AU703627B2

AU703627B2 - Interleuken-3 (IL-3) receptor agonists

Info

Publication number: AU703627B2
Application number: AU73903/96A
Authority: AU
Inventors: S.C. Bauer; Charles M. Baum; Maire H. Caparon; Yiging Feng; John P. Mckearn; Charles A. Mcwherter; Linda L. Zurfluh
Original assignee: GD Searle LLC
Current assignee: GD Searle LLC
Priority date: 1995-10-05
Filing date: 1996-10-04
Publication date: 1999-03-25
Anticipated expiration: 2016-10-04
Also published as: EP0859844A1; WO1997012979A1; CA2234045A1; JP2000510684A; AU7390396A

Description

WO 97/12979 PCT/US96/15941 1 INTERLEUKIN-3 (IL-3) RECEPTOR

AGONISTS

The present application claims priority under 35 USC c §119(e) of United States provisional application Serial No.

60/004,835 filed October 05, 1995.

FIELD OF THE INVENTION The present invention relates to receptor agonists of human interleukin-3 (hIL-3). These hIL-3 agonists 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 and/or have improved physical properties which may include increased solubility, stability and refold efficiency.

BACKGROUND OF THE INVENTION 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.

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 SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 2 amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and chemotherapy.

Interleukin-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 to support the growth and differentiation of progenitor cells committed to all, or virtually all, blood cell lineages; to interact with early multipotential stem cells; to sustain the growth of pluripotent precursor cells; to stimulate proliferation of chronic myelogenous leukemia (CML) cells; to stimulate proliferation of mast cells, eosinophils and basophils; to stimulate DNA synthesis by human acute myelogenous leukemia (AML) cells; to prime cells for production of leukotrienes and histamines; to induce leukocyte chemotaxis; and to induce cell surface molecules needed for leukocyte adhesion.

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).

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, 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.

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.

The gibbon IL-3 sequence was obtained using a gibbon SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 3 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.

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 Pro 8 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.

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 (Pro 8 sequence.

U.S. 4,877,729 and U.S. 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.

Clark-Lewis, et al., SCIENCE 231:134, 1986) performed a functional analysis of murine IL-3 analogs 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 1/500th the activity as the native molecule. Replacement of two of the four Cys residues by Ala(Cys 79 Cysl40 Ala 79 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- SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 4 Lewis, et al., PROC. NATL. ACAD. SCI. USA 85:7897, 1988).

International Patent Application (PCT) WO 88/00598 discloses gibbon- and human-like IL-3. The hIL-3 contains a Ser 8 Pro 8 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.

EP-A-0275598 (WO 88/04691) illustrates that Alal can be deleted while retaining biological activity. Some mutant hIL-3 sequences are provided, two double mutants, Alal Aspl, Trpl 3 Argl 3 (pGB/IL-302) and Alal Aspl, Met 3 Thr 3 (pGB/IL-304) and one triple mutant Alal Aspl, Leu 9 Pro 9 Trpl 3 Argl 3 (pGB/IL-303).

WO 88/05469 describes how deglycosylation mutants can be obtained and suggests mutants of Arg5 4 Arg55 and Argl0 8 Argl0 9 LysllO might avoid proteolysis upon expression in Saccharomvces 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.

WO 88/06161 mentions various mutants which theoretically may be conformationally and antigenically neutral. The only actually performed mutations are Met 2 Ile 2 and Ilel 3 1 Leul 3 1. It is not disclosed whether the contemplated neutralities were obtained for these two mutations.

WO 91/00350 discloses nonglycosylated hIL-3 analog proteins, for example, hIL-3 (Pro8Aspl5Asp70), Met3 hIL-3 (Pro 8 Aspl5Asp 7 Thr 4 hIL-3 (Pro8Aspl5Asp 7 0)and Thr6 hIL-3 (Pro 8 Aspl5Asp70). 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 Met 3 Thr 4 or Thr6.

WO 91/12874 discloses cysteine added variants (CAVs) of IL-3 which have at least one Cys residue substituted for a SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 naturally occurring amino acid residue.

WO 94/12639 discloses novel variants of IL-3 which contain one to three amino acid substitutions and optionally deletions from the N-terminus and C-terminus which have increased potency and-improved therapeutic window.

WO 94/12638 discloses novel variants of IL-3 which contain from four to forty-four amino acid substitutions and optionally deletions from the N-terminus and C-terminus which have increased potency and improved therapeutic window.

WO 95/27732 describes, but does not show that the molecule has biological activity, a circularly permuted

G-CSF

ligand with a breakpoint at positions 68/69 creating a circularly permuted G-CSF ligand with a new N-terminus at the original position 69 of G-CSF and a new C-terminus at the original position 68 of G-CSF. WO 95/27732 also discloses circularly permuted GM-CSF, IL-2 and IL-4.

Rearrangement of Protein Sequences In evolution, rearrangements of DNA sequences serve an important role in generating a diversity of protein structure and function. Gene duplication and exon shuffling provide an important mechanism to rapidly generate diversity and thereby provide organisms with a competitive advantage, especially since the basal mutation rate is low (Doolittle, Protein Science 1:191-200, 1992).

The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al., Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222, 1979; Teather Erfle, J. Bacteriol. 172: 3837-3841, 1990; SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 6 Schimming et al., Eur. J. Biochem. 204: 13-19, 1992; Yamiuchi and Minamikawa, FEBS Lett. 260:127-130, 1991; MacGregre et al., FEBS Lett. 378:263-266, 1996). The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton Mol.

Biol. 165:407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.

This approach has been applied to proteins which range in size from 58 to 462 amino acids (Goldenberg Creighton, J. Mol. Biol. 165:407-413, 1983; Li Coffino, Mol. Cell.

Biol. 13:2377-2383, 1993). The proteins examined have represented a broad range of structural classes, including proteins that contain predominantly a-helix (interleukin-4; Kreitman et al., Cytokine 7:311-318, 1995), (-sheet (interleukin-l; Horlick et al., Protein Eng. 5:427-431, 1992), or mixtures of the two (yeast phosphoribosyl anthranilate isomerase; Luger et al., Science 243:206-210, 1989). Broad categories of protein function are represented in these sequence reorganization studies: Enzymes T4 lysozyme Zhang et al., Biochemistry 32:12311-12318, 1993; Zhang et al., Nature Struct. Biol. 1:434-438 SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 (1995) dihydrofolate r educ tas e ribonuclease Ti Bacillus 1-glucanse aspartate transcarbamoylase phosphoribosy.

anthrani late isomerase Buchwalder et al., Biochemistry 31:1621-1630, 1994; Protasova et Prot. Eng. 7:1373-1377, 1995) Mullins et al., J. Am. Chem. Sac.

116:5529-5533, 1994; Garrett et al., Protein Science 5:204-211, 1996) Hahn et al., Proc. Natl. Acad. Sci.

U.S.A. 91:10417-10421, 1994) Yang Schachman, Proc. Natl. Acad.

Sdi. U.S.A. 90:11980-11984, 1993) Luger et al., Science 243:206-210 (1989; Luger et al., Prot. Eng.

3:249-258, 1990) Lin et al., Protein Science 4:159- 166, 1995) Vignais et al., Protein Science 4:994-1000, 1995) Li Coffino, Mol. Cell. Biol.

13:2377-2383, 1993) Ritdo-Voflsovjcj et al., Biochemistry 34:16543-16551, 1995) pepsin/peps inogen glyceraldehyde-3 phosphate dehydrogenase ornithine decarboxylase yeast phosphoglycerate dehydrogenase Enzyme Inhibitor SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCTIUS96/1 5941 basic pancreatic trypsin inhibitor Goldenberg Creighton, J. Moi.

Biol. 165:407-413, 1983) Cytokines interleukin-13 interleukin-4 Horlick et al., Protein Eng. 5:427- 431, 1992) Kreitman et al., Cytokine 7:311- 318, 1995) Tyrosine Kinase Recognition Domain cL-spectrin SH3 domain Transmembrane Protein omp A Viguera, et al., J.

Mci. Biol. 247:670-681, 1995) Koebnik Kramer, J. Mol. Biol.

250:617-626, 1995) Chimeric Protein interleukin-4- Pseudornonas exotoxin Kreitman et al., Proc. Nati. Acad.

Sdi. U.S.A. 91:6889-6893, 1994).

The results of these studies have been highly variable.

In many cases substantially lower activity, solubility or thermodynamic stability were observed coli dihydrofolate reductase, aspartate trans carbamoy lase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ornithine decarboxylase, amp A, yeast SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 9 phosphoglycerate dehydrogenase). In other cases, the sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease T1, Bacillus Pglucanase, interleukin-lp, a-spectrin SH3 domain, pepsinogen, interleukin-4). In exceptional cases, an unexpected improvement over some properties of the natural sequence was observed, the solubility and refolding rate for rearranged a-spectrin SH3 domain sequences, and the receptor affinity and anti-tumor activity of transposed interleukin-4- Pseudomonas exotoxin (Kreitman et al., Proc. Natl. Acad. Sci.

U.S.A. 91:6889-6893, 1994; Kreitman et al., Cancer Res.

55:3357-3363, 1995).

The primary motivation for these types of studies has been to study the role of short-range and long-range interactions in protein folding and stability. Sequence rearrangements of this type convert a subset of interactions that are long-range in the original sequence into short-range interactions in the new sequence, and vice versa. The fact that many of these sequence rearrangements are able to attain a conformation with at least some activity is persuasive evidence that protein folding occurs by multiple folding pathways (Viguera, et al., J. Mol. Biol. 247:670-681, 1995).

In the case of the SH3 domain of a-spectrin, choosing new termini at locations that corresponded to 3-hairpin turns resulted in proteins with slightly less stability, but which were nevertheless able to fold.

The positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original Nterminus to the breakpoint is ca. 10 to 80% of the total sequence length). The linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues.

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 In one case (Yang Schachman, Proc. Natl. Acad. Sci. U.S.A.

90:11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original N-terminus. Flexible-hydrophilic residues such as Gly and Ser are frequently used in the linkers. Viguera, et al.(J.

Mol. Biol. 247:670-681, 1995) compared joining the original N- and C- termini with 3- or 4-residue linkers; the 3-residue linker was less thermodynamically stable. Protasova et al.

(Protein Eng. 7:1373-1377, 1994) used 3- or 5-residue linkers in connecting the original N-termini of E. coli dihydrofolate reductase; only the 3-residue linker produced protein in good yield.

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 11 Summary of the Invention The present invention relates to novel recombinant human interleukin-3 (hIL-3) receptor agonists. These hIL-3 receptor agonists may also contain amino acid substitutions and/or deletions at either/or both the N- and C- termini.

The invention also relates to pharmaceutical compositions containing the hIL-3 receptor agonists, and methods for using the receptor agonists. Additionally, the present invention relates to DNA coding for the receptor agonists, and recombinant expression vectors comprising nucleotide sequences encoding the hIL-3 receptor agonists, related microbial expression systems, and processes for making the hIL-3 receptor agonists using the microbial expression systems. These hIL-3 receptor agonists 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 receptor agonists may have a better therapeutic index than native hIL-3.

The present invention also provides molecules which may function as IL-3 antagonists or as discrete antigenic fragments for the production of antibodies useful in immunoassay and immunotherapy protocols. 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 SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 12 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.

The modified human interleukin-3 receptor agonists of the present invention can be represented by the Formula:

X

1 -(L)a-X 2 wherein; a is 0 or 1;

X

1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues nl through J;

X

2 is a peptide comprising an amino acid sequence corresponding to the sequence of residues 1 through n; n is an integer ranging from 1 to J-1; and L is a linker.

In the formula above the constituent amino acids residues of human IL-3 are numbered sequentially 1 through J from the amino to the carboxyl terminus. A pair of adjacent amino acids within this protein may be numbered n and n+1 respectively where n is an integer ranging from 1 to J-1. The residue n+1 becomes the new N-terminus of the new interleukin-3 receptor agonist and the residue n becomes the new C-terminus of the new interleukin-3 receptor agonist.

A preferred embodiment of the present invention are modified human interleukin-3 receptor agonists of the Formula: Ala Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn 1 5 10 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa SUBSTITUTE SHEET (RULE 26) WO 97/12979 WO 9712979PCTIUS96/1 5941 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 110 Gin 125 Xaa Xaa Xaa Xaa Xaa Xaa xaa xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa 40 Xaa 55 Xaa 70 Xaa 85 Xaa 100 Xaa 115 Leu 130 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 105 Xaa 120 Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gin Thr Thr Leu Ser Ala Ile

(SEQ

Phe ID NO: 1) wherein Xaa at position 17 Xaa at position 18 is Asn, Xaa at position 19 is Met, Xaa at position 20 is Ile, Xaa at position 21 is Asp, Thr, Ser or Val; Xaa at position 22 is Giu, Leu, Val or Gly; Xaa at position 23 is Ile, Leu, Ser, or Arg; Xaa at position 24 is Ile, is Ser, Lys, Gly, Asp, Met, Gin, or Arg; His, Leu, Ile, Phe, Arg, or Gin; Phe, Ile, Arg, Gly, Ala, or Cys; Cys, Gin, Giu, Arg, Pro, or Ala; Phe, Lys, Arg, Ala, Gly, Giu, Gin, Asn, Trp, Pro, Ser, Ala, His, Asp, Asn, Gin, Val, Ala, Gly, Trp, Lys, Phe, Giy, Val, Arg, Ser, Phe, or Leu; SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Arg, Ala, Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Val, Glu, Xaa at position Gin, Arg, Xaa at position Glu, Asn, Xaa at position Trp, Asp, Xaa at position Lys, His, Xaa at position Xaa at position Lys, Thr, Xaa at position Xaa at position Ala, Ile, Xaa at position Xaa at position 25 is Thr, 26 is His, 27 is Leu, 28 is Lys, 29 is Glrr; 30 is Pro, 31 is Pro, 32 is Leu, 33 is Pro, 34 is Leu, His, Thr, Gly, Arg, Asn, His, Asp, Val, Leu, Val, Gly, Phe, Arg, Leu, Leu, Thr, Gly, Arg, Gin, Gly, Gin, Gly, Thr, Gin, Pro, Gly, Ala, Gin, Ala, Ser, Arg, Arg, Ser, Gly, Arg, Asp, Arg, Asn, Thr, Lys, Pro, or Ala; Ala, or Trp; or Ala; Pro, Val or Trp; or Val; Gin, Ser, Leu, or Lys; Leu, or Gin; Gly, Ala, or Glu; or Glu; Glu, Gin, Thr, Phe, Ile or Met; 35 is Leu, Ala, Gly, Asn, Pro, Gin, or Val; Asp, Phe, Asn, Leu, Asn, Gly, Leu, or Val; Ser, Pro, Trp, or Ala; Trp, or Arg; Cys, Arg, Leu, Asp, Ser, Cys, or Ile; His, Asn, Met, Lys, Phe, Tyr, Ile, Met or Ala; 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Thr, Gly or Ser; 44 is Asp, Ser, Leu, Arg, Lys, Thr, Gin, Ala or Pro; 45 is Gin, Pro, Phe, Val, Met, Leu, Asn, Arg, Ser, Ala, Ile, Glu or His; 46 is Asp, Phe, Ser, Thr, Cys, Glu, Ala, Tyr, Ile, Val or Gly; 47 is Ile, Gly, Val, Ser, Arg, Pro, 48 is Leu, Ser, Cys, Arg, Ile, His, Ala, Met, Val or Asn; 49 is Met, Arg, Ala, Gly, Pro, Asn, 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Val, His, Phe, Met or Gin; 51 is Asn, Arg, Met, Pro, Ser, Thr, 52 is Asn, His, Arg, Leu, Gly, Ser, or Pro; Thr, Leu, Ala, Cys, Met, Trp, Thr, Lys, Asn, Gin, or His; Phe, Glu, His, Asn, or Asp; Ser, or His; or Thr; SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 1 1 2 2 3( Xaa at position 53 is Leu, Thr, A Xaa at position 54 is Arg, Asp, I Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, V Xaa at position 56 is Pro, Gly, C Thr, Ala, Tyr, Phe, Leu, Va Xaa at position 57 is Asn or Gly; Xaa at position 58 is Leu, Ser, A Xaa at position 59 is Glu, Tyr, H 0 Xaa at position 60 is Ala, Ser, P Xaa at position 61 is Phe, Asn, G Xaa at position 62 is Asn, His, V Xaa at position 63 is Arg, Tyr, T: Xaa at position 64 is Ala, Asn, P: 5 Xaa at position 65 is Val, Thr, P: Xaa at position 66 is Lys, Ile, A: Xaa at position 67 is Ser, Ala, P] Xaa at position 68 is Leu, Val, T: Xaa at position 69 is Gin, Ala, P: 0 Xaa at position 70 is Asn, Leu, V, Xaa at position 71 is Ala, Met, Li Trp, or Asn; Xaa at position 72 is Ser, Glu, M Xaa at position 73 is Ala, Glu, A! 5 Xaa at position 74 is Ile, Met, Xaa at position 75 is Glu, Lys, G Gin, or Leu; Xaa at position 76 is Ser, Val, A: Xaa at position 77 is Ile, Ser, A 0 Xaa at position 78 is Leu, Ala, S< Xaa at position 79 is Lys, Thr, As Xaa at position 80 is Asn, Trp, V4 Xaa at position 81 is Leu, Gin, G Xaa at position 82 is Leu, Gin, L la, Gly, le, Ser, al, Ser, ys, Ser, 1 or Lys; Glu, Val, Leu, Gin, Pro, Lys, Thr, Gln, or Gly; Glu, Arg, Ser, or Met; Asn, His, sp, is, ro, lu, al, rp, ro, ro, rg, he, rp, ro, al, eu, et, sp, Arg, Leu, Tyr, Pro, Arg, Lys, Ser, His, Val, Val, Ser, Thr, Trp, Pro, Ala, Leu, Gin, Val, or Cys; Pro, or Arg; Asn, or Thr; Lys, Arg, or Ser; Pro, Thr, Asp, or Ile; Ser, His, Pro, or Val; or Lys; Leu, Phe, or Ser; Asn, Glu, or Ser; Gly, Asn, Ile, Pro, or His; Ile, Phe, Thr, or His; Glu, Arg, Trp, Gly, or Leu; Pro, or Ala; Arg, Glu, Thr, Gin, His, Ser, Asn, Arg, or Gly, Thr, or Asp; Arg; rhr, Pro, Arg, Gly, Ala; ly, Asp, Pro, Trp, Arg, Ser, la, rg, er, sn, al, ly, ys, Asn, Thr, Glu, Met, Gly, Ala, Trp, Trp, Glu, or Leu; Phe, Gly, Arg, Ile, Thr, Leu, Trp, Arg, Arg, Asp, Pro, Gly, or Asp; or Arg; Gly, or Asp; Glu, or Arg; Val, or Lys; Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 Xaa at position 83 is Xaa at position 84 is Xaa at position 85 is Xaa at position 86 is Xaa at position 87 is Xaa at position 88 is Xaa at position 89 is Xaa at position 90 is Xaa at position 91 is Xaa at position 92 is Leu; Xaa at position 93 is Xaa at position 94 is Ala, or Pro; Xaa at position 95 is Lys, Ser, Ala, I Xaa at position 96 is Xaa at position 97 is Xaa at position 98 is Glu, Gin, Ser, P Xaa at position 99 is Gly, Ser, Phe, c Xaa at position 100 is Xaa at position 101 is Tyr, Glu, Asn, S Xaa at position 102 is Xaa at position 103 is Xaa at position 104 is Gin, Lys, Ala, P Xaa at position 105 is Leu, Lys, Ile, A Xaa at position 106 is Xaa at position 108 is Ala or Pro; Pro Cys Leu, Pro, Le, Ala, Thr, Ala, Ala, Pro, Thr, Arg, His, rp, Pro, Ile, His, Ala, Glu, Asn, Cys, Ser, Lys, Asp, Pro, Pro, Phe, Asp, Ile, Gin, Thr, Gly, Val, Arg, Trp, Arg, Cys, Ser, Ser, Arg, Ser, Ser, Trp, Arg, or M Arg, Met, or V or Gin; Ala, or Lys; or Gly; Val, or Trp; Leu, Val, Glu, Thr, Gly, Asp, Thr, Phe, Leu, Ser, Lys, His, et; al; His, Ile, Asp, Ala, Leu, Gin, Asn, or Ser; or Met; or His; Gly, Ile or or Arg; Lys, His, Asn, Glu, Pro, Leu, Ala, Val, Pro, Arg, Val, Leu, Gly, Thr, Asn, Phe, Ile, or Tyr; Lys, Tyr, Gly, Ile, or Thr; Val, Lys, Ala, or Asn; Ile, Asn, Leu, Asp, Ala, Thr, he, Met, Val, Lys, Arg, Tyr or Pro; Ile, Leu, Arg, Asp, Val, Pro, Gin, >r His; Lys, Tyr, Leu, His, Arg, Ile, Ser, Asp, Pro, Met, Lys, His, Thr, Val, er, Ala, Gly, Ile, Leu, or Gin; Gly, Leu, Glu, Lys, Ser, Tyr, or P Asp, or Ser; Trp, Val, Cys, Tyr, Thr, Met, Pro, he, or Gly; Asn, Pro, Ala, Phe, Ser, Trp, Gin, %sp, or His; Glu, Ser, Ala, Lys, Thr, Ile, Gly, Arg, Lys, Asp, Leu, Thr, Ile, Gin, Gin, or Pro; ro; Leu, Tyr, or Pro; His, Ser, Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gin, His, Glu, Ser, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gin, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Pne, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, lie, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gin, or Ile; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gin; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gin, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 0 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1- 133) human interleukin-3; wherein optionally 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; wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-terminus at amino acids; 26-27 27-28 28-29 29-30 30-31 31-32 32-33 49-50 50-51 51-52 52-53 53-54 54-55 64-65 83-84 84-85 85-86 86-87 87-88 88-89 89-90 SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 18 33-34 65-66 90-91 34-35 66-67 91-92 35-36 67-68 92-93 36-37 68-69 97-98 37-38 69-70 98-99 38-39 70-71 99-100 39-40 71-72 100-101 40-41 72-73 101-102 41-42 82-83 102-103 or 103-104; and additionally said modified human interleukin-3 receptor agonist can be immediately preceded by (methionine- 1 (alanine-1) or (methionine 2 alanine- 1 The more preferred breakpoints at which new C-terminus and N-terminus can be made are; 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 66- 67, 67-68, 68-69, 69-70, 70-71, 84-85, 85-86, 86-87, 87-88, 88-89, 89-90, 90-91, 98-99, 99-100, 100-101 and 101-102.

