AU722759B2 - Novel C-MPL receptor agonists - Google Patents

Description

WO 97/12978 PCT/US96/15938 1 NOVEL c-mpl RECEPTOR AGONISTS The present application claims priority under 35 USC §119(e) of United States provisional application Serial No.

60/004,384 filed October 05, 1995.

Field of the Invention The present invention relates to human c-mpl receptor agonists (thrombopoietin) with activity on hematopoietic differentiation and expansion.

Background of the Invention Megakaryocyte (MK) maturation and platelet production has been long thought to be regulated by lineage specific humoral growth factors in a manner similar to cytokines that induce erythrocyte (erythropoietin) and granulocyte (G-CSF) expansion and maturation. Platelets are responsible for the prevention of bleeding in response to vascular injury.

Therefore, platelet production is a vital component of hematopoietic regulation. Patients undergoing chemotherapy or bone marrow transplantation usually experience severely depressed platelet levels (thrombocytopenia) which may result in life threatening bleeding episodes. Several known growth factors and cytokines have been found to stimulate megakaryocytes and platelet production but most are pleiotropic both in vitro and in vivo (IL-3, IL-6, IL-11, SCF). Plasma, serum and urine from thrombocytopenic dogs and humans have been found to contain growth factors that have specific megakarypoietic and thrombopoietic activities distinct from all known cytokines. These factors have been termed Meg-CSF, MK-CSF, megakaryocyte growth and development factor (MGDF), megakaryopoietin, and thrombopoietin but the molecular structure has not been identified until recently.

WO 97/12978 PCT/US96/15938 2 The identification of the thrombopoietic cytokine, c-mpl ligand, originated with the identification of a myeloproliferative leukemia virus (MPLV, Wending et al., Virology 149:242-246, 1986). Mice infected with this virus gave rise to multi-lineage myeloproliferation. Subsequent studies (Souyri et al., Cell 63:1137-1147, 1990) demonstrated that the retrovirus encoded an oncogene (v-mpl) that when fused with viral envelope gene gave rise to a membrane anchored protein that resembles the cytoplasmic domain of the hematopoietic growth factor receptor family. V-mpl was used to probe both human and murine RNA libraries for homologous genes. Clones were identified in both species and termed cmpl (Vigon et al., PNAS USA 89:5640-5644, 1992; Vigon et al., Oncogene 8:2607-2615, 1993). C-mpl is a member of the cytokine receptor super-family with regions of homology to IL3rc, IL4rc, mEPOrc and mGCSFrc. A chimera of the intracellular domain of c-mpl and the extracellular domain of hIL4rc was transfected into a growth factor dependent cell line (BaF3). Once transfected, the cells proliferated in response to hIL4 indicating that the c-mpl cytoplasmic domain was fully sufficient to transduce a proliferative signal (Skoda et al., EMBO J. 12(7):2645-2653, 1993).

Message for c-mpl was found in a number of hematopoietic cell lines using reverse transcriptase polymerase chain reaction (RT PCR) including the pluripotential cell lines TF- 1, Mo-7E, UT-7 and KU812; and erythro/megakaryocytic cell lines HEL, DAMI and K153. Transcripts were also identified in bone marrow, fetal liver, megakaryocytes, platelets and CD34+ enriched cells (Methia et al., Blood 82(5):1395-1401, 1993).

The identification of a putative receptor triggered several investigative teams to search for a naturally occurring ligand for c-mpl. In June of 1994 several WO 97/12978 PCT/US96/15938 3 simultaneous publications reported on a ligand that bound to c-mpl and had megakaryocytopoietic properties (de Sauvage et al., Nature 369:533-539, 1994; Lok et al., Nature 369:565- 568, 1994; Wendling et al., Nature 369:571 574, 1994) and Bartley et al., Cell 77:1117-1124, 1994). The ligand named c-mpl ligand or thrombopoietin is a peptide with a predicted molecular mass of 35,000 kDa. The protein has a two domain structure with an amino-terminal domain (153 amino acids) with homology to erythropoietin and a carboxy-terminus rich in serine, threonine and proline residues which also contains several glycosylation sites. There are two potential arginine cleavage sites resulting in two shorter peptides of kDa and 31 kDA forms both of which are biologically active. There is high inter species homology between human, murine, porcine, canine, rat and rabbit c-mpl ligand and most forms are active on all species tested.

C-mpl ligand has been shown to stimulate the differentiation of CD34+ cells into cells megakaryocyte characteristics. CD34+ cells in the presence of c-mpl ligand underwent endomitosis (Kaushansky et al., Nature 369:568-571, 1994), expressed the megakaryocyte lineage specific cell surface antigen CD41a and had morphology characteristic of megakaryocytes. In vivo administration of c-mpl ligand have given rise to increased circulating platelets in normal mice (Lok et al., Nature 369:565-568, 1994). C-mpl deficient mice generated by gene targeting demonstrated a 85% decrease in circulating platelets and megakaryocytes but had normal amounts of other hematopoietic lineages (Gurney et al., Science 265:1445-1447, 1994). Absolute thrombocytopenia was not observed in these animals indicating that other cytokines may have some activity in expansion of the MK lineage.

Studies to date show that c-mpl ligand is a cytokine with specific activity on the maturation of megakaryocytes WO 97/12978 PCT/US96/15938 4 and in platelet production. Other cytokines have been shown to have activity on megakaryocyte expansion and differentiation, including IL-3, IL-6, IL-11 and c-kit ligand. Recent studies have demonstrated that these cytokines (with the exception of IL-3) act by stimulating the production of c-mpl ligand and do not have megakaryocyte stimulating activity by themselves (Kaushansky et al., PNAS USA 92:3234-3236, 1995).

The ability of c-mpl ligand to stimulate the proliferation and maturation of megakaryocytes and production of platelets indicates that c-mpl ligand may have therapeutic use in restoring circulating platelets to normal amounts in those cases where the number of platelets have been reduced due to diseases or therapeutic treatments such as radiation and/or chemotherapy.

EP 675,201 Al relates to the c-mpl ligand (Megakaryocyte growth and development factor or MGDF), allelic variations of c-mpl ligand and c-mpl ligand attached to water soluble polymers such as polyethylene glycol.

WO 95/21920 provides the murine and human c-mpl ligand and polypeptide fragments thereof. The proteins are useful for in vivo and ex vivo therapy for stimulating platelet production.

WO 95/27732 discloses circularly permuted GM-CSF, G- CSF, IL-2 and IL-4 and fusions with Pseudomonas exotoxin.

A previously published abstract (Eaton et al., Blood 84(10) Suppl. abstract 948, 1994) reported c-DNA for an alternative splice form of c-mpl ligand identified in man, dog and mouse. The encoded protein has 4 amino deletion at position aall2-115. Although this molecule showed no activity WO 97/12978 PCT/US96/15938 in their bioassays, mRNA for this variant was found to be abundant in all three species indicating that it may be a naturally occurring alternative form of c-mpl ligand.

Rearrangement of Protein Seuences 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; Schimming et al., Eur. J. Biochem. 204: 13-19, 1992; Yamiuchi and Minamikawa, FEBS Lett. 260:127-130, 1991, MacGregor 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 WO 97/12978 PCT/US96/15938 6 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 c-helix (interleukin-4; Kreitman et al., Cytokine 7:311-318, 1995), P -sheet (interleukin-1; 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 dihydrofolate reductase Zhang et al., Biochemistry 32:12311-12318 (1993); Zhang et al., Nature Struct. Biol. 1:434-438 (1995) Buchwalder et al., Biochemistry 31:1621-1630 (1994); Protasova et al., Prot. Eng. 7:1373-1377 (1995) Mullins et al., J. Am. Chem. Soc.

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) ribonuclease T1 Bacillus P-glucanse WO 97/12978 WO 9712978PCTIUS96/15938 aspartate transcarbamoylase phosphoribosyl anthranilate isomerase peps in/peps inogen Yang Schachnan, Proc. Natl1. Acad.

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

3 :24 9-25 8 (19 Lin et al., Protein Science 4:159- 166 (1995) Vignais et al. Protein Science 4: 99 4-10 00 (19 glyceraldehyde-3 phosphate dehydrogenase ornithine decarboxylase yeast phosphoglycerate dehydrogenase Enzyme Inhibitor basic pancreatic trypsin inhibitor Li Cof fino, Mol. Cell. Biol.

13: 23 77- 23 83 (1993) Ritco-vonsovici et al., Biochemistry 34: 16543 -1655 1 (19 Goldenberg Creighton, J. Mol.

Biol. 165:407-413 (1983) Cytokines interleukin-13 Horlick et al., Protein Eng. 5:427- 431 (1992) Kreitman et al., Cytokine 7:311- 318 (1995) interleukin-4 Tyrosine Kinase WO 97/12978 PCT/US96/15938 8 Recognition Domain a-spectrin SH3 Viguera, et al., J.

domain Mol. Biol. 247:670-681 (1995) Transmembrane Protein omp A Koebnik Kramer, J. Mol. Biol.

250:617-626 (1995) Chimeric Protein interleukin-4- Kreitman et al., Proc. Natl. Acad.

Pseudomonas Sci. U.S.A. 91:6889-6893 (1994).

exotoxin fusion molecule 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 transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ornithine decarboxylase, omp A, yeast 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 Tl, Bacillus 3 -glucanase, interleukin-l, 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 fusion molecule (Kreitman WO 97/12978 PCT/US96/15938 9 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 P-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.

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 Ctermini 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 WO 97/12978 PCT/US96/15938 reductase; only the 3-residue linker produced protein in good yield. More systematic studies of linker length and composition have not been reported.

WO 97/12978 WO 9712978PCTIUS96/15938 SUMMARY OF THE IUVENTION The present invention relates to novel c-mpl receptor agonists of the following formulas: 1. A c-mpl receptor agonist of the Formula: S erProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer 1 5 10 HisValLeuHisSerArgLeuSerGlnCysProGluValHi sProLeuProThrPro 25 30 ValLeuLeuProAlaValAspPheSerLeuGlyGluTrpLySThrGlniMetGluGlu 45 50 ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla 65 70 AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly 85 90 GlnValArgLeuLeuLeuGlyAlaLeuG lnSerLeuLeuGlyThrGlaXaaXaaXaa 100 105 110 XaaGlyArgThrThrAlaHisLysAspProAsnAlal ePheLeuSerPheGlnHis 115 120 125 130 LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal 135 140 145 150 ArgArgAlaProProThrThrAlaVal ProSerArgThrSerLeuVa lLeuThrLeu 155 160 165 170 WO 97/12978 WO 9712978PCTIUS96/15938 12 AsnG luLeuProAsnArgThrS erGlyLeuLeuGluThrAsnPheThrAlaSerAla 175 180 185 190 ArgThrThrGlySerGlyLeuLeuLysTrpGlnGlnGlyPheArgAlaLysllePro 195 200 205 GlyLeuLeuAsnGlnThrSerArgSerLeUASpGlnhl eProGlyTyrLeuAsnArg 210 215 220 225 IleHisGluLeuLeuAsnGlyThrArgGlyLeuPheProGlyProSerArgArgThr 230 235 240 245 LeuGlyAlaProAspI leSerSerGlyThrSerAspThrGlySerLeuProProAsn 250 255 260 265 LeuG inProG lyTyrS erProS erProThrHi sProProThrGlyG lnTyrThrLeu 270 275 280 285 PheProLeuProProThrLeuProThrProValVa iGinLeuHisProLeuLeuPro 290 295 300 AspProSerAlaProThrProThrProThrS erProLeuLeuAsnThrSerTyrThr 305 310 315 320 HisSerGlnAsnLeuSerGlnGluGly (SEQ ID NO:1) 325 330 332 wherein; Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or Met; xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; WO 97/12978 PCT/US96/15938 13 Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn; wherein from 1 to 179 amino acids can be deleted from the Cterminus; 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 51-52 108-109 27-28 52-53 109-110 28-29 53-54 110-111 29-30 54-55 111-112 30-31 55-56 112-113 32-33 56-57 113-114 33-34 57-58 114-115 34-35 58-59 115-116 36-37 59-60 116-117 37-38 78-79 117-118 38-39 79-80 118-119 40-41 80-81 119-120 41-42 81-82 120-121 42-43 82-83 121-122 43-44 83-84 122-123 44-45 84-85 123-124 46-47 85-86 124-125 47-48 86-87 125-126 48-49 87-88 126-127 50-51 88-89 or 127-128; and additionally said c-mpl receptor agonist can be immediately preceded by (methionine- 1 (alanine 1) or (methionine 2 alanine- 1 2. A c-mpl receptor agonist of the Formula: SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer 1 5 10 WO 97/12978 PCTIUS96/15938 14 HisValLeuHisSerArgLeuSerGlnCysProGluValHi sProLeuProThrPro 25 30 ValLeuLeuProAlaValAspSerLeuG lyGluTrpLysThrGlnMetGiuGlu 40 45 50 ThrLysAlaGlnAspI leLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla 65 70 AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly 85 90 GlnValArgLeuLeuLeuGlyAlaLeuG lnSerLeuLeuGlyThrG lrXaaXaaXaa 100 1.05 110 XaaGlyArgThrThrAlaHisLysAspProAsnAlallePheLeuSerPheGlnHis 115 120 125 130 LeuLeuArgGlyLysValArgpheLeuMetLeuVa iGlyGlyS erThrLeuCysVal 135 140 145 150 Arg (SEQ ID NO:2) 153 wherein; Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or Met; Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn; 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 51-52 108-109 27-28 52-53 109-110 28-29 53-54 110-111 29-30 54-55 111-112 WO 97/12978 PCTIUS96/15938 30-31 55-56 112-113 32-33 56-57 113-114 33-34 57-58 114-115 34-35 58-59 115-116 36-37 59-60 116-117 37-38 78-79 117-118 38-39 79-80 118-119 40-41 80-81 119-120 41-42 81-82 120-121 42-43 82-83 121-122 43-44 83-84 122-123 44-45 84-85 123-124 46-47 85-86 124-125 47-48 86-87 125-126 48-49 87-88 126-127 50-51 88-89 or 127-128; and additionally said c-mpl 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 in the amino acid sequence of (SEQ ID NO:2) above are; 80-81, 81-82, 82-83, 83-84, 84-85, 85-86, 86-87, 108-109, 109-110, 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119, 119-120, 120- 121, 121-122, 122-123, 123-124, 124-125, 125-126 and 126-127.

The most preferred breakpoints at which new C-terminus and N-terminus can be made in the amino acid sequence of (SEQ ID NO:2) above are; 81-82, 108-109, 115-116, 119-120, 122-123 and 125-126.

These human c-mpl receptor agonists may contain amino acid substitutions, deletions and/or insertions and may also have amino acid deletions at either/or both the N- and Ctermini.

The modified human c-mpl receptor agonists of the present invention can be represented by the Formula: WO 97/12978 PCT/US96/15938 16 X- 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 n+1 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 forumla above the constituent amino acids residues of human c-mpl ligand are numbered sequentially 1 through J from the amino to the carboxy terminus. A pair of adjacent amino acids within this protein may be numbered n and n+l respectively where n is an integer ranging from 1 to J-l. The residue n+1 becomes the new N-terminus of the new cmpl receptor agonist and the residue n becomes the new Cterminus of the the new c-mpl receptor agonist.

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:73); GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:74); GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID SerGlyGlySerGlyGlySer (SEQ ID NO:76); GluPheGlyAsnMetAla (SEQ ID NO:77); GluPheGlyGlyAsnMetAla (SEQ ID NO:78); GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79); and GlyGlySerAspMetAlaGly (SEQ ID The present invention also encompasses recombinant human c-mpl receptor agonists co-administrated with one or more additional colony stimulating factors (CSF) including, WO 97/12978 PCT/S96/15938 17 cytokines, lymphokines, interleukins, hematopoietic growth factors (herein collectively referred to as "colony stimulating factors") each of which may act through a different and specific cell receptor to initiate complementary biological activities.

These co-administrated molecules may be characterized by having the usual activity of both of the peptides or it may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of human c-mpl receptor agonist or the second colony stimulating factor alone. The coadministration may also unexpectedly provide an enhanced effect on the activity or an activity different from that expected by the presence of human c-mpl receptor agonist or the second colony stimulating factor or human c-mpl ligand variant. The co-administration may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human c-mpl ligand or native cytokine.

In addition to the use of co-administration of the present invention in vivo, it is envisioned that in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.

WO 97/12978 PCT/US96/15938 18 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 Nterminus 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 Cterminus 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.

WO 97/12978 PCT/US96/15938 19 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 Cterminus 174) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.

WO 97/12978 PCT/US96/15938 DETAILED DESCRIPTION OF THE INVENTION Receptor agonists of the present invention may be useful in the treatment of diseases characterized by a decreased levels of megakaryocyte cells of the hematopoietic system.

A c-mpl receptor agonist may be useful in the treatment or prevention of thrombocytopenia. Currently the only therapy for thrombocytopenia is platelet transfusions which are costly and carry the significant risks of infection (HIV, HBV) and alloimunization. A c-mpl receptor agonist may alleviate or diminish the need for platelet transfusions.

Severe thrombocytopenia may result from genetic defects such as Fanconi's Anemia, Wiscott-Aldrich, or May-Hegglin syndromes. Acquired thrombocytopenia may result from auto- or allo-antibodies as in Immune Thrombocytopenida Purpura, Systemic Lupus Erythromatosis, hemolytic anemia, or fetal maternal incompatibility. In addition, splenomegaly, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, infection or prosthetic heart valves may result in thrombocytopenia. Severe thrombocytopenia may also result from chemotherapy and/or radiation therapy or cancer. Thrombocytopenia may also result from marrow invasion by carcinoma, lymphoma, leukemia or fibrosis. The c-mpl receptor agonists of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells into 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 procedure by decreasing the number of plasmaphereses WO 97/12978 PCT/US96/15938 21 required. The c-mpl receptor agonists may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.

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 c-mpl receptor agonists 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.

c-mpl ligand may be useful in treating such hematopoietic deficiency.

