CA2234059A1 - Novel c-mpl receptor agonists - Google Patents

Abstract

Disclosed are c-mpl ligand receptor agonists proteins, DNAs which encode the c-mpl ligand receptor agonists proteins, methods of making the c-mpl receptor agonists proteins and methods of using the c-mpl receptor agonists proteins.

Description

CA 022340~9 1998-04-06 .1 NOVEL c-mpl RECEP~OR 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.

B~ck~round 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-ll, SCF). Plasma, serum and urine from thrombocytopenic dogs and hllm~n~ 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.

CA 022340~9 1998-04-06 W O 97/12978 PCTAUS96/lS938 The identification of the thrombopoietlc 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 c-mpl (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 mIL-5rc, 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 2S 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 CA 022340~9 1998-04-06 W O 97tl2978 PCT~US96/15938 simultaneous publications reported on a ligand that bound to c-mpl and had megakaryocytopoietic properties tde 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 sartley 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 25 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 a6s:l44s-l447, 1994). Absolute thrombocytopenia was not observed in these ~n;m~l S 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 CA 022340~9 1998-04-06 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 CA 022340~9 1998-04-06 in their bioassays, mRNA for this variant was found to be abundant in all three specles indicating that it may be a ~ naturally occurring alternative form of c-mpl ligand.

Rearran~ement of Protein Seouences 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;
S~himming et al., Eur. J. Biochem. 204: 13-19, 1992; Yamiuchi and ~in~mikawa, 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 (J. Mol. Biol. 16S:407-413, 1983). A new N-terminus is selected at an internal site tbreakpoint) 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 se~uence 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 CA 022340~9 1998-04-06 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, . Mol. Biol. 165:407-413, 1983; Li & Coffino, Mol. Cell.
Biol . 13:2377-2383, 1993). The proteins examined have represented a broad range of structural classes, including proteins that contain predominantly a -helix (interleukin-4;
Kreitman et dl., Cytokine 7 :311-318, 1995), ~ -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 Zhang et al., Biochemistry 32:12311-12318 (1993); Zhang et al., Nature Struct. Biol . 1:434-438 (1995) dihydrofolate Buchwalder et al., siochemistry 25 reductase 31:1621-1630 (1994); Protasova et al., Prot. Eng. 7:1373-1377 (1995) ribonuclease T1 Mullins et al., J. Am. Chem. Soc.
116:5529-5533 (1994); Garrett et al., Protein Science 5:204-211 (1996) Bacillus ~-glucanse Hahn et al., Proc. Natl. Acad. Sci.
U.S.A. 91:10417-10421 (1994) CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 aspartate Yang & Schachman, Proc. Natl. Acad.
transcarbamoylase Sci. U.S.A. 90:11980-11984 (1993) ., phosphoribosyl Luger et al., Science 243:206-210 5 anthranilate (1989); Luger et al., Prot . Eng.
isomerase 3:249-258 (1990) pepsin/pepsinogen Lin et al., Protein Science 4:159-166 (1995) glyceraldehyde-3- Vignais et al., Protein Science phosphate dehydro- 4:994-1000 (1995) genase 15 ornithine Li & Coffino, Mol . Cell . Biol .
decarboxylase 13:2377-2383 (1993) yeast Ritco-Vonsovici et al., Biochemistry phosphoglycerate 34:16543-16551 (1995) 20 dehydrogenase Enzyme Inhibitor basic pancreatic Goldenberg & Creighton, J. Mol.
25 trypsin inhibitor Biol . 165:407-413 (1983) Cytokines interleukin-1~ Horlick et al., Protein Eng. 5:427-431 (1992) interleukin-4 Kreitman et al., Cytokine 7:311-318 (1995) Tyrosine Rinase CA 022340~9 1998-04-06 Recognition Domain ~-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 (E. coli dihydrofolate reductase, aspartate transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ornithine decarboxylase, omp A, yeast phosphoglycerate dehydrogenase~. In other cases, the se~uence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease Tl, Bacillus -glucanase, interleukin-1~, a-spectrin SH3 domain, pepsinogen, interleukin-4). In exceptional cases, an unexpected improvement over some properties of the natural sequence was observed, e.g., the solubility and refolding rate for rearranged a-spectrin SH3 ~m~in sequences, and the receptor affinity and anti-tumor activity of transposed interleukin-4- Pseudomonas exotoxin fusion molecule (Kreitman CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 g 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 se~uence into short-range interactions in the new se~uence, 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 ~-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 N-terminus to the breakpoint is ca. 10 to 80% of the total se~Iuence 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 se~uence 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 fre~uently used in the linkers. Viguera, et al. ~J. Mol. Biol.
247:670-681, 1995) compared joining the original N- and C-termini with 3- or 4-residue linkers; the 3-residue linker was less thermodynamically stable. Protasova et al. (Protein Eng. 7:1373-1377, 1994) used 3- or 5-residue linkers in connecting the original N-termini of E. coli dihydrofolate W O 97/12978 PCTrUS96/lS938 reductase; only the 3-residue linker produced protein in good yield. More systematic studies o~ linker length and composition have not been reported.