The most preferred breakpoints at which new C-terminus and N-terminus can be made are; 34-35, 69-70 and 90-91.

In a preferred embodiment of the present invention the linker joining the N-terminus to the C-terminus is a polypeptide selected from the group consisting of: GlyGlyGlySer (SEQ ID NO:2); GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:33); GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:34); SerGlyGlySerGlyGlySer (SEQ ID GluPheGlyAsnMet (SEQ ID NO:36); GluPheGlyGlyAsnMet (SEQ ID NO:37); GluPheGlyGlyAsnGlyGlyAsnMet (SEQ ID NO:38); and GlyGlySerAspMetAlaGly (SEQ ID NO:39).

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 19 BRIEF DESCRIPTION OF THE FIGURES Figure 1 schematically illustrates the sequence rearrangement of a protein. The N-terminus and the Cterminus of the native protein are joined through a linker, or joined directly. The protein is opened at a breakpoint creating a new N-terminus (new N) and a new Cterminus (new-C) resulting in a protein with a new linear amino acid sequence. A rearranged molecule may be synthesized de novo as linear molecule and not go through the steps of joining the original N-terminus and the C-terminus and opening of the protein at the breakpoint.

Figure 2 shows a schematic of Method I, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different

N-

terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original

C-

terminus 174) joined to the amino acid 11 1- are deleted) through a linker region and a new C-terminus created at amino acid 96 of the original sequence.

Figure 3 shows a schematic of Method II, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined without a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new Nterminus created at amino acid 97 of the original protein, the original C-terminus 174) joined to the original Nterminus and a new C-terminus created at amino acid 96 of the original sequence.

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 Figure 4 shows a schematic of Method III, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original

C-

terminus 174) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 21 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel receptor agonists of human interleukin-3 (hIL-3) in which the sequence has been rearranged to have a new N-terminus and a new C-terminus, and which may also have amino acid substitutions and/or insertions and/or deletions, and which have substantially the same structure and substantially the same biological activity. The novel receptor agonists of human interleukin-3 (hIL-3) may also contain the amino acid substitutions of naturally occurring variants of hIL-3 polypeptide amino acids (for example, the allele in which proline rather than serine is at position 8 in the hIL-3 polypeptide sequence) or the IL-3 variants described in WO 94/12639, WO 94/12638,

WO

95/20976, WO 95/21197, WO 95/20977, WO 95/21254, as are hIL-3 receptor agonists which are modified post-translationally glycosylation).

The present invention also includes the DNA sequences which code for the receptor agonists, DNA sequences which are substantially similar and perform substantially the same function, and DNA sequences which differ from the DNAs encoding the receptor agonists of the invention only due to the degeneracy of the genetic code.

Also included in the present invention are the DNA sequences coding for the receptor agonists of the present invention; the oligonucleotide intermediates used to construct the receptor agonists 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.

Human IL-3 can be characterized by its ability to stimulate colony formation by human hematopoietic progenitor SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCTIUS96/15941 22 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.

et al., 1990; Ganser, A, et al., 1990; Falk, 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. Receptor agonists 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 physical properties, 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 receptor agonists. Since hIL-3 functions by binding to its receptor(s) and triggering second messages resulting in competent signal transduction, hIL-3 receptor agonists of this invention may be useful in helping to determine which specific amino acid sequences are responsible for these activities.

The novel hIL-3 receptor agonists 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 SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 23 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.

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 factordependent 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.

Some receptor agonists 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.

Biological activity of hIL-3 and hIL-3 receptor agonists of the present invention is determined by DNA synthesis by human acute myelogenous leukemia cells (AML). The factordependent cell line AML 193 was adapted for use in testing biological activity.

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 24 "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.

One potential advantage of the hIL-3 receptor agonists 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 receptor agonists 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 receptor agonists 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 receptor agonists of the present invention can be administered parenterally, intravenously, or subcutaneously.

The modified hIL-3 receptor agonists of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells in peripheral blood. Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation. Hematopoietic growth factors including

G-CSF

and GM-CSF have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 procedure by decreasing the number of pheresis required. The modified hIL-3 receptor agonist may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.

The modified hIL-3 receptor agonists of the present invention may also be useful in the ex vivo expansion of hematopoietic progenitors and stem cells. Colony stimulating factors (CSFs), such as hIL-3, have been administered alone, co-administered with other CSFs, or in combination with bone marrow transplants subsequent to high dose chemotherapy to treat the neutropenia and thrombocytopenia which are often the result of such treatment. However the period of severe neutropenia and thrombocytopenia may not be totally eliminated. The myeloid lineage, which is comprised of monocytes (macrophages), granulocytes (including neutrophils) and megakaryocytes, is critical in preventing infections and bleeding which can be life-threatening. Neutropenia and thrombocytopenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.

Bone marrow transplants have been used to treat this patient population. However, several problems are associated with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow, spleen or peripheral blood is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible contamination with tumor cells. Stem cells make up a very small percentage of the nucleated cells in the bone marrow, spleen and peripheral blood. It is clear that a dose response exists such that a greater number of stem cells will enhance hematopoietic recovery. Therefore, the in vitro expansion of stem cells should enhance hematopoietic recovery and patient survival. Bone marrow from an allogeneic donor has been used to provide bone marrow for transplant. However, Graft Versus SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 26 Host Disease and graft rejection limit bone marrow transplantation even in recipients with HLA-matched sibling donors. An alternative to allogeneic bone marrow transplants is autologous bone marrow transplants. In autologous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back into the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection. However, autologous bone marrow transplants still present problems in terms of the limited number of stems cells in the marrow and possible contamination with tumor cells. The limited number of stem cells may be overcome by ex-vivo expansion of the stem cells. In addition, stem cells can be specifically isolated, based on the presence of specific surface antigens such as CD34+ in order to decrease tumor cell contamination of the marrow graft.

The following patents contain further details on separating stem cells, CD34+ cells, culturing the cells with hematopoietic factors, the use of the cells for the treatment of patients with hematopoietic disorders and the use of hematopoietic factors for cell expansion and gene therapy.

5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separating the stem cells from dedicated cells.

5,199,942 describes a method for autologous hematopoietic cell transplantation comprising: obtaining hematopoietic progenitor cells from a patient; ex-vivo expansion of cells with a growth factor selected from the group consisting of IL-3, flt3 ligand, c-kit ligand, GM-CSF, IL-1, GM-CSF/IL-3 fusion protein and combinations thereof; administering cellular preparation to a patient.

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 27 5,240,856 relates to a cell separator that includes an apparatus for automatically controlling the cell separation process.

WO 91/16116 describes devices and methods for selectively isolating and separating target cells from a mixture of cells.

WO 91/18972 describes methods for in vitro culturing of bone marrow, by incubating suspension of bone marrow cells, using a hollow fiber bioreactor.

WO 92/18615 relates to a process for maintaining and expanding bone marrow cells, in a culture medium containing specific mixtures of cytokines, for use in transplants.

WO 93/08268 describes a method for selectively expanding stem cells, comprising the steps of separating CD34+ stem cells from other cells and incubating the separated cells in a selective medium, such that the stem cells are selectively expanded.

WO 93/18136 describes a process for in vitro support of mammalian cells derived from peripheral blood.

WO 93/18648 relates to a composition comprising human neutrophil precursor cells with a high content of myeloblasts and promyelocytes for treating genetic or acquired neutropenia.

WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 28 WO 94/11493 describes a stem cell population that are CD34+ and small in size, which are isolated using a counterflow elutriation method.

WO 94/27698 relates to a method combining immunoaffinity separation and continuous flow centrifugal separation for the selective separation of a nucleated heterogeneous cell population from a heterogeneous cell mixture.

WO 94/25848 describes a cell separation apparatus for collection and manipulation of target cells.

The long term culturing of highly enriched CD34+ precursors of hematopoietic progenitor cells from human bone marrow in cultures containing IL-la, IL-3, IL-6 or GM-CSF is discussed in Brandt et al J. Clin. Invest. 86:932-941, 1990).

One aspect of the present invention provides a method for selective ex-vivo expansion of stem cells. The term "stem cell" refers to the totipotent hematopoietic stem cells as well as early precursors and progenitor cells which can be isolated from bone marrow, spleen or peripheral blood. The term "expansion" refers to the differentiation and proliferation of the cells. The present invention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of: separating stem cells from other cells, culturing the separated stem cells with a selective culture medium which contains hIL-3 receptor agonist(s) of the present invention and harvesting the cultured cells. Stem cells, as well as committed progenitor cells destined to become neutrophils, erythrocytes, platelets, etc. may be distinguished from most other cells by the presence or absence of particular progenitor marker antigens, such as CD34, that are present on the surface of these cells and/or by morphological characteristics. The SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 29 phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-l+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population. The CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affinity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+. Further, physical separation methods such as counterflow elutriation may be used to enrich hematopoietic progenitors. The CD34+ progenitors are heterogeneous, and may be divided into several sub-populations characterized by the presence or absence of co-expression of different lineage associated cell surface associated molecules. The most immature progenitor cells do not express any known lineage associated markers, such as HLA-DR or CD38, but they may express Other surface antigens such as CD33, CD38, CD41, CD71, HLA-DR or c-kit can also be used to selectively isolate hematopoietic progenitors. The separated cells can be incubated in selected medium in a culture flask, sterile bag or in hollow fibers. Various colony stimulating factors may be utilized in order to selectively expand cells.

Representative factors that have been utilized for ex-vivo expansion of bone marrow include, c-kit ligand, IL-3, G-CSF, GM-CSF, IL-1, IL-6, IL-11, flt-3 ligand or combinations thereof. The proliferation of the stem cells can be monitored by enumerating the number of stem cells and other cells, by standard techniques hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent to incubation.

Several methods for ex-vivo expansion of stem cells have been reported utilizing a number of selection methods and expansion using various colony stimulating factors including c-kit ligand (Brandt et al., Blood 83:1507-1514, 1994; McKenna et al., Blood 86:3413-3420, 1995), IL-3 (Brandt et SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 al., Blood 83:1507-1514, 1994; Sato et al., Blood 82:3600- 3609, 1993), G-CSF (Sato et al., Blood 82:3600-3609, 1993), GM-CSF (Sato et al., Blood 82:3600-3609, 1993), IL-I (Muench et al., Blood 81:3463-3473, 1993), IL-6 (Sato et al., Blood 82:3600-3609, 1993), -L-11 (Lemoli et al., Exp. Hem.

21:1668-1672, 1993; Sato et al., Blood 82:3600-3609, 1993), flt-3 ligand (McKenna et al., Blood 86:3413-3420, 1995) and/or combinations thereof (Brandt et al., Blood 83:1507- 1514, 1994; Haylock et al., Blood 80:1405-1412, 1992; Koller et al., Biotechnology 11:358-363, 1993; Lemoli et al., Exp.

Hem. 21:1668-1672, 1993; McKenna et al., Blood 86:3413-3420, 1995; Muench et al., Blood 81:3463-3473,1993; Patchen et al., Biotherapy 7:13-26, 1994; Sato et al., Blood 82:3600-3609, 1993; Smith et al., Exp. Hem. 21:870-877, 1993; Steen et al., Stem Cells 12:214-224, 1994; Tsujino et al., Exp. Hem.

21:1379-1386, 1993). Among the individual colony stimulating factors, hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609, 1993; Kobayashi et al., Blood 73:1836-1841, 1989). However, no single factor has been shown to be as effective as the combination of multiple factors. The present invention provides methods for ex vivo expansion that utilize modified hIL-3 receptor agonists that are more effective than a single factor alone.

Another aspect of the invention provides methods of sustaining and/or expanding hematopoietic precursor cells which includes inoculating the cells into a culture vessel which contains a culture medium that has been conditioned by exposure to a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a modified hIL-3 receptor agonist of the present invention.

Another projected clinical use of growth factors has SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 31 been in the in vitro activation of hematopoietic progenitors and stem cells for gene therapy. Due to the long life-span of hematopoietic progenitor cells and the distribution of their daughter cells throughout the entire body, hematopoietic progenitor cells are-good candidates for ex vivo gene transfection. In order to have the gene of interest incorporated into the genome of the hematopoietic progenitor or stem cell one needs to stimulate cell division and DNA replication. Hematopoietic stem cells cycle at a very low frequency which means that growth factors may be useful to promote gene transduction and thereby enhance the clinical prospects for gene therapy. Potential applications of gene therapy (review Crystal, Science 270:404-410, 1995) include: 1) the treatment of many congenital metabolic disorders and immunodeficiencies (Kay and Woo, Trends Genet. 10:253-257, 1994), 2) neurological disorders (Friedmann, Trends Genet.

10:210-214, 1994), 3) cancer (Culver and Blaese, Trends Genet. 10:174-178, 1994) and 4) infectious diseases (Gilboa and Smith, Trends Genet. 10:139-144, 1994). Due to the long life-span of hematopoietic progenitor cells and the distribution of their daughter cells throughout the entire body, hematopoietic progenitor cells are good candidates for ex vivo gene transfection.

There are a variety of methods, known to those with skill in the art, for introducing genetic material into a host cell. A number of vectors, both viral and non-viral have been developed for transferring therapeutic genes into primary cells. Viral based vectors include; 1) replication deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr. Opin. Genet. Dev. 3:102-109, 1993; Boris-Lawrie and Temin, Annal. New York Acad. Sci. 716:59-71, 1994; Miller, Current Top. Microbiol. Immunol. 158:1-24, 1992) and replication-deficient recombinant adenovirus (Berkner, BioTechniques 6:616-629, 1988; Berkner, Current Top.

Microbiol. Immunol. 158:39-66, 1992; Brody and Crystal, SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 32 Annal. New York Acad. Sci. 716:90-103, 1994). Non-viral based vectors include protein/DNA complexes (Cristiano et al., PNAS USA. 90:2122-2126, 1993; Curiel et al., PNAS USA 88:8850-8854, 1991; Curiel, Annal. New York Acad. Sci.

716:36-58; 1994), electroporation and liposome mediated delivery such as cationic liposomes (Farhood et al., Annal.

New York Acad. Sci. 716:23-35, 1994).

The present invention provides an improvement to the existing methods of expanding hematopoietic cells, into which new genetic material has been introduced, in that it provides methods utilizing hIL-3 receptor agonists that have improved biological activity, including an activity not seen by any single colony stimulation factor and/or physical properties.

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, gancyclovir, daunomycin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, analgesics such as aminopyrine and dipyrone, anti-convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics. The modified hIL-3 receptor agonists 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.

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, dialysis.

The modified hIL-3 receptor agonists of the present invention may be useful in treating such hematopoietic deficiencies.

The treatment of hematopoietic deficiency may include administration of a pharmaceutical composition containing the modified hIL-3 receptor agonists to a patient. The modified hIL-3 receptor agonists of the present invention may also be SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 33 useful for the activation and amplification of hematopoietic precursor cells by treating these cells in vitro with the modified hIL-3 receptor agonist proteins of the present invention prior to injecting the cells into a patient.

Various immunodeficiencies, in T and/or

B

lymphocytes, or immune disorders, rheumatoid arthritis, may also be beneficially affected by treatment with the modified hIL-3 receptor agonists of the present invention.

Immunodeficiencies may be the result of viral infections, HTLVI, HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or the result of other medical treatment. The modified hIL-3 receptor agonists of the present invention may also be employed, alone or in combination with other colony stimulating factors, in the treatment of other blood cell deficiencies, including thrombocytopenia (platelet deficiency), or anemia. Other uses for these novel polypeptides are the in vivo and ex vivo treatment of patients recovering from bone marrow transplants, and in the development of monoclonal and polyclonal antibodies generated by standard methods for diagnostic or therapeutic use.