As another aspect of the present invention, there is provided a novel method for producing the novel family of human c-mpl receptor agonists. The method of the present invention involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel c-mpl receptor agonist polypeptide. Suitable cells or cell lines may include various strains of E. coli, yeast, mammalian cells, or insect cells may be utilized as host cells in the method of the present invention.

Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel c-mpl receptor agonists. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Appropriate vectors which can WO 97/12978 PCT/US96/15938 22 transform microorganisms capable of expressing the c-mpl receptor agonists include expression vectors comprising nucleotide sequences coding for the c-mpl 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 c-mpl receptor agonist polypeptides.

The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.

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 c-mpl 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.

The c-mpl receptor agonists of the present invention may be useful in the mobilization of multipotential hematopoietic progenitors 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 WO 97/12978 PCTIUS96/15938 23 and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of pheresis required. The c-mpl receptor agonists may be useful in mobilization of multipotential hematopoietic cells and further enhance the efficacy of peripheral blood cell transplantation.

The c-mpl receptor agonists of the present invention may also be useful in the ex vivo expansion of multipotential hematopoietic cells. Colony stimulating factors (CSFs), such as hIL-3, have been administered alone, co-administered with other colony stimulating factors, 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 or other 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 exits such that a greater number of stem cells will enhance hematopoietic recovery. Therefore, the in vitro expansion of WO 97/12978 PCT/US96/15938 24 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 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 selected based on the presence of specific surface antigen 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 WO 97/12978 PCT/US96/15938 of IL-3, flk3 ligand, c-kit ligand, GM CSF, IL-1, GM-CSF/IL-3 fusion protein and combinations thereof; administering cellular preparation to a patient.

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 97/12978 PCT/US96/15938 26 WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.

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 multipotential hematopoietic cells as well as early progenitor and precursors 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 said separated stem cells with a selected medium which contains a c-mpl receptor agonist and optionally a colony stimulating factor; and harvesting said stems cells. Stem cells as well as committed progenitor cells destined to become neutrophils, erythrocytes, WO 97/12978 PCTIUS96/15938 27 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 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.

WO 97/12978 PCT/US96/15938 28 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 ligana (Brandt et al., Blood 83:1507-1514, 1994; McKenna et al., Blood 86:3413-3420, 1995); IL-3 (Brandt et 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-1 (Muench et al., Blood 81:3463-3473, 1993), IL-6 (Sato et al., Blood 82:3600-3609, 1993), IL-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; 3mith 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 c-mpl receptor agonists that are more effective.

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 WO 97/12978 PCT/US96/15938 29 been supplemented with a c-mpl receptor agonist of the present invention.

Another projected clinical use of growth factors has 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 immunodifiencies (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).

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, WO 97/12978 PCTIUS96/1 5938 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, which new genetic material has been introduced, in that it provides methods utilizing c-mpl receptor agonists that have improved biological activity, including an activity not seen by any single colony stimulation factor and/or physical properties.

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 cf 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 c-mpl receptor agonists protein per kilogram of body weight. Dosages would be adjusted relative to the activity of a given receptor agonist 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 c-mpl receptor agonist would be adjusted higher or lower than the range of 0.2 150 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 c-mpl receptor agonists; and various patient-related issues mentioned earlier in this section. As indicated above, the therapeutic method and WO 97/12978 PCT/US96/15938 31 compositions may also include co-administration with other human factors. A non-exclusive list of other appropriate hematopoietins, CSFs and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, G-CSF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL-5, IL 6, IL-7, IL-8, IL-9, 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, (herein collectively referred to as "colony stimulating factors"), or combinations thereof. In addition to the list above, IL-3 variants taught in WO 94/12639 and WO 94/12638 can be co-administered with the polypeptides of the present invention. The c-mpl receptor agonists of the present invention can be co-administered as with another "colony stimulating factor" as discussed above in a fashion taught in WO 95/20976 and WO 95/20977. 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 and the like.

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 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.

483-489, 1983; Kyte Doolittle, J. Mol. Biol. 157:105- 132, 1992; solvent exposed surface area, Lee Richards, J.

WO 97/12978 PCTfUS96/15938 32 Mol. Biol. 55:379-400, 1971) and the ability to adopt the necessary conformation without deranging the conformation of the c-mpl 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 30 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 (SEQ ID NO:3) repeated n times, where n is i, 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 threedimensional 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 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 WO 97/12978 PCT/US96/15938 33 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:3) 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 of c-mol licrand Sequences of c-mpl ligand 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 will recognize that selections of termini anywhere within the region may function, and that these will effectively lead to WO 97/12978 PCT/US96/15938 34 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 (Gentile Salvatore, Eur. J. Biochem. 218:603-621, 1993) WO 97/12978 PCT/US96/15938 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.

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

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).

Methods for creation of aenes with new N-terminus/C-terminus WO 97/12978 PCT/US96/15938 36 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 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 2.

In the first step, the first primer set ("new start" and "Linker start") is used to create and amplify, from the 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 second primer set ("new stop" and "linker stop") is used to create and amplify, from the 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" 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; 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 72 0 C 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 Ix PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, units AmpliTaq DNA polymerase and 2 mM MgCl2. PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT).

WO 97/12978 PCT/US96/1 5938 37 "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 94°C denaturation for one minute, 60 0 C annealing for one minute and 72 0 C extension for one minute; plus one cycle 72 0

C

extension fo-: 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, 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 3. In the first step, the first primer set ("new start" and "P-bl start") is used to create and amplify, from the original gene sequence, the DNA WO 97/12978 PCT/US96/15938 38 fragment ("Fragment Start") that contains the sequence encoding the new N-terminal portion of the new protein. In the second step, the second primer set ("new stop" and "P-bl stop") is used to create and amplify, from 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; 25 cycles 94°C denaturation for one minute, 50 0 C annealing for 45 seconds and 72 0 C 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 lx 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, 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 WO 97/12978 PCT/US96/15938 39 pair NcoI/HindIII vector fragment of pMON3934 by heating at 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 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 4.

The tandemly-duplicated template DNA is created by cloning and contains two similar, but not necessarily identical, copies of the gene separated by DNA sequence encoding a linker connecting the original C- and N-terminal ends of the two copies of the gene. Specific primer sets are used to create and amplify a full-length new N terminus/Cterminus 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 melting for two minutes; 25 cycles 94C denaturation for one minute, 50 0 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; WO 97/12978 PCT/US96/15938 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).

The construction of the PCR templates involves the steps outlined below; 1. Generation of the c-mpl ligand gene with and without amino acid codons 112-115 via reverse transcriptase/polymerase chain reaction (RT/PCR). Human liver contains c-mpl ligand mRNA with and without a deletion of amino acids 112-115.

2. Subcloning the PCR products into an mammalian expression vector.

3. Assembly of dimer templates as genel/linker/genell from; i) a gene encoding c-mpl ligand amino acids 1-153 for the genel position; ii) unique synthetic linkers for separation of the two genes; and iii) a gene encoding c-mpl ligand amino acids 1-153, with or without amino acids 112-115 for the genell position.

Step 1: Reverse transcriotase/polvmerase chain reaction The two forms of c-mpl ligand, one with a deletion of amino acids 112-115 and one without the deletion, can be isolated RT/PCR technology. Synthetic primers are designed so that they would anneal to either c-mpl ligand DNA or mRNA (cmpl ligand sequence based on Genebank accession #L33410 or de Sauvage et al., Nature 369, 1994, pp.533 538) for priming first-strand complementary DNA (cDNA) synthesis. The resulting cDNA is used as a template in PCR (Saiki, 1985) to generate double-stranded DNA (dsDNA or DNA) which can be used in additional PCR or digested with appropriate restriction WO 97/12978 PCTIUS96/15938 41 enzymes for transfer to E. coli or mammalian expression plasmids.

For the reveise transcriptase (RT) reaction, human fetal (lot #38130) and adult liver (lot #46018) A+ RNA can be obtained from Clontech (Palo Alto, CA). The RT reactions are carried out using a cDNA Cycle T Kit obtained from Invitrogen (San Diego, CA). One microgram (ug) of each RNA sample is combined and denatured at 65 0 C for 10 min. in the presence of either random primers, oligo dT primer or a specific 3' antisense primer. Following denaturation, the samples are cooled for 2 min. on ice and spun down for 10 sec. at 10,000 x g.

RNAse inhibitor, reverse transcriptase buffer, deoxynucleotides, sodium pyrophosphate and reverse transcriptase are added as described by manufacturer, and the microliter reaction is incubated at 42 0 C tor 1 hr.

For PCR a specific 5' sense primer and 3' anti-sense primers are added to the RT reactions and the PCR is carried out using reagents from Boehringer Mannheim (Indianapolis, IN) or Perkin-Elmer (Norwalk, CT) as described by the manufacturers using Taq polymerase. The PCR reactions are subjected to cycles of the following; 1 min. 94 0 C, 1 min. 58 0 C, sec. 72 0 C. An equal volume of loading dye (0.01% each bromophenol blue and xylene cyanole blue) is added to microliters of the final product for electrophoresis through a 1% SeaKemR LE agarose (FMC, Rockland, ME) gel in the presence of Ix TBE/EtBr (Tris-borate-EDTA plus ethidium bromide;Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Horbor, NY, 1989). For molecular weight standards, 1 microgram of phiX174 phage DNA digested with HaeIII restriction enzyme (New England Biolabs, Beverly, MA) is loaded onto the gel. The product (about 1090 base pairs) is visualized using a short-wave UV light source. The reactions WO 97/12978 PCT/US96/15938 42 are purified using a Wizard T PCR Preps kit from Promega (Madison, WI). Briefly, the PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 minute incubation at 24 0 C, the supernatant is removed by vacuum filtration through a filtration column.

Two mLs of 80% isopropanol is used to wash the resin via vacuum filtration. The column containing the resin is then subjected to centrifugation at 10,000 x g for 30 seconds to remove residual isopropanol. The PCR product is eluted with microliters of 10 mM Tris-Cl, 1 mM EDTA, pH7.4, via centrifugation at 10,000 x g for 30 seconds followed by transfer of supernatant to a new tube.

Step 2: Subcloning the PCR products into a mammalian expression vector The c-mpl ligand PCR products are digested with the appropriate restriction enzymes for ligation to a mammalian expression vector. The mammalian expression vector is a derivative of pMON3359 which is a pUC18-based vector containing a mammalian expression cassette. The cassette includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 secretion signal sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker (Hippenmeyer et al., Bio/Technology, 1037-1041, 1993). Restriction enzyme digestions are incubated for 1 hour at 37 0 C as described by the manufacturer prior to electrophoresis through a 1% agarose/lx TBE/EtBr gel. Fragments are first visualized by long-wave UV and gel-purified using a Qiaex DNA Extraction kit (Qiagen, Chatsworth, CA). The DNA fragments are purified from the resin by agarose solublization, addition of a DNAbinding resin, and extensive washing of the resin prior to elution with water. The purified DNA products are combined at WO 97/12978 PCT/US96/15938 43 a molar excess of PCR product to vector fragment and the ligation reactions are carried out according to the manufacturer's recommended conditions for T4 DNA ligase. An E. coli strain is then transformed with the ligation, plated out onto LB-agar plus ampicillin (100 ug/ml). The colonies are screened for presence of a c-mpl ligand gene and DNA is isolated for DNA sequencing to identify both forms c-mpl ligand, one with amino acids 112-115 deleted and one with them present.

3. Assembly of dimer PCR templates The linkers that join c-mpl (1-153) ligand genes are created by annealing 200 picomoles each of a pair of complementary synthetic oligonucleotides in 5 microliters of ligation buffer (Boehringer-Mannheim #1243 292). Each linker, which has flanking EcoRI and AflIII sites, is ligated overnight with a 3.7 Kbp EcoRI/BstXI fragment from a form of c-mpl ligand with amino acids 1-153 (step and a 1 Kbp NcoI/BstXI fragment from the either of the two types of clones in step 2 above, one with and one without a deletion of amino acids 112-115. The resulting DNA is used to transform E. coli DH5a T M cells. Transformed cells are selected on LB agar plates containing ampicillin (100 ug/ml). Plasmid DNA is obtained from single colonies of several clones and sequenced to verify correct assembly of the dimer via the linker. The resulting plasmid DNA template can be used in making novel c-mpl ligand molecules via the Horlick method (Prot. Eng. 5:427-433, 1992).

B. Horlick Method sense ana 3' anti-sense primers are combined with a dimer template for PCR using reagents from Boehringer Mannheim (Indianapolis, IN) or Perkin-Elmer (Norwalk, CT) as described by the manufacturersusing Taq polymerase. The PCR reactions are subjected to 30 cycles of the following; 1 min. 94 0 C, 1 WO 97/12978 PCT/US96/15938 44 min. 58 0 C, 90 sec. 72 0 C. The product (about 480 base pairs) is visualized using a short-wave UV light source. The reactions are purified using a Wizard T PCR Preps kit from Promega (Madison, WI). Briefly, the PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 minute incubation at 24 0 C, the supernatant is removed by vacuum filtration through a filtration column. Two mLs of 80% isopropanol is used to wash the resin via vacuum filtration. The column containing the resin is then subjected to centrifugation at 10,000 x g for seconds to remove residual isopropanol. The PCR product is eluted with 50 microliters of 10 mM Tris-Cl, 1 mM EDTA, pH7.4, via centrifugation at 10,000 x g for 30 seconds followed by transfer of supernatant to a new tube.

Subcloning novel c-mpl receptor agonists into expression vectors The novel c-mpl receptor agonists PCR products are digested with the appropriate restriction enzymes for ligation to either mammalian or E. coli expression vectors.

Mammalian Expression Vectors The mammalian expression vectors are derivatives of pMON3359 which is a pUC18-based vector containing a mammalian expression cassette. The cassette includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 signal peptide sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker [See Hippenmeyer et al., Bio/Technology, 1993, pp.1037-1041].

Restriction enzyme digestions are incubated for 1 hour at 37 0 C as described by the manufacturer prior to electrophoresis through a 1% agarose/lx TBE/EtBr gel.

WO 97/12978 PCT/US96/15938 Fragments are first visualized by long-wave UV and gelpurified using a Qiaex DNA Extraction kit (Qiagen, Chatsworth, CA). The DNA fragments are purified from the resin by agarose solublization, addition of a DNA-binding resin, and extensive washing of the resin prior to elution with water. The purified DNA products are combined at a molar excess of PCR product to vector and the ligation reactions are carried out according to the manufacturer's recommended conditions for T4 DNA ligase.

E. coli Expression Vectors The E. coli expression vectors that direct high-level production of heterologous proteins in the cytoplasm are derivatives of that described elsewhere (Olins et al., Methods Enzym., 185:115-119, 1988 and Rangwala et al., Gene, 122: 263-269, 1992). The expression cassette consists of the recA promoter and T7 gene 10 ribosome binding site (RBS) as well as an M13 origin of replication or a tandem inverted repeat of a phage P22 gene which acts as a transcription terminator. These cassettes are on a plasmid with the pBR327 origin of replication and encode a gene either for spectinomycin or ampicillin resistance.

Transformation of E. coli strains E. coli strains DH5a TM (Life Technologies, Gaithersburg, MD) and TG1 (Amersham Corp., Arlington Heights, IL) are used for all transformation of ligation reactions and are the source of plasmid DNA for transfecting mammalian cells. E.

coli strain MON105 can be obtained from the American Type Culture Collection (ATCC, Rockville, MD) and is the host for expressing alternate forms of c-mpl ligand in the cytoplasm or periplasmic space, respectively, of E. coli.

MON105 ATCC#55204: lamda-,IN(rrnD, rrE)l, rpoD+, rpoH358 WO 97/12978 PCT/US96/15938 46 phi80dlacZdeltaMl5, delta(lacZYA-argF)U169, deoR, recAl, endAl, hsdR17(rk-,mk+), phoA, supE441amda-, thi-l, gyrA96, relAl TG1: delta(lac-pro), supE, thi-l, hsdD5/F'(traD36, proA+B+, lacIq, T 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 mLs of cells are grown in LB medium bacto-tryptone, 0.5% bacto-yeast extract, 150 millimolar NaCl) to a density of approximately 1.0 optical density units 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 CaC12 solution (50 millimolar CaCl2, 10 millimolar Tris-Cl, pH7.4) and are held at 4'C for 30 minutes. The cells are again collected by centrifugation and resuspended in one tenth culture volume of CaCl2 solution. Ligated DNA is added to 0.2 mL of these cells, and the samples are held at 4'C for 1 hour. The samples are shifted to 42'C for two minutes and 1.0 mL of LB is added prior to shaking the samples at 37'C for one hour. Cells from these samples are spread on plates (LB medium plus bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin (75 ug/mL) when selecting for spectinomycinresistant transformants. The plates are incubated overnight at 37'C. Single colonies are picked, grown in LB supplemented with appropriate antibiotic for 6-16 hours at 37'C with shaking.

Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL WO 97/12978 PCT/US96/15938 47 spectinomycin) and are grown at 37 0 C while shaking. Before harvesting the cultures, 1 ul of cells are analyzed by PCR for the presence of a c-mpl ligand gene. The PCR is carried out using a combination of primers that anneal to the c-mpl ligand gene and/or vector. After the PCR is complete, loading dye is added to the sample followed by electrophoresis as described earlier. A gene has been ligated to the vector when a PCR product of the expected size is observed.

DNA isolation and characterization Plasmid DNA is isolated using the Promega Wizard

T

Miniprep kit (Madison, WI) or the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA). Both kits follow the same general procedure for plasmid DNA isolation. Briefly, cells are pelleted by centrifugation (5000 x plasmid DNA released with sequential NaOH acid treatment, and cellular debris is removed by centrifugation (10000 x 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 are inoculated into 100 mls of LB plus appropriate antibiotic for overnight growth at 37 0 C in an air incubator while shaking.

Plasmid DNA is isolated using the Qiagen Plasmid Midi kit (Chatsworth, CA) which is a scaled-up version of the Qiagen QIAwell Plasmid isolation kit described earlier. The DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into mammalian or E. coli cells.