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 SUM~RY OF THE INVF.NTION

The present invention relates to novel c-mpl receptor agonists of the ~ollowing formulas:
1. A c-mpl receptor agonist o~ the Formula:

SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrPro ValLeuLeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGlu ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa XaaGlyArgThrThrAlaHisLysAspProAsnAlaIlePheLeuSerPheGlnHis LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal ArgArgAlaProProThrThrAlaValProSerArgThrSerLeuValLeuThrLeu CA 022340~9 l998-04-06 . 12 AsnGluLeuProAsnArgThrSerGlyLeuLeuGluThrAsnPheThrAlaSerAla ArgThrThrGlySerGlyLeuLeuLysTrpGlnGlnGlyPheArgAlaLysIlePro GlyLeuLeuAsnGlnThrSerArgSerLeuAspGlnIleProGlyTyrLeuAsnArg IleHisGluLeuLeuAsnGlyThrArgGlyLeuPheProGlyProSerArgArgThr LeuGlyAlaProAspIleSerSerGlyThrSerAspThrGlySerLeuProProAsn LeuGlnProGlyTyrSerProSerProThrHisProProThrGlyGlnTyrThrLeu PheProLeuProProThrLeuProThrProValValGlnLeuHisProLeuLeuPro AspProSerAlaProThrProThrProThrSerProLeuLeuAsnThrSerTyrThr HisSerGlnAsnLeuSerGlnGluGly (SEQ ID NO:1) 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;

CA 022340~9 1998-04-06 Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met;
Xaa at position 115 is deleted or Gln, Gly, Ser, Thr, Tyr, or Asn;
S

wherein from 1 to 179 amino acids can be deleted from the C-terminus;

wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new c- and N-termini at amino acids;

50-51 88-89 or 127-128; and additionally said c-mpl receptor agonist can be immediately preceded by (methionine~1), (~l~n;ne~1) or (methionine~2, alanine~l).

2. A c-mpl receptor agonist of the Formula:

SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer CA 022340~9 l998-04-06 W 097/l2978 PCTAUS96/15938 HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrPro ValLeuLeuProAlaValAspSerLeuGlyGluTrpLysThrGlnMetGluGlu ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa XaaGlyArgThrThrAlaHisLysAspProAsnAlaIlePheLeuSerPheGlnHis LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal Arg (SEQ ID NO: 2) 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 Gln, 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;

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 ~ 15 50-51 88-89 or 127-128; and additionally said c-mpl receptor agonist can be immediately preceded by (methionine~l), (alanine~l) or (methionine~2, alanine~l).

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 se~uence 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 C-termini.