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 modified hIL-3 receptor agonists 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.

Another aspect is to provide DNA sequence that encode SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCTIUS96/15941 34 for the novel hIL-3 receptor agonists and DNA sequences which are the same except for the degeneracy of the genetic code.

It is well known to those skilled in the art that the DNA sequence can be altered to remove "rare" codons, modified to alter the GC content of the DNA sequences, modified to increase RNA message stability and modified at the N-terminus to improve expression in a particular host cell.

Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel hIL-3 receptor agonists. 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 receptor agonists include expression vectors comprising nucleotide sequences coding for the hIL-3 receptor agonists joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.

Vectors incorporating modified sequences as described above are included in the present invention and are useful in the production of the hIL-3 receptor agonists. 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.

As another aspect of the present invention, there is provided a method for producing the novel modified hIL-3 receptor agonists. The method of the present invention involves culturing the suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel modified hIL-3 receptor agonist. Suitable cells or cell lines may be bacterial cells. For example, the various strains of E. coli are wellknown as host cells in the field of biotechnology. Examples SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 of such strains include E. coli strains JM101 (Yanish-Perron et al. Gene 33:103-119, 1985) and MON105 (Obukowicz et al., Applied Environmental Microbiology 58:1511-1523, 1992). Also included in the present invention is the expression of the modified hIL-3 receptor agonist protein utilizing a chromosomal expression vector for E. coli based on the bacteriophage Mu (Weinberg et al., Gene 126:25-33, 1993).

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. When expressed in the E. coli cytoplasm, the gene encoding the modified hIL-3 receptor agonists of the present invention may also be constructed such that at the 5' end of the gene codons are added to encode Met -Ala or Met 1 at the N-terminus of the protein. The N termini of proteins made in the cytoplasm of E. coli are affected by post-translational processing by methionine aminopeptidase (Ben Bassat et al., J. Bac.

169:751-757, 1987) and possibly by other peptidases so that upon expression the methionine is cleaved off the N-terminus.

The modified hIL-3 receptor agonists of the present invention may include modified hIL-3 receptor agonist polypeptides having Met Ala or Met-2Ala at the N-terminus. These mutant modified hIL-3 receptor agonists may also be expressed in E. coli by fusing a secretion signal peptide to the Nterminus. This signal peptide is cleaved from the polypeptide as part of the secretion process.

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. 1987) 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 SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 36 secretion signal peptide coding region, which is translationally joined to the coding region for the modified hIL-3 receptor agonist. For example, plasmids such as pcDNA I/Neo, pRc/RSV, and pRc/CMV (obtained from Invitrogen Corp., San Diego, California) can be used. The eukaryotic secretion signal peptide coding region can be from the gene itself or it can be from another secreted mammalian protein (Bayne,

M.

L. et al., Proc. Natl. Acad. Sci. USA 84: 2638-2642, 1987).

After construction of the vector containing the 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 polypeptide secreted into the media can be recovered by standard biochemical approaches following transient expression for 24 72 hours after transfection of the cells or after establishment of stable cell lines following selection for antibiotic 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.

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. 1987) A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555. An SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 37 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 joined to the coding region for the modified hIL-3 receptor agonist polypeptide. For example, the plasmid pVL1392 (obtained from Invitrogen Corp., San Diego, California) can be used. After construction of the vector carrying the gene encoding the modified hIL-3 receptor agonist polypeptide, 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 modified hIL-3 receptor agonist gene is used to infect cells cultured, for example, in Excell 401 serum-free medium (JRH Biosciences, Lenexa, Kansas). The modified hIL-3 receptor agonist secreted into the medium can be recovered by standard biochemical approaches. Supernatants from mammalian or insect cells expressing the modified hIL-3 receptor agonist protein can be first concentrated using any of a number of commercial concentration units.

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 gg/kg of nonglycosylated 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 SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 38 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 receptor agonist 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 receptor agonists; 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, colony stimulating factors and interleukins, collectively referred to herein as "colony stimulating factors", for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, G- CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL-5, IL 6, IL- 7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and 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, differential cell count.

Determination of the Linker The length of the amino acid sequence of the linker can be selected empirically or with guidance from structural information, or by using a combination of the two approaches.

When no structural information is available, a small SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 39 series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp Woods, Mol. Immunol.

20: 483-489,1983, Kyte Doolittle, J. Mol. Biol. 157:105- 132, 1982); solvent exposed surface area, Lee Richards, J.

Mol. Biol. 55:379-400, 1971) and the ability to adopt the necessary conformation without deranging the conformation of the IL-3 receptor agonist(conformationally flexible; Karplus Schulz, Naturwissenschaften 72:212-213, 1985). Assuming an average of translation of 2.0 to 3.8 A per residue, this would mean the length to test would be between 0 to residues, with 0 to 15 residues being the preferred range.

Exemplary of such an empirical series would be to construct linkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated n times, where n is 1, 2, 3 or 4. Those skilled in the art will recognize that there are many such sequences that vary in length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor short Sandhu, Critical Rev.

Biotech. 12: 437-462, 1992); if they are too long, entropy effects will likely destabilize the three-dimensional fold, and may also make folding kinetically impractical, and if they are too short, they will likely destabilize the molecule because of torsional or steric strain.

Those skilled in the analysis of protein structural information will recognize that using the distance between the chain ends, defined as the distance between the c-alpha carbons, can be used to define the length of the sequence to be used, or at least to limit the number of possibilities that must be tested in an empirical selection of linkers.

They will also recognize that it is sometimes the case that the positions of the ends of the polypeptide chain are illdefined in structural models derived from x-ray diffraction SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 or nuclear magnetic resonance spectroscopy data, and that when true, this situation will therefore need to be taken into account in order to properly estimate the length of the linker required. From those residues whose positions are well defined are selected two residues that are close in sequence to the chain ends, and the distance between their calpha carbons is used to calculate an approximate length for a linker between them. Using the calculated length as a guide, linkers with a range of number of residues (calculated using 2 to 3.8A per residue) are then selected. These linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic .as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly-Gly-Ser (SEQ ID NO:2) cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used.

Determination of the Amino and Carboxvl Termini Sequences capable of folding to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while using the linker sequence as described above. Amino and carboxyl termini are selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below. A novel amino acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. In many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus. However, those skilled in the art SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 41 will recognize that selections of termini anywhere within the region may function, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence.

It is a central tenet of molecular biology that the primary amino acid sequence of a protein dictates folding to the three-dimensional structure necessary for expression of its biological function. Methods are known to those skilled in the art to obtain and interpret three-dimensional structural information using x-ray diffraction of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions. Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops; Kabsch Sander, Biopolymers 22: 2577-2637, 1983), the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, Ann.

Rev. Biochem. 53:537-572, 1984) and the static and dynamic distribution of conformations along the polypeptide chain (Alber Mathews, Methods Enzymol. 154: 511-533, 1987). In some cases additional information is known about solvent exposure of residues; one example is a site of posttranslational attachment of carbohydrate which is necessarily on the surface of the protein. When experimental structural information is not available, or is not feasible to obtain, methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops. Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 42 (Gentile Salvatore, Eur. J. Biochem. 218:603-621, 1993) Thus using either the experimentally derived structural information or predictive methods Srinivisan Rose Proteins: Struct., Funct. Genetics, 22: 81-99, 1995) the parental amino acid sequence is inspected to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure. The occurrence of sequences within regions that are known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) are regions that should be avoided. Similarly, regions of amino acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the socalled hydrophobic core of the protein and should also be avoided for selection of amino and carboxyl termini. In contrast, those regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity, are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence that are preferred based on the above criteria are referred to as a breakpoint region.

Materials And Methods Unless noted otherwise, all specialty chemicals were obtained from Sigma, Co. (St. Louis, MO). Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN).

Transformation of E. coli strains E. coli strains, such as DH5a T M (Life Technologies, Gaithersburg, MD) and TG1 (Amersham Corp., Arlington Heights, IL) are used for transformation of ligation reactions and are SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 43 the source of plasmid DNA for transfecting mammalian cells.

E. coli strains, such as JM101 (Yanisch-Perron, et al., Gene, 33: 103-119, 1985) and MON105 (Obukowicz, et al., Appl. and Envir. Micr., 58: 1511-1523, 1992) can be used for expressing the modified hIL-3 receptor agonist of the present invention in the cytoplasm or periplasmic space.

MON105 ATCC#55204: lambda-,IN(rrnD, rrE)l, rpoD+, rpoH358 DH5aTM: phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recAl, endAl, hsdRl7(rk-,mk+), phoA, supE441amda-, thi-1, gyrA96, relAl TG1: delta(lac-pro), supE, thi-1, hsdD5/F'(traD36, proA+B+, lacIq, JM101 ATCC#33876: delta (pro lac), supE, thi, F'(traD36, proA+B+, laclq, DH5a T M Subcloning efficiency cells are purchased as competent cells and are ready for transformation using the manufacturer's protocol, while both E. coli strains TG1 and MON105 are rendered competent to take up DNA using a CaC12 method. Typically, 20 to 50 mL of cells are grown in LB medium bacto-tryptone, 0.5% bacto-yeast extract, 150 mM NaC1) to a density of approximately 1.0 optical density unit at 600 nanometers (OD600) as measured by a Baush Lomb Spectronic spectrophotometer (Rochester, NY). The cells are collected by centrifugation and resuspended in one-fifth culture volume of CaCl2 solution (50 mM CaCl2, 10 mM Tris-Cl, pH7.4) and are held at 4'C for 30 minutes. The cells are again collected by centrifugation and resuspended in onetenth culture volume of CaC12 solution. Ligated DNA is added to 0.2 mL of these cells, and the samples are held at 4'C for 30-60 minutes. The samples are shifted to 42'C for two minutes and 1.0 mL of LB is added prior to shaking the SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 44 samples at 37'C for one hour. Cells from these samples are spread on plates (LB medium plus 1.5% bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin ug/mL) when selecting for spectinomycin-resistant transformants. The plates are incubated overnight at 37'C.

Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and are grown at 37 0 C while shaking.

Methods for creation of aenes with new N-terminus/C-terminus Method I. Creation of genes with new N-terminus/C-terminus which contain a linker region.

Genes with new N-terminus/C-terminus which contain a linker region separating the original C-terminus and Nterminus can be made essentially following the method described in L. S. Mullins, et al J. Am. Chem. Soc. 116, 5529-5533, 1994). Multiple steps of polymerase chain reaction (PCR) amplifications are used to rearrange the DNA sequence encoding the primary amino acid sequence of the protein. The steps are illustrated in Figure 1.

In the first step, the primer set ("new start" and "linker start") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Start") that contains the sequence encoding the new N-terminal portion of the new protein followed by the linker that connects the C-terminal and N-terminal ends of the original protein. In the second step, the primer set ("new stop" and "linker stop") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Stop") that encodes the same linker as used above, followed by the new C-terminal portion of the new protein. The "new start" SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 and "new stop" primers are designed to include the appropriate restriction sites which allow cloning of the new gene into expression plasmids. Typical PCR conditions are one cycle 95 0 C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50 0 C annealing for one minute and 72 0 C extension for one minute; plus one cycle 720C extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgC12. PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT).

"Fragment Start" and "Fragment Stop", which have complementary sequence in the linker region and the coding sequence for the two amino acids on both sides of the linker, are joined together in a third PCR step to make the fulllength gene encoding the new protein. The DNA fragments "Fragment Start" and "Fragment Stop" are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen). These fragments are combined in equimolar quantities, heated at 70 0 C for ten minutes and slow cooled to allow annealing through their shared sequence in "linker start" and "linker stop". In the third PCR step, primers "new start" and "new stop" are added to the annealed fragments to create and amplify the full-length new Nterminus/C-terminus gene. Typical PCR conditions are one cycle 95 0 C melting for two minutes; 25 cycles 940C denaturation for one minute, 600C annealing for one minute and 720C extension for one minute; plus one cycle 720C extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains 100 pmole of each primer and approximately 0.5 ug of DNA; and Ix PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 46 units AmpliTaq DNA polymerase and 2 mM MgC12. PCR reactions are purified using a Wizard PCR Preps kit (Promega).

Method II. Creation of genes with new N-terminus/C-terminus without a linker region.

New N-terminus/C-terminus genes without a linker joining the original N-terminus and C-terminus can be made using two steps of PCR amplification and a blunt end ligation. The steps are illustrated in Figure 2. In the first step, the primer set ("new start" and "P-bl start") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Start") that contains the sequence encoding the new N-terminal portion of the new protein. In the second step, the primer set ("new stop" and "P-bl stop") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Stop") that contains the sequence encoding the new C-terminal portion of the new protein. The "new start" and "new stop" primers are designed to include appropriate restriction sites which allow cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95 0 C melting for two minutes; cycles 94°C denaturation for one minute, 50 0 C annealing for 45 seconds and 720C extension for 45 seconds. Deep Vent polymerase (New England Biolabs) is used to reduce the occurrence of overhangs in conditions recommended by the manufacturer. The "P-bl start" and "P-bl stop" primers are phosphorylated at the 5' end to aid in the subsequent blunt end ligation of "Fragment Start" and "Fragment Stop" to each other. A 100 ul reaction contained 150 pmole of each primer and one ug of template DNA; and Ix Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit Deep Vent polymerase. PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 47 Norwalk, CT). PCR reaction products are purified using a Wizard PCR Preps kit (Promega).

The primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typically "Fragment Start" is designed to create NcoI restriction site, and "Fragment Stop" is designed to create a HindIII restriction site.

Restriction digest reactions are purified using a Magic DNA Clean-up System kit (Promega). Fragments Start and Stop are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen). These fragments are combined with and annealed to the ends of the 3800 base pair NcoI/HindIII vector fragment of pMON3934 by heating at 50 0 C for ten minutes and allowed to slow cool. The three fragments are ligated together using T4 DNA ligase (Boehringer Mannheim). The result is a plasmid containing the full-length new N-terminus/C-terminus gene. A portion of the ligation reaction is used to transform E. coli strain cells (Life Technologies, Gaithersburg, MD). Plasmid DNA is purified and sequence confirmed as below.

Method III. Creation of new N-terminus/C-terminus genes by tandem-duplication method New N-terminus/C-terminus genes can be made based on the method described in R. A. Horlick, et al Protein Eng. 5:427- 431, 1992). Polymerase chain reaction (PCR) amplification of the new N-terminus/C-terminus genes is performed using a tandemly duplicated template DNA. The steps are illustrated in Figure 3.

The tandemly-duplicated template DNA is created by cloning and contains two copies of the gene separated by DNA sequence encoding a linker connecting the original C- and N- SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 48 terminal ends of the two copies of the gene. Specific primer sets are used to create and amplify a full-length new N terminus/C-terminus gene from the tandemly-duplicated template DNA. These primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95 0 C melting for two minutes; 25 cycles 94 0

C

denaturation for one minute, 50°C annealing for one minute and 72°C extension for one minute; plus one cycle 72 0

C

extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit (Perkin Elmer Corporation, Norwalk, CT) is used.

A 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl 2 PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT). PCR reactions are purified using a Wizard PCR Preps kit (Promega).

Clonina of new N-terminus/C-terminus genes into expression vectors.

The new N-terminus/C-terminus gene is digested with restriction endonucleases to create ends that are compatible to insertion into an expression vector. This expression vector is likewise digested with restriction endonucleases to form compatible ends. After purification, the gene and the vector DNAs are combined and ligated using T4 DNA ligase. A portion of the ligation reaction is used to transform E.

coli. Plasmid DNA is purified and sequenced to confirm the correct insert. The correct clones are grown for protein expression.

DNA isolation and characterization SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 49 Plasmid DNA can be isolated by a number of different methods and using commercially available kits known to those skilled in the art. A few such methods are shown herein.

Plasmid DNA is isolated using the Promega Wizard T M Miniprep kit (Madison, WI), the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA) or Qiagen Plasmid Midi kit. These kits follow the same general procedure for plasmid DNA isolation.

Briefly, cells are pelleted by centrifugation (5000 x g), plasmid DNA released with sequential NaOH/acid treatment, and cellular debris is removed by centrifugation (10000 x g).

The supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA-binding resin, the column is washed, and plasmid DNA eluted with TE. After screening for the colonies with the plasmid of interest, the E. coli cells of selected transformants are inoculated into 50-100 mLs of LB plus appropriate antibiotic for overnight growth at 37 0 C in an air incubator while shaking. The purified plasmid DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into mammalian, E. coli or other cells.

Sequence confirmation.

Purified plasmid DNA is resuspended in dH20 and quantitated by measuring the absorbance at 260/280 nm in a Bausch and Lomb Spectronic 601 UV spectrometer. DNA samples are sequenced using ABI PRISM T DyeDeoxym terminator sequencing chemistry (Applied Biosystems Division of Perkin Elmer Corporation, Lincoln City, CA) kits (Part Number 401388 or 402078) according to the manufacturers suggested protocol usually modified by the addition of 5% DMSO to the sequencing mixture. Sequencing reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) following the recommended amplification conditions. Samples are purified to remove excess dye terminators with Centri- SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 SepT spin columns (Princeton Separations, Adelphia, NJ) and lyophilized. Fluorescent dye labeled sequencing reactions are resuspended in deionized formamide, and sequenced on denaturing 4.75% polyacrylamide-8M urea gels using an ABI Model 373A automated DNA sequencer. Overlapping DNA sequence fragments are analyzed and assembled into master DNA contigs using Sequencher v2.1 DNA analysis software (Gene Codes Corporation, Ann Arbor, MI).

Expression of hIL-3 receptor aaonists in mammalian cells Mammalian Cell Transfection/Production of Conditioned Media The BHK-21 cell line can be obtained from the ATCC (Rockville, MD). The cells are cultured in Dulbecco's modified Eagle media (DMEM/high-glucose), supplemented to 2 mM (mM) L-glutamine and 10% fetal bovine serum (FBS). This formulation is designated BHK growth media. Selective media is BHK growth media supplemented with 453 units/mL hygromycin B (Calbiochem, San Diego, CA). The BHK-21 cell line was previously stably transfected with the HSV transactivating protein VP16, which transactivates the IE110 promoter found on the plasmid pMON3359 (See Hippenmeyer et al., Bio/Technology, pp.1037-1041, 1993). The VP16 protein drives expression of genes inserted behind the IE110 promoter. BHK- 21 cells expressing the transactivating protein VP16 are designated BHK-VP16. The plasmid pMON1118 (See Highkin et al., Poultry Sci., 70: 970-981, 1991) expresses the hygromycin resistance gene from the SV40 promoter. A similar plasmid is available from ATCC, pSV2-hph.

