1
34ile2
NcoRV3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 252
SEQ ID NO: 524

NcoRV4
34ile5
NcoRV6

SEQ ID NO: 525
SEQ ID NO: 253
SEQ ID NO: 527

42 gly
lys
AAA
NcoRV1
NcoRv2
42lys3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 268

NcoRV4
NcoRV5
42lys6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 269

42 gly
asn
AAC
NcoRV1
NcoRv2
42asn3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 260

NcoRV4
NcoRV5
42asn6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 261

42 gly
thr
ACC
NcoRV1
NcoRv2
42thr3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 274

NcoRV4
NcoRV5
42thr6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 275

42 gly
leu
CTG
NcoRV1
NcoRv2
42leu3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 266

NcoRV4
NcoRV5
42leu6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 267

42 gly
val
GTT
NcoRV1
NcoRv2
42val3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 278

NcoRV4
NcoRV5
42val6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 279

42 gly
glu
GAA
NcoRV1
NcoRv2
42glu3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 262

NcoRV4
NcoRV5
42glu6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 263

42 gly
phe
TTC
NcoRV1
NcoRv2
42phe3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 272

NcoRV4
NcoRV5
42phe6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 273

42 gly
tyr
TAC
NcoRV1
NcoRv2
42tyr3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 276

NcoRV4
NcoRV5
42tyr6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 277

42 gly
ile
ATC
NcoRV1
NcoRv2
42lle3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 264

NcoRV4
NcoRV5
42ile6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 265

42 gly
met
ATG
NcoRV1
NcoRv2
42met3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 270

NcoRV4
NcoRV5
42met6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 271

43 glu
gln
CAG
NcoRV1
NcoRv2
43gln3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 282

NcoRV4
NcoRV5
43gln6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 283

43 glu
arg
CGT
NcoRV1
NcoRv2
43arg3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 280

NcoRV4
NcoRV5
43arg6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 281

43 glu
thr
ACC
NcoRV1
NcoRv2
43thr3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 286

NcoRV4
NcoRV5
43thr6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 287

43 glu
gly
GGT
NcoRV1
NcoRv2
43gly3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 284

NcoRV4
NcoRV5
43gly6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 285

44 asp
glu
GAA
NcoRV1
NcoRv2
44glu3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 294

NcoRV4
NcoRV5
44glu6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 295

44 asp
asn
AAC
NcoRV1
NcoRv2
44asn3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 290

NcoRV4
NcoRV5
44asn6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 291

44 asp
gln
CAG
NcoRV1
NcoRv2
44gln3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 292

NcoRV4
NcoRV5
44gln6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 293

44 asp
ala
GCT
NcoRV1
NcoRv2
44ala3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 288

NcoRV4
NcoRV5
44ala6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 289

45 gln
asp
GAC
NcoRV1
NcoRv2
45asp3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 302

NcoRV4
NcoRV5
45asp6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 303

45 gln
asn
AAC
NcoRV1
NcoRv2
45asn3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 300

NcoRV4
NcoRV5
45asn6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 301

45 gln
arg
CGT
NcoRV1
NcoRv2
45arg3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 298

NcoRV4
NcoRV5
45arg6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 299

45 gln
ser
TCC
NcoRV1
NcoRv2
45ser3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 310

NcoRV4
NcoRV5
45ser6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 311

45 gln
ala
GCT
NcoRV1
NcoRv2
45ala3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 296

NcoRV4
NcoRV5
45ala6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 297

45 gln
ile
ATC
NcoRV1
NcoRv2
45ile3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 308

NcoRV4
NcoRV5
45ile6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 309

45 gln
glu
GAA
NcoRV1
NcoRv2
45glu3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 304

NcoRV4
NcoRV5
45glu6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 305

45 gln
his
CAC
NcoRV1
NcoRv2
45his3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 306

NcoRV4
NcoRV5
45his6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 307

46 asp
glu
GAA
NcoRV1
NcoRv2
46glu3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 318

NcoRV4
NcoRV5
46glu6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 319

46 asp
asn
AAC
NcoRV1
NcoRv2
46asn3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 314

NcoRV4
NcoRV5
46asn6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 315

46 asp
gln
CAG
NcoRV1
NcoRv2
46gln3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 316