Purified recombinant double-stranded DNA is sequenced using the Applied Biosystems Inc. (ABI, Foster City, CA)

PRISM

T M Ready Reaction DyeDeoxy T M Terminator Sequencing system. The ABI system relies on incorporation of four fluorescence labelled dideoxy nucleotides into single- WO 97/12978 PCT/US96/15938 48 stranded DNA during multiple rounds of amplification.

Plasmid DNA and a sequencing primer are added to the reaction mixture (including Taq DNA polmerase, buffer and nucleotides), which is subjected to 25 cycles of amplification (30 seconds at 96 0 C, 15 seconds at 50 0 C, 4 minutes at 60 0 Following amplification, unincorporated nucleotides are removed using Centri-Sep spin columns (equilibrated in water) as described by Princeton Separations, Inc. (Adelphia, NJ). Briefly, the samples are loaded onto a column that has excess water removed by centrifugation (700 x g) for 2 minutes, and the purified sequencing product is eluted by centrifugation (700 x g) for 4 minutes. The samples are then dried down in a Speed Vac (Savant, Hicksville, NY) prior to addition of loading solution. The samples are electrophoresed through a 4.75% polyacrylamide sequencing gel containing 7M urea in 1X TBE at watts constant power. The ABI system uses a detector that recognizes each differntially labelled PCR product as they are being subjected to electrophoresis.

Production of novel c-mpl receptor agonists 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 millimolar (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 (Hippenmeyer et al., Bio/Technology, 1037-1041, 1993). The VP16 protein drives expression of genes inserted behind the IE110 WO 97/12978 PCT/US96/15938 49 promoter. BHK-21 cells expressing the transactivating protein VP16 is designated BHK-VP16. The plasmid pMON1118 (Highkin et al., Poultry Sci., 70:970-981, 1991) expresses the hygromycin resistance gene from the promoter. A similar plasmid is available from ATCC, pSV2hph.

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"T M (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug 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 confluency, the conditioned media is reassayed, and positive clones are expanded into growth media.

Expression and purification of recombinant protein from E.

coli E. coli strain MON105 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 WO 97/12978 PCT/US96/15938 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 gg/mL. The cultures are then shaken at 37 0 C for thr:e 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 (Maniatis et al. Molecular Cloning: A Laboratory Manual, 1982). After centrifugation (5000 x g) to pellet the cells, the first step in purification of the protein is either sonication or homogenization of the cells. For sonication, the cells are resuspended in one-tenth volume (based on culture size) sonication buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA). These resuspended cells are subjected to several repeated sonication iirsts using the microtip from a Sonicator cell disrupter, Model W-375 obtained from Heat Systems-Ultrasonics Inc. (Farmingdale, New York). The extent of sonication is monitored by examining the homogenates under a light microscope. After all of the cells are disrupted, the homogenates are fractionated by centrifugation at 10000 x g for 20 minutes at 4°C in a JA-20 rotor and J2-21 centrifuge (Beckman, Fullerton, CA). Alternatively, the IBs are released from the cells by lysing the cells in sonication buffer with a Manton-Gaulin homogenizer (Holland) followed by centrifugation as above. The IB pellets, which are highly enriched for the recombinant protein, are then subjected to another round of sonication and centrifugation as described above. The recombinant protein is purified by a variety of standard methods. The most common methods involve solublization of the IBs with 4-6 molar urea or guanidine-HCl buffers at pH 9-12, and air oxidation/folding in the presence WO 97/12978 PCT/US96/15938 51 of cataytic concentrations of cysteine, beta-mercaptoethanol or dithiothreitol for 24 to 72 hours. The protein is purified from E. coli contaminants using ion-exhange chomotography, such as Q-sepharose (anion) and S-sepharose (cation), gel filtration, hydrophobic chromatography or reversed phase HPLC. After dialysis against a low ionic strength buffer, the purified protein is stored frozen or lyophilized.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

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 United States Patent Applications WO 94/12639, WO 94/12638, WO 95/20976, WO 95/21197, WO 95/20977, WO 95/21254, and US Serial No. 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 Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory (1982) and references cited therein, incorporated herein by reference; and in J.

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

WO 97/12978 PCT/US96/15938 All references, patents or applications cited herein are incorporated by reference in their entirety.

WO 97/12978 WO 9712978PCTIUS96/15938 53 TABLE 1 OLIGONIJCLEOTIDES c -mplNcoI ECOMpi C-mplHindIII 4L-51 4L-3' 5L-51 5L-31 8L-51 8L-31 31-5' 31-3' 35-5, 35-3, 39-5, 39-3' 43-5, 43-3' 45-5, 45-3, 49-5' 49-3, 82-5' 82-3'

ACGTCCATGGCNTCNCCNGCNCCNCCTGCTTGTGCACTCCGAGTC

(SEQ ID NO:4) N=A,C,G or T ATGCACGAATTCCCTGACGCAGAGGGTGGA (SEQ ID

TGACAAGCTTACCTGACGCAGAGGGTGGACCCT

(SEQ ID NO:6) AATTCGGCAA (SEQ ID N0:7) CATGTTGCCG (SEQ ID NO:8) AATTCGGCGGCAA (SEQ ID NO:9) CATGTTGCCGCCG (SEQ ID AATTCGGCGGCAACGGCGGCAA (SEQ ID NO:11) CATGTTGCCGCCGTTGCCGCCG (SEQ ID NO:12) CGATCCATGGAGGTTCACCCTTTGCCT (SEQ ID NO:13) GATCAAGCTTATGGGCACTGGCTCAGTCT (SEQ ID NO:14) CGATACATGTTGCCTACACCTGTCCTG (SEQ ID GATCAAGCTTAAGGGTGAACCTCTGGGCA (SEQ ID NO:16) CGATCCATGGTCCTGCTGCCTGCTGTG (SEQ ID NO:17) GATCAAGCTTAAGGTGTAGGCAAAGGGTG (SEQ ID NO:18) CGATCCATGGCTGTGGACTTTAGCTTGGGA (SEQ ID NO:19) GATCAAGCTTAAGGCAGCAGGACAGGTGT (SEQ ID CGATCCATGGACTTTAGCTTGGGAGAA (SEQ ID NO:21) GATCAAGCTTACACAGCAGGCAGCAGGAC (SEQ ID NO:22) CGATCCATGGGAGAATGGAAAACCCAG (SEQ ID NO:23) GATCAAGCTTACAAGCTAAAGTCCACAGC (SEQ ID NO: 24) CGATCCATGGGACCCACTTGCCTCTCA (SEQ ID -GATCAAGCTTACAGTTGTCCCCGTGCTGC (SEQ ID NO:26) WO 97/12978PC/S/153 PCTIUS96/15938 109-5' 109-3' 116-5' 116-3' 120-5' 120-3' 123-5' 123-3' 126-5' 126-3 NO: 36) CAGTCCATGGGAACCCAGCTTCCTCCA (SEQ ID NO:27) GATCAAGCTTAAAGGAGGCTCTGCAGGGC (SEQ ID NO:28) CGATCCATGGGCAGGACCACAGCTCAC (SEQ ID NO:29) GATCAAGCTTACTGTGGAGGAAGCTGGGTT (SEQ ID CGATCCATGGCTCACAAGGATCCCAATGCC (SEQ ID NO:31) GATCAAGCTTATGTGGTCCTGCCCTGTGG (SEQ ID NO:32) CGATCCATGGATCCCAATGCCATCTTCCTG (SEQ ID NO:33) GATCAAGCTTACTTGTGAGCTGTGGTCCT (SEQ ID NO:34) CGATCCATGGCCATCTTCCTGAGCTTCCAA (SEQ ID GATCAAGCTTAATTGCGATCCTTGTGAGCTGT (SEQ ID WO 97/12978 WO 97/ 2978PCT/US96/15938 TABLE 2 GENE SEQUENCES PM0N26458

TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG

TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT

GCCTGCTGTGGACTTTAGCTTGGGAGA.ATGGAAAACCCAGATGGAGGAGACCAAGGCACAG

GACATTCTGGGA Y-AGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG

GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG

GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA

TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC (SEQ ID NO:37); PM0N28548

TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG

TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT

GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG

GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG

GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG

GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA

TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGC

TCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCAC

AGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTG

TGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCT

GGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT

TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGC

AGAGCCTCCTTGGAACCCAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCT

GAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACC

CTCTGCGTCAGG (SEQ ID NO:38); PM0N32132

TCTCCCGCTCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG

TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT

GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG

GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG

GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG

GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA

TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:82) PMON3 2133 TCTCCCGCTCCGCCTGCTTGTGACCTCC GAGTCCTCAGTAAACTGCTTCGTGACTCCCATG

TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT

GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAA.ACCCAGATGGAGGAGACCAAGGCACAG

GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG

WO 97/12978 PCT/US96/15938 1 56 GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTC CGTCTCCTCCTTGG GGCCCTGCAGAGCC -TCCTTGGAACCCAGGGCAGGACCACAGCTCACAAGGATCCCAATGCC

ATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAG

GGTCCACCCTCTGCGTCAGG (SEQ ID NO:83) PMON28500

TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG-CTTCGTGACTCCCATG

TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT

GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG

GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG

GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG

GGCCCTGCAGAGCCTC CTTGGAACCCAGCTTCCTC CACAGGGCAGGACCACAGCTCACAAG

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA

TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCAACATGGCGTCTCCCGCTCC

GCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC

AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGG

ACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGG

AGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC

CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCC CTGCAGA

GCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGC

CATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGA

GGGTCCACCCTCTGCGTCAGG (SEQ ID NO:39); PM0N28501

TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG

TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT

GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG

GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG

GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG

GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA

TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGC

TCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCAC

AGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTG

TGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCT

GGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACA.ACTGGGACCCACT

TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGC

AGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAA

TGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA

GGAGGGTCCACCCTCTGCGTCAGG (SEQ ID PM0N3213 6 TCCCCAGCGCCgCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG TCCTTCACAGCA 3-ACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT

GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG

GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACA.ACTGG

GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTC

CTCCTTGG

GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA

WO 97/12978 WO 9712978PCTIUS96/15938 57

TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACGGCGGCAACATGGC

GTCCCCAGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCAT

GTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGC

TGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACA

CGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG

GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTG

GGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAA

GGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG

ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:4 1); PMON3 03 73

GAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAA

TGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTG

CTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTG

GGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACC

CAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGC

TTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTC

TGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGA

GTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCA

(SEQ ID NO:61); PMON3 0374

TTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAG

ATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTG

ATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCT

GGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCA

CAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTG

CTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAA

TTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAA

CTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCT

(SEQ ID NO:62); PM0N30375

GTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAA-ACCCAGATGGAGGAGACC

AAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGG

GGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGT

CTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACC

ACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAG

GTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAAC

ATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGAC

TCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCT

(SEQ ID NO:63); PM0N30376

GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC

ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA

WO 97/12978 WO 9712978PCTIUS96/15938 58

CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG

GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACpAG

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG

ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG

GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT

CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT

(SEQ ID NO:64); PMON3 0377

GACTTTAGCTTGGGAGAATGGAAAAICCCAGATGGAGGAGACCAAGGCACAGGACATTCTG

GGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT

TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTG

CAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCC

AATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTT

GTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCG

CCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC

AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTG

(SEQ ID PMON3 0378

GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC

CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATC

C

CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT

GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC

CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC

ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCNTCTCCGGCGCCGCCTGCTTGTGAC

CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG

TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG

(SEQ ID NO:66); PMON3 0379

GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTT

GGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAG3ACCACAGCTCAC

AAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTC

CTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCT

CCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTcCCATGTC

CTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTG

CCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCkA.GGCACAG

GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG

(SEQ ID NO:67); PMON30380

GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC

CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC

ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGAC

CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG

TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTCCTGTGGACTTTAGCTTG

WO 97/12978 WO 9712978PCTIUS96/1 5938 59

GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC

CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC

CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT

(SEQ ID NO:68); PMON3 03 81

GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC

CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC

GGCGGCAACATG,;:GTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG

CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG

CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG

GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATG

GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGA

CAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAG

(SEQ ID NO:69) PMON3 0382

GCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTG

CGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATG

GCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCC

CATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTC

CTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAG

GCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGA

CAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTC

CTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACA

(SEQ ID PM0N30383

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG

ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG

GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT

CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT

GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC

ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA

CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG

GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG

(SEQ ID NO:71); PMON3 0384

GCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA

GGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGCTCCGCCT

GCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGA

CTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCrGCTGTGGAC

TTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGA

GCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC

CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAG

AGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACcACAGCTCACAAGGATCCCAAT (SEQ ID NO:72).