BHK-VP16 cells are seeded into a 60 millimeter (mm) tissue culture dish at 3 X 105 cells per dish 24 hours prior to transfection. Cells are transfected for 16 hours in 3 mL of "OPTIMEM"

O

t (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 51 hygromycin resistance plasmid, pMON1118, and 80 ug of Gibco- BRL "LIPOFECTAMINE"T per dish. The media is subsequently aspirated and replaced with 3 mL of growth media. At 48 hours post-transfection, media from each dish is collected and assayed for activity (transient conditioned media). The cells are removed from the dish by trypsin-EDTA, diluted 1:10 and transferred to 100 mm tissue culture dishes containing 10 mL of selective media. After approximately 7 days in selective media, resistant cells grow into colonies several millimeters in diameter. The colonies are removed from the dish with filter paper (cut to approximately the same size as the colonies and soaked in trypsin/EDTA) and transferred to individual wells of a 24 well plate containing 1 mL of selective media. After the clones are grown to confluence, the conditioned media is re-assayed, and positive clones are expanded into growth media.

Expression of hIL-3 receptor aaonists in E. coli E. coli strain MON105 or JM101 harboring the plasmid of interest are grown at 37 0 C in M9 plus casamino acids medium with shaking in a air incubator Model G25 from New Brunswick Scientific (Edison, New Jersey). Growth is monitored at OD600 until it reaches a value of 1.0 at which time Nalidixic acid (10 milligrams/mL) in 0.1 N NaOH is added to a final concentration of 50 (Ig/mL. The cultures are then shaken at 37 0 C for three to four additional hours. A high degree of aeration is maintained throughout culture period in order to achieve maximal production of the desired gene product. The cells are examined under a light microscope for the presence of inclusion bodies One mL aliquots of the culture are removed for analysis of protein content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (see Maniatis et al. Molecular Cloning: A Laboratory Manual, 1982). The culture is SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 52 centrifuged (5000 x g) to pellet the cells.

Inclusion Body preparation, Extraction. Refolding, Dialysis.

DEAE Chromatoaraphv, and Characterization of the modified hIL-3 receptor aaonists which accumulate as inclusion bodies in E. coli.

Isolation of Inclusion Bodies: The cell pellet from a 330 mL E. coli culture is resuspended in 15 mL of sonication buffer (10 mM 2-amino-2- (hydroxymethyl) 1,3-propanediol hydrochloride (Tris-HCl), pH 1 mM ethylenediaminetetraacetic acid (EDTA). These resuspended cells are sonicated using the microtip probe of a Sonicator Cell Disruptor (Model W-375, Heat Systems- Ultrasonics, Inc., Farmingdale, New York). Three rounds of sonication in sonication buffer followed by centrifugation are employed to disrupt the cells and wash the inclusion bodies The first round of sonication is a 3 minute burst followed by a 1 minute burst, and the final two rounds of sonication are for 1 minute each.

Extraction and refolding of proteins from inclusion body pellets: Following the final centrifugation step, the IB pellet is resuspended in 10 mL of 50 mM Tris-HCl, pH 9.5, 8 M urea and 5 mM dithiothreitol (DTT) and stirred at room temperature for approximately 45 minutes to allow for denaturation of the expressed protein.

The extraction solution is transferred to a beaker containing 70 mL of 5 mM Tris-HCl, pH 9.5 and 2.3 M urea and gently stirred while exposed to air at 4°C for 18 to 48 hours to allow the proteins to refold. Refolding is monitored by analysis on a Vydac (Hesperia, Ca.) C18 reversed phase high SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 53 pressure liquid chromatography (RP-HPLC) column (0.46x25 cm).

A linear gradient of 40% to 65% acetonitrile, containing 0.1% trifluoroacetic acid (TFA), is employed to monitor the refold. This gradient is developed over 30 minutes at a flow rate of 1.5 mL per minute. Denatured proteins generally elute later in the gradient than the refolded proteins.

Purification: Following the refold, contaminating E. coli proteins are removed by acid precipitation. The pH of the refold solution is titrated to between pH 5.0 and pH 5.2 using 15% (v/v) acetic acid (HOAc). This solution is stirred at 4 0 C for 2 hours and then centrifuged for 20 minutes at 12,000 x g to pellet any insoluble protein.

The supernatant from the acid precipitation step is dialyzed using a Spectra/Por 3 membrane with a molecular weight cut off (MWCO) of 3,500 daltons. The dialysis is against 2 changes of 4 liters (a 50-fold excess) of 10 mM Tris-HCl, pH 8.0 for a total of 18 hours. Dialysis lowers the sample conductivity and removes urea prior to DEAE chromatography. The sample is then centrifuged (20 minutes at 12,000 x g) to pellet any insoluble protein following dialysis.

A Bio-Rad Bio-Scale DEAE2 column (7 x 52 mm) is used for ion exchange chromatography. The column is equilibrated in a buffer containing 10 mM Tris-HCl, pH 8.0, and a 0-to-500 mM sodium chloride (NaC1) gradient, in equilibration buffer, over 45 column volumes is used to elute the protein. A flow rate of 1.0 mL per minute is used throughout the run. Column fractions (2.0 mL per fraction) are collected across the gradient and analyzed by RP HPLC on a Vydac (Hesperia, Ca.) C18 column (0.46 x 25 cm). A linear gradient of 40% to acetonitrile, containing 0.1% trifluoroacetic acid (TFA), is employed. This gradient is developed over 30 minutes at a SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 54 flow rate of 1.5 mL per minute. Pooled fractions are then dialyzed against 2 changes of 4 liters (50-to-500-fold excess) of 10 mM ammonium acetate (NH4Ac), pH 4.0 for a total of 18 hours. Dialysis is performed using a Spectra/Por 3 membrane with a MWCO of 3,500 daltons. Finally, the sample is sterile filtered using a 0.22pm syringe filter (4Star LB syringe filter, Costar, Cambridge, and stored at 4 0

C.

In some cases the folded proteins can be affinity purified using affinity reagents such as mAbs or receptor subunits attached to a suitable matrix. Alternatively, (or in addition) purification can be accomplished using any of a variety of chromatographic methods such as: ion exchange, gel filtration or hydrophobic chromatography or reversed phase

HPLC.

These and other protein purification methods are described in detail in Methods in Enzymology, Volume 182 'Guide to Protein Purification' edited by Murray Deutscher, Academic Press, San Diego, CA, 1990).

Protein Characterization: The purified protein is analyzed by RP-HPLC, electrospray mass spectrometry, and SDS-PAGE. The protein quantitation is done by amino acid composition, RP-HPLC, and Bradford protein determination. In some cases tryptic peptide mapping is performed in conjunction with electrospray mass spectrometry to confirm the identity of the protein.

AML Proliferation Assay for Bioactive Human Interleukin-3 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 SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 (Lange, et al., Blood 70: 192, 1987) Valtieri, et al., J. Immunol. 138:4042, 1987). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, et al., J. Immunol. 139: 348, 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 are then replated at 1x10 5 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 are maintained as AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.

AML 193 1.3 cells are washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, NY) by centrifuging cell suspensions at 250 x g for 10 minutes followed by decantation of the supernatant. Pelleted cells are resuspended in HBSS and the procedure is repeated until six wash cycles are completed. Cells washed six times by this procedure are resuspended in tissue culture medium at a density ranging from 2 x 105 to 5 x 105 viable cells/mL.

This medium is prepared by supplementing Iscove's modified Dulbecco's Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrin, lipids and 2 -mercaptoethanol. Bovine albumin (Boehringer-Mannheim, Indianapolis, IN) is added at 500 gg/mL; human transferrin (Boehringer-Mannheim, Indianapolis, IN) is added at 100 gg/mL; soybean lipid (Boehringer- Mannheim, Indianapolis, IN) is added at 50 gg/mL; and 2mercaptoethanol (Sigma, St. Louis, MO) is added at 5 x 10-

M.

Serial dilutions of human interleukin-3 or modified hIL- 3 receptor agonist proteins are made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 p.

of medium containing interleukin-3 or modified hIL-3 receptor SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 56 agonist proteins once serial dilutions are completed.

Control wells contained tissue culture medium alone (negative control). AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting 50 tl (2.5 x 104 cells) into each well. Tissue culture plates are incubated at 37 0 C with CO2 in humidified air for 3 days. On day 3, 0.5 RCi 3

H-

thymidine (2 Ci/mM, New England Nuclear, Boston, MA) is added in 50 gl of tissue culture medium. Cultures are incubated at 37 0 C with 5% C02 in humidified air for 18-24 hours. Cellular DNA is harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, MD) using a TOMTEC cell harvester (TOMTEC, Orange, CT) which utilized a water wash cycle followed by a ethanol wash cycle. Filter mats are 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) is added. Beta emissions of samples from individual tissue culture wells are counted in a LKB BetaPlate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, MD) and data is expressed as counts per minute of 3 H-thymidine incorporated into cells from each tissue culture well.

Activity of each human interleukin-3 preparation or modified hIL-3 receptor agonist proteins preparation is quantitated by measuring cell proliferation 3 H-thymidine incorporation) induced by graded concentrations of interleukin-3 or modified hIL-3 receptor agonist. 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 modified hIL-3 receptor agonist protein which provides 50% of maximal proliferation (EC50 0.5 x (maximum average counts per minute of 3 H-thymidine incorporated per well among triplicate cultures of all concentrations of interleukin-3 tested background proliferation measured by 3 H-thymidine incorporation observed in triplicate cultures lacking interleukin-3). This EC50 value is also equivalent to 1 unit SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 57 of bioactivity. Every assay is performed with native interleukin-3 as a reference standard so that relative activity levels could be assigned.

Typically, the modified hIL-3 receptor agonist proteins are tested in a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions.

Activity for each sample is determined by the concentration which gave 50% of the maximal response by fitting a four-parameter logistic model to the data. It was observed that the upper plateau (maximal response) for the sample and the standard with which it was compared did not differ. Therefore relative potency calculation for each sample is determined from EC50 estimations for the sample and the standard as indicated above.

Methvlcellulose Assay This assay reflects the ability of colony stimulating factors to stimulate normal bone marrow cells to produce different types of hematopoietic colonies in vitro (Bradley et al., Aust. Exp Biol. Sci. 44:287-300, 1966), Pluznik et al., J.

Cell Comp. Physio 66:319-324, 1965).

Methods Approximately 30 mL of fresh, normal, healthy bone marrow aspirate are obtained from individuals following informed consent. Under sterile conditions samples are diluted with a 1X PBS (#14040.059 Life Technologies, Gaithersburg, MD.) solution in a 50 mL conical tube (#25339-50 Corning, Corning MD). Ficoll (Histopaque 1077 Sigma H-8889) is layered under the diluted sample and centrifuged, 300 x g for min. The mononuclear cell band is removed and washed two times in IX PBS and once with 1% BSA PBS (CellPro Co., Bothel, WA). Mononuclear cells are counted and CD34+ cells are selected using the Ceprate LC (CD34) Kit (CellPro Co., SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 58 Bothel, WA) column. This fractionation is performed since all stem and progenitor cells within the bone marrow display CD34 surface antigen.

Cultures are set up in triplicate with a final volume of mL in a 35 X 10 mm petri dish (Nunc#174926). Culture medium is purchased from Terry Fox Labs. (HCC-4230 medium (Terry Fox Labs, Vancouver, Canada) and erythropoietin (Amgen, Thousand Oaks, CA.) is added to the culture media. 3,000- 10,000 CD34+ cells are added per dish. Recombinant IL-3, purified from mammalian cells or E. coli, and modified hIL-3 receptor agonist proteins, in conditioned media from transfected mammalian cells or purified from conditioned media from transfected mammalian cells or E. coli, are added to give final concentrations ranging from .001 nM to 10 nM.

Recombinant hIL-3, GM-CSF, c-mpl ligand and modified hIL-3 receptor agonist are supplied in house. G-CSF (Neupogen) is from Amgen (Thousand Oaks Calf.). Cultures are resuspended using a 3cc syringe and 1.0 mL is dispensed per dish.

Control (baseline response) cultures received no colony stimulating factors. Positive control cultures received conditioned media (PHA stimulated human cells: Terry Fox Lab.

H2400). Cultures are incubated at 37 0 C, 5% CO2 in humidified air.

Hematopoietic colonies which are defined as greater than cells are counted on the day of peak response (days 10-11) using a Nikon inverted phase microscope with a 40x objective combination. Groups of cells containing fewer than 50 cells are referred to as clusters. Alternatively colonies can be identified by spreading the colonies on a slide and stained or they can be picked, resuspended and spun onto cytospin slides for staining.

Human Cord Blood HematoDoietic Growth Factor Assays SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 59 Bone marrow cells are traditionally used for in vitro assays of hematopoietic colony stimulating factor (CSF) activity. However, human bone marrow is not always available, and there is considerable variability between donors.

Umbilical cord blood -is comparable to bone marrow as a source of hematopoietic stem cells and progenitors (Broxmeyer et al., PNAS USA 89:4109-113, 1992; Mayani et al., Blood 81:3252-3258, 1993). In contrast to bone marrow, cord blood is more readily available on a regular basis. There is also a potential to reduce assay variability by pooling cells obtained fresh from several donors, or to create a bank of cryopreserved cells for this purpose. By modifying the culture conditions, and/or analyzing for lineage specific markers, it should be possible to assay specifically for granulocyte macrophage colonies (CFU-GM), for megakaryocyte CSF activity, or for high proliferative potential colony forming cell (HPP-CFC) activity.

Methods Mononuclear cells (MNC) are isolated from cord blood within 24 hr. of collection, using a standard density gradient (1.077 g/mL Histopaque). Cord blood MNC have been further enriched for stem cells and progenitors by several procedures, including immunomagnetic selection for CD14-, CD34+ cells; panning for SBA-, CD34+ fraction using coated flasks from Applied Immune Science (Santa Clara, CA); and CD34+ selection using a CellPro (Bothell, WA) avidin column.

Either freshly isolated or cryopreserved CD34+ cell enriched fractions are used for the assay. Duplicate cultures for each serial dilution of sample (concentration range from 1 pM to 1204 pM) are prepared with 1x104 cells in lml of 0.9% methycellulose containing medium without additional growth factors (Methocult H4230 from Stem Cell Technologies, Vancouver, In some experiments, Methocult H4330 containing erythropoietin (EPO) is used instead of Methocult H4230, or Stem Cell Factor (SCF), 50 ng/mL (Biosource SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 International, Camarillo, CA) is added. After culturing for 7-9 days, colonies containing >30 cells are counted. In order to rule out subjective bias in scoring, assays are scored blind.

Additional details about recombinant DNA methods which may be used to create the variants, express them in bacteria, mammalian cells or insect cells, purification and refold of the desired proteins and assays for determining the bioactvity of the proteins may be found in co-filed Applications WO 95/00646, WO 94/12639, WO 94/12638,

WO

95/20976, WO 95/21197, WO 95/20977, WO 95/21254 and US 08/383,035 which are hereby incorporated by reference in their entirety.

Further details known to those skilled in the art may be found in T. Maniatis, et al., Molecular Clonin., A Laboratory Manual, Cold Spring Harbor Laboratory (1982) and references cited therein, incorporated herein by reference; and in J.

Sambrook, et al., Molecular Clonin., A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (1989) and references cited therein, incorporated herein by reference.

All references, patents or applications cited herein are incorporated by reference in their entirety as if written herein.

SUBSTITUTE SHEET (RULE 26) WO 97/12979 WO 9712979PCTIUS96/15941 Lisyn. f or Llsyn. rev L3syn.for OL IGONUCLEOT IDES GTTACCCTTG AGCAAGCGCA GGAACAACAG GGTGGtGGCT CTAACTGCTC TATAATGAT (SEQ ID NO:3) CGATCA.TTAT AGAGCAGTTA GAGCCACCAC

CCTGTTGTTC

CTGCGCTTGC TCAAGC (SEQ ID NO:4) L3syn. rev 3 5start seq 3 4 stop.seq 7Ostart .seq 69stop. seq 9 1start.seq seq seq GTTACCCTTG AGCAAGCGCA CTGGCGGTGG

CAGCGGCGGC

GAT (SEQ ID CGATCATTAT AGAGCAGTTA GCCAGAGCCA

CCACCCTGTT

(SEQ ID NO:6) GATCGACCAT GGCTCTGGAC (SEQ ID NO:7) CGATCGAAGC TTATTACAAA (SEQ ID NO:8) GATCGACCAT GGCTAATGCA (SEQ ID NO:9) CGATCGAAGC

TTATTATTCT

(SEQ ID GATCGACCAT GGCTGCACCC (SEQ ID NO:11) CGATCGAAGC TTATTAGGCC (SEQ ID NO:12) GATCGACCAT GGCTGCAGGT (SEQ ID NO:13)

GGTTCTAACT

GAACCGCCGC

GTTCCTGCGC

CC GAACAACC

GGTGCAGGTG

TCAGGTATTG

AAGTTCTTGA

TCTCGACATC

GTGGCAGAGG

GACTGGCAAG

GGAACAACAG GGTGGTGGCT

GCTCTATAAT

CGCTGCCACC

TTGCTCAAGG

TC

GTCTCT

AG

CAGCCC

CA

GCAGAC

AA

100stop.seq CGATCGAAGC TTATTACTTG ATGATGATTG

GATGTC

(SEQ ID NO:14) SUBSTITUTE SHEET (RULE 26) WO 97/12979 PTU9/54 PCT/US96/15941 62 TABLE 2 DNA Seciuences Syntani 1 51 101 151 201 251 301 351 401 451 501 551 601 651 CATGGCTAAC

TGCTCTATAA

GACCACCTGC

ACCTTTGCTG

TCTATCCTGA TGGA CCGAAA AAGGGCTGTC

AAGAACTTAG

GTAATCTCCA

ACCATGTCTG

CCAATCATCA

TCAAGGCAGG

GTTCTATCTG

GTTACCCTTG

CTAACTGCTC

TATAATGATC

CCTGCACCTT

TGCTGGACCC

CCTGATGGAC

CGAA.ACCTTC

CTGTCAAGAA

CTTAGAAAAT

CTCCAACCAT

GTCTGCCCTC

CATCATCAAG

GCAGGTGACT

ATCTGGTTAC

CCTTGAGCAA

(SEQ ID NO:15)

TGATCGATGA

GACCCGAACA

CCTTCGACTT

AAAATGCATC

CCCTCTGCCA

TGACTGGCAA

AGCA.AGCGCA

GATGAAATTA

GAACAACCTC

GACTTCCAAA

GCATCAGGTA

TGCCACGGCC

GGCAAGAATT

GCGCAGGAAC

AATTATACAT

ACCTCAATGA

CCAAACCTGG

AGGTATTGAG

CGGCCGCACC

GAATTCCGGG

GGAACAACAG

TACATCACTT

AATGACGAAG

CCTGGAGAGC

TTGAGGCAAT

GCACCCTCTC

CCGGGAAAAA

AACAGTAC

CACTTAAAGA

CGAAGACGTC

AGAGCTTCGT

GCAATTCTTC

CTCTCGACAT

AAAAACTGAC

GGTGGTGGCT

AAAGAGACCA

ACGTCTCTAT

TTCGTAAGGG

TCTTCGTAAT

GACATCCAAT

CTGACGTTCT

Synt an3 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701

CATGGCTAAC

GACCACCTGC

TCTATCCTGA

AAGGGCTGTC

G TAATC T CCA

CCAATCATCA

GTTCTATCTG

CTGGCGGTGG

ATTATACATC

CCTCAATGAC

CAAACCTGGA

GGTATTGAGG

GGCCGCACCC

AATTCCGGGA

GAACAACAGT

TGCTCTATAA

ACCTTTGCTG

TGGACCGAA.A

AAGAACTTAG

ACCATGTCTG

TCAAGGCAGG

GTTACCCTTG

CAGCGGCGGC

ACTTAAAGAG

GAAGACGTCT

GAGCTTCGTA

CAATTCTTCG

TCTCGACATC

AAAACTGACG

AC (SEQ ID

TGATCGATGA

GACCCGAACA

CCTTCGACTT

AAAAT GC AT C

CCCTCTGCCA

TGACTGGCAA

AGCAAGCGCA

GGTTCTAACT

ACCACCTGCA

CTATCCTGAT

AGGGCTGTCA

TAATCTCCAA

C AAT CAT CAT

TTCTATCTGG

NO: 16)