NcoRV4
NcoRV5
46gln6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 317

46 asp
lys
AAA
NcoRV1
NcoRv2
46lys3

pMON13356
Ncol, EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 326

NcoRV4
NcoRV5
46lys6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 327

46 asp
his
CAC
NcoRV1
NcoRv2
46his3

pMON13356
Ncol,EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 320

NcoRV4
NcoRV5
46his6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 321

46 asp
ala
GCT
NcoRV1
NcoRv2
46ala3

pMON13356
Ncol,EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 312

NcoRV4
NcoRV5
46ala6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 313

46 asp
tyr
TAC
NcoRV1
NcoRv2
46tyr3

pMON13356
Ncol,EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 328

NcoRV4
NcoRV5
46tyr6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 329

46 asp
lle
ATC
NcoRV1
NcoRv2
46lle3

pMON13356
Ncol,EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 322

NcoRV4
NcoRV5
46lle6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 323

46 asp
val
GTT
NcoRV1
NcoRv2
46val3

pMON13356
Ncol,EcoRV

SEQ ID NO: 522
SEQ ID NO: 523
SEQ ID NO: 330

NcoRV4
NcoRV5
46val6

SEQ ID NO: 525
SEQ ID NO: 526
SEQ ID NO: 331

48 leu
glu
GAA
48glu1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 334
SEQ ID NO: 529
SEQ ID NO: 530

48glu4
RVNsi5
RVNsi6

SEQ ID NO: 535
SEQ ID NO: 532
SEQ ID NO: 533

48 leu
lys
AAA
48lys1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 336
SEQ ID NO: 529
SEQ ID NO: 530

48lys4
RVNsi5
RVNsi6

SEQ ID NO: 337
SEQ ID NO: 532
SEQ ID NO: 533

48 leu
thr
ACC
48thr1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 340
SEQ ID NO: 529
SEQ ID NO: 530

48thr4
RVNsi5
RVNsi6

SEQ ID NO: 341
SEQ ID NO: 532
SEQ ID NO: 533

48 leu
ala
GCT
48ala1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 332
SEQ ID NO: 529
SEQ ID NO: 530

48ala4
RVNsi5
RVNsi6

SEQ ID NO: 333
SEQ ID NO: 532
SEQ ID NO: 533

48 leu
met
ATG
48met1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 338
SEQ ID NO: 529
SEQ ID NO: 530

48met4
RVNsi5
RVNsi6

SEQ ID NO: 339
SEQ ID NO: 532
SEQ ID NO: 533

48 leu
val
CAC
48val1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 342
SEQ ID NO: 529
SEQ ID NO: 530

48val4
RVNsi5
RVNsi6

SEQ ID NO: 343
SEQ ID NO: 532
SEQ ID NO: 533

50 glu
lys
AAA
50lys1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 356
SEQ ID NO: 529
SEQ ID NO: 530

50lys4
RVNsi5
RVNsi6

SEQ ID NO: 357
SEQ ID NO: 532
SEQ ID NO: 533

50 glu
asn
AAC
50asn1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 352
SEQ ID NO: 529
SEQ ID NO: 530

50asn4
RVNsi5
RVNsi6

SEQ ID NO: 353
SEQ ID NO: 532
SEQ ID NO: 533

50 glu
ser
TCC
50ser1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 358
SEQ ID NO: 529
SEQ ID NO: 530

50ser4
RVNsi5
RVNsi6

SEQ ID NO: 359
SEQ ID NO: 532
SEQ ID NO: 533

50 glu
ala
GCT
50ala1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 350
SEQ ID NO: 529
SEQ ID NO: 530

50ala4
RVNsi5
RVNsi6

SEQ ID NO: 351
SEQ ID NO: 532
SEQ ID NO: 533

50 glu
ile
ATC
50ile1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 354
SEQ ID NO: 529
SEQ ID NO: 530

50ile4
RVNsi5
RVNsi6

SEQ ID NO: 355
SEQ ID NO: 532
SEQ ID NO: 533

50 glu
val
GTT
50val1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 360
SEQ ID NO: 529
SEQ ID NO: 530