WO 97/12978 WO 9712978PCT/US96/15938 PROTEIN SEQUENCES PMON26458pep SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHi s ValLeuHisSerArgLeuSerGlnCysProGluValHi sProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGln~etGluG luThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerS erLeuLeuGlyGlnLeuSerGlyGlriValArgLeuLeu LeuG lyAlaLeuGinS erLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAl a HisLysAspProAsnAlallePheLeuSerPheGlnui sLeuLeuArgGlyLysValArg PhebeuletLeuValGlyGlySerThrLeuCysValArgGluPhe (SEQ ID NO:4 2); PMON2 854 Spep SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHi s ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnl~etGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerS erLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuG lyAlaLeuG inS erLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAl a HisLySASpProAsnAlalePheLeuSerPheGinHisLeuLeuArgGlyLysValArg PheLeuMet.LeuVa1GlyGlySerThrLeuCysVaArgGuPheGyGyAsnl4etAla SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHi s ValLeuHisS erArgLeuSerGlnCysProGluVa iisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyG luTrpLy sThrGln~etG luG2luThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGiyValMetAlaAlaArgGlyGin LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnG lyArgThrThrAlaHiSLySASpPro AsnAlal lePheLeuS erPheG inHi sLeuLeuArgGlyLysValArgPheLeuMetLeu ValGlyGlySerThrLeuCysValArg (SEQ ID NO:43); PMON3 2132 SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeUArgAspSerHi s ValLeuHisSeiArgbeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyG luTrpLysThrGlniMetGluGluThrLysAla GlnAspIleLeuGlyAiaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuG lyAlaLeuGlnS erLeuLeuGlyThrG lnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO: 44); PM0N32133 SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeUArgAspSerHi s WO 97/12978 WO 9712978PCTIUS96/15938 61 ValLeuHisSerArgLeuSerGlnCysProGluVa iHi sProLeuProThrProValLeu LeuProAlaValAspPheS erLeuGlyG luTrpLy sThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuserGlyGliValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnGlyArgThrThrAlaHisLysAspPro AsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysvalArgPheLeuMetLeu ValGlyGlySerThrLeuCysValArg (SEQ ID PM0N28500 SerProAlaProProAlaCysASpLeuArgValLeuSerLysLeuLeuArgAspSerHi s ValLeuHisSerArgLeuSerGlnCysProGluValllisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysTh-rGlnietGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuG lyAlaLeuGinS erLeuLeuGlyThrGlnLeuPro ProGlnGlyArgThrThrAl a HisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyAsnlMetAlaSer ProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHisVal LeuHisSerArgLeuSerGlnCysProGluValHi sProLeuProThrProValLeuLeu ProAlaValAspPheS erLeuG lyGluTrpLysThrGlriMetGluGluThrLysAlaGln AspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGlnLeu GlyProThrCysLeuS erSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeuLeu GlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAlaHis LysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPhe LeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:4 6); PM0N28501 SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerxi s ValLeuHisS erArgLeuSerG inCysProGluVa lisProLeuProThrProValLeu LeuProAlaValASpPheSerLeuGlyGluTrpLysThrGlnmetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGlu~lyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuG lyAlaLeuGinS erLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAl a HisLysAspProAsnAlallePheLeuSerPheGlnHi SLeuLeuArgGlyLysValArg PheLeuMetLeuValG lyGlyS erThrLeuCysVa lArgGluPheGlyG 1yAsnMetA1 a SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHi sProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyG luTrpLysThrGlniMetGluGluThrLysAla GinAspI leLeuGlyAlaValThrLeuLeuLeuGluG lyVa lMetAlaAlaArgGlyGln LeuGlyProThrcysLeuSerS erLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProASnAlallePheLeuS erPheGinHi sLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:47); pMON32136 SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHi s WO 97/12978 WO 9712978PCT/US96/15938 62 ValLeuHisSerArgLeuSerGinCysProGiuVaiHisProLeuProThrProValLeu LeuProAiaValAspPheSerLeuGlyG iuTrpLy sThrGin.MetGluGlUThrLySAi a GlnAspleLeuGiyAiaVaiThrLeuLeuLeuGiuGlyValMetAiaAiaArgGiyGln LeuG iyProThrCysLeuS erSerLeuLeuG lyGl1nLeuSerGiyGlnVaiArgLeuLeu LeuGiyAlaLeuGinSerLeuLeuclyThrGinLeuProProGinGiyArgThrThrAia HisLysAspProAsnAialePheLeuSerPheGinHi sLeuLeUArgGiyLysVaiArg PheLeuMetLeuVaiGlyciySerThrLeuCysVaiArgciuPheGiyGiyAsnGlyGiy AsniMetAiaS erProAiaProProAiaCysAspLeuArgValLeuSerLysLeuLeu~rg AspSerHisVaiLeuHisSerArgLeuSerGinCysProGiuVaiHi sProLeuProThr ProVaiLeuLeuProAiaValAspPheSerLeuGlyGiuTrpLysThrGli~etGiuGlu ThrLysAiaGinAsplieLeuGiyAiaValThrLeuLeuLeuGiuGiyVaiMetAiaAla ArgG iyGlnLeuGiyProThrCysLeuS erSerLeuLeuG iyGinLeuSerGiycinVa 1 ArgLeuLeuLeuGiyAiaLeuG inSerLeuLeuGlyThrGinLeuiProProGlnGiyArg ThrThrAiaHisLysAspProAsnAialePheLeuSerPheGinHisLeuLeuArgGiy LysVaiArgPheLeuMetLeuValciyGiySerThrLeuCysVaiArg (SEQ ID NO:48); pMON3 0373 GiuVaiHisProLeuProThrProVaiLeuLeuProAlaValAspPheSerLeuGlyGiu TrpLysThrGlniMetGiuGiuThrLySAiaGinAspl eLeuGiyAiaVaiThrLeuLeu LeuGluGlyVaiMetAiaAiaArgGiyGinLeuGlyProThrCysLeuSerSerLeuLeu GlyGinLeuS erGlyGinVaiArgLeULeuLeuGlyAlaLeuGinSerLeuLeuGiyThr GinLeuProProGinGiyArgThrThrAiaHisLysAspProAsnAialePheLeuSer PheGinHisLeuLeuArgGlyLysVaiArgPheLeuMetLeuValGiyGiySerThrLeu CysValArgG luPheG lyG lyAsnMetAlaS erProAl aProProAl aCysAspLeuArg VaiLeuSerLysLeuLeuArgAspSerHisVaiLeuHi sSerArgLeuSerGinCysPro (SEQ ID NO:49); pMON3 1074 Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Giu Trp Lys Thr Gin Met Giu Giu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu ?ro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Giu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Giu Val His Pro (SEQ ID pMON3 0375 Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Giu Trp Lys Thr Gin Met Giu Giu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Giu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Giy Gin Val Arg WO 97/12978 PCT/US96/15938 63 Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser -ro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro (SEQ ID NO:51); pMON30376 Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro (SEQ ID NO:52); pMON30377 Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val (SEQ ID NO:53); pMON30378 Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val jeu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu (SEQ ID NO:54); WO 97/12978 WO 9712978PCTIUS96115938 PMON3 0379 Gly Val Leu Ile Leu Gly Leu Ser Pro Giu Giu Pro Arg Pro Phe Met Asn Ser Gin Aia Thr G iy Thr Leu Pro Leu Leu Met Ly s Cys Val Lys Vali Cys Leu Gin Ser Val Ala Leu Pro Asp Al a Met Leu Leu Gly Phe G ly Ser Leu Glu Phe Gin Ala Ser Giy Arg Gin Gly Pro Arg Val Ser Asp Ala Ser Ala Thr His Ser Ala Asp His Leu Ile Arg Leu Leu Thr Leu Thr Pro Ser Pro G ly Leu G ly Leu Gin Ala Leu Leu Pro His Leu Giu G ly Gin Gly Ser His Arg cys Al a Val1 pro Trp Ala Leu Gln Leu Leu Leu Lys Asp Gly Lys Val Arg Cys P-sp Leu His Thr Pro Lys Thr Vai Thr (SEQ ID Ser Gly Gly Thr Pro Asn Val Arg Glu Phe Leu Arg Ser Arg Val Leu Gin Met Leu Leu NO: Gin Gin Ala Phe G ly Val Leu Leu Glu Leu pMON3 0380 Gly Pro Val Glu Leu Ser Val1 Gin Leu Thr Leu Thr Asn Arg Phe Arg Arg Leu Met Leu Cys Leu Gin Al a Phe Gly Val1 Leu Leu Glu Leu Leu Leu Leu Ile Leu G ly Leu Ser Pro Glu Giu Ser G ly Pro Phe Met Asn Ser Gin Ala Thr G ly Ser Ala Pro Leu Leu Met Lys5 Cys Val1 Lys Val Leu Leu Gin Ser Val Ala Leu Pro Asp Ala Met Leu Gin Gly Phe G ly Ser Leu Glu Phe Gin Ala G ly Ser Arg Gin G ly Pro Arg Val Ser Asp Ala Gin Leu Thr His Ser Ala Asp His Leu Ile Arg Leu Leu Thr Ala Leu Leu Thr Leu Pro Pro Ser His Pro Leu Gly Glu Leu Gly Gly Gin Ser Giy (SEQ ID His Lys Arg Gly Cys Val Ala Cys Val Leu Pro Thr Trp Lys Ala Val Leu Gly Gin Val NO: 56); Asp Lys Arg Asp His Pro Thr Thr Pro Arg pMON3O3 81 Gly Phe Gly Ser Leu Glu Phe Gin Ala Gly Ser Arg Gin G ly Pro Arg Val Ser Asp Ala Gin Leu Thr His Ser Al a Asp His Leu Ile Arg Leu Leu Thr Leu Thr Pro Ser Pro Gly Leu Gly Ser G ly Ala Leu Leu Pro His Leu Giu Gly Gin Gly Thr His Arg Cys Ala Val Pro Trp Ala Leu Gin Gin Lys G ly Val Cys Leu Thr Lys Val G ly Val Leu Asp Lys Arg Asp His Pro Thr Thr Pro Arg Pro Pro Val Glu Leu Ser Val Gin Leu Thr Leu Pro Asn Arg Phe Arg Arg Leu Met Leu Cys Leu Gin Ala Ile Phe Leu Gly Gly Val Leu Leu Ser Leu Pro Glu Giu Leu Giu Leu Ser Leu Gly (SEQ ID Phe Leu Met Leu Asn Met Ser Lys Gin Cys Ala Val Thr Lys Gly Val Ser Leu Ala Leu NO: 57); Ser Val1 Ala Leu Pro Asp Ala Met Leu Gin pMON3 0382 Ala His Lys Leu Arg Gly Leu Cys Val Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Arg Giu Phe Gly Gly Asn Met Ala Ser Pro Ala Leu Thr Pro WO 97/12978 PCT/US96/15938 Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr (SEQ ID NO:58); pMON30383 Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys (SEQ ID NO:59); pMON30384 Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn (SEQ ID The following examples will illustrate the invention in greater detail although it will be understood that the invention is not limited to these specific examples.

EXAMPLE 1 Construction of the parental plasmid containing the first gene of the dimer template WO 97/12978 PCTIUS96/15938 66 In order to generate a plasmid DNA with the coding sequence of c-mpl (1-153) ligand followed by a unique EcoRI restriction site, the gene is isolated via reverse transcriptase/polymerase chain reaction (RT/PCR). Human fetal (lot #38130) and adult liver (lot #46018) A+ RNA are obtained from Clontech (Palo Alto, CA) for source of c-mpl ligand messager RNA (mRNA). The first strand cDNA reactions are carried out using a cDNA Cycle M Kit obtained from Invitrogen (San Diego, CA). In the RT reaction, random primers and oligo dT primer are used to generate cDNA from a combination of human and fetal liver mRNA. For amplification of c-mpl ligand gene fragment encoding amino acids 1-153, the RT product serves as the template for PCR with a combination of the primers, Forward primer: c-mplNcol (SEQ ID NO:4) and Reverse primer: Ecompl (SEQ ID NO:5). The c-mplNcol (SEQ ID NO:4) primer anneals to the c-mpl ligand gene (bases #279-311 based on c-mpl ligand sequence from Genebank accession #L33410 or de Sauvage et al., Nature 369:533-538, 1994) and encodes a NcoI restriction enzyme site just 5' to the first mature codon (Serl). The NcoI restriction enzyme site codes for methionine and alanine codons prior to ser-1 and includes codon degeneracy for the Ala codon and the first four c-mpl ligand codons (Serl, Pro 2 Ala 3 Pro 4 The Ecompl (SEQ ID NO:5) primer anneals to bases #720-737 of c-mpl ligand and encodes a EcoRI recognition site in-frame with the c-mpl ligand gene immediately following Arg 153 The EcoRI site creates Glu 154 and Phe 155 codons following Arg 153 The ca.

480 bp PCR product is purified, digested with NcoI and EcoRI and ligated to the NcoI-EcoRI vector fragment of pMON3993 (ca. 4550 pMON3993 is a derivative of pMON3359. The expression cassette in pMON3359 includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 signal peptide sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker (See WO 97/12978 PCT/US96/15938 67 Hippenmeyer et al., Bio/Technology, 1037-1041, 1993). The human IL-3 signal peptide sequence, which had been subloned as a BamHI fragment into the unique BamHI site between the IE110 promoter and poly-A signal, contains an NcoI site at its 3' end and is then followed by a unique EcoRI site. The DNA sequence of the signal peptide is shown below (restriction enzyme sites are indicated above). The ATG (methionine) codon within the NcoI site is in-frame with the initiator ATG of the signal peptide (underlined); BamHI

GGATCCACCATGAGCCGCCTGCCCGTCCTGCTCCTGCTCCAACTCCTGGTCCGCCCCGC

NcoI CATGG (SEQ ID NO:81) pMON26458, coding for c-mpl ligand amino acids 1-153, is a result of this cloning.

EXAMPLE 2 Construction of the parental plasmids containing the second genes of the dimer templates For amplification of c-mpl ligand gene fragments starting at amino acid 1 (Ser) with a termination codon following amino acid 153 (Arg), the RT reaction from Example 1 serves as the template for PCR with a combination of the following primers; c-mplNcol (SEQ ID NO:4) (forward primer) and c-mplHindIII (SEQ ID NO:6) (reverse primer). The cmplNcol (SEQ ID NO:4) primer is described in Example 1. The c-mplHindIII (SEQ ID NO:6) primer, which annals to bases #716-737 of c-mpl ligand, adds both a termination codon and a HindIII restriction enzyme site immediately following the final codon, Arg 153 Two types of PCR products are generated from the RT cDNA samples, one with a deletion of the codons for amino acids WO 97/12978 PCT/US96/15938 68 112-115 and one without the deletion of these codons. The cmpl ligand PCR products (ca. 480 bp) are digested with NcoI and HindIII restriction enzymes for transfer to a mammalian expression vector, pMON3934, which is a derivative of pMON3359 (see Example pMON3934 is digested with NcoI and HindIII (ca. 3800 bp) and will accept the PCR products.

The plasmid, pMON32132, contians the DNA sequence of (SEQ ID NO:82) which encodes for amino acids 1-153 of c-mpl ligand (SEQ ID NO:j4) was a result of this cloning. The plasmid, pMON32133, contains the DNA sequence of (SEQ ID NO:83) which encodes amino acids 1-153 of c-mpl ligand with a deletion of codons 112-115 (A112-115) (SEQ ID NO:45) was also a result of this cloning.

EXAMPLE 3 Generation of PCR 5L dimer template containing A112-115 in the second gene A PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32133 (containing a deletion of amino acids 112-115) along with the EcoRI/AflIII 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:9) and 5L-3' (SEQ ID The EcoRI end of the linker will ligate to the EcoRI end of pMON26458. The AflIII end of the linker will ligate to the NcoI site of pMON32133, and neither restriction site will be retained upon ligation. The BstXI sites of pMON26458 and pMON32133 will ligate as well. Plasmid, pMON28548, is a result of the cloning and contains the DNA sequence of (SEQ ID NO:38) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker to amino acids 1-153 c-mpl ligand that contains a deletion of amino acids 112-115 (SEQ ID N0:43).

WO 97/12978 PCT/US96/15938 69 EXAMPLE 4 Generation of PCR 4L dimer template pMON28500 A PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132 along with the EcoRI/AflIII 4L synthetic oligonucleotide linker 4L-5' (SEQ ID NO:7) and 4L-3' (SEQ ID NO:8).

The EcoRI end of the linker will ligate to the EcoRI end of pMON26458. The AflIII end of the linker will ligate to the NcoI site of pMON32132, and neither restriction site will be retained upon ligation. The BstXI sites of-pMON26458 and pMON32132 will ligate as well. Plasmid, pMON28500 is a result of the cloning and contains the DNA sequence of (SEQ ID NO:39) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyAsnMetAla (SEQ ID NO:77) linker (4L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:46).

EXAMPLE Generation of PCR 5L dimer template pMON28501 A PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132 along with the EcoRI/AflIII 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:9) and 5L-3' (SEQ ID The ECORI end of the linker will ligate to the EcoRI end of pMON26458. The AflIII end of the linker will ligate to the NcoI site of pMON32132, and neither restriction site will be retained upon ligation. The BstXI sites of pMON26458 and pMON32132 will ligate as well. PLasmid, pMON28501 is a result of the cloning and contains the DNA sequence of (SEQ ID NO: WO 97/12978 PCT/US96/15938 which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker (5L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:47).

EXAMPLE 6 Generation of PCR 8L dimer template pMON32136 A PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132 along with the EcoRI/AflIII 8L synthetic oligonucleotide linker 8L-5' (SEQ ID NO:11) and 8L-3' (SEQ ID NO:12).

The EcoRI end of the linker will ligate to the EcoRI end of pMON26458. The AflIII end of the linker will ligate to the NcoI site of pMON32132, and neither restriction site will be retained upon ligation. The BstXI sites of pMON26458 and pMON32132 will ligate as well. Plasmid, pMON32136 is a result of the cloning which contains the DNA sequence of (SEQ ID NO:41) and encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79) linker (8L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:48).

EXAMPLES 7-18 Generation of novel c-mpl receptor agonists A. PCR generation of novel c-mpl receptor aaonists.

Novel c-mpl ligand genes are generated using the Horlick method. The PCR reaction was carried out using dimer template pMON28501 and one of the sets of synthetic primer sets below (number refers to first amino acid of new molecule).

WO 97/12978 PCT/US96/15938 71 31-5' (SEQ ID NO:13) and 31-3' (SEQ ID NO:14), 35-5' (SEQ ID and 35-3' (SEQ ID NO:16), 39-5' (SEQ ID NO:17) and 39- 3' (SEQ ID NO:18), 43-5' (SEQ ID NO:19) and 43-3' (SEQ ID 45-5' (SEQ ID NO:21) and 45-3' (SEQ ID NO:22), 49-5' (SEQ ID NO:23) and 49-3' (SEQ ID NO:24), 82-5' (SEQ ID and 82-3' (SEQ ID NO:26), 109-5' (SEQ ID NO:27) and 109-3' (SEQ ID NO:28), 116-5' (SEQ ID NO:29) and 116-3' (SEQ ID ,120-5' (SEQ ID NO:31) and 120-3' (SEQ ID NO:32), 123- (SEQ ID NO:33) and 123-3' (SEQ ID NO:34), 126-5' (SEQ ID NO:35) and 126-3' (SEQ ID NO:36) The products that are generated are about 480 bp and are purified via Magic PCR Clean up kits (Promega). Table 4 shows the template, the primer set used in the PCR reaction and the breakpoint for each Example.

B. Subclonina of novel c-mDl receptor aaonists into mammalian expression vector The c-mpl receptor agonist PCR products are digested with NcoI and HindIII or AflIII and HindIII restriction enzymes (ca. 470 bp) for transfer to a mammalian expression vector.

The expression vector, pMON3934, is digested with NcoI and HindIII (ca. 3800 bp) and accepts the PCR products as NcoI- HindIII or AflIII-HindIII fragments. Table 4 shows the restriction digest of the PCR product and the resulting expression plasmid pMON designation.

WO 97/12978 WO 9712978PCTIUS96/1 5938 72 TABLE 4 Example 7 8 9 11 12 13 14 16 17 18

PCR

template pMON2 8501 pMON2B5O1 pMON2 8501 pMON2Z 501 pM0M28501 pM0N2 8501 pM0M2 8501 pM0N2 8501 pMON2 8501 pMON2 8501 pM0N28501 pMON2 8501

PCR

Primer set 31 35 39 43 45 49 82 109 116 120 123 126 Breakpoint 30-31 34-3 5 38-39 42-43 44-45 48-49 81-82 108-109 115-116 119-120 122- 123 125-126 PCR Product Resulting Linker Restriction Plasmid Digest pM0N 5L NcoI/HindIII pM0N30373 5L AflIII/HindIII pM0N30374 5L NcoI/HindIII pM0N30375 5L NcoI/HindIII pM0N30376 5L NcoI/HindIII pM0N30377 5L NcoI/HindIII pM0N30378 5L NcoI/HindIII pM0N30379 5L NcoI/HindIII pM0N30380 5L NcoI/HindIII pM0N30381 5L NcoI/HindIII pM0N30382 5L NcoI/HindIII pM0N30383 5L NcoI/HindIII pM0N30384 in a similar manner the dimer templates pMON28500, pMON32l36 and pMON28548 could be used in the PCR reaction as described in Examples 7-18.

Bioactivity determination of c-mpl receptor agonits Transfected cell lines: Cell lines such as Baf/3 cell line can be transfected with a colony stimulating factor receptor, such as the human IL-3 receptor or human c-mpl receptor, which the cell line does not normally have. These transfected cell lines can be used to determine the activity of the ligand for which the receptor has been transfected into the cell line.

one such transfected Baf /3 cell line was made by cloning the cDNA encoding c-mpl from a library made from a cmpl responsive cell line and cloned into the multiple cloning site of the plasmid pcDNA3 (Invitrogen, San Diego Baf /3 WO 97/12978 WO 9712978PCTIUS96/15938 73 cells were transfected with the plasmid via electroporation.

The cells were grown under G418 selection in the presence of mouse IL-3 in Wehi conditioned media. Clones were established through limited dilution.

The BHK expression levels and bioactivity data of some of the c-mpl receptor agonists of the present invention are shown in Table 5. The supernatant from the transfected BHK cells was evaluated for expression of the c-mpl receptor agonists by western analysis using an antobody raised against c-mpl ligand. Constructs that expressed at a 11 level were assayed in the Baf-3/c-mpl cell proliferation assay.