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

X1-(L)a-X2 wherein;
a is 0 or 1;
X1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues n+1 through J;
X2 is a peptide comprising an amino acid sequence corresponding to the sequence of residues 1 through n;
n is an integer ranging from 1 to J-1; and L is a linker.
In the formula above the constituent amino acids residues of Human 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+1 respectively where n is an integer ranging from 1 to J-1. The residue n+1 becomes the new N-terminus of the new c-mpl receptor agonist and the residue n becomes the new C-terminus of the the new c-mpl recepter agonist.
In a preferred embodiment of the present invention the linker (L) 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 NO:75);
SerGlyGlySerGlyGlySer (SEQ ID NO:76);
GluPheGlyAsnMetAla (SEQ ID NO:77);
GluPheGlyGlyAsnMetAla (SEQ ID NO:78);
GluPheGlyGlyAsnGlyGlYAsnMetAla (SEQ ID NO:79); and GlyGlyGlySerAspMetAlaGly (SEQ ID NO:80).
The present invention also encompasses recombinant human c-mpl receptor agonists co-administrated with one or more additional colony stimulating factors (CSF) including, CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 , 17 cytokines, lymphokines, interleukins, hematopoietic growth factors (herein collectively referred to as "colony stimulating factorsn) 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 co-administration 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.

CA 022340~9 1998-04-06 . 18 BRTFF DF~CRIPTION OF THF FIGURFS

Figure 1 schematically illustrates the sequence rearrangement of a protein. The N-terminus (N) and the C-terminus (C) 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 C-terminus (new-C) resulting in a protein with a new linear amino acid sequence. A rearranged molecule may be synthesized de novo as linear molecule and not go through the steps of joining the original N-terminus and the C-terminus and opening of the protein at the breakpoint.

Figure 2 shows a schematic of Method I, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C-terminus (a~a. 174) joined to the amino acid 11 (a.a. 1- 10 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 N-terminus created at amino acid 97 of the original protein, the original C-terminus (a.a. 174) joined to the original N-terminus and a new C-terminus created at amino acid 96 of the original se~uence.

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/1~938 ~ 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 C-terminus (a.a. 17~) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 . 20 D~TATTl~n DFSCRIPTION OF TH~ INVF.~TION

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 ~;m;n;sh the need for platelet transfusions.
Severe thrombocytopenia may result from genetic defects such as Fanconi's Anemia, Wiscott-Aldrich, or May-Hegglin syndromes. Ac~uired 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 CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 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, e.g., 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
se~uence 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 CA 022340~9 1998-04-06 . 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 :
CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 2~

and stem cel's 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 ~m; n; stered alone, co-administered with other colony stimulating factors, or in combination with bone marrow transplants subse~uent to high dose chemotherapy to treat the neutropenia and thrombocytopenia which are often the result o' 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: l) the number of stem cells in bone marrow or other is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible cont~min~tion 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 CA 022340~9 1998-04-06 . 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: (1) obtaining hematopoietic progenitor cells from a patient; (2) ex-vivo expansion of cells with a growth factor selected from the group consisting CA 022340~9 l998-04-06 W O 97/12978 PCTAUS96/1~938 of IL-3, flk3 ligand, c-kit ligand, GM CSF, IL-l, GM-CSF/IL-3 fusion protein and combinations thereof; ( 3) 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 (a) separating CD34+ stem cells from other cells and (b) 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 m~mm~l ian cells derived from peripheral blood.

30 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.

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.

3 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 ~. 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; (a) separating stem cells from other cells, (b) culturing said separated stem cells with a selected medium which contains a c-mpl receptor agonist and optionally a colony stimulating factor; and (c) harvesting said stems cells. Stem cells as well as committed progenitor cells destined to become neutrophils, erythrocytes, CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 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-1+ 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 CD90(thy-1).
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 ~nllmerating the number of stem cells and other cells, by standard techni~ues (e.g. hemacytometer, CFU, LTCIC) or by flow cytometry prior and subse~uent to incubation.

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 . 28 Several methods for ex-vivo expanslon 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 (srandt et al., Blood ~3:1507-1514, 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), fIt-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., B~ood 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; ~ith 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., Blooa 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 ~ stromal cell line such as HS-5 (WO 96/02662, 35 Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/lS938 . 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 ;mmllnodifiencies (Kay and Woo, Trends Genet. 10:253-257, 20 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, BioTechni~ues 6:616-629, 1988; Berkner, Current Top.
Microbiol. Immunol. 158:39-66, 1992; Brody and Crystal, CA 022340~9 1998-04-06 W O 97/1~978 PCTAUS96/15938 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 c r 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 ~g/kg of non-glycosylated 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 CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 compositions may also include co-~ministration 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-CS~, 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-10, IL-ll, IL-12, IL-13, IL-15, LIF, flt3iflk2 ligand, human growth hormone, s-cell growth factor, s-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 factorsn), 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, e.g., differential cell count and the like.
Determination of the J,inker.
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 50 A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp & Woods, Mol. Immunol.
20: 483-489, 1983; Kyte & Doolittle, ~. Mol . Biol . 157:105-132, 1992; solvent exposed surface area, Lee & Richards, J.
.