50val4
RVNsi5
RVNsi6

SEQ ID NO: 361
SEQ ID NO: 532
SEQ ID NO: 533

50 glu
his
CAC
50his1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 344
SEQ ID NO: 529
SEQ ID NO: 530

50his4
RVNsi5
RVNsi6

SEQ ID NO: 345
SEQ ID NO: 532
SEQ ID NO: 533

50 glu
phe
TTC
50phe1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 348
SEQ ID NO: 529
SEQ ID NO: 530

50phe4
RVNsi5
RVNsi6

SEQ ID NO: 349
SEQ ID NO: 532
SEQ ID NO: 533

50 glu
met
ATG
50met1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 346
SEQ ID NO: 529
SEQ ID NO: 530

50met4
RVNsi5
RVNsi6

SEQ ID NO: 347
SEQ ID NO: 532
SEQ ID NO: 533

54 arg
asn
AAC
54asn1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 364
SEQ ID NO: 529
SEQ ID NO: 530

54asn4
RVNsi5
RVNsi6

SEQ ID NO: 365
SEQ ID NO: 532
SEQ ID NO: 533

54 arg
lys
AAA
54lys1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 368
SEQ ID NO: 529
SEQ ID NO: 530

54lys4
RVNsi5
RVNsi6

SEQ ID NO: 369
SEQ ID NO: 532
SEQ ID NO: 533

54 arg
his
CAC
54his1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 366
SEQ ID NO: 529
SEQ ID NO: 530

54his4
RVNsi5
RVNsi6

SEQ ID NO: 367
SEQ ID NO: 532
SEQ ID NO: 533

54 arg
ala
GCT
54ala1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 362
SEQ ID NO: 529
SEQ ID NO: 530

54ala4
RVNsi5
RVNsi6

SEQ ID NO: 363
SEQ ID NO: 532
SEQ ID NO: 533

56 pro
glu
GAA
56glu1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 376
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56glu5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 377
SEQ ID NO: 533

56 pro
gln

56gln1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 374
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56gln5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 375
SEQ ID NO: 533

56 pro
arg
CGT
56arg1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 372
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56arg5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 373
SEQ ID NO: 533

56 pro
his
CAC
56his1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 378
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56his5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 379
SEQ ID NO: 533

56 pro
thr
ACC
56thr1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 384
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56thr5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 385
SEQ ID NO: 533

56 pro
ala
GCT
56ala1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 370
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56ala5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 371
SEQ ID NO: 533

56 pro
tyr
TAC
56tyr1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 386
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56tyr5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 387
SEQ ID NO: 533

56 pro
phe
TTC
56phe1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 382
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56phe5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 383
SEQ ID NO: 533

56 pro
leu
CTG
56leu1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 380
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56leu5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 381
SEQ ID NO: 533

56 pro
val
GTT
56val1
RVNsi2
RVNsi3

pMON13357
EcoRV,Ncol

SEQ ID NO: 388
SEQ ID NO: 529
SEQ ID NO: 530

RVNsi4
56val5
RVNsi6

SEQ ID NO: 531
SEQ ID NO: 389
SEQ ID NO: 533

82 leu
glu
GAA
NsiEco1
82glu2
NsiEco3
NsiEco4

pMON13358
Nsil,EcoRI

SEQ ID NO: 534
SEQ ID NO: 394
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82glu6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 395
SEQ ID NO: 542
SEQ ID NO: 545

82 leu
asn
AAC
NsiEco1
82asn2
NsiEco3
NsiEco4

pMON13358
Nsil,EcoRI

SEQ ID NO: 534
SEQ ID NO: 392
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82asn6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 393
SEQ ID NO: 542
SEQ ID NO: 545

82 leu
his
CAC
NsiEco1
82his2
NsiEco3
NsiEco4

pMON13358
Nsil,EcoRI

SEQ ID NO: 534
SEQ ID NO: 396
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82his6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 397
SEQ ID NO: 542
SEQ ID NO: 545

82 leu
thr
ACC
NsiEco1
82thr2
NsiEco3
NsiEco4

pMON13358
Nsil,EcoRI

SEQ ID NO: 534
SEQ ID NO: 406
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82thr6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 407
SEQ ID NO: 542
SEQ ID NO: 545