TABLE Express'Lon and Bioactivitv' of c-mpl rece tor agonist pMON# c-mpl receptor BH-K expression Baf-3/c-mpl agonist levels cell proliferation IDMON30373 _31-153/5L/1-30 nd TpMON30374 35-153/5L/1-34 rid pM0N30375 39-153/5L/1-38 -nd pMON30376 43-153/5L/1-42 -nd PMON30377 45-153/5L/1-44 nd vMQN30378 49-153/5L/1-48 nd 3 79 82-153/5L/1-81 -nd PMON30380 109-153/5L/1-108 PMON30381 l16-153/5L/1-115 TpMON30382 120-153/5L/1-119 rid PM0N30383 123-153/5L/1-122 r- rid 2. Bone marrow proliferation assay a. CD34+ Cell Purification: Between 15-20 rnL bone marrow aspirates are obtained from normal allogeneic marrow donors after informed consent. Cells are diluted 1:3 in phosphate buffered saline (PBS, Gibco- BRL), 30 mL are layered over 15 mL Histopaque-1077 (Sigma) and centrifuged for 30 minutes at 300 RCF. The mononuclear interface layer is collected and washed in PBS. CD34+ cells WO 97/12978 PCTIUS96/15938 74 are enriched from the mononuclear cell preparation using an affinity column per manufacturers instructions (CellPro, Inc, Bothell WA). After enrichment, the purity of CD34+ cells is on average as determined by using flow cytometric analysis using anti CD34 monoclonal antibody conjugated to fluorescein and anti-CD38 conjugated to phycoerythrin (Becton Dickinson, San Jose CA).

Cells are resuspended at 40,000 cells/mL in X-Vivo media (Bio-Whittaker, Walkersville, MD) and 1 mL is plated in 12-well tissue culture plates (Costar). Human IL-3 variant, pMON13288, is used at 10 ng/mL or 100 ng/mL. Conditioned media from BHK cells transfected with plasmid encoding c-mpl ligand are tested by addition of 100 p. of supernatant added to 1 mL cultures (approximately a 10% dilution). Cells are incubated at 37 0 C for 8-14 days at 5% C02 in a 37°C humidified incubator.

b. Cell Harvest and Analysis: At the end of the culture period a total cell count is obtained for each condition. For fluorescence analysis and ploidy determination cells, are washed in megakaryocyte buffer (MK buffer, 13.6 mM Sodium Citrate, 1 mM Theophylline, 2.2 pm PGE1, 11 mM Glucose, 3% w/v BSA, in PBS, pH 7.4,) (Tomer et al., Blood 70(6): 1735-42 [1987]) resuspended in 500 pR of MK buffer containing anti-CD41a FITC antibody (1:200, AMAC, Westbrook, ME) and washed in MK buffer. For DNA analysis cells are made permeable in MK buffer containing Tween 20 (Fisher, Fair Lawn NJ) for 20 minutes on ice followed by fixation in 0.5% Tween-20 and 1% paraformaldehyde (Fisher Chemical) for 30 minutes followed by incubation in Propidium Iodide (Calbiochem La Jolla Ca) (50 pg/mL) with RNAase (400 U/mL) in 55% v/v MK buffer (200 mOsm) for 1-2 hours on ice. Cells are analyzed on a FACScan or Vantage flow cytometer (Becton Dickinson, San Jose, CA). Green fluorescence (CD41a-FITC) is collected along with linear and log signals for red fluorescence (PI) to determine DNA ploidy. All cells are collected to determine the percent of cells that are CD41+. Data analysis is performed using LYSIS software (Becton Dickinson, San Jose, CA). Percent of cells expressing the CD41 antigen is obtained from flow cytometry analysis (Percent). Absolute (Abs) number of CD41+ cells/mL is calculated by: (Abs)=(Cell Count)*(Percent)/100.

3. Megakaryocyte fibrin clot assay.

CD34+ enriched population are isolated as described above. Cells are suspended at 25,000 cells/mL with/without cytokine(s) in a media consisting of a base Iscoves

IMDM

media supplemented with 0.3% BSA, 0.4 mg/mL apo-transferrin, 6.67pM FeC12, 25gg/mL CaC12, 25 gg/mL L asparagine, 500 gg/mL E-amino-n-caproic acid and Penicillin/Streptomycin. Prior to plating into 35 mm plates, thrombin is added (0.25 units/mL).

to initiate clot formation. Cells are incubated at 37 0 C for 13 days at 5% C02 in a 37°C humidified incubator. At the end of the culture period plates are fixed with methanol:acetone air dried and stored at -200C until staining.

A

peroxidase immunocytochemistry staining procedure is used (Zymed, Histostain-SP. San Francisco, CA) using a cocktail of primary monoclonal antibodies consisting of anti CD41a, CD42 and CD61. Colonies are counted after staining and classified as negative, CFU-MK (small colonies, 1-2 foci and less that 25 approx. 25 cells), BFU MK (large, multi-foci colonies with cells) or mixed colonies (mixture of both positive and negative cells).

Various other examples will be apparent to the person skilled in the art after reading the present disclosure 30 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.

With reference to the use of the word(s) "comprise" or "comprises" or "comprising" in the foregoing description and/or in the following claims, we note that unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and A t hat we intend each of those words to be so interpreted in construing the foregoing description and/or the following claims.

WO 97/12978 PCT/US96/15938 76 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: G. D. Searle STREET: P.O. Box 5110 CITY: Chicago STATE: Illinois COUNTRY: United States of America POSTAL CODE (ZIP): 60680 TELEPHONE: (708) 470-6501 TELEFAX: (708) 470-6881 NAME: Monsanto Company STREET: 88 North Lindbergh Boulevard CITY: St. Louis STATE: Missouri COUNTRY: United States of America POSTAL CODE (ZIP): 63167 TELEPHONE: (314) 694-3131 TELEFAX: (314) 694-5435 (ii) TITLE OF INVENTION: c-mpl ligand Receptor Agonists (iii) NUMBER OF SEQUENCES: 83 (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 C-2908 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/004,824 FILING DATE: 05-OCT-1995 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 332 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Modified-site WO 97/12978 WO 9712978PCT/US96/15938 77 LOCATION:l12 OTHER INFORMATION:/note= "position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or (ix) FEATURE: NAME/KEY: Modified-site LOCATION:113 OTHER INFORMATION:/note= "position 113 is deleted or Pro, Phe, Ala, Leu, Ile, Trp, or Met" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:114 OTHER INFORMATION:/note= "position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp or Met" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:115 OTHER INFORMATION: /note= "Position 115 is deleted or Gin, Gly, Ser, Thr, Tyr or Asn" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg His Gly Gly Leu Val Xaa Leu Asp Pro Glu Ala Gly Arg Xaa Ser 130 Ser Leu Trp Val Pro Leu Xaa 115 Phe His Pro Lys Thr Thr Leu 100 Gly Gin Val Thr Thr Leu Cys Leu Arg His Leu Pro Gin Leu 70 Leu Gly Thr Leu His Val Met 55 Leu Ser Ala Thr Leu 135 Ser Leu 40 Giu Glu Ser Leu Ala 120 Arg Arg 25 Leu Glu Gly Leu Gin 105 His Gly Leu Pro Thr Val Leu 90 Ser Lys Lys Ser Ala Lys Met 75 Gly Leu Asp Val Gin Cys Val Asp Ala Gin Ala Ala Gin Leu Leu Gly Pro Asn 125 Arg Phe 140 Pro Phe Asp Arg Ser Thr 110 Ala Leu Glu Ser Ile Gly Gly Gin Ile Met Val Leu Leu Gin Gin Xaa Phe Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala 145 150 155 160 WO 97/12978 WO 9712978PCT/US96/15938 Val Pro Ser Arg Thr Ser Leu Val 165 Arg Thr Pro Tyr 225 Pro Thr Pro Pro Asp 305 Thr Gly Gly 210 Leu Gly Ser Ser Pro 290 Pro Ser Ser 195 Leu Asn Pro Asp Pro 275 Thr Ser Gly 180 Giy Leu Arg Ser Thr 260 Thr Leu Ala Leu Leu Asn Ile Arg 245 Gly His Pro Pro Leu Leu Gin His 230 Arg Ser Pro Thr Thr 310 Giu Lys Thr 215 Giu Thr Leu Pro Pro 295 Pro Thr Trp 200 Ser Leu Leu Pro Thr 280 Val1 Thr Leu Thr Leu 170 Asn Phe Thr 185 Gin Gin Gly Arg Ser Leu Leu Asn Gly 235 Gly Ala Pro 250 Pro Asn Leu 265 Gly Gin Tyr Val Gin Leu Pro Thr Ser 315 Asn Ala Phe Asp 220 Thr Asp Gin Thr His 300 Pro Giu Ser Arg 205 Gin Arg Ile Pro Leu 285 Pro Leu Leu Ala 190 Ala Ile Gly Ser Gly 270 Phe Leu Leu Pro 175 Arg Lys Pro Leu Ser 255 Tyr Pro Leu Asn Asn Thr Ile Giy Phe 240 Gly Ser Leu Pro Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu Gly INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 153 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Modified-site LOCATION:112 OTHER INFORMATION:/note= "position 112 is deleted or Leu, Aia,VA1, Ile, Pro, Phe, Trp or Met" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:ii3 WO 97/12978 WO 9712978PCT/US96/15938 OTHER INFORMATION:/note= "positoin 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp or Met" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:114 OTHER INFORMATION:/note= "Position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp or Met, (ix) FEATURE: NAME/KEY: Modified-site LOCATION:115 OTHER INFORMATION:/note= "positon 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 Arg His Gly Gly Leu Val Xaa Leu Asp Pro Giu Ala Gly Arg Xaa Ser 130 Ser Leu Trp Val1 Pro Leu Xaa 115 Phe His Val Pro Thr Lys Thr Thr Leu Thr Cys Leu Leu 100 Gly Arg Gin His Leu Pro Gin Leu 70 Leu Gly Thr Leu His Val Met 55 Leu Ser Ala Thr Leu 135 Ser Leu 40 Glu Glu Ser Leu Ala 120 Arg Arg 25 Leu Glu Gly Leu Gin 105 His Gly Leu Pro Thr Vai Leu 90 Ser Lys Lys Ser Gin Ala Val Lys Ala Met Ala 75 Gly Gin Leu Leu Asp Pro Val Arg 140 Cys Pro Asp Phe Gin Asp Ala Arg Leu Ser Gly Thr 110 Asn Ala 125 Phe Leu Giu Ser Ile Gly G ly Gin Ile Met Val1 Leu Leu Gin Gin Xaa Phe Leu Val Gly Gly Ser Thr Leu Cys Val Arg 145 150 INFORMATION FOR SEQ ID NO: 3- SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: unknown WO 97/12978 PCT/US96/15938 TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 45 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: ACGTCCATGG CNTCNCCNGC NCCNCCTGCT TGTGCACTCC GAGTC INFORMATION FOR SEQ ID NO: 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: ATGCACGAAT TCCCTGACGC AGAGGGTGGA INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs WO 97/12978 PCT/US96/15938 81 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: TGACAAGCTT ACCTGACGCA GAGGGTGGAC CCT 33 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 10 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:

AATTCGGCAA

INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 10 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:

CATGTTGCCG

INFORMATION FOR SEQ ID NO: 9: WO 97/12978 PCT/US96/15938 82 SEQUENCE CHARACTERISTICS: LENGTH: 13 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: AATTCGGCGG CAA 13 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 13 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: CATGTTGCCG CCG 13 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 22 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: WO 97/12978 PCT/US96/15938 83 AATTCGGCGG CAACGGCGGC AA 22 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 22 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: CATGTTGCCG CCGTTGCCGC CG 22 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 27 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: CGATCCATGG AGGTTCACCC TTTGCCT 27 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" WO 97/12978 PCT/US96/15938 84 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GATCAAGCTT ATGGGCACTG GCTCAGTCT 29 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 27 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: CGATACATGT TGCCTACACC TGTCCTG 27 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 27 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: 16: CGATCCATGG TCCTGCTGCC TGCTGTG 27 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" WO 97/12978 PCT/US96/15938 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CGATCCATGG TCCTGCTGCC TGCTGTG 27 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 29 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: GATCAAGCTT AAGGTGTAGG CAAAGGGTG 29 INFORMATION FOR SEQ ID NO: 19: 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: 19: CGATCCATGG CTGTGGACTT TAGCTTGGGA INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/12978 PCT/US96/15938 86 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATCAAGCTT AAGGCAGCAG GACAGGTGT 29 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 27 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: 21: CGATCCATGG ACTTTAGCTT GGGAGAA 27 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 29 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: 22: GATCAAGCTT ACACAGCAGG CAGCAGGAC 29 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs WO 97/12978 PCT/US96/15938 87 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CGATCCATGG GAGAATGGAA AACCCAG 27 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 29 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: 24: GATCAAGCTT ACAAGCTAAA GTCCACAGC 29 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 27 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: CGATCCATGG GACCCACTTG CCTCTCA 27 INFORMATION FOR SEQ ID NO: 26: WO 97/12978 PCT/US96/15938 88 SEQUENCE CHARACTERISTICS: LENGTH: 29 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: 26: GATCAAGCTT ACAGTTGTCC CCGTGCTGC 29 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 29 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: 27: GATCAAGCTT AAAGGAGGCT CTGCAGGGC 29 INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 29 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: 28: WO 97/12978 PCT/US96/15938 89 GATCAAGCTT AAAGGAGGCT CTGCAGGGC 29 INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 27 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: 29: CGATCCATGG GCAGGACCAC AGCTCAC 27 INFORMATION FOR SEQ ID NO: 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: GATCAAGCTT ACTGTGGAGG AAGCTGGGTT INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetis)" WO 97/12978 PCT/US96/15938 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GATCAAGCTT ACTGTGGAGG AAGCTGGGTT INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 29 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: 32: GATCAAGCTT ATGTGGTCCT GCCCTGTGG 29 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (syntheitc)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CGATCCATGG ATCCCAATGC CATCTTCCTG INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" WO 97/12978 PCT/US96/15938 91 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: GATCAAGCTT ACTTGTGAGC TGTGGTCCT 29 INFORMATION FOR SEQ ID NO: 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: CGATCCATGG CCATCTTCCT GAGCTTCCAA INFORMATION FOR SEQ ID NO: 36: 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: 36: GATCAAGCTT AATTGGGATC CTTGTGAGCT GT 32 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 465 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/12978 PCT/US96/15938 92 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC 120 CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA 180 CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC 240 CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT 300 CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG 360 CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG 420 TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTC 465 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 927 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

TGACTCCCAT

ACCTGTCCTG

GACCAAGGCA

ACGGGGACAA

CCGTCTCCTC

GACCACAGCT

AAAGGTGCGT

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120 WO 97/12978 WO 9712978PCTIUS96/15938

CTGCCTGCTG

180

CAGGACATTC

240

CTGGGACCCA

300

CTTGGGGCCC

360

CACAAGGATC

420

TTCCTGATGC

480

TCTCCCGCTC

540

GTCCTTCACA.

600

CTGCCTGCTG

660

CAGGACATTC

720

CTGGGACCCA

780

CTTGGGGCCC

840

AATGCCATCT

900

TGGACTTTAG

TGGGAGCAGT

CTTGCCTCTC

TGCAGAGCCT

CCAATGCCAT

TTGTAGGAGG

CGCCTGCTTG

GCAGACTGAG

TGGACTTTAG

TGGGAGCAGT

CTTGCCTCTC

TGCAGAGCCT

CTTGGGAGAA

GACCCTTCTG

ATCCCTCCTG

CCTTGGAACC

CTTCCTGAGC

GTCCACCCTC

TGACCTCCGA

CCAGTGCCCA

CTTGGGAGAA

GACCCTTCTG

ATCCCTCCTG

CCTTGGA.ACC

TGGAAAACCC AGATGGAGGA CTGGAGGGAG TGATGGCAGC GGGCAGCTTT CTGGAC!AGGT CAGCTTCCTC CACAGGGCAG TTCCAACACC TGCTCCGAGG TGCGTCAGGG AATTCGGCGG GTCCTCAGTA AACTGCTTCG

GACCAAGGCA

ACGGGGACAA

CCGTCTCCTC

GACCACAGCT

AAAGGTGCGT

CAACATGGCG

TGACTCCCAT

ACCTGTCCTG

GACCAAGGCA

ACGGGGACAA

CCGTCTCCTC

CAAGGATCCC

GAGGTTCACC

TGGAAAACCC

CTGGAGGGAG

GGGCAGCTTT

CAGGGCAGGA

CTTTGCCTAC

AGATGGAGGA

TGATGGCAGC

CTGGACAGGT

CCACAGCTCA

TCCTGAGCTT CCAACP ~CCTG CTCCGAGGAA AGGTGCGTTT CCTGATGCTT GTAGGAGGGT CCACCCTCTG CGTCAGG 927 INFORMATION FOR SEQ ID NO: 39: Mi SEQUENCE CHARACTERISTICS: LENGTH: 936 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" WO 97/12978 WO 9712978PCTIUS96/15938 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT

GTCCTTCACA

120

CTGCCTGCTG

180

CAGGACATTC

240

CTGGGACCCA

300

CTTGGGGCCC

360

CACAAGGATC

420

TTCCTGATGC

480

CCCGCTCCGC

540

CTTCACAGCA

600

CCTGCTGTGG

660

GACATTCTGG

720

GGACCCACTT

780

GGGGCCCTGC

840

AAGGATCCCA

900

GCAGACTGAG

TGGACTTTAG

TGGGAGCAGT

CTTGCCTCTC

TGCAGAGCCT

CCAATGCCAT

TTGTAGGAGG

CTGCTTGTGA

GACTGAGCCA

ACTTTAGCTT

GAGCAGTGAC

GCCTCTCATC

AGAGCCTCCT

.ATGCCATCTl CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG

CTTGGGAGAA

GACCCTTCTG

ATCCCTCCTG

CCTTGGAACC

CTTCCTGAGC

GTCCACCCTC

CCTCCGAGTC

GTGCCCAGAG

GGGAGAATGG

CCTTCTGCTG

CCTCCTGGGG

TGGAACCCAG

CCTGAGCTTC

TGGAAAACCC AGATGGAGGA CTGGAGGGAG TGATGGCAGC GGGCAGCTTT CTGGACAGGT CAGCTTCCTC CACAGGGCAG TTCCAACACC TGCTCCGAGG TGCGTCAGGG A.ATTCGGCAA CTCAGTAAAC TGCTTCGTGA GTTCACCCTT TGCCTACACC AAAACCCAGA TGGAGGAGAC GAGGGAGTGA TGGCAGCACG CAGCTTTC!TG GACAGGTCCG CTTCCTCC!AC AGGGCAGGAC CAACACCTGC TCCGAGGAAA