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 . 32 Mol. Biol. 55:379-400, 1971) and the ability to adopt the necessary conformation without deranging the con~ormation of the c-mpl receptor agonist (con~ormationally 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 pre~erred 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 1, 2, 3 or 4. Those skilled in the art will recognize that there are many such se~uences that vary in length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor short (cf., Sandhu, Critical Re~r. Biotech. 12: 437-462, 1992); if they are too long, entropy effects will likely destabilize the three-dimensional fold, and may also make folding kinetically impractical, and if they are too short, they will likely destabilize the molecule because of torsional or steric strain.

Those skilled in the analysis of protein structural information will recognize that using the distance between the chain ends, defined as the distance between the c-alpha carbons, can be used to define the length of the sequence to be used, or at least to limit the number o~ 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 ill-defined 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 CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 . 33 sequence to the chain ends, and the distance between their c-alpha 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 seauence 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.

Determin~tion of the Amino and C~rboxYl ~ermini of c-~nl lia~nd 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 2S 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 CA 022340~9 1998-04-06 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-~;m~n~ional structure necessary for expression of its biological function. Methods are known to those skilled in the art to obtain and interpret three-~im~n~ional 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 &
15 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 20 (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 post-translational 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) CA 022340~9 1998-04-06 .
W097/12978 PCT~S96/15938 ~5 Thus using either the experimentally derived structural information or predictive methods (e.g., 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-lO
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 so-called 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 acti~ity, are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence tha~ 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.
M~terials 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).

Metho~ for creation of ~enes with new N-terminus/C-terminus CA 022340~9 1998-04-06 W O 97/12978 PCT~US96tl5938 ~ 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 N-terminus can be made essentially following the method described in L. S. Mullins, et al (.J. Am. Chem. Soc. 116, 5529-5533, 1994). Multiple steps of polymerase chain reaction ( PCR) amplifications are used to rearrange the DNA
10 sequence encoding the primary amino acid sequence of the protein. The steps are illustrated in Figure 2.

n the first step, the first primer set ("new start~ and IlLinker start") is used to create and amplify, from the gene 15 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 20 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 25 cloning of the new gene into expression plasmids. Typical PCR
conditions are one cycle 95~C melting for two minutes; 25 cycles 94~C denaturation for one minute, 50~C annealing for one minute and 72~C extension for one minute; plus one cycle 72~C extension for seven minutes. A Perkin Elmer GeneAmp PCR
30 Core Reagents kit is used. A 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR
buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2. PCR
reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT).

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/1~938 Fragment Start" and "Fragment Stop", which have complementary se~uence in the linker region and the coding se~uence fo~ the two amino acids on both sides o~ the linker, are joined together in a third PCR step to make the full-length gene encoding the new protein. The DNA fragments IlFragment Start~ and ~Fragment stop~ are resolved on a 196 TAE
gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen). These fragments are combined in e~uimolar quantities, heated at 70~C for ten minutes and slow cooled to allow annealing through their shared sequence in "Linker start" and Illinker 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 N-terminus/C-terminus gene. Typical PCR conditions are one cycle 95~C melting for two minutes; 25 cycles 94~C
denaturation for one minute, 60~C annealing for one minute and 72~C extension for one minute; plus one cycle 72~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 lx PCR
buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2. 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 se~uence, the DNA

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 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 se~uence, the DNA fragment ("Fragment Stop'~) that contains the se~uence 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 cGnditions are one cycle 95~C melting for two minutes; 25 cycles 94~C
denaturation for one minute, 50~C annealing for 45 seconds and 72~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 siolabs), 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 CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 pair NcoI/HindIII vector fragment of pMON3934 by heating at 50~C for ten minutes and allowed to slow cool. The three fragments are ligated together using T~ DNA ligase (Boehringer ~annheim). The result is a plasmid cont~ining the full-length new N-terminus/C-terminus gene. A portion of the ligation reaction is used to transform E. coli strain DH5a cells (Life Technologies, Gaithersburg, MD). Plasmid DNA is purified and se~uence confirmed as below.