82 leu
ser
TCC
NsiEco1
82ser2
NsiEco3
NsiEco4

pMON13358
Nsil,EcoRI

SEQ ID NO: 534
SEQ ID NO: 404
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82ser6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 405
SEQ ID NO: 542
SEQ ID NO: 545

82 leu
ala
GCT
NsiEco1
82ala2
NsiEco3
NsiEco4

pMON13358
Nsil,EcoRI

SEQ ID NO: 534
SEQ ID NO: 390
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82ala6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 391
SEQ ID NO: 542
SEQ ID NO: 545

82 leu
tyr
TAC
NsiEco1
82tyr2
NsiEco3
NsiEco4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 408
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82tyr6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 409
SEQ ID NO: 542
SEQ ID NO: 545

82 leu
phe
TTC
NsiEco1
82phe2
NsiEco3
NsiEco4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 402
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82phe6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 403
SEQ ID NO: 542
SEQ ID NO: 545

82 leu
ile
ATC
NsiEco1
82ile2
NsiEco3
NsiEco4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 398
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82ile6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 399
SEQ ID NO: 542
SEQ ID NO: 545

82 leu
met
ATG
NsiEco1
82met2
NsiEco3
NsiEco4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 400
SEQ ID NO: 536
SEQ ID NO: 539

NsiEco5
82met6
NsiEco7
NsiEco8

SEQ ID NO: 540
SEQ ID NO: 401
SEQ ID NO: 542
SEQ ID NO: 545

92 pro
ala
GCT
NsiEco1
NsiEco2
92ala3A
NsiEco3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 410
SEQ ID NO: 538
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
92ala7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 411
NsiEco8

SEQ ID

NO: 545

92 pro
gly
GGT
NsiEco1
NsiEco2
92gly3A
NsiEco3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 412
SEQ ID NO: 538
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
92ala7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 413
NsiEco8

SEQ ID

NO: 545

92 pro
ile
ATC
NsiEco1
NsiEco2
92ile3A
NsiEco3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 414
SEQ ID NO: 538
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
92ala7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 415
NsiEco8

SEQ ID

NO: 545

94 arg
gln
CAG
NsiEco1
NsiEco2
NsiEco3A
94gln3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 418
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
94gln7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 419
NsiEco8

SEQ ID

NO: 545

94 arg
lys
AAA
NsiEco1
NsiEco2
NsiEco3A
94lysB
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 422
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
94lys7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 423
NsiEco8

SEQ ID

NO: 545

94 arg
his
CAC
NsiEco1
NsiEco2
NsiEco3A
94his3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 420
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
94his7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 536
SEQ ID NO: 543
SEQ ID NO: 421
NsiEco8

SEQ ID

NO: 545

94 arg
ala
GCT
NsiEco1
NsiEco2
NsiEco3A
94ala3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 416
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
94ala7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 417
NsiEco8

SEQ ID

NO: 545

95 his
asn
AAC
NsiEco1
NsiEco2
NsiEco3A
95 asn3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 426
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
95asn7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 427
NsiEco8

SEQ ID

NO: 545

95 his
lys
AAA
NsiEco1
NsiEco2
NsiEco3A
95lys3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 432
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
95lys7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 433
NsiEco8

SEQ ID

NO: 545

95 his
ser
TCC
NsiEco1
NsiEco2
NsiEco3A
95ser3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 438
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
95ser7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 439
NsiEco8

SEQ ID

NO: 545

95 his
ala
GCT
NsiEco1
NsiEco2
NsiEco3A
95ala3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 424
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
95ala7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 425
NsiEco8

SEQ ID

NO: 545

95 his
trp
TGG
NsiEco1
NsiEco2
NsiEco3A
95trp3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 440
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
95trp7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 441
NsiEco8

SEQ ID

NO: 545

95 his
phe
TTC
NsiEco1
NsiEco2
NsiEco3A
95phe3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO11
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
95phe7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 437
NsiEco8

SEQ ID

NO: 545

95 his
ile
ATC
NsiEco1
NsiEco2
NsiEco3A
95ile3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 430
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
95ile7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 431
NsiEco8