AATTATACAT

ACCTCAATGA

CCAAACCTGG

AGGTATTGAG

CGGCC GCACC

GAATTCCGGG

GGAACAACAG

GCTCTATAAT

CCTTTGCTGG

GGACCGAAAC

AGAACTTAGA

CCATGTCTGC

CAAGGCAGGT

TTACC CTTGA

CACTTAA-AGA

CGAAGACGTC

AGAGCTTCGT

GCAATTCTTC

CTCTCGACAT

AAAAACTGAC

GGTGGTGGCT

GATCGATGAA

ACCCGAACAA

CTTCGACTTC

AAATGCATCA

CCTCTGCCAC

GACTGGCAAG

GCA-AGCGCAG

pMON3 1155.seq 1 51 101 151 201 251 301 ATGGCTCTGG ACCCGAACAA

CCTCAATGAC

GGAGCGAAAC CTTCGACTTC

CAAACCTGGA

AGAACTTAGA AAATGCATCA

GGTATTGAGG

CCATGTCTGC CCTCTGCCAC GGCCGCACCC CAAGGCAGGT GACTGGCAAG

AATTCCGGGA

TTACCCTTGA GCAAGCGCAG

GAACAACAGG

ATAATGATCG ATGAAATTAT

ACATCACTTA

GTAATAA (SEQ ID NO:17) GAAGAC GTCT

GAGCTTCGTA

CAATTCTTCG

TCTCGACATC

AAACTGACG

GTGGTGGCTC

AAGAGACCAC

CTATCCTGAT

AGGGCTGTCA

TAATCTCCAA

CAATCATCAT

TTCTATCTGG

TAACTGCTCT

CTGCACCTTT

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 pMON3 1156. seq 1 51 101 151 201 251 301 351 ATGGCTA.ATG CATCAGGTAT

TGAGGCAATT

TCTGCCCTCT GCCACGGCCG

CACCCTCTCG

CAGGTGACTG GCAAGAATTC

CGGGAAAAAC

CTTGAGCAAG CGCAGGAACA

ACAGGGTGGT

GATCGATGAA ATTATACATC

ACTTAAAGAG

ACCCGAACAA CCTCAATGAC

GAAGACGTCT

CTTCGACTTC CAAACCTGGA

GAGCTTCGTA

ATAATAA (SEQ ID NO:18)

CTTCGTAATC

ACATCCAATC

TGACGTTCTA

GGCTCTAACT

ACCACCTGCA

CTATCCTGAT

AGGGCTGTCA

TCCAACCATG

TCTGGTTACC

GCTCTATAAT

CCTTTGCTGG

GGAGCGAAkC

AGAACTTAGA

pMON3 1157.seq 1 51 101 151 201 251 301 351 ATGGCTGCAC CCTCTCGACA

TCCAATCATC

AGAATTCCGG GAAAAACTGA

CGTTCTATCT

AGGAACAACA GGGTGGTGGC

TCTAACTGCT

ATACATCACT TAAAGAGACC

ACCTGCACCT

CAATGACGAA GACGTCTCTA

TCCTGATGGA

ACCTGGAGAG CTTCGTAAGG

GCTGTCAAGA

ATTGAGGCAA TTCTTCGTALA

TCTCCAACCA

CTAATAA (SEQ ID NO:19)

ATCAAGGCAG

GGTTAC

CCTT

CTATAATGAT

TTGCTGGACC

CCGAAACCTT

ACTTAGAAAA

TGTCTGCCCT

GTGACTGGCA

GAGCAAGCGC

CGATGAAATT

CGAACAACCT

CGACTTCCAA

TGCATCAGGT

CTGCCACGGC

PMON31158.seq 1 51 101 151 201 251 301 351 ATGGCTGCAG GTGACTGGCA

AGAATTCCGG

GGTTACCCTT GAGCAAGCGC

AGGAACAACA

CTATAATGAT CGATGAAATT

ATACATCACT

TTGCTGGACC CGAACAACCT

CAATGACGAA

CCGAAACCTT CGACTTCCAA

ACCTGGAGAG

ACTTAGAAAA TGCATCAGGT

ATTGAGGCAA

TGTCTGCCCT CTGCCACGGC

CGCACCCTCT

GTAATAA (SEQ ID NO:20)

GAAAAACTGA

GGGTGGTGGC

TAAAGAGACC

GACGTCTCTA

CTTCGTAAGG

TTCTTCGTAA

CGACATCCAA

CGTTCTATCT

TCTA.ACTGCT

ACCTGCACCT

TCCTGATGGA

GCTGTCAAGA

TCTCCAACCA

TCATCATCA

pMON3 1159 .seq 1 51 101 151 201 251 301 351

ATGGCTCTGG

GGAGCGAAAC

AGAACTTAGA

CC ATGT CTG C

CAAGGCAGGT

TTACCCTTGA

AGCGGCGGCG

CTTAAAGAGA

ACCCGAACAA

CTTCGACTTC

AAATGCATCA

CCTCTGCCAC

GACTGGCAAG

GCAAGCGCAG

GTTCTAACT.G

CCACCTGCAC

CCTCAATGAC

CAAACCTGGA

GGTATTGAGG

GGCCGCACCC

AATTCCGGGA

GAACAACAGG

CTCTATAATG

CTTTGTAATA

GAAGACGTCT

CTATCCTGAT

GAGCTTCGTA

AGGGCTGTCA

CAATTCTTCG

TAATCTCCAA

TCTCGACATC

CAATCATCAT

AAAACTGACG

TTCTATCTGG

GTGGTGGCTC

TGGCGGTGGC

ATCGATGAA

TTATACATCA

A (SEQ ID NO:21) pMON3 1160. seq 1 51 101

ATGGCTAATG

TCTGCCCTCT

CAGGTGACTG

CATCAGGTAT

CCAC GGC C

GCAAGAATTC

TGAGGCAATT

CGGGAAAAC

C TT CGT AAT C

ACATCCAATC

TGACGTTCTA

TCCAACCATG

ATCATCAAGG

TCTGGTTACC

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCTIUS96/1 5941 151 201 251 301 351

CTTGAGCAAG

CGGCGGTTCT

AGAGACCACC

GTCTCTATCC

CGTAAGGGCT

CGCAGGAACA

AACTGCTCTA

TGCACCTTTG

TGATGGACCG

GTCAAGAACT

ACAGGGTGGT

TAATGATCGA

CTGGACCCGA

AAACCTTCGA

TAGAATAATA

CGCTCTGGCG

GTGGCAGCGG

TGAAATTATA

CATCACTTA

ACAACCTCA

TGACGAAGAC

CTTCCAAACC

TGGAGAGCTT

A (SEQ ID NO:22) pMON3 1161. seq 1 51 101 151 201 251 301 351

ATGGCTGCAC

AGAATTCCGG

AGGAACAA.CA

TGCTCTATAA

ACCTTTGCTG

TGGACCGAAA

AAGAACTTAG

ACCATGTCTG

CCTCTCGACA

GAAAAACTGA

GGGTGGTGGC

TGATCGATGA

GACCCGAACA

CCTTCGACTT

AAAATGCATC

CCCTCTGCCA

TCCAATCATC

CGTTCTATCT

TCTGGCGGTG

AATTATACAT

ACCTCAATGA

CCAA.ACCTGG

AGGTATTGAG

CGGCCTAATA

ATCAAGGCAG

GTGACTGGCA

GGTTACCCTT

GAGCAAGCGC

GCAGCGGCGG

CGGTTCTAAC

CACTTAAAGA

GACCACCTGC

CGAAGACGTC

TCTATCCTGA

AGAGCTTCGT

AAGGGCTGTC

CCAATTCTTC

GTAATCTCCA

A (SEQ ID NO:23) PMON31162.seq 1 51 101 151 201 251 301 351

ATGGCTGCAG

GGTTACCCTT

GCAGCGGCGG

CACTTAAAGA

CGAAGACGTC

AGAGCTTCGT

GCAATTCTTC

CTCTCGACAT

GTGACTGGCA

GAG CAAGCG C

CGGTTCTAAC

GACCACCTGC

TCTATCCTGA

AAGGGCTGTC

GTAATCTCCA

CCAATCATCA

AGAATTCCGG

AGGAACAACA

TGCTCTATAA

ACCTTTGCTG

TGGACCGAAA

AAGAACTTAG

ACCATGTCTG

TCAAGTAATA

GAAAA.ACTGA

CGTTCTATCT

GGGTGGTGGC

TCTGGCGGTG

TGATCGATGA

AATTATACAT

GACCCGAACA

ACCTCAATGA

CCTTCGACTT

CCAAACCTGG

AAAATGCATC

AGGTATTGAG

CCCTCTGCCA

CGGCCGCACC

A (SEQ ID NO:24) SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 TABLE 3 PROTEIN SEQUENCES pMON31155.Pep Leu Asp Pro Asn Asp Arg Asn Leu Val Lys Asn Leu Asn Leu Gin Pro His Pro Ile Ile Lys Leu Thr Phe Gin Gly Gly Gly His His Leu Lys Asn Arg Glu Cys Ile Tyr Ser Arg Leu Leu Asn Leu Lys Leu Asn Pro Asn Pro Ala Pro Ala Val Cys Pro Asp Glu Asp Val Ser Ile Leu Asn Leu Giu Ser Phe Val Arg Ser Gly Ile Giu Aia Ile Leu Ser Ala Thr Ala Ala Pro Ser Gly Asp Trp Gin Giu Phe Arg Thr Leu Giu Gin Aia Gin Glu Ser Ile Met Ile Asp Giu Ile Ala Pro Leu (SEQ ID Met Ala Arg Arg Glu Gin Ile pMON31156 Pep Asn Ala Leu Pro Lys Ala Leu Val Asn Cys Pro Pro Val Ser Ser Phe Ser Gly Ile Glu Ala Ser Ala Thr Ala Ala Gly Asp Trp Gin Glu Thr Leu Giu Gin Ala Ser Ile Met Ile Asp Ala Pro Leu Leu Asp Ile Leu Met Asp Arg Val Arg Ala Val Lys Ile Pro Phe Gin Glu Pro Asn Asn Leu Arg Ser Arg Arg Glu Glu Gin lie Ile Asn Asn Leu Arg Leu Glu Asn Leu Gin Pro His Pro Ile Ile Lys Leu Thr Phe Gin Gly Gly Gly His His Leu Lys Leu Asn Asp Glu Leu Pro Asn Leu (SEQ ID NO:26) Cys Ile Tyr Ser Arg Asp Glu pMON31157.Pep Ala Pro Glu Phe Ala Gin Asp Glu Asp Pro Arg Asn Lys Asn Leu Gin Ser Arg Glu Ile Asn Leu Leu Pro Arg Glu Gin Ile Asn Arg Glu Cys His Pro Ile Lys Leu Thr Gin Gly Gly His His Leu Leu Asn Asp Leu Pro Asn Asn Ala Ser Leu Pro Ser Ile Phe Gly Lys Glu Leu Gly Ala Ile Lys Tyr Leu Ser Asn Arg Pro Asp Val Glu Ser Ile Glu Thr Ala Ala Gly Val Thr Cys Ser Pro Ala Ser Ile Phe Val Ala Ile (SEQ ID Asp Trp Gin Leu Giu Gin Ile Met Ile Pro Leu Leu Leu Met Asp Arg Ala Vai Leu Arg Asn NO: 27) PMON31158.Pep Ala Gly Asp Trp Val Thr Leu Glu Cys Ser Ile Met Pro Ala Pro Leu Ser Ile Leu Met Phe Val Arg Ala Ala Ile Leu Arg Ala Pro Ser Arg Gin Glu Gin Ala Ile Asp Leu Asp Asp Arg Val Lys Asn Leu His Pro Phe Gin Glu Pro Asn Asn Gin Ile Arg Glu Ile Asn Leu Leu Pro Ile Glu Lys Leu Thr Phe Tyr Gin Gin Gly Gly Giy Ser Ile His His Leu Lys Arg Asn Leu Asn Asp Giu Asp Arg Leu Pro Asn Leu Glu Glu Asn Ala Ser Gly Ile Cys Leu Pro Ser Ala Thr Ile Lys (SEQ ID NO:28) Leu Asn Pro Val Ser Glu Ala pMON31159.Pep SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCTIUS96/15941 Leu ASP Pro ASP Arg Asn Val Lys Asn Asn Leu Gin His Pro Ile Lys Leu Thr Gin Gly Gly Ser Ile met Ala Pro Leu Asn Asn Leu Asn Leu Arg Leu Pro Leu Giu Asn Ala Pro Cys Leu Pro Ile Ile Lys Ala Phe Tyr Leu Val Gly Ser Giy Gly Ile Asp Giu Ile (SEQ ID NO:29) Asp GiU Asp Asn Leu Giu Ser Gly Ile Ser Ala Thr Gly Asp Trp Thr Leu Giu Giy Ser Giy Ile His His Val Ser Glu Ala Gin Gin Gly Leu S er Phe Ala Ala Glu Ala G ly Lys Ile Val lie Pro Phe Gin Ser Arg Leu Arg Leu Ser Arg Giu Asn Pro Met Ala Arg Arg Giu Gin Cys Pro PMON3 1160. Pep Asn Ala Leu Pro Lys Ala Leu Val Gly Giy Giu lie Pro Asn Asn Leu Asn Leu Ser Giy Ile Ser Ala Thr Gly Asp Trp, Thr Leu Giu Gly Ser Gly le.His His Asn Leu Asn Arg Leu Pro Giu (SEQ ID Giu Ala Ile Leu Ala Ala Pro Ser Gin Giu Phe Arg Gin Ala Gin Giu Gly Gly Ser Asn Leu Lys Arg Pro Asp Giu Asp Val Asn Leu Giu Ser NO: 30) Arg Asn Arg His Giu Lys Gin Gin Cys Ser Pro Ala Ser Ile Phe Val Leu Gin Pro Ile Leu Thr Gly Gly Ile Met Pro Leu Leu Met Arg Ala Pro Cys Ile Ile Phe Tyr Gly Ser Ile ASP LeU Asp Asp Arg Val Lys PMON3 1161. Pep Ala Pro Ser Giu Phe Arg Ala Gin Glu Gly Ser Asn Lys Arg Pro Giu ASP Val Leu Giu Ser Giy Ile Giu Ala Thr Ala Arg His Pro Ile Giu Lys Leu Thr Gin Gin Gly Gly Cys Ser Ile Met Pro Ala Pro Leu Ser Ile Leu Met Phe Val Arg Ala Ala Ile Leu Arg (SEQ ID NO:31) Ile Ile Lys Phe Tyr Leu Gly Ser Gly Ile Asp Giu Leu Asp Pro Asp Arg Asn Val Lys Asn Asn Leu Gin Ala Val Gly Ile Asn Leu Leu Pro Gly Asp Thr Leu Gly Ser Ile His Asn Leu Arg Leu Giu Asn CYS Leu Trp Giu Gly His Asn Pro Ala Pro Gin Gin G ly Leu Asp Asn Ser Ser pMON3 ii 6 2.Pep Ala Gly Asp Trp Gin Giu Phe Arg Val Thr Leu Giu Gin Ala Gin Giu Gly Gly Ser Gly Gly Gly Ser Asn Ile Ile His His Leu Lys Arg Pro Asn Asn Leu Asn ASP Giu Asp Val Leu Arg Leu Pro Asn Leu Giu Ser Leu Giu Asn Ala Ser Giy Ile Giu Pro Cys Leu Pro Ser Ala Thr Ala Ile Ile Lys (SEQ ID NO:32) Giu Lys Leu Gin Gin Giy Cys Ser Ile Pro Ala Pro Ser Ile Leu Phe Val Arg Ala Ile Leu Ala Pro Ser Thr Phe Gly Gly Met Ile Leu Leu Met Asp Ala Val Arg Asn Arg His Ser Asp Asp Arg Lys Leu Pro Leu Gly Giu Pro Asn Asn Gin Ile SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 67 The following examples will illustrate the invention in greater detail although it will be understood that the invention is not limited to this specific example.

EXAMPLE1 Const rcion of t n l. i a S n a To create the tandemly-duplicated hIL-3 receptor agonist pMON13416 template, Syntanl (SEQ ID NO:15), three DNAs were joined by means of ligation using T4 DNA ligase (Boehringer Mannheim). The three DNAs are: 1) pMON13046, containing hIL-3 receptor agonist pMON13416, digested with BstEII and SnaBI; 2) the annealed complimentary pair of synthetic oligonucleotides, Llsyn.for (SEQ ID NO:3) and Llsyn.rev

(SEQ

ID NO:4), which contain sequence encoding the linker that connects the C-terminal and N-terminal ends of the original protein and a small amount of surrounding pMON13416 sequence and which when properly assembled result in BstEII and ClaI ends; and 3) a portion of hIL-3 receptor agonist pMONI3416 igested from pMON3046 with ClaI (DNA had been grown in the dam- cells, DM1 (Life Technologies) and SnaBI. The digested DNAs were resolved on a 0.9% TAE gel, stained with ethidium bromide and isolated using Geneclean (Biol01).

A portion of the ligation reaction was used to transform

E.

coli strain DH5a cells (Life Technologies, Gaithersburg,

MD).

Miniprep DNA was isolated from the transformants, and the transformants were screened using a PCR based assay. Plasmid DNA from selected transformants was sequenced to obtain the correct template. The resulting plasmid was designated syntanl.

EXAMPLE 2 SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 68 Construction of tandemlv-duplicated template, svntan3.

To create the tandemly-duplicated hIL-3 receptor agonist pMON13416 template, syntan3 (SEQ ID NO:16), three DNAs were joined by means of ligation using T4 DNA ligase (Boehringer Mannheim). The three DNAs are: 1) pMON13046, containing hIL-3 receptor agonist pMON13416, digested with BstEII and SnaBI; 2) the annealed complimentary pair of synthetic oligonucleotides, L3syn.for (SEQ ID NO:5) and L3syn.rev

(SEQ

ID NO:6), which contain sequence encoding the linker that connects the C-terminal and N-terminal ends of the original protein and a small amount of surrounding pMON13416 sequence and which when properly assembled result in BstEII and SnaBI ends; and 3) a portion of hIL-3 receptor agonist pMON13416 digested from pMON13046 with ClaI (DNA had been grown in the dam- cells, DM1 (Life Technologies) and SnaBI. The digested DNAs were resolved on a 0.9% TAE gel, stained with ethidium bromide and isolated using Geneclean (BiolOl).