GACCAAGGCA

ACGGGGACAA

CCGTCTCCTC

GACCACAGCT

AAAGGTGCGT

CATGGCGTCT

CTCCCATGTC

TGTCCTGCTG

CAAGGCACAG

GGGACAACTG

TCTCCTCCTT

CACAGCTCAC

GGTGCGTTTC

CTGATGCTTG TAGGAGGGTC CACCCTCTGC GTCAGG 936 INFORMATION FOR SEQ ID NO: WO 97/12978 WO 9712978PCT/US96/15938 SEQUENCE CHARACTERISTICS: LENGTH: 939 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: TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT

GTCCTTCACA

120

CTGCCTGCTG

180

CAGGACATTC

240

CTGGGACCCA

300

CTTGGGGCCC

360

CACAAGGATC

420

TTCCTGATGC

480

TCTCCCGCTC

540

GTCCTTCACA

600

GCAGACTGAG

TGGACTTTAG

TGGGAGCAGT

CTTGCCTCTC

TGCAGAGCCT

CCAATGCCAT

TTGTAGGAGG

CGCCTGCTTG

GCAGACTGAG

CCAGTGCCCA

CTTGGGAGAA

GACCCTTCTG

ATCCCTCCTG

CCTTGGAACC

CTTCCTGAGC

GTCCACCCTC

TGACCTCCGA

CCAGTGCCCA

GAGGTTCACC CTTTGCCTAC ACCTGTCCTG

TGGAAAACCC

CTGGAGGGAG

GGGCAGCTTT

CAGCT CCTC

TTCCAACACC

TGCGTCAGGG

GTCCTCAGTA

GAGGTTCACC

AGATGGAGGA

TGATGGCAGC

CTGGACAGGT

CACAGGGCAG

TGCTCCGAGG

A.ATTCGGCGG

A.ACTGCTTCG

CTTTGCCTAC

AGATGGAGGA

GACCAAGGCA

ACGGGGACAA

CCGTCTCCTC

GACCACAGC

AAAGGTGCGT

CAACATGGCG

TGACTCCCAT

ACCTGTCCTG

GACCAAGGCA

CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC 660 CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 720

CTGGGACCCA

780 CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC WO 97/12978 WO 9712978PCT/US96/I 5938 96 CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 840 CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 900 TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG 939 INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS: LENGTH: 948 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: 41: TCCCCAGCGC CGCCTGCTTG TGACCTCCGA GTCCTCAGTA A.ACTGCTTCG TGACTCCCAT

GTCCTTCACA

120

CTGCCTGCTG

180

CAGGACATTC

240

CTGGGACCCA

300

CTTGGGGCCC

360

CACAAGGATC

420

TTCCTGATGC

480

AACATGGCGT

GCAGACTGAG

TGGACTTTAG

TGGGAGCAGT

CTTGCCTCTC

TGCAGAGCCT

CCAATGCCAT

TTGTAGGAGG

CCCCAGCGCC

CCAGTGCCCA

CTTGGGAGAA

GACCCTTCTG

ATCCCTCCTG

CCTTGGAACC

CTTCCTGAGC

GTCCACCCTC

GCCTGCTTGT

GAGGTTCACC

TGGAAAACCC

CTGGAGGGAG

GGGCAGCTTT

CAGCTTCCTC

TTCCAACACC

TGCGTCAGGG

GACCTCCGAG

CTTTGCCTAC

AGATGGAGGA

TGATGGCAGC

CTGGACAGGT

CACAGGGCAG

TGCTCCGAGG

AATTCGGCGG

TCCTCAGTAA

ACCTGTCCTG

GACCAAGGCA

ACGGGGACAA

CCGTCTCCTC

GACCACAGCT

AAAGGTGCGT

CAACGGCGGC

ACTGCTTCGT

GACTCCCATG

600 TCCTTCACAG CAGACTGAGC CAGTGCCCAG AGGTTCACCC TTTGCCTACA WO 97/12978 WO 9712978PCTIUS96/15938 97 CCTGTCCTGC TGCCTGCTGT GGACTTTAGC TTGGGAGAAT GGAAAACCCA GATGGAGGAG 660 ACCAAGGCAC AGGACATTCT GGGAGCAGTG ACCCTTCTGC TGGAGGGAGT GATGGCAGCA 720 CGGGGACAAC TGGGACCCAC TTGCCTCTCA TCCCTCCTGG GGCAGCTTTC TGGACAGGTC 780 CGTCTCCTCC TTGGGGCCCT GCAGAGCCTC CTTGGAACCC AGCTTCCTCC ACAGGGCAGG 840 ACCACAGCTC ACAAGGATCC CAATGCCATC TTCCTGAGCT TCCAACACCT GCTCCGAGGA 900 AAGGTGCGTT TCCTGATGCT TGTAGGAGGG TCCACCCTCT GCGTCAGG 948 INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: 155 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Giu Val 25 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 40 Gly Giu Trp Lys Thr Gin Met Glu Giu Thr Lys Ala Gin Asp Ile Leu 55 Gly Ala Val Thr Leu Leu Leu Giu Gly Val Met Ala Ala Arg Gly Gin 70 75 Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 90 Vai Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110 WO 97/12978 PCT/US96/15938 Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125 Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140 Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe 145 150 155 INFORMATION FOR SEQ ID NO: 43: SEQUENCE CHARACTERISTICS: LENGTH: 309 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 Arg His Gly Gly Leu Val Pro Leu Val 145 Asp Pro Glu Ala Gly Arg Pro Ser 130 Ser Leu Trp Val Pro Leu Gin 115 Phe His Pro Lys Thr Thr Leu 100 Gly Gin Val Thr Thr Leu Cys Leu Arg His Leu Pro Gin Leu 70 Leu Gly Thr Leu His Val Met 55 Leu Ser Ala Thr Leu 135 Ser Leu 40 Glu Glu Ser Leu Ala 120 Arg Arg Leu 25 Leu Pro Glu Thr Gly Val Leu Leu 90 Gin Ser 105 His Lys Gly Lys Ser Ala Lys Met 75 Gly Leu Asp Val Gin Cys Pro Glu Val Val Asp Phe Ser Leu Ala Gln Asp Ile Leu Ala Ala Arg Gly Gin Gin Leu Ser Gly Gin Leu Gly Thr Gin Leu 110 Pro Asn Ala Ile Phe 125 Arg Phe Leu Met Leu 140 Gly Gly Asn Met Ala 160 Gly Gly Ser Thr Leu Cys Val Arg Glu Phe 150 155 WO 97/12978 WO 9712978PCT/US96/15938 Ser Arg His Gly Gly 225 Leu Val Arg His Pro Asp Pro Glu 210 Ala Gly Arg Thr Leu 290 Ala Ser Leu 195 Trp Val Pro Leu Thr 275 Leu Pro His 180 Pro Lys Thr Thr Leu 260 Ala Arg Pro 165 Val Thr Thr Leu Cys 245 Leu His Gly Ala Leu Pro Gln Leu 230 Leu Gly Lys Lys Cys His Val Met 215 Leu Ser Ala Asp Val 295 Asp Ser Leu 200 Glu Glu Ser Leu Pro 280 Arg Leu Arg 185 Leu Glu Gly Leu Gln 265 Asn Phe Arg 170 Leu Pro Thr Val Leu 250 Ser Ala Leu Val Ser Ala Lys Met 235 Gly Leu I le Met Leu Gln Val Ala 220 Ala Gln Leu Phe Leu 300 Lys Pro 190 Phe Asp Arg Ser Thr 270 Ser Gly Leu 175 Glu Ser Ile Gly Gly 255 Gln Phe Gly Leu Val Leu Leu Gln 240 Gln Gly Gln Ser Thr Leu Cys Val Arg 305 INFORMATION FOR SEQ ID NO: 44: SEQUENCE CHARACTERISTICS: LENGTH: 153 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 25 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 40 WO 97/12978 PCT/US96/15938 100 Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 55 Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 70 75 Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 90 Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110 Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125 Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140 Val Gly Gly Ser Thr Leu Cys Val Arg 145 150 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 149 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 25 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 40 Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 55 Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 70 75 Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 90 WO 97/12978 PCT/US96/15938 101 Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Gly 100 105 110 Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin 115 120 125 His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser 130 135 140 Thr Leu Cys Val Arg 145 INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: 312 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 25 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 40 Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 55 Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 70 75 Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 90 Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110 Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125 Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140 WO 97/12978 WO 9712978PCTIUS96/15938 Val Gly 145 Pro Ala Asp Ser Pro Leu Glu Trp 210 Ala Val 225 Gly Pro Arg Leu Pro Gin Ser Phe 290 Gly Pro His Pro 195 Lys Thr Thr Leu Gly 275 Gin Ser Pro Val 180 Thr Thr Leu Cys Leu 260 Arg His Thr Ala 165 Leu Pro Gin Leu Leu 245 Gly Thr Leu Leu 150 Cys His Val Met Leu 230 Ser Ala Thr Leu Cys Asp Ser Leu Glu 215 Glu Ser Leu Ala Arg 295 Val1 Leu Arg Leu 200 Glu Gly Leu Gin His 280 Gly Arg Giu Phe Gly Asn Met Ala Ser 155 Arg Leu 185 Pro Thr Val Leu Ser 265 Lys Val Leu 170 Ser Gin Ala Val Lys Ala Met Ala 235 Gly Gin 250 Leu Leu Asp Pro Ser Cys Asp Gin 220 Ala Leu Gly Asn Lys Pro Phe 205 Asp Arg Ser Thr Ala 285 Leu Glu 190 Ser Ile Gly Gly Gin 270 Ile Leu 175 Val Leu Leu Gin Gin 255 Leu Phe 160 Arg His Gly Gly Leu 240 Vai Pro Leu Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg 305 310 INFORMATION FOR SEQ ID NO: 47: SEQUENCE CHARACTERISTICS: LENGTH: 313 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Vai Leu Ser Lys Leu Leu 1 5 10 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Giu Val 25 WO 97/12978 PTU9/53 PCTIUS96/15938 His Gly Gly Leu Val Pro Leu Val 145 Ser Arg His Gly Gly 225 Leu Val Pro Leu Val 305 Pro Glu Ala Gly Arg Pro Ser 130 Gly Pro Asp Pro Giu 210 Ala Gly Arg Pro Ser 290 Leu Trp Val Pro Leu Gin 115 Phe Gly Ala Ser Leu 195 Trp Val1 Pro Leu Gin 275 Phe ?ro -lys ['hr rhr Leu 1.00 Gly Gin Ser Pro His 180 Pro Lys Thr Thr Leu 260 Gly Gln Thr Pro Val I Thr Gin Met( 55 Leu Leu Leu 70 Cys Leu Ser Leu Gly Ala Arg Thr Thr His Leu Leu 135 Thr Leu Cys 150 Pro Ala Cys 165 Val Leu His Thr Pro Val Thr Gin Met 215 Leu Leu Leu 230 Cys Leu Ser 245 Leu Gly Ala Arg Thr Thr His Leu Leu 295 ~eu 31U Ser Leu Ala 120 Arg Val1 Asp Ser Leu 200 Giu Glu Ser Leu Ala 280 Arg Leu Glu Gly Leu Gin 105 His Gly Arg Leu Arg 185 Leu Glu Gly Leu Gin 265 His Gly Pro Ala Val Asp Phe Ser Leu rhr VIal Lieu 90 Ser Lys Lys Glu Arg 170 Leu Pro Thr Val Leu 250 Ser Lys Lys Lys Met 75 Gly Leu Asp Val Phe 155 Val Ser Ala Lys Met 235 Gly Leu Asp Val Ala Ala Gin Leu Pro Arg 140 G ly Leu Gin Val1 Ala 220 Ala Gin Leu Pro Arg Gin Asp Ala Arg Leu Ser Gly Thr 110 Asn Ala 125 Phe Leu Gly Asn Ser Lys Cys Pro 190 Asp Phe 205 Gin Asp Ala Arg Leu Ser Gly Thr 270 Asn Ala 285 Phe Leu Ile Gly Gly Gin Ile Met Met Leu 175 Giu Ser Ile Gly Gly 255 Gin Ile Met Leu Gin Gin Leu Phe Leu Ala 160 Leu Val Leu Leu Gin 240 Gin *Leu Phe *Leu Giy Gly Ser Thr Cys Val Arg WO 97/12978 PCT/US96/15938 104 INFORMATION FOR SEQ ID NO: 48: SEQUENCE CHARACTERISTICS: LENGTH: 316 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 25 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 40 Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 55 Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 70 75 Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 90 Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110 Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125 Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140 Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Gly Gly 145 150 155 160 Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser 165 170 175 Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys 180 185 190 Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp 195 205 WO 97/12978 PCT/US96/15938 105 Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin 210 215 220 Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala 225 230 235 240 Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu 245 250 255 Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly 260 265 270 Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn 275 280 285 Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe 290 295 300 Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg 305 310 315 INFORMATION FOR SEQ ID NO: 49: SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe 1 5 10 Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp 25 Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg 40 Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser 55 Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr 70 75 Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala 90 WO 97/12978 WO 9712978PCTIUS96/15938 Ile Phe Leu Ser Phe Gin His 100 Met Leu Val Giy Gly Ser Thr 115 Met Ala Ser Pro Ala Pro Pro 130 135 Leu Leu. Arg Asp Ser His Val 145 150 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTIC! LENGTH: 160 amino i TYPE: amino acid STRANDEDNESS: unknc TOPOLOGY: unknown (ii) MOLECULE TYPE: protein Leu. Leu. Arg Gly Lys Val Arg Phe Leu 105 110 Leu. Cys Val Arg Giu Phe Gly Giy Asn 120 125 Ala Cys Asp Leu Arg Val Leu Ser Lys 140 Leu His Ser Arg Leu Ser Gin Cys Pro 155 160 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Leu Pro Thr Pro Vai Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Giu 1 Trp Val Pro Leu Gin Phe Gly Ala 5 10 Lys Thr Thr Leu Gly Gin Ser Pro Thr Leu Cys Leu Arg His Thr 115 Pro Gin Leu Leu Gly Thr Leu.

100 Leu.

Ala Met Leu Ser Ala Thr Leu Cys Cys Giu.