SEQ ID

NO: 545

98 his
glu
GAA
NsiEco1
NsiEco2
NsiEco3A
98glu3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 446
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
98glu7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 447
NSiEco8

SEQ ID

NO: 545

98 his
gln
CAA
NsiEco1
NsiEco2
NsiEco3A
98gln3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 444
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
98gln7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 445
NsiEco8

SEQ ID

NO: 545

98 his
ser
TCC
NsiEco1
NsiEco2
NsiEco3A
98ser3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 452
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
98ser7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 453
NsiEco8

SEQ ID

NO: 545

98 his
phe
TTC
NsiEco1
NsiEco2
NsiEco3A
98phe3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 450
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
98phe7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 451
NsiEco8

SEQ ID

NO: 545

98 his
met
ATG
NsiEco1
NsiEco2
NsiEco3A
98met3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 448
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
98met7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 449
NsiEco8

SEQ ID

NO: 545

98 his
val
GTA
NsiEco1
NsiEco2
NsiEco3A
98val3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 454
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
98val7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 455
NsiEco8

SEQ ID

NO: 545

98 his
lys
AAA
NsiEco1
NsiEco2
NsiEco3A
98lys3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 458
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
98lys7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO :459
NsiEco8

SEQ ID

NO: 545

98 his
arg
CGT
NsiEco1
NsiEco2
NSiEco3A
98arg3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 456
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
98arg7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 457
NsiEco8

SEQ ID

NO: 545

98 his
tyr
TAC
NsiEco1
NsiEco2
NSiEco3A
98tyr3B
NsiEco4
pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 537
SEQ ID NO: 460
SEQ ID

NsiEco5
NsiEco6
NsiEco7A
98tyr7B
NO: 539

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 543
SEQ ID NO: 461
NsiEco8

SEQ ID

NO: 545

101 asp
glu
GAA
NsiEco1
NsiEco2
NsiEco3
101glu4

pMON13358
Nsil, EcoRl

SEQ ID NQ: 534
SEQ ID NO: 535
SEQ ID NO: 536
SEQ ID NO: 466

NsiEco5
NsiEco6
NsiEco7
101glu8

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 542
SEQ ID NO: 467

101 asp
asn
AAC
NsiEco1
NsiEco2
NsiEco3
101asn4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 536
SEQ ID NO: 464

NsiEco5
NsiEco6
NsiEco7
101asn8

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 542
SEQ ID NO: 465

101 asp
ser
TCC
NsiEco1
NsiEco2
NsiEco3
101ser4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 536
SEQ ID NO: 476

NsiEco5
NsiEco6
NsiEco7
101ser8

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 542
SEQ ID NO: 477

101 asp
ala
GCT
NsiEco1
NsiEco2
NsiEco3
101ala4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 536
SEQ ID NO: 462

NsiEco5
NsiEco6
NsiEco7
101ala8

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 542
SEQ ID NO: 463

101 asp
gly
GGT
NsiEco1
NsiEco2
NsiEco3
101gly4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 536
SEQ ID NO: 468

NsiEco5
NsiEco6
NsiEco7
101gly8

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 542
SEQ ID NO: 469

101 asp
ile
ATC
NsiEco1
NsiEco2
NsiEco3
101ile4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 536
SEQ ID NO: 470

NsiEco5
NsiEco6
NsiEco7
101ile8

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 542
SEQ ID NQ: 471

101 asp
leu
CTG
NsiEco1
NsiEco2
NsiEco3
101leu4

pMON13358
Nsil, EcoRl

SEQ ID NO: 534
SEQ ID NO: 535
SEQ ID NO: 536
SEQ ID NO: 472

NsiEco5
NsiEco6
NsiEco7
101leu8

SEQ ID NO: 540
SEQ ID NO: 541
SEQ ID NO: 542
SEQ ID NO: 473

108 arg
gln
CAG
108gln1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 480
SEQ ID NO: 547

108gln3
EcoHin4

SEQ ID NO: 481
SEQ ID NO: 549

108 arg
his
CAC
108his1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 482
SEQ ID NO: 547