Method III. Creation of new N-terminus/C-terminus genes b~
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 ofthe 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 b~
cloning and contains two similar, but not necessarily identical, copies of the yene separated by DNA se~uence 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/C-terminus gene from the tandemly-duplicated template DNA.
These primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95~C
melting for two minutes; 25 cycles 94~C denaturation for one minute, 50~C annealing for one minute and 72C' extension for one minute; plus one cycle 72~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;

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 . 40 and lx PCR buf~er, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgC12.
PCR reactions are performed in a Model 480 DNA thermal cycler r (Perkin Elmer Corporation, Norwalk, CT). PCR reactions are purified using a Wizard PCR Preps kit (Promega).

The construction o~ 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 geneI/linker/geneII
from; i) a gene encoding c-mpl ligand amino acids 1-153 for the geneI position; ii) uni~ue 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 geneII position.

Step 1: Reverse tr~nscri~tase/~olvmerase 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 (c-mpl ligand sequence based on Genebank accession #L33410 or deSauvage 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 CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 enzymes for transfer to E. coli or m~mmAlian expression plasmids.

For the revelse transcriptase (RT) reaction, human fetal (lot 5 ~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~ Kit obtained from Invitrogen (San Diego, CA). One microgram (ug) of each RNA sample is combined and denatured at 65~C for 10 min. in the presence of either random primers, oligo dT primer or a speci~ic 3' anti-sense 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 20 microliter reaction is incubated at 42~C for 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 Tag polymerase. The PCR reactions are subjec~ed to 30 cycles of the following; 1 min. @ 94~C, l min. @ 58~C, 90 sec. @ 72~C. An equal volume of loading dye (0.01% each bromophenol blue and xylene cyanole blue) is added to 10 microliters of the final product for electrophoresis through a 1% SeaKemR LE agarose (FMC, Rockland, ME) gel in the presence of lx 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 stAn~Ards, 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 3 5 visualized using a short-wave W light source. The reactions CA 022340~9 1998-04-06 are purified using a WizardTM 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 5 mixture. After 1 minute incubation at 24~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 10 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.

Step 2: Subcloning the PCR products into a m~3mm~3 lian expression vector The c-mpl ligand PCR products are digested with the appropriate restriction enzymes for ligation to a mammalian 20 expression vector. The m~mm~ 1 ian 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 25 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~C as described by the manufacturer prior to electrophoresis through a 19~
30 agarose/lx TBE/EtBr gel. Fragments are first visualized by long-wave W 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 DNA-binding resin, and extensive washing of the resin prior to 35 elution with water. The purified DNA products are combined at CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 ~ 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 flankins ~coRI and AflIII sites, is ligated overnight with a 3.7 Kbp EcoRI/sstXI fragment from a form of c-mpl ligand with amino acids 1-153 (step 2), 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 DH5~~ 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 5~ 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~C, 1 CA 022340~9 1998-04-06 min. @ 58~C, 90 sec. ~ 72~C. The product (about 480 base pairs) is visualized using a short-wave W light source. The reactions are purified using a Wizard~ 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~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 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. col i expression vectors.

Mammalian Expression Vectors The mammalian expression vectors are derivatives of pMON3359 which is a pUC18-based vector containing a m~mm~l ian expression cassette. The cassette includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 signal peptide se~uence 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~C as described by the manufacturer prior to electrophoresis through a 1% agarose/lx TsE/EtBr gel.