A portion of the ligation reaction was used to transform

E.

coli strain DH5 cells (Life Technologies, Gaithersburg,

MD).

Miniprep DNA was isolated from the transformants, and the transformants were screened using a PCR based assay. Plasmid DNA from selected transformants was sequenced to obtain the correct template. The resulting plasmid was designated syntan3.

EXAMPLE 3 Construction of DMON31155 The new N-terminus/C-terminus gene in pMON31155 is created using Method III as described in Materials and Methods. The full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 is created and amplified SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCTIUS96/15941 69 from the intermediate plasmid, Syntanl, using the primer set (SEQ ID NO:7) and 3 4stop (SEQ ID NO:8).

The resulting DNA fragment which contains the new gene is digested with restriction endonucleases NcoI and HindIII.

The digested DNA fragment is resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Biol0a, Vista, CA). The purified digested DNA fragment is ligated into an expression vector using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). The expression vector DNA is digested with NcoI and HindIII. A portion of the ligation reaction is used to transform E. coli strain cells (Life Technologies, Gaithersburg,

MD).

Transformant bacteria are selected on ampicillin-containing plates. Plasmid DNA is isolated and sequenced to confirm the correct insert. The resulting plasmid is designated pMON31155.

E. coli strain JM101 was transformed with pMON31155 for protein expression and protein isolation from inclusion bodies.

The plasmid, pMON31155 containing the gene of (SEQ ID NO:17) encodes the following amino acid sequence: Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gin Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gin Ala Gln Glu Gln Gln Gly Gly Gly Ser Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Ala Pro Leu (SEQ ID Examples 4-10 Examples 4-10 are constructed in a similar manner as SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US9615941 described in Example 3 using the Primers indicated in Table 4. The resulting proteins have new N-terminus and C-terminus as indicated in Table 4.

templat~e primer pair new gene pmoN# flew

PMON#

N/-trinus flew protei- -Q ID NO:9 6 9stop (SEQ ID NO:10) sYntanl 9 1start (SEQ ID NO:11(I 9sOtOP(SEQ ID NO:12) sYntanl 101srtart(SEQ ID NO:13)/ ID NO:14) sYfltan3 3 5srtart (SEQ ID NO:7)/ 3 4stop(SEQ ID NO:8) sYntan3 7 0start (SEQ ID NO:9)/ 6 9 stop(SEQ ID NO:10) sYfltan3 9 1srtarc (SEQ ID NO:11(/ 9 Ostop(SEQ ID NO:12) sYntan3 101start (SEQ ID NO:13)/ PMON3 1156 (SEQ ID NO;18) PMON3 1157 (SEQ ID NO:19) PMON3 1158 (SEQ ID NQ:20) PMON3 1159 (SEQ ID NO:21) PMON3 1160 (SEQ ID NO:22) PMON3 1161 (SEQ ID NO:23) 7 0/69 9 1/90 101/100 35/34 70/69 9 1/90 PMON3 1156 .pep (SEQ ID NO:26} PM0N31157 .pep (SEQ ID NO:27) PmON31 158 Pep (SEQ ID NO:28) PM0N31159pep (SEQ ID NO:29) PMON3 1160 .Pep (SEQ ID PM0N31161 .pep (SEQ ID NO:31) PMON3 1162 .pap 100 stOP(SEQ) ID NO:14) (SEQ 1D NO:24)10/0 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.

SUBSTITUTE SHEET (RULE 26) WO 97/12979 PCT/US96/15941 71 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: G. D. Searle Co.

STREET: P. O. Box 5110 CITY: Chicago STATE: Illinois COUNTRY: United States of POSTAL CODE (ZIP): America TELEPHONE: (708)470-6501 TELEFAX: (704)470-6881 NAME: Monsanto Company STREET: 800 North Lindbergh Boulevard CITY: St. Louis STATE: Missouri COUNTRY: United States of America POSTAL CODE (ZIP): 63167 TELEPHONE: (314)647-3131 TELEFAX: (314)694-5435 (ii) TITLE OF INVENTION: IL-3 RECEPTOR

AGONISTS

(iii) NUMBER OF SEQUENCES: 39 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM:

PC-DOS/MS-DOS

SOFTWARE: PatentIn Release Version #1.30 (EPO) CURRENT APPLICATION

DATA:

APPLICATION NUMBER: US 2909 (vi) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 60/004,835 FILING DATE: 05-OCT-1995 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 133 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Modified-site WO 97/12979 PCT/US96/15941 72 LOCATION:17 OTHER INFORMATION:/note= "Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gin, or Arg;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:18 OTHER INFORMATION:/note= "Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gin;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:19 OTHER INFORMATION:/note= "Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 20 is Ile, Cys, Gin, Glu, Arg, Pro, or Ala;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:21 OTHER INFORMATION:/note= "Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gin, Asn, Thr, Ser or Val;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:22 OTHER INFORMATION:/note= "Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gin, Leu, Val or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:23 OTHER INFORMATION:/note= "Xaa at position 23 is Ile, Val, Ala, Gly, Trp, Lys, Phe, Leu, Ser, or Arg;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:24 OTHER INFORMATION:/note= "Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, Leu;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 25 is Thr, His, Gly, Gin, Arg, Pro, or Ala;" wn Q711"aa VYv PCT/US96/15941 73 (ix) FEATURE: NAME/KEY: Modified-site LOCATION:26 OTHER INFORMATION:/note= "Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, Trp;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:27 OTHER INFORMATION:/note= "Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:28 OTHER INFORMATION:/note= "Xaa at position 28 is Lys, Arg, Leu, Gin, Gly, Pro, Val or Trp;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:29 OTHER INFORMATION:/note= "Xaa at position 29 is Gin, Asn, Leu, Pro, Arg, or Val;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gin, Ser, Leu, or (ix) FEATURE: NAME/KEY: Modified-site LOCATION:31 OTHER INFORMATION:/note= "Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gin;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:32 OTHER INFORMATION:/note= "Xaa at position 32 is Leu, Val, Arg, Gin, Asn, Gly, Ala, or Glu;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:33 OTHER INFORMATION:/note= "Xaa at position 33 is Pro, Leu, Gin, Ala, Thr, or Glu;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:34 OTHER INFORMATION:/note= "Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gin, Thr, Arg, Ala, Phe, Ile ti ur met; WO 97/12979 PCT/US96/15941 74 (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gin, or Val;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:36 OTHER INFORMATION:/note= "Xaa at position 36 is Asp, Leu, or Val;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:37 OTHER INFORMATION:/note= "Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:38 OTHER INFORMATION:/note= or Ala;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= Trp, or Arg;" "Xaa at position 38 is Asn, "Xaa at position 40 is Leu, (ix) FEATURE: NAME/KEY: Modified-site LOCATION:41 OTHER INFORMATION:/note= "Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or pro;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:42 OTHER INFORMATION:/note= "Xaa at Asp, Ser, Cys, Asn, Lys, Thr, Ile, Met or Ala;" position 42 is Gly, Leu, Val, Glu, Phe, Tyr, (ix) FEATURE: NAME/KEY: Modified-site LOCATION:43 OTHER INFORMATION:/note= "Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gin, Arg, Thr, Gly, or Ser;" (ix) FEATURE: NAME/KEY: Modified-site WO 97/12979 PCT/US96/15941 LOCATION:44 OTHER INFORMATION:/note= "Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 45 is Gin, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:46 OTHER INFORMATION:/note= "Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:47 OTHER INFORMATION:/note= "Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:48 OTHER INFORMATION:/note= "Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:49 OTHER INFORMATION:/note= "Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gin;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:51 OTHER INFORMATION:/note= "Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or his;" WO 97/12979 PCT/US96/15941 76 (ix) FEATURE: NAME/KEY: Modified-site LOCATION:52 OTHER INFORMATION:/note= "Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:53 OTHER INFORMATION:/note= "Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or (ix) FEATURE: NAME/KEY: Modified-site LOCATION:54 OTHER INFORMATION:/note= "Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gin, Asn, Lys, His, Ala or Leu; (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:56 OTHER INFORMATION:/note= "Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:57 OTHER INFORMATION:/note= "Xaa at position 57 is Asn or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:58 OTHER INFORMATION:/note= "Xaa at position 58 is Leu, Ser, Asp, Arg, Gin, Val, or Cys;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:59 OTHER INFORMATION:/note= "Xaa at position 59 is Glu, Tyr, His, Leu, Pro, or Arg;" (ix) FEATURE: NAME/KEY: Modified-site WO 97/12979 PCT/US96/15941 77 OTHER INFORMATION:/note= "Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:61 OTHER INFORMATION:/note= "Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:62 OTHER INFORMATION:/note= "Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:63 OTHER INFORMATION:/note= "Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:64 OTHER INFORMATION:/note= "Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:66 OTHER INFORMATION:/note= "Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:67 OTHER INFORMATION:/note= "Xaa at postion 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:68 OTHER INFORMATION:/note= "Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;" (ix) FEATURE: NAME/KEY: Modified-site WO 97/12979 PCT/US96/15941 78 LOCATION:69 OTHER INFORMATION:/note= "Xaa at position 69 is Gin, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 70 is Asn, Leu, Val, Trp, pro, or Ala;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:71 OTHER INFORMATION:/note= "Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gin, Trp, or Asn;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:72 OTHER INFORMATION:/note= "Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:73 OTHER INFORMATION:/note= "Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:74 OTHER INFORMATION:/note= "Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gin, or Leu;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:76 OTHER INFORMATION:/note= "Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or (ix) FEATURE: NAME/KEY: Modified-site LOCATION:77 OTHER INFORMATION:/note= "Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;" (ix) FEATURE: NAME/KEY: Modified-site WO 97/12979 PCT/US96/15941 79 LOCATION:78 OTHER INFORMATION:/note= "Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:79 OTHER INFORMATION:/note= "Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa position at 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:81 OTHER INFORMATION:/note= "Xaa at position 81 is Leu, Gin, Gly, Ala, Trp, Arg, Val, or Lys;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:82 OTHER INFORMATION:/note= "Xaa at position 82 is Leu, Gin, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:83 OTHER INFORMATION:/note= "Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:84 OTHER INFORMATION:/note= "Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 85 is Leu, Asn, Val, or Gin;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:86 OTHER INFORMATION:/note= "Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;" WO 97/12979 PCTIUS96/15941 (ix) FEATURE: NAME/KEY: Modified-site LOCATION:87 OTHER INFORMATION:/note= "Xaa at position 87 is Leu, Ser, Trp, or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:88 OTHER INFORMATION:/note= "Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:89 OTHER INFORMATION:/note= "Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or ,Met;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:91 OTHER INFORMATION:/note= "Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:92 OTHER INFORMATION:/note= "Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:93 OTHER INFORMATION:/note= "Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:94 OTHER INFORMATION:/note= "Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gin, Lys, His, Ala, or Pro;" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/note= "Xaa at position 95 is His, Gin, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, WO 97/12979 PCT/US96/15941 81 Ile, or Tyr;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:96 OTHER INFORMATION:/note= "Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:97 OTHER INFORMATION:/note= "Xaa at position 97 is Ile, Val, Lys, Ala, or Asn;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:98 OTHER INFORMATION:/note= "Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gin, Ser, Phe, Met, Val, Lys, Arg, Tyr, or Pro;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:99 OTHER INFORMATION:/note= "Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gin, Gly, Ser, Phe, or His;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:100 OTHER INFORMATION:/note= "Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gin, or (ix) FEATURE: NAME/KEY: Modified-site LOCATION:101 OTHER INFORMATION:/note= "Xaa at position is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gin;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:102 OTHER INFORMATION:/note= "Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:103 OTHER INFORMATION:/note= "Xaa at position 103 is Asp, or Ser;" WO 97/12979 PCT/US96/15941 82 (ix) FEATURE: NAME/KEY: Modified-site LOCATION:104 OTHER INFORMATION:/note= "Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gin, Lys, Ala, Phe, or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:105 OTHER INFORMATION:/note= "Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gin, Tyr, Leu, Lys, Ile, Asp, or His;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:106 OTHER INFORMATION:/note= "Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:108 OTHER INFORMATION:/note= "Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gin, His, Ser, Ala or Pro;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:109 OTHER INFORMATION:/note= "Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:110 OTHER INFORMATION:/note= "Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gin, His, Glu, Ser, or Trp;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:111 OTHER INFORMATION:/note= "Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:112 OTHER INFORMATION:/note= "Xaa at position 112 is Thr, Val, Gin, Tyr, Glu, His, Ser, or Phe;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:113 OTHER INFORMATION:/note= "Xaa at position 113 is Phe, WO 97/12979 PCT/US96/15941 83 Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:114 OTHER INFORMATION:/note= "Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:115 OTHER INFORMATION:/note= "Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:116 OTHER INFORMATION:/note= "Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gin, or Ile;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:117 OTHER INFORMATION:/note= "Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:118 OTHER INFORMATION:/note= "Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:119 OTHER INFORMATION:/note= "Xaa at position 119 is Glu, Ser, Lys, Pro, leu, Thr, Tyr, or Arg;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:120 OTHER INFORMATION:/note= "Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gin;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:121 OTHER INFORMATION:/note= "Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;" (ix) FEATURE: WO 97/12979 PCT/US96/15941 84 NAME/KEY: Modified-site LOCATION:122 OTHER INFORMATION:/note= "Xaa at position 122 is Gin, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:123 OTHER INFORMATION:/note= "Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Ala Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 70 75 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 90 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gin Gin Thr Thr Leu 115 120 125 Ser Leu Ala Ile Phe 130 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 97/12979 PCT/US96/15941 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTTACCCTTG AGCAAGCGCA GGAACAACAG GGTGGTGGCT CTAACTGCTC TATAATGAT 59 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 56 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CGATCATTAT AGAGCAGTTA GAGCCACCAC CCTGTTGTTC CTGCGCTTGC TCAAGG 56 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" WO 97/12979 PCT/US96/15941 86 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTTACCCTTG AGCAAGCGCA GGAACAACAG GGTGGTGGCT CTGGCGGTGG

CAGCGGCGGC

GGTTCTAACT GCTCTATAAT INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CGATCATTAT AGAGCAGTTA GAACCGCCGC CGCTGCCACC GCCAGAGCCA

CCACCCTGTT

GTTCCTGCGC TTGCTCAAGG INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GATCGACCAT GGCTCTGGAC CCGAACAACC

TC

32 INFORMATION FOR SEQ ID NO: 8: WO 97/12979 PCT/US96/15941 87 SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CGATCGAAGC TTATTACAAA GGTGCAGGTG

GTCTCT

36 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GATCGACCAT GGCTAATGCA TCAGGTATTG

AG

32 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGATCGAAGC TTATTATTCT AAGTTCTTGA

CAGCCC

36 WO 97/12979 PCT/US96/15941 88 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GATCGACCAT GGCTGCACCC

TCTCGACATC

INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CGATCGAAGC TTATTAGGCC GTGGCAGAGG

GCAGAC

36 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: WO 97/12979 PCT/US96/15941 89 GATCGACCAT GGCTGCAGGT GACTGGCAAG

AA

32 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CGATCGAAGC TTATTACTTG ATGATGATTG

GATGTC

36 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 688 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CATGGCTAAC TGCTCTATAA TGATCGATGA AATTATACAT

CACTTAAAGA

ACCTTTGCTG GACCCGAACA ACCTCAATGA CGAAGACGTC

TCTATCCTGA

120 CCTTCGACTT CCAAACCTGG AGAGCTTCGT AAGGGCTGTC

AAGAACTTAG

180 AGGTATTGAG GCAATTCTTC GTAATCTCCA ACCATGTCTG

CCCTCTGCCA

240 CTCTCGACAT CCAATCATCA TCAAGGCAGG TGACTGGCAA

GAATTCCGGG

300

GACCACCTGC

TGGACCGAAA

AAAATGCATC

CGGCCGCACC

AAAAACTGAC

WO 97/12979 PCTIUS96/15941 GTTCTATCTG GTTACCCTTG AGCAAGCGCA GGAACAACAG

GGTGGTGGCT

360 TATAATGATC GATGAAATTA TACATCACTT AAAGAGACCA

CCTGCACCTT

420 GAACAACCTC AATGACGAG ACGTCTCTAT CCTGATGGAC

CGAAACCTTC

480 CCTGGAGAGC TTCGTAAGGG CTGTCAAGA CTTAGAAAAT

GCATCAGGTA

540 TCTTCGTAAT CTCCAACCAT GTCTGCCCTC TGCCACGGCC

GCACCCTCTC

600 CATCATCAAG GCAGGTGACT GGCAAGAATT CCGGGAAAA

CTGACGTTCT

660 CCTTGAGCAA GCGCAGGAAC

AACAGTAC

688 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 712 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

CTAACTGCTC

TGCTGGACCC

GACTTCCAAA

TTGAGGCAAT

GACATCCAAT

ATCTGGTTAC

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CATGGCTA-AC TGCTCTATAA TGATCGATGA AATTATACAT

CACTTAAAGA

ACCTTTGCTG GACCCGAACA ACCTCAATGA CGAAGACGTC

TCTATCCTGA

120 CCTTCGACTT CCAAACCTGG AGAGCTTCGT AAGGGCTGTC

AAGAACTTAG

180 AGGTATTGAG GCAATTCTTC GTAATCTCCA ACCATGTCTG

CCCTCTGCCA

240 CTCTCGACAT CCAATCATCA TCAAGGCAGG TGACTGGCAA

GAATTCCGGG

300 GTTCTATCTG GTTACCCTTG AGCAAGCGCA GGAACAACAG

GGTGGTGGCT

360

GACCACCTGC

TGGACCGAAA

AAAATGCATC

CGGCCGCACC

AAAAACTGAC

CTGGCGGTGG

WO 97/12979 PCT/US96/15941 CAGCGGCGGC GGTTCTAACT GCTCTATAAT GATCGATGAA ATTATAcATC 420 ACCACCTGCA CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT 480 GGACCGAAAC CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA 540 AAATGCATCA GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC 600 GGCCGCACCC TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG 660 AAAACTGACG TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT 712 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 357 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

ACTTAAAGAG

CTATCCTGAT

AGAACTTAGA

CCTCTGCCAC

AATTCCGGGA

AC

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: ATGGCTCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA 120 GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC 180 TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA 240 TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGG GTGGTGGCTC 300 ATAATGATCG ATGAAATTAT ACATCACTTA AAGAGACCAC CTGCACCTTT 357 INFORMATION FOR SEQ ID NO: 18:

GGAGCGAAAC

AAATGCATCA

GGCCGCACCC

AAAACTGACG

TAACTGCTCT

GTAATAA

WO 97/12979 PCT/US96/15941 92 SEQUENCE CHARACTERISTICS: LENGTH: 357 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: ATGGCTAATG CATCAGGTAT TGAGGCAATT CTTCGTAATC TCCAACCATG GCCACGGCCG CACCCTCTCG ACATCCAATC ATCATCAAGG CAGGTGACTG 120 CGGGAAAAAC TGACGTTCTA TCTGGTTACC CTTGAGCAAG CGCAGGAACA 180 GGCTCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG 240 CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT 300 CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA 357 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 357 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