108his3
EcoHin4

SEQ ID NO: 483
SEQ ID NO: 549

108 arg
ser
TCC
108ser1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 484
SEQ ID NO: 547

108ser3
EcoHin4

SEQ ID NO: 485
SEQ ID NO: 549

108 arg
ala
GCT
108ala1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 478
SEQ ID NQ: 547

108ala3
EcoHin4

SEQ ID NO: 479
SEQ ID NO: 549

110 lys
arg
CGT
110arg1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 486
SEQ ID NO: 547

110arg3
EcoHin4

SEQ ID NO: 487
SEQ ID NO: 549

110 lys
his
CAC
110his1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 490
SEQ ID NO: 547

110his3
EcoHin4

SEQ ID NO: 491
SEQ ID NO: 549

110 lys
glu
GAA
110glu1
EcoHin2

pMON13359
EcoRl, HiDlll

SEQ ID NO: 488
SEQ ID NO: 547

110glu3
EcoHin4

SEQ ID NO: 489
SEQ ID NO: 549

110 lys
ser
TCC
110ser1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 494
SEQ ID NO: 547

110ser3
EcoHin4

SEQ ID NO: 495
SEQ ID NO: 549

110 lys
ala
GCT
110ala1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 492
SEQ ID NO: 547

110ala3
EcoHin4

SEQ ID NO: 493
SEQ ID NO: 549

113 phe
asp
GAC
113asp1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 496
SEQ ID NO: 547

113asp3
EcoHin4

SEQ ID NO: 497
SEQ ID NO: 549

113 phe
lys
AAA
113lys1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 502
SEQ ID NO: 547

113lys3
EcoHin4

SEQ ID NO: 503
SEQ ID NO: 549

113 phe
leu
CTG
113leu1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 500
SEQ ID NO: 547

113leu3
EcoHin4

SEQ ID NO: 501
SEQ ID NO: 549

113 phe
ile
ATC
113ile1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 498
SEQ ID NO: 547

113ile3
EcoHin4

SEQ ID NO: 499
SEQ ID NQ: 549

113 phe
val
GTT
113val1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 504
SEQ ID NO: 547

113val3
EcoHin4

SEQ ID NO: 505
SEQ ID NO: 549

116 lys
asn
AAC
116asn1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 510
SEQ ID NO: 547

116asn3
EcoHin4

SEQ ID NO: 511
SEQ ID NO: 549

116 lys
arg
CGT
116arg1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 508
SEQ ID NO: 547

116arg3
EcoHin4

SEQ ID NO: 509
SEQ ID NO: 549

116 lys
his
CAC
116his1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 514
SEQ ID NO: 547

116his3
EcoHin4

SEQ ID NO: 515
SEQ ID NO: 549

116 lys
ala
GCT
116ala1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO506
SEQ ID NO: 547

116ala3
EcoHin4

SEQ ID NO: 507
SEQ ID NO: 549

116 lys
tyr
TAC
116tyr1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 520
SEQ ID NO: 547

116tyr3
EcoHin4

SEQ ID NO: 521
SEQ ID NO: 549

116 lys
phe
TTC
116phe1
EcoHin2

pMON13369
EcoRl, HinDlll

SEQ ID NO: 518
SEQ ID NO: 547

116phe3
EcoHin4

SEQ ID NO: 519
SEQ ID NO: 549

116 lys
gln
CAG
116gln1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 512
SEQ ID NO: 547

116gln3
EcoHin4

SEQ ID NO: 513
SEQ ID NO: 549

116 lys
met
ATG
116met1
EcoHin2

pMON13359
EcoRl, HinDlll

SEQ ID NO: 516
SEQ ID NO: 547

116met3
EcoHin4

SEQ ID NO: 517
SEQ ID NO: 549

[1052] Table 9 shows the biological activity of (15-125)hIL-3 mutant polypeptides of the present invention expressed from intracellular expression vectors. Upon expression these muteins may have Met- or Met-Ala- preceding the initial (15-125)hIL-3 amino acid. The relative biological activity of IL-3 mutants is calculated by dividing the EC50 (1-133) hIL-3 by the EC50 of the mutant.