Giu Ser Leu, 70 Ala Arg Val Asp Giu Gly Leu 55 Gln His Gly Arg Leu Thr Val 40 Leu Ser Lys Lys Giu 120 Arg Lys Ala 25 Met Aia Gly Gin Leu Leu Asp Pro 90 Val Arg 105 Phe Gly Val Leu Gin Ala Leu Gly 75 Asn Phe Gly Ser Asp Arg Ser Thr Ala Leu Asn Lys Ile Gly Gly Gin Ile Met Met 125 Leu Leu Gin Gin Leu Phe Leu 110 Ala Leu Gly Ala Leu Giy Val Arg Pro Pro Leu Ser Val Gly Ser Pro Arg Asp WO 97/12978 PCT/US96/15938 107 130 135 140 Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro 145 150 155 160 INFORMATION FOR SEQ ID NO: 51: SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin 1 Met Glu Leu Glu Ser Ser Ala Leu Thr Ala Leu Arg Cys Val Cys Asp 130 Glu Gly Leu Gln His Gly Arg 115 Leu Thr Lys Val Met Leu Gly Ser Leu Lys Asp Lys Val 100 Glu Phe Arg Val Ala Gn Ala Ala Gin Leu 55 Leu Gly 70 Pro Asn Arg Phe Gly Gly Leu Ser Asp Arg 40 Ser Thr Ala Leu Asn 120 Lys Ile 25 Gly Gly Gin Ile Met 105 Met Leu 10 Leu Gin Gin Leu Phe 90 Leu Ala Leu Gly Leu Val Pro 75 Leu Val Ser Arg His 155 Ala Gly Arg Pro Ser Gly Pro Asp 140 Val Pro Leu Gin Phe Gly Ala 125 Ser Thr Thr Leu Gly Gin Ser 110 Pro His Leu Cys Leu Arg His Thr Pro Val Leu Leu Gly Thr Leu Leu Ala Leu 135 His Ser Arg Leu Ser Gin Cys Pro Glu Val 145 150 INFORMATION FOR SEQ ID NO: 52: Pro Leu Pro Thr WO 97/12978 108 SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein PCT/US9615938 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: Ala Val Asp Phe Ser Leu Gly Giu Trp Lys Thr Gin Met Giu Giu. Thr Lys Met Gly Leu Asp Val Phe Val Ser Ala Ala Gln Leu Pro Arg Gly Leu 130 Gin Gin Ala Leu Gly Asn Phe Gly 115 Ser Cys Asp Arg Ser Thr Ala Leu 100 Asn Lys Pro Ile Gly Gly Gin Ile Met Met Leu Glu Leu Gin Gin Leu 70 Phe Leu Ala Leu Gly Leu Val 55 Pro Leu Val Ser Arg 135 Ala Gly 40 Arg Pro Ser Gly Pro 120 Asp Val 25 Pro Leu Gin Phe Gly 105 Ala Ser Thr Thr Leu G ly Gin 90 Ser Pro His Leu Cys Leu Arg 75 His Thr Pro Val Leu Leu G ly Thr Leu Leu Ala Leu 140 Leu Ser Ala Thr Leu Cys Cys 125 His Giu Gly Ser Leu Leu Gin Ala His Arg Gly Val Arg 110 Asp Leu Ser Arg Val Leu Ser Lys Lys Giu Arg Leu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro INFORMATION FOR SEQ ID NO: 53: SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein WO 97/12978 PCT/US96/15938 109 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala 1 5 10 Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala 25 Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin 40 Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu 55 Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro 70 75 Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg 90 Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly 100 105 110 Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu 115 120 125 Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin 130 135 140 Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val 145 150 155 160 INFORMATION FOR SEQ ID NO: 54: SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 1 5 10 WO 97/12978 PCT/US96/15938 110 Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 25 Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 40 Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 55 Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 70 75 Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 90 Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala 100 105 110 Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 115 120 125 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 130 135 140 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 145 150 155 160 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val 1 5 10 Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro 25 Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu 40 Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val 55 WO 97/12978 PCT/US96/15938 111 Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser 70 75 Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg 90 Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His 100 105 110 Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly 115 120 125 Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly 130 135 140 Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu 145 150 155 160 INFORMATION FOR SEQ ID NO: 56: SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro 1 5 10 Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg 25 Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly 40 Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu 55 Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin 70 75 Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val 90 Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala WO 97/12978 PCT/US96/15938 112 100 105 110 Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala 115 120 125 Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin 130 135 140 Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu 145 150 155 160 INFORMATION FOR SEQ ID NO: 57: SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe 1 5 10 Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly 25 Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala 40 Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser 55 His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu 70 75 Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp 90 Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val 100 105 110 Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro 115 120 125 Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu 130 135 140 WO 97/12978 PCT/US96/15938 113 Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin 145 150 155 160 INFORMATION FOR SEQ ID NO: 58: SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu 1 5 10 Arg Val Asp Ser Leu Glu Glu Ser Leu Gly Arg Leu Arg Leu Glu Gly Leu 130 Gin Lys Glu Arg Leu Pro Thr Val 115 Leu Ser Val Phe Val Ser Ala Lys 100 Met Gly Arg Gly Leu Gin Val Ala Ala Gin Phe Gly Ser Cys 70 Asp Gin Ala Leu Leu Asn Lys 55 Pro Phe Asp Arg Ser 135 Met Leu 25 Met Ala 40 Leu Leu Glu Val Ser Leu Ile Leu 105 Gly Gin 120 Gly Gin Val Ser Arg His Gly 90 Gly Leu Val Gly Pro Asp Pro 75 Glu Ala Gly Arg Gly Ala Ser Leu Trp Val Pro Leu 140 Ser Pro His Pro Lys Thr Thr 125 Leu Thr Pro Val Thr Thr Leu 110 Cys Leu Leu Ala Leu Pro Gin Leu Leu Gly Cys Cys His Val Met Leu Ser Ala Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr INFORMATION FOR SEQ ID NO: 59: SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids WO 97/12978 114 TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein PCTIUS96/15938 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu. Arg Gly Lys Val Phe Val Ser Ala Lys Met Gly Leu Arg Giy Leu Gin Val Aia Aia Gin 130 Leu Phe Gly Ser Cys Asp Gin Ala 115 Leu Gly Leu Asn Lys Pro Phe Asp 100 Arg Ser Thr Met Met Leu Giu Ser Ile Gly Gly Gin Leu Ala Leu Vai 70 Leu Leu Gin Gin Leu Val Ser Arg 55 His Gly Giy Leu Vai 135 Pro Giy Pro 40 Asp Pro Giu Ala Gly 120 Arg Pro Giy Ser Thr 25 Ala Pro Pro Ser His Val Leu Pro Thr 75 Trp Lys Thr 90 Vai Thr Leu 105 Pro Thr Cys Leu Leu Leu Leu Cys Aia Cys Leu His Pro Val Gin Met Leu Leu Leu Ser 125 Gly Ala 140 Val Arg Asp Leu Ser Arg Leu Leu Giu Giu Giu Gly 110 Ser Leu Leu Gin Glu Arg Leu Pro Thr Val Leu Ser Lys 160 Gin Gly Arg 155 Thr Thr Ala His INFORMATION FOR SEQ ID NO: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 160 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein WO 97/12978 WO 9712978PCTIUS96/15938 115 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Asn Lys Pro Phe Asp Arg Ser Thr Met Met Leu Glu Ser Ile Gly Gly 130 Gin Leu Ala Leu Vai Leu Leu Gin 115 Gin Leu Vai Ser Arg His Gly Gly 100 Leu Vai Pro Gly Pro Asp Pro Glu Ala Gly Arg Pro Gly Ala Ser Leu 70 Trp Val1 Pro Leu Gin Ser Pro His 55 Pro Lys Thr Thr Leu 135 G ly Thr Pro 40 Val Thr Thr Leu Cys 120 Leu Arg Leu 25 Ala Leu Pro Gin Leu 105 Leu Gly Thr Cys Cys His Vai Met 90 Leu Ser Ala Thr Val Asp Ser Leu 75 Glu Giu Ser Leu Ala 155 Arg Leu Arg Leu Giu Gly Leu Gin 140 His Giu Arg Leu Pro Thr Val Leu 125 Ser Lys Phe Val Ser Ala Lys Met 110 Gly Leu Asp Gly Leu Gin Val Ala Ala Gin Leu Pro Gly Ser Cys Asp Gin Ala Leu Gly Asn 160 INFORMATION FOR SEQ ID NO: 61: SEQUENCE CHARACTERISTICS: LENGTH: 480 base pairs TYPE: nucleic acid STRAN'DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: GAGGTTCACC CTTTGCCTAC ACCTGTCCTG CTGCCTGCTG TGGACTTTAG CTTGGGAGAA WO 97/12978 WO 9712978PCT/US961 15938 TGGAAAACCC AGATGGAGGA GACCAAGGCA CAGGACATTC TGGGAGCAGT 120 CTGGAGGGAG TGATGGCAGC ACGGGGACAA CTGGGACCCA CTTGCCTCTC 180 GGGCAGCTTT CTGGACAGGT CCGTCTCCTC CTTGGGGCCC TGCAGAGCCT 240 CAGCTTCCTC CACAGGGCAG GACCACAGCT CACAAGGATC CCAATGCCAT 300 TTCCAACACC TGCTCCGAGG AAAGGTGCGT TTCCTGATGC TTGTAGGAGG 360 TGCGTCAGGG AATTCGGCGG CAACATGGCG TCTCCGGCGC CGCCTGCTTG 420 GTCCTCAGTA AACTGCTTCG TGACTCCCAT GTCCTTCACA GCAGACTGAG 480 INFORMATION FOR SEQ ID NO: 62: Wi SEQUENCE CHARACTERISTICS: LENGTH: 480 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc *DNA (Synthetic)"

GA.CCCTTCTG

ATCCCTCCTG

CCTTGGAACC

CTTCCTGAGC

GTCCACCCTC

TGACCTCCGA

CCAGTGCCCA

(xi) SEQUENCE DESCRIPTION: SEQ, ID NO: 62: TTGCCTACAC CTGTCCTGCT GCCTGCTGTG GACTTTAGCT TGGGAGAATG ATGGAGGAGA CCAAGGCACA GGACATTCTG, GGAGCAGTGA CCCTTCTGCT 120 ATGGCAGCAC GGGGACAACT GGGACCCACT TGCCTCTCAT CCCTCCTGGG 180 GGACAGGTCC GTCTCCTCCT TGGGGCCCTG CAGAGCCTCC TTGGAACCCA 240 CAGGGCAGGA CCACAGCTCA CAAGGATCCC AATGCCATCT TCCTGAGCTT 300

GAAAACCCAG

GGAGGGAGTG

GCAGCTTTCT

GCTTCCTCCA

CCAACACCTG

WO 97/12978 WO 9712978PCTIUS96/1 5938 117 CTCCGAGGAA AGGTGCGTTT CCTGATGCTT GTAGGAGGGT CCACCCTCTG CGTCAGGGAA 360 TTCGGCGGCA ACATGGCGTC TCCGGCGCCG CCTGCTTGTG ACCTCCGAGT CCTCAGTAAA 420 CTGCTTCGTG ACTCCCATGT CCTTCACAGC AGACTGAGCC AGTGCCCAGA GGTTCACCCT 480 INFORMATION FOR SEQ, ID NO: 63: SEQUENCE CHARACTERISTICS: LENGTH: 480 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: 63: GTCCTGCTGC CTGCTGTGGA CTTTAGCTTG GGAGAATGGA A.AACCCAGAT AAGGCACAGG ACATTCTGGG AGCAGTGACC CTTCTGCTGG AGGGAGTGAT 120 GGACAACTGG GACCCACTTG CCTCTCATCC CTCCTGGGGC AGCTTTCTGG 180 CTCCTCCTTG GGGCCCTGCA GAGCCTCCTT GGAACCCAGC TTCCTCCACA 240 ACAGCTCACA AGGATCCCAA TGCCATCTTC CTGAGCTTCC ALACACCTGCT 300 GTGCGTTTCC TGATGCTTGT AGGAGGGTCC ACCCTCTGCG TCAGGGAATT 360 ATGGCGTCTC CGGCGCCGCC TGCTTGTGAC CTCCGAGTCC TCAGTAAACT 420 TCCCATGTCC TTCACAGCAG ACTGAGCCAG TGCCCAGAGG TTCACCCTTT 480 INFORMATION FOR SEQ ID NO: 64: Wi SEQUENCE CHARACTERISTICS:

GGAGGAGACC

GGCAGCACGG

ACAGGTCCGT

GGGCAGGACC

CCGAGGAAAG

CGGCGGCAAC

GCTTCGTGAC

GCCTACACCT

WO 97/12978 PCT/US96/15938 118 LENGTH: 480 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: 64: GCTGTGGACT TTAGCTTGGG AGAATGGAAA ACCCAGATGG AGGAGACCAA ATTCTGGGAG CAGTGACCCT TCTGCTGGAG GGAGTGATGG CAGCACGGGG 120 CCCACTTGCC TCTCATCCCT CCTGGGGCAG CTTTCTGGAC AGGTCCGTCT 180 GCCCTGCAGA GCCTCCTTGG AACCCAGCTT CCTCCACAGG GCAGGACCAC 240 GATCCCAATG CCATCTTCCT GAGCTTCCAA CACCTGCTCC GAGGAAAGGT 300 ATGCTTGTAG GAGGGTCCAC CCTCTGCGTC AGGGAATTCG GCGGCAACAT 360 GCGCCGCCTG CTTGTGACCT CCGAGTCCTC AGTAAACTGC TTCGTGACTC 420 CACAGCAGAC TGAGCCAGTG CCCAGAGGTT CACCCTTTGC CTACACCTGT 480 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 480 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

GGCACAGGAC

ACAACTGGGA

CCTCCTTGGG

AGCTCACAAG

GCGTTTCCTG

GGCGTCTCCG

CCATGTCCTT

CCTGCTGCCT

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: WO 97/12978 WO 9712978PCTIUS96/15938 GACTTTAGCT TGGGAGAATG GAAAACCCAG ATGGAGGAGA CCAAGGCACA GGAGCAGTGA CCCTTCTGCT GGAGGGAGTG ATGGCAGCAC GGGGACAACT 120 TGCCTCTCAT CCCTCCTGGG GCAGCTTTCT GGACAGGTCC GTCTCCTCCT 180 CAGAGCCTCC TTGGAACCCA GCTTCCTCCA CAGGGCAGGA CCACAGCTCA 240 AATGCCATCT TCCTGAGCTT CCAACACCTG CTCCGAGGAA AGGTGC!GTTT 300 GTAGGAGGGT CCACCCTCTG CGTCAGGGAA TTCGGCGGCA ACATGGCGTC 360 CCTGCTTGTG ACCTCCGAGT CCTCAGTAAA CTGCTTCGTG ACTCCCATGT 420 AGACTGAGCC AGTGCCCAGA GGTTCACCCT TTGCCTACAC CTGTCCTGCT 480 INFORMATION FOR SEQ ID NO: 66: SEQUENCE CHARACTERISTICS: LENGTH: 479 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

GGACATTCTG

GGGACCCACT

TGGGGCCCTG

CAAGGATCCC

CCTGATGCTT

TCCGGCGCCG

CCTTCACAGC

GCCTGCTGTG

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: GGAGAATGGA AAACCCAGAT GGAGGAGACC AAGGCACAGG ACATTCTGGG AGCAGTGACC 4 CTTCTGCTGG AGGGAGTGAT GGCAGCACGG GGACAACTGG GACCCACTTG CCTCTCATCC 120 CTC!CTGGGGC AGCTTTCTGG ACAGGTCCGT CTCCTC!CTTG GGGCCCTGCA GAGCCTCCTT 180 GGAACCCAGC TTCCTCCACA GGGCAGGACC ACAGCTCACA AGGATCCCAA TGCCATCTTC 240 WO 97/12978 WO 9712978PCT/US96/15938 120 CTGAGCTTCC AACACCTGCT CCGAGGAAAG GTGCGTTTCC TGATGCTTGT AGGAGGGTCC 300 ACCCTCTGCG TCAGGGAATT CGGCGGCAAC ATGGCTCTCC GGCGCCGCCT GCTTGTGACC 360 TCCGAGTCCT CAGTAAACTG CTTCGTGACT CCCATGTCCT TCACAGCAGA CTGAGCCAGT 420 GCCCAGAGGT TCACCCTTTG CCTACACCTG TCCTGCTGCC TGCTGTGGAC TTTAGCTTG 479 INFORMATION FOR SEQ ID NO: 67: SEQUENCE CHARACTERISTICS: LENGTH: 480 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synithetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: GGACCCACTT GCCTCTCATC CCTCCTGGGG CAGCTTTCTG GACAGGTCCG TCTCCTCCTT

GGGGCCCTGC

120

AAGGATCCCA

180

CTGATGCTTG

240

CCGGCGCCGC

300

CTTCACAGCA

360

CCTGCTGTGG

420

GACATTCTGG

480

AGAGCCTCCT

ATGCCATCTT

TAGGAGGGTC

CTGCTTGTGA

GACTGAGCCA

ACTTTAGCTT

GAGCAGTGAC

TGGAACCCAG

CCTGAGCTTC

CACCCTCTGC

CCTCCGAGTC

GTGCCCAGAG

GGGAGAATGG

CCTTCTGCTG

CTTCCTCCAC

CAACACCTGC

GTCAGGGAAT

CTCAGTAAAC

GTTCACCCTT

AAAACCCAGA

GAGGGAGTGA

AGGGCAGGAC

TCCGAGGAA.A

TCGGCGGCAA

TGCTTCGTGA

TGCCTACACC

TGGAGGAGAC

TGGCAGCACG

CACAGCTCAC

GGTGCGTTTC

CATGGCGTCT

CTCCCATGTC

TGTCCTGCTG

CAAGGCACAG

GGGACAACTG

INFORMATION FOR SEQ ID NO: 68: WO 97/12978 PCT/US96/15938 121 SEQUENCE CHARACTERISTICS: LENGTH: 480 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: 68: GGAACCCAGC TTCCTCCACA GGGCAGGACC ACAGCTCACA AGGATCCCAA CTGAGCTTCC AACACCTGCT CCGAGGAAAG GTGCGTTTCC TGATGCTTGT 120 ACCCTCTGCG TCAGGGAATT CGGCGGCAAC ATGGCGTCTC CGGCGCCGCC 180 CTCCGAGTCC TCAGTAAACT GCTTCGTGAC TCCCATGTCC TTCACAGCAG 240 TGCCCAGAGG TTCACCCTTT GCCTACACCT GTCCTGCTGC CTGCTGTGGA 300 GGAGAATGGA AAACCCAGAT GGAGGAGACC AAGGCACAGG ACATTCTGGG 360 CTTCTGCTGG AGGGAGTGAT GGCAGCACGG GGACAACTGG GACCCACTTG 420 CTCCTGGGGC AGCTTTCTGG ACAGGTCCGT CTCCTCCTTG GGGCCCTGCA 480 INFORMATION FOR SEQ ID NO: 69: SEQUENCE CHARACTERISTICS: LENGTH: 480 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

TGCCATCTTC

AGGAGGGTCC

TGCTTGTGAC

ACTGAGCCAG

CTTTAGCTTG

AGCAGTGACC

CCTCTCATCC

GAGCCTCCTT

WO 97/12978 PCTIUS96/15938 (xi) SEQUENCE DESCRIPTION: SEQ ID-NO: 69: GGCAGGACCA CAGCTCACAA GGATCCCAAT GCCATCTTCC TGAGCTTCCA CGAGGAAAGG TGCGTTTCCT GATGCTTGTA GGAGGGTCCA CCCTCTGCGT 120 GGCGGCAACA TGGCGTCTCC GGCGCCGCCT GCTTGTGACC TCCGAGTCCT 180 CTTCGTGACT CCCATGTCCT TCACAGCAGA CTGAGCCAGT GCCCAGAGGT 240 CCTACACCTG TCCTGCTGCC TGCTGTGGAC TTTAGCTTGG GAGAATGGAA 300 GAGGAGACCA AGGCACAGGA CATTCTGGGA GCAGTGACCC TTCTGCTGGA 360 GCAGCACGGG GACAACTGGG ACCCACTTGC CTCTCATCCC TCCTGGGGCA 420 CAGGTCCGTC TCCTCCTTGG GGCCCTGCAG AGCCTCCTTG GAACCCAGCT 480