TCTGCCCTCT

GCAAGAATTC

ACAGGGTGGT

ACCACCTGCA

GGAGCGAAAC

ATAATAA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: ATGGCTGCAC CCTCTCGACA TCCAATCATC ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA CGTTCTATCT GGTTACCCTT GAGCAAGCGC AGGAACAACA GGGTGGTGGC 120 WO 97/12979 PCTIUS96/15941 TCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC

ACCTGCACCT

180 TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA

CCGAAACCTT

240 CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA

TGCATCAGGT

300 ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC

CTAATA

357 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 357 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATGGCTGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA

CGTTCTATCT

GAGCAAGCGC AGGAACAACA GGGTGGTGGC TCTAACTGCT

CTATAATGAT

120 ATACATCACT TAAAGAGACC ACCTGCACCT TTGCTGGACC

CGAACAACCT

180 GACGTCTCTA TCCTGATGGA CCGAAACCTT CGACTTCCAA

ACCTGGAGAG

240 GCTGTCAAGA ACTTAGAAAA TGCATCAGGT ATTGAGGCAA

TTCTTCGTAA

300 TGTCTGCCCT CTGCCACGGC CGCACCCTCT CGACATCCAA

TCATCATCAA

357 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 381 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

GGTTACCCTT

CGATGAAATT

CAATGACGAA

CTTCGTAAGG

TCTCCAACCA

GTAATAA

WO 97/12979 PCTIUS96/15941 94 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: ATGGCTCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT

CTATCCTGAT

CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA

AGAACTTAGA

120 GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC

CCTCTGCCAC

180 TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG

AATTCCGGGA

240 TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGG

GTGGTGGCTC

300 AGCGGCGGCG GTTCTAACTG CTCTATAATG ATCGATGAAA

TTATACATCA

360 CCACCTGCAC CTTTGTAATA A 381 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 381 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

GGAGCGAAAC

AAATGCATCA

GGCCGCACCC

AAAACTGACG

TGGCGGTGGC

CTTAAAGAGA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: ATGGCTAATG CATCAGGTAT TGAGGCAATT CTTCGTAATC TCCAACCATG

TCTGCCCTCT

GCCACGGCCG CACCCTCTCG ACATCCAATC ATCATCAAGG CAGGTGACTG

GCAAGAATTC

120 CGGGAAAAAC TGACGTTCTA TCTGGTTACC CTTGAGCAAG CGCAGGAACA ACAGGGTGGT 180 WO 97/12979 PCT/US96/15941 GGCTCTGGCG GTGGCAGCGG CGGCGGTTCT AACTGCTCTA TAATGATCGA

TGAAATTATA

240 CATCACTTAA AGAGACCACC TGCACCTTTG CTGGACCCGA ACAACCTCAA

TGACGAAGAC

300 GTCTCTATCC TGATGGACCG AAACCTTCGA CTTCCAAACC TGGAGAGCTT

CGTAAGGGCT

360 GTCAAGAACT TAGAATAATA

A

381 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 381 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: ATGGCTGCAC CCTCTCGACA TCCAATCATC ATCAAGGCAG GTGACTGGCA GAAAAACTGA CGTTCTATCT GGTTACCCTT GAGCAAGCGC

AGGAACAACA

120 TCTGGCGGTG GCAGCGGCGG CGGTTCTAAC TGCTCTATAA

TGATCGATGA

180 CACTTAAAGA GACCACCTGC ACCTTTGCTG GACCCGAACA ACCTCAATGA 240 TCTATCCTGA TGGACCGAAA CCTTCGACTT CCAAACCTGG

AGAGCTTCGT

300 AAGAACTTAG AAAATGCATC AGGTATTGAG GCAATTCTTC

GTAATCTCCA

360 CCCTCTGCCA CGGCCTAATA A 381 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 381 base pairs

AGAATTCCGG

GGGTGGTGGC

AATTATACAT

CGAAGACGTC

AAGGGCTGTC

ACCATGTCTG

WO 97/12979 PCT/US96/15941 96 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: ATGGCTGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA

CGTTCTATCT

GAGCAAGCGC AGGAACAACA GGGTGGTGGC TCTGGCGGTG GCAGCGGCGG 120 TGCTCTATAA TGATCGATGA AATTATACAT CACTTAAAGA GACCACCTGC 180 GACCCGAACA ACCTCAATGA CGAAGACGTC TCTATCCTGA TGGACCGAAA 240 CCAAACCTGG AGAGCTTCGT AAGGGCTGTC AAGAACTTAG AAAATGCATC 300 GCAATTCTTC GTAATCTCCA ACCATGTCTG CCCTCTGCCA CGGCCGCACC 360 CCAATCATCA TCAAGTAATA A 381 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 115 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein

GGTTACCCTT

CGGTTCTAAC

ACCTTTGCTG

CCTTCGACTT

AGGTATTGAG

CTCTCGACAT

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp 1 5 10 Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys 25 WO 97/12979 PCT[US96/15941 97 Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gin 40 Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile 55 Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr 70 75 Leu Val Thr Leu Glu Gin Ala Gin Glu Gin Gin Gly Gly Gly Ser Asn 90 Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro 100 105 110 Ala Pro Leu 115 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 115 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gin Pro Cys Leu 1 5 10 Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala 25 Gly Asp Trp Gin Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr 40 Leu Glu Gin Ala Gin Glu Gin Gin Gly Gly Gly Ser Asn Cys Ser Ile 55 Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Ala Pro Leu 70 75 Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp 90 Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys 100 105 110 WO 97/12979 PCT/US96/15941 98 Asn Leu Glu 115 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 115 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gin Glu 1 5 10 Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gin Ala Gin 25 Glu Gin Gin Gly Gly Gly Ser Asn Cys Ser Ile Met Ile Asp Glu Ile 40 Ile His His Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn 55 Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu 70 75 Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala 90 Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser 100 105 110 Ala Thr Ala 115 INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 115 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 97/12979 PCT/US96/15941 99 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Ala Gly Asp Trp Gin Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val 1 5 10 Thr Leu Glu Gin Ala Gin Glu Gin Gin Gly Gly Gly Ser Asn Cys Ser 25 Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Ala Pro 40 Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met 55 Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val Arg Ala Val 70 75 Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu 90 Gin Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile 100 105 110 Ile Ile Lys 115 INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 123 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp 1 5 10 Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys 25 Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gin 40 Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile 55 WO 97/12979 PCT/US96/15941 100 Ile Lys Ala Gly Asp Trp Gin Glu Phe Arg Glu Lys Leu Thr Phe Tyr 70 75 Leu Val Thr Leu Glu Gin Ala Gin Glu Gin Gin Gly Gly Gly Ser Gly 90 Gly Gly Ser Gly Gly Gly Ser Asn Cys Ser Ile Met Ile Asp Glu Ile 100 105 110 Ile His His Leu Lys Arg Pro Pro Ala Pro Leu 115 120 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 123 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Asn Ala Ser Gly 1 Pro Gly Leu Gly Leu Glu Glu Ser Asp Glu Gly Lys Asp Ser Ala Trp Gin Gly Arg Val Phe Thr Gin Ala Ser Pro Ser 100 Val Ile 5 Ala Glu Gin Asn Pro Ile Ala Phe Glu Cys 70 Ala Leu Pro Arg Gin 55 Ser Pro Met Ser Glu 40 Gin Ile Leu Asp Arg 25 Lys Gly Met Leu Arg 105 10 His Leu Gly Ile Asp 90 Asn Pro Thr Gly Asp 75 Pro Leu Ile Phe Ser Glu Asn Arg Ile Tyr Gly Ile Asn Leu Ile Leu Gly Ile Leu Pro 110 Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Lys Val Gly His Asn Asn Ala Thr Ser His Asp Leu Arg Ala Val Lys Asn Leu Glu 120 115 INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: WO 97/12979 101 LENGTH: 123 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein PCTIUS96/15941 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Phe Glu Cys Ala Leu Ala Asn Arg Gin Ser Pro Met Val Leu Glu Gin Ile Leu Asp Lys Gln Lys Gly Met Leu Arg Asn 100 Leu Gly Ile Asp Asn Leu Thr Gly Asp Pro 70 Leu Glu Phe Ser Glu 55 Asn Arg Asn Tyr Gly 40 Ile Asn Leu Ala Leu 25 Gly Ile Leu Pro Ser 105 Thr Ser His Asp 75 Leu Ile Gly Leu Gly Leu Glu Glu Glu Asp Glu Gly Lys Asp Ser Ala Trp Gin Gly Arg Val Phe Ile 110 Gin Ala Ser Pro Ser Val Leu Glu Gin Asn Pro Ile Arg Arg Pro Cys Leu Pro Ser Ala Thr Ala 120 INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 123 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Ala Gly Asp Trp Gin Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val 1 5 10 WO 97/12979 PCT/US96/15941 102 Thr Leu Glu Gin Ala Gin Glu Gin Gin Gly Gly Gly Ser Gly Gly Gly 25 Ser Gly Gly Gly Ser Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His 40 His Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn 55 Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn 70 75 Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly 90 Ile Glu Ala Ile Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr 100 105 110 Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys 115 120 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: Gly Gly Gly Ser Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: WO 97/12979 PCT/US96/15941 103 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ser Gly Gly Ser Gly Gly Ser 1 INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: Glu Phe Gly Asn Met 1 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: WO 97/12979 104 Glu Phe Gly Gly Asn Met 1 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Glu Phe Gly Gly Asn Gly Gly Asn Met 1 INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: Gly Gly Ser Asp Met Ala Gly 1 PCT/US96/15941

Claims

1. A human interleukin-3 receptor agonist polypeptide, comprising a modified interleukin-3 amino acid sequence of the Formula: Ala Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser 1 5 10 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Trp Val Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 Xaa Phe Xaa Xaa Xaa 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 Xaa Xaa Xaa Gin Gin Thr Thr Leu Ser Leu Ala Ile Phe 125 130 (SEQ ID NO:1) WO 97/12979 PCT/US96/15941 wherein Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gin, or Arg; Xaa at position 18 Xaa at position 19 Xaa at position 20 Xaa at position 21 Ser or Val; Xaa at position 22 Val or Gly; Xaa at position 23 Leu, Ser, or Xaa at position 24 Xaa at position 25 Xaa at position 26 Xaa at position 27 Xaa at position 28 Xaa at position 29 Xaa at position 30 Xaa at position 31 Xaa at position 32 Xaa at position 33 Xaa at position 34 Arg, Ala, Phe Xaa at position 35 Xaa at position 36 Xaa at position 37 Xaa at position 38 Xaa at position 40 Xaa at position 41 Xaa at position 42 Val, Glu, Phe Xaa at position 43 Gin, Arg, Thr Xaa at position 44 35 Glu, Asn, Gin is Asn, is Met, is lie, is Asp, His, Phe, Cys, Phe, Leu, lie, Gin, Lys, Ile, Arg, Glu, Arg, Phe, Gly, Arg, Ala, Arg, Ala, Pro, Gly, is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gin, Leu, is lie, Val, Arg; is lie, Gly, is Thr, His, is His, Thr, is Leu, Gly, is Lys, Arg, is Gin, Asn, is Pro, His, is Pro, Asp, is Leu, Val, is Pro, Leu, is Leu, Val, Ile or Met; is Leu, Ala, is Asp, Leu, is Phe, Ser, is Asn, or Al is Leu, Trp, is Asn, Cys, is Gly, Asp, Ala, Gly, Trp, Lys, Phe, or Gin; or Cys; or Ala; Glu, Gin, Asn, Thr, Val, Gly, Phe, Arg, Leu, Leu, Thr, Gly, Arg, Gin, Gly, Arg, Gin, Gly, Thr, Gin, Pro, Gly, Ala, Gin, Ala, Ser, Ser, Arg, Arg, Ser, Gly, Arg, Asp, Arg, Asn, Thr, Lys, Phe, or Leu; Pro, or Ala; Ala, or Trp; or Ala; Pro, Val or Trp; or Val; Gin, Ser, Leu, or Lys; Leu, or Gin; Gly, Ala, or Glu; or Glu; Glu, Gin, Thr, Gly, Asn, Pro, Gin, or Val; or Val; Pro, Trp, a; or Arg; Arg, Leu, Ser, Cys, or Ile; His, Asn, Met, Lys, or Pro; Thr, Leu, Tyr, lie, Met or Ala; is Glu, Asn, Tyr, Leu, Phe, Gly or Ser; is Asp, Ser, Leu, Arg, Lys, Ala or Pro; Asp, Ala, Cys, Thr, Met, Trp, a WO 97/12979 PCT/US96/15941 Xaa at position 4E Trp, Asp, As Xaa at position 4E Lys, His, Al Xaa at position 47 Xaa at position 48 Lys, Thr, Al Xaa at position 49 Xaa at position 50 Ala, Ile, Va Xaa at position 51 Xaa at position 52 Xaa at position 53 Xaa at position 54 Lys, His, Al Xaa at position 55 Xaa at position 56 Thr, Ala, Ty Xaa at position 57 Xaa at position 58 Xaa at position 59 Xaa at position 60 Xaa at position 61 Xaa at position 62 Xaa at position 63 Xaa at position 64 Xaa at position 65 Xaa at position 66 Xaa at position 67 Xaa at position 68 Xaa at position 69 Xaa at position 70 Xaa at position 71 Trp, or Asn; Xaa at position 72 5 is Gin, Pro, Phe, Val, Met, Leu, Thr, Lys, sn, Arg, Ser, Ala, Ile, Glu or His; 6 is Asp, Phe, Ser, Thr, Cys, Tyr, Ile, Val or Gly; is Ile, Gly, Val, Ser, Arg, is Leu, Ser, Cys, Arg, Ile, a, Met, Val or Asn; is Met, Arg, Ala, Gly, Pro, is Glu, Leu, Thr, Asp, Tyr, His, Phe, Met or Gin; is Asn, Arg, Met, Pro, Ser, is Asn, His, Arg, Leu, Gly, is Leu, Thr, Ala, Gly, Glu, is Arg, Asp, Ile, Ser, Val, a or Leu; is Arg, Thr, Val, Ser, Leu, is Pro, Gly, Cys, Ser, Gin, r, Phe, Leu, Val or Lys; is Asn or Gly; is Leu, Ser, Asp, Arg, Gin, is Glu Tyr, His, Leu, Pro, is Ala, Ser, Pro, Tyr, Asn, is Phe, Asn, Glu, Pro, Lys, is Asn His, Val, Arg, Pro, is Arg, Tyr, Trp, Lys, Ser, is Ala, Asn, Pro, Ser, or L is Val, Thr, Pro, His, Leu, is Lys, Ile, Arg, Val, Asn, is Ser, Ala, Phe, Val, Gly, is Leu, Val, Trp, Ser, Ile, is Gin, Ala, Pro, Thr, Glu, is Asn, Leu, Val, Trp, Pro, is Ala, Met, Leu, Pro, Arg, Glu, Asn, Gin, Pro, His, Asn, Lys, Thr, Ser, Pro, Thr, or His; Phe, Glu, His, Asn, or Asp; Ser, or His; or Thr; Lys, Ser or Met; Gin, Asn, or Gly; Glu, Arg, His, Val, or Cys; or Arg; or Thr; Arg, or Ser; rhr, Asp, or Ile; His, Pro, or Val Is; Phe, or Ser; Glu, or Ser; Asn, Ile, Pro or Phe, Thr or His; Arg, Trp, Gly or or Ala; Glu, Thr, Gln. His; Leu; Glu, Thr, Gin. is Ser, Glu, Met, Ala, His, Asn, Arg or Asp; WO 97/12979 PCT/US96/15941 Xaa at position 73 Xaa at position 74 is Ala, Glu, Asp, Leu, Ser, Gly, Thr or Arg; is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gin, or Leu; Xaa at position 76 Xaa at position 77 Xaa at position 78 is Ser, is Ile, is Leu, Xaa at position Xaa at position Xaa at position Xaa at position His, Thr, Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position Xaa at position or Leu; Xaa at position Xaa at position or Pro; Xaa at position Ser, Ala, Xaa at position Xaa at position Xaa at position Glu, Gin, Xaa at position Gly, Ser, Xaa at position 79 is Lys, 80 is Asn, 81 is Leu, 82 is Leu, Ser, Ala, 83 is Pro, 84 is Cys, 85 is Leu, 86 is Pro, 87 is Leu, 88 is Ala, 89 is Thr, 90 is Ala, 91 is Ala, 92 is Pro, 93 is Thr, 94 is Arg, 95 is His, Trp, Phe, 96 is Pro, 97 is Ile, 98 is His, Val, Ser, Ala, Thr, Trp, Gin, Gin, Tyr, Ala, Glu, Asn, Cys, Ser, Lys, Asp, Pro, Pro, Phe, Asp, Ile, Ala, Arg, Ser, Asn, Val, Gly, Lys, Phe, Thr, Gly, Val, Arg, Trp, Arg, Cys, Ser, Ser, Arg, Ser, Ser, Asn, Trp, Glu, Pro, Gly or Asp; Thr, or Leu; Glu, Phe, Gly, or Arg; -Met, Arg, Ile, Gly or Asp; Gly, Thr, Leu, Glu or Arg; Ala, Trp, Arg, Val or Lys; Trp, Arg, Asp, Glu, Asn, Ile, Met or Val; Trp, Arg, or Met; Arg, Met, or Val; or Gin; Ala, or Lys; or Gly; Val, or Trp; Leu, Val, Glu, His, Asn or Ser; Thr, Gly, Asp, Ile or Met; Thr, Phe, Leu, Asp, or His; Ser, Lys, His, Ala, Gly, Ile Asn, Glu, Gin, Pro, Arg, Ile, or Tyr; Lys, Tyr, Gly, Val, Lys, Ala, Ile, Asn, Leu, Pro, Ala, Leu, or Arg; Leu, Val, Gin, Lys, His, Ala, Val, Leu, Gly, Thr, Asn, Lys, Ile, or Thr; or Asn; Asp, Ala, Thr, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; 99 is Ile, Leu, Arg, Asp, Val, Pro, Gin, Phe, or His; 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gin or Pro; WO 97/12979 PCT/US96/15941 109 Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu. Asn, Ser, Ala, Gly, Ile, Leu, or Gin; Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser; Xaa at position 104 is Trp, Val, Cys, Gin, Lys, Ala, Phe, or Gly; Xaa at position 105 is Asn, Pro, Ala, Leu, Lys, Ile, Asp, or His; Tyr, Thr, Met, Pro, Leu, Phe, Ser, Trp, Gin, Tyr, Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gin, His, S< Ala or Pro; Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gin, His, G] Ser, Ala, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gin, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gin, or Ile; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gin; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp or Gly; Xaa at position 122 is Gin, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; LU, wherein from 0 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1- 133) human interleukin-3; wherein from 1 to 14 amino acids WO 97/12979 PCT/US96/15941 110 can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C- terminus of said modified interleukin-3 amino acid sequence; wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;