EXAMPLE 66

[1053] The variants in Table 9 were constructed by cassette mutagenesis using methods described in the Materials and Methods and the Examples contained herein, particularly Examples 54-57. Parental plasmid DNA (Table 9), digested with the appropriate restriction enzymes (Table 9), was ligated with the indicated annealed pairs of complementary oligonucleotides (Table 9). The assembled oligonucleotides create appropriate restriction ends and a portion of the (15-125) hIL-3 gene sequence Individual isolates were screened by restriction analysis and DNA sequenced to confirm that the desired changes in the (15-125) hIL-3 variant gene were made. The oligonucleotides create change(s) in the (15-125) hIL-3 gene which enclode the corresponding amino acid substitution in the variant polypeptide (Table 9). The amino acids substitutions in polypeptide #1 (SEQ ID NO:65) are indicated in Table 9.

42TABLE 9(15-125) hIL-3 MUTANTPARENTALSEQ IDaa positionAAcodonAANO:codonBIOL ACTIVITY21/7 ASPGATASN19AAC0.0121/7 ASPGATGLN19CAA0.0721/7 ASPGATGLU19GAA0.521/7 ASPGATSER19AGC0.121/7 ASPGATTHR19ACC0.122/8 GLUGAAASN19AAC*22/8 GLUGAAASP19GAC*22/8 GLUGAAGLN19CAG<0.0122/8 GLUGAALEU19CTG*22/8 GLUGAAVAL19GTT*34/20LEUTTGALA19GCT2.234/20LEUTTGARG19CGT2.234/20LEUTTGGLN19CAG1.134/20LEUTTGGLU19GAA1.534/20LEUTTGILE19ATC1.334/20LEUTTGPHE19TTC1.834/20LEUTTGTHR19ACC1.142/28GLYGGGASN19AAC 1.3 (3) 0.2842/28GLYGGGILE19ATC1042/28GLYGGGLEU19CTG10.1 (3) 7.5742/28GLYGGGMET19ATG 2.2 (3) 1.1442/28GLYGGGTYR19TAC 11 (2) 8.9 42/28GLYGGGVAL19GTT0.3343/29GLUGAAARG19CGT*43/29GLUGAAGLN19CAG<0.00443/29GLUGAAGLY19GGT*43/29GLUGAATHR19ACC0.00544/30ASPGACALA19GCT*44/30ASPGACASN19AAC*44/30ASPGACGLN19CAG*44/30ASPGACGLU19GAA0.6645/31GLNCAAALA19GCT145/31GLNCAAASN19AAC15.845/31GLNCAAGLU19GAA2.345/31GLNCAAILE19ATC4.945/31GLNCAASER19TCC0.746/32ASPGACALA19GCT6.346/32ASPGACASN19AAC0.66, 1.146/32ASPGACGLN19CAG6.346/32ASPGACGLU19GAA1.97 (3) 2.1446/32ASPGACHIS19CAC 3.2, 1.446/32ASPGACILE19ATC0.546/32ASPGACLYS19AAA0.546/32ASPGACTYR19TAC0.6646/32ASPGACVAL19GTT6.348/34LEUCTGGLU19GAA*48/34LEUCTGHIS19*48/34LEUCTGLYS19AAA*48/34LEUCTGTHR19ACC*48/34LEUCTGVAL19CAC*50/36GLUGAAALA19GCT0.550/36GLUGAAASN19AAC1.750/36GLUGAAHIS19CAC*50/36GLUGAALYS19AAA*50/36GLUGAASER19TCC1.350/36GLUGAAVAL19GTT*54/40ARGCGAALA19GCT0.954/40ARGCGAASN19AAC*54/40ARGCGAHIS19CAC0.0154/40ARGCGALYS19AAA0.256/42PROCAAALA19GCT1.856/42PROCAAASN190.656/42PROCAAARG19CGT1.256/42PROCAAGLU19GAA0.956/42PROCAAHIS19CAC0.456/42PROCAALEU19CTG1.256/42PROCAAPHE19TTC56/42PROCAATHR19ACC0.656/42PROCAAVAL19GTT1.182/68LEUCTGALA19GCT0.582/68LEUCTGASN19AAC2.982/68LEUCTGGLU19GAA4.57 (3) 5.0 82/68LEUCTGHIS19CAC2.282/68LEUCTGILE19ATC0.882/68LEUCTGMET19ATG1.182/68LEUCTGPHE19TTC3.282/68LEUCTGSER19TCC2.282/68LEUCTGTHR19ACC1.682/68LEUCTGTYR19TAC2.794/80ARGCGAGUN19CAG0.0394/80ARGCGAHIS19CAC0.0194/80ARGCGALYS19AAA*95/81HISCATASN19AAC 2.7 (2) 2.3 95/81HISCATILE19ATC0.3395/81HISCATLYS19AAA0.995/81HISCATMET19ATG195/81HISCATPHE19TTC0.6695/81HISCATSER19TTC495/81HISCATTRP19TGG*98/84HISCATARG19CGT3.298/84HISCATGLN19CAA2.298/84HISCATGLU19GAA1.55 (2) 0.1598/84HISCATLYS19AAA498/84HISCATMET19ATG2.298/84HISCATPHE19TTC198/84HISCATSER19TTC498/84HISCATTHR192.298/84HISCATVAL19GTA 2.4 (2) 0.8 101/87 ASPGACASN19AAC7101/87 ASPGACGLU19GAA*101/87 ASPGACILE19ATC3.2101/87 ASPGACLEU19CRG3.2108/94 ARGCGGALA19GCT4108/94 ARGCGGGLN19CAG0.4108/94 ARGCGGHIS19CAC*108/94 ARGCGGSER19TCC3.7110/96 LYSAAAGLU19GAA*110/96 LYSAAAHIS19CAC*110/96 LYSAAAILE19ATC*113/99 PHETTCASP19GAC*113/99 PHETTCILE19ATC*113/99 PHETTCLEU19CTG*113/99 PHETTCLYS19AAA*116/102LYSAAAALA19GCT5116/102LYSAAAARG19CGT0.03116/102LYSAAAASN19AAC0.22116/102LYSAAAGLN19CAG0.33116/102LYSAAAHIS19CAC3.2116/102LYSAAAMET19ATG0.9116/102LYSAAAPHE19TTC2.5116/102LYSAAATYR19TAC 5.4 (2) 0.3