ACACCTGCTC

CAGGGAATTC

CAGTAAACTG

TCACCCTTTG

AACCCAGATG

GGGAGTGATG

GCTTTCTGGA

TCCTCCACAG

INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 480 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: GCTCACAAGG ATCCCAATGC CATCTTCCTG AGCTTCCAAC ACCTGCTCCG AGGAAAGGTG CGTTTCCTGA TGCTTGTAGG AGGGTCCACC CTCTGCGTCA GGGAATTCGG CGGCAACATG 120 GCGTCTCCGG CGCCGCCTGC TTGTGACCTC CGAGTCCTCA GTAAACTGCT TCGTGACTCC 180 CATGTCCTTC ACAGCAGACT GAGCCAGTGC CCAGAGGTTC ACCCTTTGCC TACACCTGTC 240 WO 97/12978 PCTIUS96/15938 CTGCTGCCTG CTGTGGACTT TAGCTTGGGA GAATGGAAAA CCCAGATGGA GGAGACCAAG 300 GCACAGGACA TTCTGGGAGC AGTGACCCTT CTGCTGGAGG GAGTGATGGC AGCACGGGGA 360 CAACTGGGAC CCACTTGCCT CTCATCCCTC CTGGGGCAGC TTTCTGGACA GGTCCGTCTC 420 CTCCTTGGGG CCCTGCAGAG CCTCCTTGGA ACCCAGCTTC CTCCACAGGG CAGGACCACA 480 INFORMATION FOR SEQ ID NO: 71: SEQUENCE CHARACTERISTICS: LENGTH: 480 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: 71: GATCCCAATG CCATCTTCCT GAGCTTCCAA CACCTGCTCC GAGGAAAGGT ATGCTTGTAG GAGGGTCCAC CCTCTGCGTC AGGGAATTCG GCGGCAACAT 120 GCGCCGCCTG CTTGTGACCT CCGAGTCCTC AGTAAACTGC TTCGTGACTC 180 CACAGCAGAC TGAGCCAGTG CCCAGAGGTT CACCCTTTGC CTACACCTGT 240 GCTGTGGACT TTAGCTTGGG AGAATGGAAA ACCCAGATGG AGGAGACCAA 300 ATTCTGGGAG CAGTGACCCT TCTGCTGGAG GGAGTGATGG CAGCACGGGG 360 CCCACTTGCC TCTCATCCCT CCTGGGGCAG CTTTCTGGAC AGGTCCGTCT 420 GCCCTGCAGA GCCTCCTTGG AACCCAGCTT CCTCCACAGG GCAGGACCAC 480

GCGTTTCCTG

GGCGTCTCCG

CCATGTCCTT

CCTGCTGCCT

GGCACAGGAC

ACAACTGGGA

CCTCCTTGGG

AGCTCACAAG

WO 97/12978 PCT/US96/15938 124 INFORMATION FOR SEQ ID NO: 72: SEQUENCE CHARACTERISTICS: LENGTH: 480 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: 72: GCCATCTTCC TGAGCTTCCA ACACCTGCTC CGAGGAAAGG TGCGTTTCCT GGAGGGTCCA CCCTCTGCGT CAGGGAATTC GGCGGCAACA TGGCGTCTCC 120 GCTTGTGACC TCCGAGTCCT CAGTAAACTG CTTCGTGACT CCCATGTCCT 180 CTGAGCCAGT GCCCAGAGGT TCACCCTTTG CCTACACCTG TCCTGCTGCC 240 TTTAGCTTGG GAGAATGGAA AACCCAGATG GAGGAGACCA AGGCACAGGA 300 GCAGTGACCC TTCTGCTGGA GGGAGTGATG GCAGCACGGG GACAACTGGG 360 CTCTCATCCC TCCTGGGGCA GCTTTCTGGA CAGGTCCGTC TCCTCCTTGG 420 AGCCTCCTTG GAACCCAGCT TCCTCCACAG GGCAGGACCA CAGCTCACAA 480 INFORMATION FOR SEQ ID NO: 73: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide

GATGCTTGTA

CGCTCCGCCT

TCACAGCAGA

TGCTGTGGAC

CATTCTGGGA

ACCCACTTGC

GGCCCTGCAG

GGATCCCAAT

WO 97/12978 PCT/US96/15938 125 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO: 74: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: Gly Gly Gly Ser Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 INFORMATION FOR SEQ ID NO: 76: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 97/12978 PCT/US96/15938 126 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: Ser Gly Gly Ser Gly Gly Ser 1 INFORMATION FOR SEQ ID NO: 77: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: Glu Phe Gly Asn Met Ala 1 INFORMATION FOR SEQ ID NO: 78: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: Glu Phe Gly Gly Asn Met Ala 1 INFORMATION FOR SEQ ID NO: 79: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 97/12978 PCT/US96/15938 127 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: Glu Phe Gly Gly Asn Gly Gly Asn Met Ala 1 5 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gly Gly Ser Asp Met Ala Gly 1 INFORMATION FOR SEQ ID NO: 81: 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: 81: GGATCCACCA TGAGCCGCCT GCCCGTCCTG CTCCTGCTCC AACTCCTGGT CCGCCCCGC 59 INFORMATION FOR SEQ ID NO: 82: SEQUENCE CHARACTERISTICS: LENGTH: 459 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" WO 97/12978 PCTIUS96/15938 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: TCTCCCGCTC CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120 CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180 CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 240 CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 300 CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 360 CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 420 TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG 459 INFORMATION FOR SEQ ID NO: 83: SEQUENCE CHARACTERISTICS: LENGTH: 936 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: 83: TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120 CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180 WO 97/12978 WO 9712978PCTIUS96/1 5938

CAGGACATTC

240

CTGGGACCCA

300

CTTGGGGCCC

360

CACAAGGATC

420

TTCCTGATGC

480

CCCGCTCCGC

540

CTTCACAGCA

600

CCTGCTGTGG

660

GACATTCTGG

720

GGACCCACTT

780

GGGGCCCTGC

840

AAGGATCCCA

900

CTGATGCTTG

936

TGGGAGCAGT

CTTGCCTCTC

TGCAGAGCCT

CCAATGCCAT

TTGTAGGAGG

CTGCTTGTGA

GACTGAGCCA

ACTTTAGCTT

GAGCAGTGAC

GCCTCTCATC

AGAGCCTCCT

ATGCCATCTT

TAGGAGGGTC

GACCCTTCTG

ATCCCTCCTG

CCTTGGAACC

CTTCCTGAGC

GTCCACCCTC

CCTCCGAGTC

GTGCCCAGAG

GGGAGAATGG

CCTTCTGCTG

CCTCCTGGGG

TGGAACCCAG

CCTGAGCTTC

CACCCTCTGC

CTGGAGGGAG TGATGGCAGC ACGGGGACAA GGGCAGCTTT CTGGACAGGT CCGTCTCCTC CAGCTTCCTC CACAGGGCAG GACCACAGCT TTCCAACACC TGCTCCGAGG AAAGGTGCGT TGCGTCAGGG AATTCGGCAA CATGGCGTCT CTCAGTAAAC TGCTTCGTGA CTCCCATGTC GTTCACCCTT TGCCTACACC TGTCCTGCTG AAAACCCAGA TGGAGGAGAC CAAGGCACAG GAGGGAGTGA TGGCAGCACG GGGACAACTG CAGCTTTCTG GACAGGTCCG TCTCCTCCTT CTTCCTCCAC AGGGCAGGAC CACAGCTCAC CAACACCTGC TCCGAGGAAA GGTGCGTTTC

GTCAGG

Claims (21)

1. A c-mpl receptor agonist polypeptide comprising, a modified c-mpl ligand amino acid sequence of the Formula: SerProAiaProProAlaCysAspLeuArgVaiLeuS erLysLeuLeuArgAspSer 1 5 10 HisValLeuHisSerArgLeuSerGlnCysProGluValHi sProLeuProThrPro 20 25 30 ValLeuLeuProAiaVaiAspPheS erLeuGlyGluTrpLy sThrGlnMetGluGlu 45 50 ThrLysAlaGlnAsplleLeuGlyAlaVaiThrLeuLeuLeuGluGlyVaiMetAla 65 70 AiaArgG lyG inLeuG iyProThrCysLeuSerSerLeuLeuGlyGlnLeuS erGly 85 90 GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuG iyThrGinXaaXaaXaa 100 105 110 XaaGlyArgThrThrAiaHisLysAspProAsnAlal lePheLeuSerPheGinHis 115 120 125 130 LeuLeuArgG lyLysVaiArgPheLeuMetLeuValG lyGlySerThrLeuCysVai 135 140 145 150 Arg (SEQ ID NO:2) 153 wherein; Xaa at position 112 is deleted or Leu, Ala, Vai, Ile, Pro, Phe, Trp, or Met; xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn; WO 97/12978 WO 9712978PCTIUS96/1 5938 131 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 51-52 108-109

27-28 52-53 109-110

28-29 53-54 110-111

29-30 54-55 111-112

30-31 55-56 112-113

32-33 56-57 113-114

33-34 57-58 114-115

34-35 58-59 115-116

36-37 59-60 116-117

37-38 78-79 117-118

38-39 79-80 118-119

40-41 80-81 119-120

41-42 81-82 120-121

42-43 82-83 121-122

43-44 83-84 122-123

44-45 84-85 123-124

46-47 85-86 124-125

47-48 86-87 125-126

48-49 87-88 126-127

50-51 88-89 or 127-128; and additionally said polypeptide can be immediately preceded by (methionine- 1 (alanine- 1 or (methionine- 2 alanine- 1 2. The c-mpl receptor agonist polypeptide, as recited in claim 1, wherein said linker is selected from the group consisting of; GlyGlyGlySer (SEQ ID NO:73); GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:74) GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID SerGlyGlySerGlyGlySer (SEQ ID NO:76); GluPheGlyAsnIetAla (SEQ ID NO:77) GluPheGlyGlyAsnMetAla (SEQ ID NO:78); GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79); and GlyGlySerAspMetAlaGly (SEQ ID WO 97/12978 PCT/US96/15938 132 3. The c-mpl receptor agonist polypeptide as recited in claim 1 wherein said polypeptide is selected from the group consisting of: Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Gilu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro (SEQ ID NO:49); Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro (SEQ ID Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro (SEQ ID NO:51); Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly WO 97/12978 PCT/US9615938 133 Ser Thr Leu Cys Val Arg Giu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro (SEQ ID NO:52); Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Giu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Vai Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Giu Phe Giy Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Giu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val (SEQ ID NO:53); Gly Glu Trp Lys Thr Gin Met Giu Giu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Giu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Giy Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Giy Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Vai Arg Phe Leu Met Leu Vai Gly Giy Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Vai Leu His Ser Arg Leu Ser Gin Cys Pro Giu Vai His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu (SEQ ID NO:54); Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Giy Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Giu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Giu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu (SEQ ID Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala le Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Vai Leu His Ser Arg Leu Ser Gin Cys Pro Glu Vai His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Giu Trp Lys Thr WO 97/12978 PCT/US96/15938 134 Gin Met Giu Giu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu (SEQ ID NO:56); Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Giu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin (SEQ ID NO:57); Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Giu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr (SEQ ID NO:58); Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Giu Trp Lys Thr Gin Met Glu Giu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Giu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys (SEQ ID NO:59); and Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Giu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Giu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Giy Giu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu WO 97/12978 PCT/US96/15938 135 Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn (SEQ ID 4. The c-mpl receptor agonist polypeptide as recited in claim 3 wherein said polypeptide is selected from the group consisting of: Gly Thr Pro Asn Val Arg Glu Phe Leu Arg Ser Arg Val Leu Gin Met Leu Leu Thr Cys Leu Leu Gly Arg Phe Gin Gly Gly Ser Pro Leu Arg Glu Val Phe Ser Gin Asp Ala Ala Gly Gin Ser Leu Gin Leu Pro Pro Gin Ala Ile Phe Leu Ser Phe Leu Met Leu Val Gly Gly Asn Met Ala Val Leu Ser Lys Leu Leu Ser Gin Cys Pro Leu Pro Ala Val Asp Glu Glu Thr Lys Ala Leu Glu Gly Val Met Leu Ser Ser Leu Leu Leu Gly Ala Leu Gin Gly Arg Thr Thr Ala Phe Gin His Leu Leu Gly Gly Ser Thr Leu Ser Pro Ala Pro Pro Leu Arg Asp Ser His Glu Val His Pro Leu Phe Ser Leu Gly Glu Gin Asp Ile Leu Gly Ala Ala Arg Gly Gin Gly Gin Leu Ser Gly Ser Leu Leu (SEQ ID His Lys Asp Arg Gly Lys Cys Val Arg Ala Cys Asp Val Leu His Pro Thr Pro Trp Lys Thr Ala Val Thr Leu Gly Pro Gin Val Arg NO;56); and Thr Thr His Leu Ser Thr Ala Pro Asp Ser His Pro Leu Gly Ile Leu Arg Gly Leu Ser Leu Gly Ala Leu Leu Pro His Leu Glu Gly Gin Gly Thr His Lys Arg Gly Cys Val Ala Cys Val Leu Pro Thr Trp Lys Ala Val Leu Gly Gin Val Gin Leu Asp Pro Asn Ala Ile Lys Val Arg Phe Leu Arg Glu Phe Gly Gly Asp Leu Arg Val Leu His Ser Arg Leu Ser Pro Val Leu Leu Pro Thr Gin Met Glu Glu Thr Leu Leu Leu Glu Pro Thr Cys Leu Ser Arg Leu Leu Leu Gly Pro Pro Gin (SEQ ID Phe Met Asn Ser Gin Ala Thr Gly Leu Ser Leu Val Met Ala Lys Leu Cys Pro Val Asp Lys Ala Val Met Ser Leu Leu Ala Leu Gin NO:57). A nucleic acid molecule, encoding the c-mpl receptor agonist polypeptide of claim 1. 6. A nucleic acid molecule encoding the c-mpl receptor agonist polypeptide of claim 2. 7. A nucleic acid molecule encoding the c-mpl receptor agonist polypeptide of claim 3. WO 97/12978 WO 9712978PCTIUS96/15938 136 8. A nucleic acid molecule encoding the c-mpl receptor agonist polypeptide of claim 4. 9. A nucleic acid molecule according to claim 7 selected from the group consisting of: GAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAA TGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTG CTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTG GGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACC CAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGC TTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTC TGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGA GTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCA (SEQ ID NO:61); TTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAG ATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTG ATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATC CCTCCTGGGGCAGCTTTCT GGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCA CAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTG CTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTC CACCCTCTGCGTCAGGGAA TTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAA CTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCT (SEQ ID NO:62); GTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACC AAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGG GGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGT CTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACC ACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAG GTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAAC ATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGAC TCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCT (SEQ ID NO:63); GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT WO 97/12978 WO 9712978PCTIUS96/15938 137 CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT (SEQ ID NO:64); GACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGA GACCAAGGCACAGGACATTCTG GGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTG CAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCC AATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTT GTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCG CCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTG (SEQ ID GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCNTCTCC GGCGCCGCCTGCTTGTGAC CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG (SEQ ID NO:66); GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTT GGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCAC AAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTC CTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCT CCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTC CTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTG CCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG (SEQ ID NO:67); GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCC GGCGCCGCCTGCTTGTGAC CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT (SEQ ID NO:68); GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC GGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG WO 97/12978 WO 9712978PCT/US96/1 5938 138 GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGArG GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGA CAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAG (SEQ ID NO:69); GCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTG CGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATG GCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCC CATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTC CTGCTGCCTGCrI GTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAG GCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGA CAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTC CTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACA (SEQ ID GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG GCCCTGCAGAGCCTCCTTGGALACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG (SEQ ID NO:71); and GCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA GGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGCTCCGCCT GCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGA CTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGAC TTTAGCTTGGGAGA-ATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGA GCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAG AGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAAT (SEQ ID NO:72). A nucleic acid molecule according to claim 8 selected from the group consisting of: GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGAC CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT (SEQ ID NO:68); and WO 97/12978 WO 9712978PCTIUS96/1 5938 139 GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC GGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATG GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGA CAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAG (SEQ ID NO:69). 11. A method of producing a human c-mpl receptor agonist polypeptide comprising, growing under suitable nutrient conditions, a host cell transformed or transfected with a replicable vector comprising a nucleic acid molecule of claim 5, 6, 7 or 8 in a manner allowing expression of said human c-mpl receptor agonist polypeptide and recovering said human c-mpl receptor agonist polypeptide. 12. A composition comprising a polypeptide of claim 1, 2, 3 or 4 and a pharmaceutically acceptable carrier. 13. A composition comprising; a polypeptide of claim 1, 2, 3 or 4; a colony stimulating factor; and a pharmaceutically acceptable carrier. 14. A composition comprising a polypeptide of claim 1, 2, 3 or 4; a colony stimulating factor selected from the group consisting of; GM-CSF, G-CSF, G-CSF Serl 7 M-CSF, erythropoietin (EPO), IL-l, IL-4, IL-2, IL-3, IL-5, IL 6, IL- 7, IL-8, IL-9, IL-10, IL-li, IM-12, IL-13, IL-iS, LIF, fit3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil. differentiation factor and stem cell factor; and a pharmaceutically acceptable carrier. 140 Use of a polypepide according to any of claims 1, 2, 3 or 4 for preparing a medicament for stimulating the production of hematopoietic cells in a patient. 16. Use of a composition of claim 13 for preparing a medicament for stimulating the production of hematopoietic cells in a patient. 17. Use of a composition of claim 14 for preparing a medicament for stimulating the production of hematopoietic cells in a patient. 18 A method of stimulating the production of hematopoietic cells in a patient comprising the step of administering said polypeptide of claim 1, 2, 3 or 4 to said patient. 19 A method of stimulating the production of hematopoietic cells in a patient comprising .the step of administering the composition of claim 13 to said patient. 20 A method of stimulating the production of hematopoietic cells in a patient comprising the step of administering the composition of claim 14 to said patient. *9* 21. A method for selective ex vivo expansion of stem cells, comprising the steps of; 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; and harvesting said cultured cells. *22 Use of a polypeptide of claim 1, 2,3 or 4 for preparing a medicament for treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) 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; and transplanting said cultured cells into said patient. L u- 23. Use of a hematopoietic protein of claim 1, 2, 3 or 4 for preparing a 30 dicament for human gene therapy, comprising the steps of; 141 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; and transplanting said transduced cells into said patient. 24 A method for treatment of a patient having a hematopoietic disorder, comprising the steps of; removing stem cells; separating stem cells from other cells; (c) culturing said separated stem cells with a selected culture medium comprising the polypeptide of claim 1, 2, 3 or 4; harvesting said cultured cells; and transplanting said cultured cells into said patient. 9 5 25. A method of human gene therapy, comprising the steps of; removing stem cells from a patient; separating said stem cells from other cells; 0 culturing said separated stem cells with a selected culture medium comprising the hematopoietic protein of claim 1 i, 2, 3 or 4; C: introducing DNA into said cultured cells; harvesting said transduced cells; and 5 transplanting said transduced cells into said patient. a DATED this 1 day of May 2000: G. D, SEARLE CO., By its Patent Attorneys, E F. WE INGON CO., ace Wellington).