26-27

51-52 27-28

52-53

87-88 28-29 53-54

88-89 29-30 54-55

89-90 30-31 64-65

90-91 31-32 65-66

91-92 32-33 66-67

92-93 33-34 67-68

93-94 34-35 68-69

94-95 35-36 69-70

95-96 36-37 70-71

96-97 37-38 71-72

97-98 38-39 72-73

98-99 39-40 82-83

99-100 40-41 83-84

100-101 41-42 84-85

101-102 49-50 85-86

102-103 50-51 86-87 or 103-104 respectively; and said interleukin-3 receptor agonist polypeptide can optionally be immediately preceded by (methionine- 1 (alanine-1) or (methionine- 2 alanine- 1 2. The interleukin-3 receptor agonist polypeptide, as recited in claim 1, wherein said linker is selected from the group consisting of; GlyGlyGlySer (SEQ ID NO:2); GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:33); GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:34); SerGlyGlySerGlyGlySer (SEQ ID GluPheGlyAsnMet (SEQ ID NO:36); GluPheGlyGlyAsnMet (SEQ ID NO:37); WO 97/12979 PCT/US96/15941 111 GluPheGlyGlyAsnGlyGlyAsnMet (SEQ ID NO:38); and GlyGlySerAspMetAlaGly (SEQ ID NO:39). 3. The interleukin-3 receptor agonist polypeptide, as recited in claim 1, selected from the group consisting of; Leu Asp Val Asn His Lys Gin His Asn Leu Lys Leu Asn Pro Val Ser Ala Glu Ala Asp Asp Arg Lys Leu Ala Val Cys Pro Ser Phe Ala Ala Asp Arg Lys Leu Pro Leu Gly His Ala Pro Ala Val Cys Pro Ser Phe Pro Phe Gin Glu Pro Asn Asn Gin Gly Thr Ser Ala Ile Val Ile Pro Pro Asn Asn Gin Ile Thr Gly Leu Ser Ser Gly Thr Ser Ala Ile Val Asn Leu Leu Pro Ile Phe Gly Lys Gly Ala Asp Leu Ile Pro Leu Arg Asn Arg Glu Cys Ile Tyr Ser Arg Ile Thr Trp Glu Met Leu Met Ala Leu Leu Asn Leu Lys Leu Asn Pro Glu Ala Gin Gin Ile Leu Asp Val Asn Pro Ala Pro Ala Val Cys Pro Ala Ala Glu Ala Asp Asp Arg Lys Asp Asn Ser Ser Gly Thr Ser Ala Ile Pro Phe Gin Glu Pro Asn Asn Glu Leu Gly Ala Asp Leu Ile Pro Leu Ser Arg Glu Ile Asn Leu Leu Asp Glu Ile Thr Trp Glu Met Leu Arg Arg Glu Gin Ile Asn Arg Glu Ser Arg Arg Glu Glu Gin Ile Ile Asn Asn Leu Arg Leu Glu Pro Cys Asp Trp Leu Glu Ile Met Pro Leu Leu Met Arg Ala Leu Arg Ser Arg Pro Asn Asn Leu Asn Leu His Pro Lys Leu Gin Gly His His Leu Asn Leu Pro Asn Ala Leu Pro Gin Glu Gin Ala Ile Asp Leu Asp Asp Arg Val Lys Asn Leu His Pro Asn Leu Arg Leu Glu Asn Ile Ile Thr Phe Gly Gly Leu Lys Asp Glu Asn Leu Ser Gly Ser Ala Phe Arg Gin Glu Glu Ile Pro Asn Asn Leu Asn Leu Gin Pro Ile Ile Asn Asp Pro Asn Ala Ser Ile Lys Tyr Leu Ser Asn Arg Pro Asp Val Glu Ser Ile Glu Thr Ala Glu Lys Gin Gin Ile His Asn Leu Arg Leu Glu Asn Cys Leu Ile Lys Glu Asp Leu Glu Gly Ile Val Ser Ser Phe Glu Ala Ala Ala Gin Glu Gin Ala Ile Asp (SEQ ID Asn Leu His Pro Lys Leu Gin Gly His His Leu Asn Leu Pro (SEQ ID Ala Gly Val Thr Cys Ser Pro Ala Ser Ile Phe Val Ala Ile (SEQ ID Leu Thr Gly Gly His Leu Asn Asp Pro Asn Ala Ser Pro Ser (SEQ ID Val Ser Ser Phe Glu Ala Ile Leu Val Arg Ile Leu Pro Ser Phe Arg Gin Glu Glu Ile Gin Pro Ile Ile Thr Phe Gly Gly Leu Lys Asp Glu Asn Leu NO:26); Met Ala Arg Arg Glu Gin Ile Cys Ile Tyr Ser Arg Asp Glu Asp Trp Leu Glu Ile Met Pro Leu Leu Met Arg Ala Leu Arg NO:27); Phe Tyr Gly Ser Lys Arg Glu Asp Leu Glu Gly Ile Ala Thr NO:28); Gin Gin Ile Leu Asp Val Asn Leu Asn Pro Val Ser Glu Ala Leu Asp Asp Arg Val Lys Ile Leu Met Val Arg Ala Ile Leu Arg WO 97/12979 PCT/US96/15941 Asn His Lys Gin Ser Ala Asn Leu Lys Leu Gly Glu Pro Asn Asn Ala Glu Ala Gly Lys Glu Leu Gly Ala Ala Val Gly Ile Asn Leu Leu Pro Ile Leu Pro Leu Gly Ile Pro Ala Pro Ala Val Gly Ile Asn Leu Leu Gin Ile Thr Gly Met Leu Ser Ser Gly Thr Gly Ile Asn Arg Glu Pro Cys Ile Ile Phe Tyr Gly Ser Ile Asp (SEQ ID Gly Ile Ala Thr Asp Trp Leu Glu Ser Gly His His Leu Asn Leu Pro (SEQ ID Leu Pro Lys Ala Leu Val Gly Gly Glu Ile NO:29); Glu Ala Ala Ala Gin Glu Gin Ala Gly Gly Leu Lys Asp Glu Asn Leu Ser Ala Gly Asp Thr Leu Gly Ser Ile His Thr Trp Glu Gly His Ala Gin Gin Gly Leu Ala Glu Ala Gly Lys Pro Phe Gin Ser Arg Ser Arg Glu Asn Pro Arg Glu Gin Cys Pro Ile Leu Pro Ser Phe Arg Gin Glu Ser Asn Arg Pro Asp Val Glu Ser Ile Ile Phe Tyr Gly Ser Ile Asp Leu Asp Asp Arg Val Lys Asn Leu Arg Arg Glu Gin Cys Pro Ser Phe Lys Leu Gly Glu Pro Asn Asn Gin Asn His Lys Gin Ser Ala Ile Val Ala Val Gly Ile Asn Leu Leu Pro Leu Pro Leu Gly Ile Pro Leu Arg Gly Thr Gly Ile Asn Arg Glu Cys Gin Ile Thr Gly Met Leu Met Ala Pro Ile Phe Gly Ile Leu Asp Val Cys Ile Tyr Ser Asp Asp Arg Lys Gin Gin Gly Leu Asp Asn Ser Ser Pro Ser Phe Arg Gin Glu Ser Asn Arg Pro Asp Val Glu Ser Ile Glu Thr Ala Arg Glu Gin Cys Pro Ser Phe Ala His Lys Gin Ser Ala Ile Val Ile Pro Leu Gly Ile Pro Leu Arg Leu Ile Thr Gly Met Leu Met Ala Arg Asp Trp Leu Glu Ser Gly His His Leu Asn Leu Pro Asn Ala Leu Pro (SEQ ID NO:31); and Gly Thr Gly Ile Asn Arg Glu Cys Ile Asp Leu Ser His Leu Leu Asn Leu Lys Trp Gin Glu Phe Arg Glu Gin Ala Gin Glu Gly Gly Gly Ser Asn His Leu Lys Arg Pro Asn Asp Glu Asp Val Pro Asn Leu Glu Ser Ala Ser Gly Ile Glu Pro Ser Ala Thr Ala (SEQ ID NO:32). Glu Gin Cys Pro Ser Phe Ala Ala Lys Gin Ser Ala Ile Val Ile Pro Leu Thr Gly Gly Ile Met Pro Leu Leu Met Arg Ala Leu Arg Ser Arg Phe Gly Ile Leu Asp Val Asn His Tyr Ser Asp Asp Arg Lys Leu Pro Leu Gly Glu Pro Asn Asn Gin Ile 4. A nucleic acid molecule, comprising a sequence encoding the interleukin-3 receptor agonist polypeptide of claim 1. A nucleic acid molecule, comprising a sequence encoding the interleukin-3 receptor agonist polypeptide of claim 2. WO 97/12979 PCT/US96/15941 113 6. A nucleic acid molecule, comprising a sequence encoding the interleukin-3 receptor agonist polypeptide of claim 3. 7. A nucleic acid molecule, comprising a sequence encoding the interleukin-3 receptor agonist polypeptide of claim 3, selected from the group consisting of: pMON3 1155. seq 1 51 101 151 201 251 301 351 ATGGCTCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATOCTGAT GGAGCGAAAC CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGG GTGGTGGCTC TAACTGCTCT ATAATGA TCG ATGAAATTAT AOATOAOTTA AAGAGACCAC OTGOAOOTTT GTAATAA (SEQ ID NO:17); .seq 1 51 101 151 201 251 301 351 ATGGCTAATG OATOAGGTAT TGAGGOAATT TCTOCOTCT GCCAOGGCCG CACCOTOTOG CAGGTGACTG GCAAGAATTC CGGGAAAAAC CTTGAGCAAG CGCAGGAACA ACAGGGTGGT GATCGATGAA ATTATACATC ACTTAAAGAG AOOOGAAOAA CCTCAATGAC GAAGACGTCT CTTCGACTTO CAAACCTGGA GAGCTTCGTA ATAATAA (SEQ ID NO:l8); OTT CGTAATC AOATOOAATO TGACGTTCTA GGCTCTAACT AOCAOCTGOA CTATCOTGAT AG GGO TGTC A TCCAACOATG ATCATCAAGG TCTGGTTACC GCTCTATAAT CCTTTGOTGG GGAGCGAAAO AGAACTTAGA pMON3 1157 .seq 1 51 101 151 201 251 301 351 ATGGOTGOAO COTOTOGACA TOCAATCATC ATOAAGGOAG GTGAOTGGOA AGAATTOCGG GAAAAACTGA OGTTOTATOT GGTTACTT GAGCAAGCGC AGGAACAACA GGGTGGTGGO TOTAAOTGOT OTATAATGAT CGATGAA-ATT ATACATOACT TAAAGAGACC ACOTGCAOCT TTGOTGGAOO CGAACAACCT CAATGACGAA GAOGTOTOTA TOOTGATGGA OCGAAACCTT OGAOTTOCAA ACCTGGAGAG OTTOGTAAGG GCTGTCAAGA ACTTAGAAAA TGOATOAGGT ATTGAGGOAA TTOTTOGTAA TOTOOAAOCA TGTOTGCOOT OTGOOAOGGO CTAATAA (SEQ ID NO:19); pMON31158.seq 1 ATGGOTGOAG GTGAOTGGOA AGAATTOCGG GAAAAACTGA CGTTOTATOT 51 GGTTAOOCTT GAGOAAGOGO AGGAACAAOA GGGTGGTGGC TOTAAOTGOT 101 OTATAATGAT OGATGAAATT ATACATOACT TAAAGAGACO ACCTGCAOOT WO 97/12979 PCTIUS96/1 5941 151 201 251 301 351 TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA CCGAAACCTT CGACTTCCAA ACCTGGAGAG CTTCGTAAGG ACTTAGAAAA TGCATCAGGT ATTGAGGCAA TTCTTCGTAA TGTCTGCCCT CTGCCACGGC CGCACCCTCT CGACATCCAA GTAATAA (SEQ ID NO:20); TCCTGATGGA GCTGTCAAGA TCTCCAACCA TCATCATCAA pMON3 1159. seq 1 51 101 151 201 251 301 351 ATGGCTCTGG OGAGO GIAAC AGAACTTAGA CCATGTCTGC CAAGGCAGGT TTACCCTTGA AGCGGCGGCG CTTAAAGAGA ACCCGAACAA CTTCGACTTC AAAT G CATC A CCTCTGCCAC GACTGGCAAG GCAAGC OCAG GTTCTAACTG CCACCTGCAC CCTCAATGAC C AAAC CTG GA GGTATTGAGG GGCCGCACCC AATTCCGGGA GAACAACAGG CTCTATAATG C TTTGTAATA GAAGACGTCT CTATCCTGAT GAGCTTCGTA AGGGCTGTCA CAATTCTTCG TAATCTCCAA TCTCGACATC CAATCATCAT AAAACTGACG TTCTATCTGG GTGGTGGCTC TGGCGGTGGC ATCGATGAAA TTATACATCA A (SEQ ID NO:21); pMON3116O.seq 1 51 101 151 201 251 301 351 ATGGCTAATG TCTGCCCTCT CAGGTGACTG CTTGAGCAAG CGGCGGTTCT AGAGACCACC GTCTCTATCC CGTAAGGGCT CATCAGGTAT GCCACGGCCG GCAAGAATTC CGCAGGAACA AACTGCTCTA TGCACCTTTG TGATGGACCG GTCAAGAACT TGAGGCAATT C AC C CTCT C C GGGAAAAAC ACAGGGTGGT TAATGATC GA CTGGACCCGA AAACCTTCGA TAGAATAATA CTTCGTAATC TCCAACCATG ACATCCAATC ATCATCAAGG TGACGTTCTA TCTGGTTACC GGCTCTGGCG GTGGCAGCGG TGAAATTATA CATCACTTAA ACAACCTCAA TGACGAAGAC CTTCCAAACC TGGAGAGCTT A (SEQ ID NO:22); pMON3 1161 .seq 1 51 101 151 201 251 301 351 ATGGCTGCAC AGAATTCCGG AGGAACAACA TGCTC TATAA ACCTTTGCTG TGGAC CGAAA AAGAACTTAG ACCATGTCTG CCTCTCGACA GAAAAACTGA GGGTGGTGGC TGATCGATGA GACCCGAACA CCTTCGACTT AAAATGCATC CCCTCTGCCA TCCAATCATC C GTTCTATCT TCTGGCGGTG AATTATACAT ACCTCAATGA CCAAACCTGG AGGTATTGAG CGGCCTAATA ATCAAGGCAG GTGACTGGCA GGTTACCCTT GAGCAAGCGC GCAGCGGCGG CGGTTCTAAC CACTTAAAGA GACCACCTGC CGAAGACGTC TCTATCCTGA AGAGCTTCGT AAGGGCTGTC GCAATTCTTC GTAATCTCCA A (SEQ ID NO:23); and pMON3 1162. seq 1 51 101 151 201 251 301 ATGGCTGCAG GGTTACCCTT GCAGCGGCGG CACTTAAAGA CGAAGACGTC AGAGCTTCGT GCAATTCTTC GTGACTGGCA GAGCAAGCGC CGGTTCTAAC GACCACCTGC TCTATCCTGA AAGGGCTGTC GTAATCTCCA AGAATTCCGG AGGAACAACA TGCTCTATAA ACCTTTGCTG TGGACCGAAA AAGAACTTAG ACCATGTCTG GAAAAACTGA GGGTGGTGGC TGATCGATGA GACCCGAACA CCTTCGACTT AAAATGCATC CCCTCTGCCA CGTTCTATCT TCTGGCGGTG AATTATACAT ACCTCAATGA CCAAACCTGG AGGTATTGAG CGGCCGCACC 351 CTCTCGACAT CCAATCATCA TCA-ATAATA A (SEQ !D MO:24). 8. A reolicable vector, comprising the nucleic acid molecule of claim 4, 5, 6 or 7. 9. A host-, cell, comprising said vector of claim 8. A- method of oroducing a human interleukin-3 recepto r agonist polypeptide comprising the s -eos of; cult:ivatuing said host cell of claim 9 under conditions suitable for the expcression of said nolvoeutide; and recovering said ooly-eptide. A 7omcosicion, comcris-ing; said poly-peptide of claim 1, 2, 3, or 4; and a charmaceuftcalLy acceptable carr _er. -12. A cocsition, comorising; said oolvceotide of claim 1, 2, 3, or 4; a col!ony scimulating -factor, and a pharmaceuticallv acceoQ.able carrier. 13. A comoositi41on, comprising; said polypeouide o: claim 1, 2, 3, or 4; a colony stimaulating factor selected from the group consisting of; C-M-CSF, G-CSF, C--CS.7 Ser 1 c-mpl ligand, M-CSF, erythrocoietin 7L-4, 7L-3, IL 6, !L- 7, !L-10, 7L-ll, 7L-12, !L-13, !L-15, LIF, f~1t3/_Flk2 ligand, human growtuh hormone, 3-cell growth factor, B-cell differentiation factor, eosinoohil- differentiation faccor and s~em cell factor; and a oharmaceuticallv accepcable carrier. 14. Use of a polypeptidle of claim 1, 2, 3 or 4 for preparing a medicament for stimulating the production of hematopoietic cells in a patient. LUAMNFD '3E7 1 5. Use of a composition of cl aim 11, 12 or 13 for preparing a medicament for stimulating the production of hematopoietic cells in a patient. 16. A mtethod for selactiva ex ,ivo exna~sio~j of szem cell.s, comprisin-g the Steps 0:; (a)sen~rai~stezi Cezlls rom other cells; b)cult-uring sa..Id separatci stemn ceiis with' a selected cucr n~~cr'rS 7 the polv~pepzide of clai-m 1, 2, 3, or 4; and har-vesting- said cu' -red cells. 17. k method for selec:tiv7,e ex vivar ex_-anzion Oz stern cells, comnprisIrg the steps oa:; sepazatjzi strn= cells f'=r or-h e= cells; cu~tur4ig said separated stemt cells with a selec-ed cutu~re ,aediLn corpzisjng :ec-Mposiltion of C a m ;ad har-esting said CulttureCL cells. 18. Use of a polypeptide of claim 1, 2, 3, or 4 for preparing a medicament for treatment of a patient having a hemnatopoietic disorder by removing stem cells; separating stem cells from other cells; culturing said separated stem cells with a selected culture medium comprising the polypeptide of claim 1, 2, 3, or 4; harvesting said cultured cells for use. 19. Use of a composition of claim 11, 12 or 13 for preparing a medicament for treatment of a patient having a hematopoietic disorder by removing stem cells; separating stem cells from other cells; culturing said separated stem cells with a selected culture medium -A7 1 ',omprising the composition of claim 11, 12 or 13; 117 harvesting said cultured cells for use. Use of a hematopoietic protein of claim 1, 2, 3 or 4 for preparing a medicament for human gene therapy by removing stem cells from a patient; separating said stem cells from other cells; culturing said separated stem cells with a selected culture medium comprising the hematopoietic protein of claim 1, 2,3 or 4; introducing DNA into said cultured cells; harvesting said transduced cells for use. 21. Use of a composition of claim 11, 12 or 13 for preparing a medicament for human gene therapy by removing stem cells from a patient; separating said stem cells from other cells; culturing said separated stem cells with a selected medium comprising the composition of claim 11, 12 or 13; introducing DNA into said cultured cells; harvesting said transduced cells for use. I