[1054] It will be apparent to those skilled in the art that other codons besides those shown in Table 9 can also code for the substituted amino acids in the hIL-3 muteins. The present invention includes the DNAs encoding the mutant hIL-3 polypeptides of the invention including the various codons which can code for the parental and substituted amino acids of the hIL-3 muteins of the invention due to the degeneracy of the genetic code.

[1055] hIL-3 (15-125) variant genes encoding the variants listed in Table 9 can also be expressed from intracellular expression vectors to produce large quantities of the variant protein which can be purified and assayed for biological activity. The hIL-3 variant genes, from Table 9, can be excised from the secretion expression vector, as a 345 base pair NcoI/HindIII fragment and ligated into an appropriate intrecellular expression vector, such as pMON2341 digested with NcoI and HindIII.

[1056] Table 9 shows the biological activity of (15-125)hIL-3 muteins of the present invention which have one amino acid substitutions in the (15-125)hIL-3 polypeptide and which were constructed as described in Example 66. The mutants in Table 9 were secreted into the periplasmic space in E.coli. The periplasmic content was released by osmotic shock and the material in the crude osmotic shock fraction was screened for growth promoting activity. Biological activity is the growth promoting activity of AML cells relative to (15-125) hIL-3 (pMON6458 or pMMON5988). The relative biological activity of IL-3 mutants is calculated by dividing the EC50 (1-133) hIL-3 by the EC50 of the mutant. The numbers in parentheses indicate the number of repeat assays. When a variant was assayed more than once the standard deviation is indicated. An “*” indicates that the hIL3 variant protein level was less than 1.0 μg/ml and was not screened for growth promoting activity.

	Number	Date	Country
Parent	08559390	Nov 1995	US
Child	10179940	Jun 2002	US
Parent	08411796	Apr 1995	US
Child	08559390	Nov 1995	US

	Number	Date	Country
Parent	07981044	Nov 1992	US
Child	08411796	Apr 1995	US

Interleukin-3 (IL-3) mutant polypeptides

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