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 Fragments ar~ first visualized by long-wave W 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 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 o~ 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: 2~3-269, 1992). The expression cassette consists of the recA promoter and T7 gene 10 ribosome binding site (RBS) as well as an Ml3 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 DH5~~ (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 m~mm~l ian cells. E.
coli strain MON105 can be obtained from the American Type Culture Col~ection (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: F-, lamda-,IN(rrnD, rrE)1, rpoD+, rpoH358 CA 022340~9 1998-04-06 DH5a~: F-, phi80dlacZdeltaM15, delta~lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk-,mk+), phoA, supE441amda-, thi-1, gyrA96, relA1 TG1: delta(lac-pro), supE, thi-1, hsdD5/F'(traD36, proA+B+, lacIq, lacZdeltaM15) DH5~ 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 Ls medium (1% 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 saush & Lomb Spectronic spectrophotometer (Rochester, NY).
The cells are collected by centrifugation and resuspended in one-fifth culture volume of CaC12 solution (50 millimolar CaC12, 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 CaC12 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 Ls is added prior to shaking the samples at 37 C for one hour. Cells from these samples are spread on plates (Ls medium plus 1.5%
bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin (75 ug/mL) when selecting for spectinomycin-resistant transformants. The plates are incubated overnightat 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

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 . 47 spectinomycin) and are grown at 37~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 isol~tion and characterization Plasmid DNA is isolated using the Promega Wizard~
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 g), plasmid DNA
released with sequential NaOH & acid treatment, and cellular debris is removed by centrifugation (10000 x g). The supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA-binding resin, the column is washed, and plasmid DNA eluted with TE. After screening for the colonies with the plasmid of interest, the E. coli cells are inoculated into 100 mls of LB plus appropriate antibiotic for overnight growth at 37~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 m~mm~l ian or E. coli cells.
Purified recombinant double-stranded DNA is sequenced using the Applied Biosystems Inc. (ABI, Foster City, CA) PRISM~ Ready Reaction DyeDeoxy~ Terminator Sequencing system. The ABI system relies on incorporation of four fluorescence labelled dideoxy nucleotides into single-CA 022340~9 1998-04-06 . 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 S amplification (30 seconds at 96~C, 15 seconds at 50~C, 4 minutes at 60~C). 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 lX TsE at 70 watts constant power. The ABI system uses a detector that recognizes each differntially labelled PCR product as they are being subjected to electrophoresis.
Pro~uction of novel c-mDl rece~tor a~onists 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 (EBS).
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 sHK-21 cell line was previously stably transfected with the HSV
transactivating protein VP16, which transactlvates 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 CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 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 SV40 promoter. A similar plasmid is available from ATCC, pSV2-hph.
BHK-VP16 cells are seeded into a 60 millimeter (mm) tissue culture dish at 3 X 105 cells per dish 24 hours prior to transfection. Cells are transfected for 16 hours in 3 mL
of '1OPTIMEM'VTM (Gibco-sRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug hygromycin resistance plasmid, pMON1118, and 80 ug of Gibco-sRL ~LIPOFECT~MINE~ per dish. The media is subse~uently 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.

Fxnression and ~urification of recombinant ~rotein from ~.
coli E. coli strain MON105 harboring the plasmid of interest are grown at 37~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 CA 022340~9 1998-04-06 W O 97/12978 PCTAUS96/lS938 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 ~g/mL. The cultures are then shaken at 37~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 (IB). One mL ali~uots 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 blrsts 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 e~mi~ing 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 CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 of cataytic concentrations of cysteine, beta-mercaptoethanol or dithiothreitol for 24 to 72 hours. The protein is purified from E. coli cont~min~nts 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.

Withou~ 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 ir. their entirety.

Further details known to those skilled in the art may be found in T. Maniatis, et al., Molecular Clonina, A T~horatorv M~nu~l, Cold Spring Harbor Laboratory (1982) and references ; 30 cited therein, incorporated herein by reference; and in J.
Sambrook, et al., Molecul~r Clonina, A TaboratorY Manl~l, 2nd edition, Cold Spring Harbor Laboratory (1989) and references cited therein, incorporated herein by reference.

W O 97/12978 PCTrUS96/15938 All references, patents or applications cited herein are incorporated by re~erence in their entirety.

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 . 53 TARR~ 1 OTIGONUCT.~OTIDF..S
c-mplNcoI ACGTCCATGGCNTCNCCNGCNCCNCCTGCTTGTGCACTCCGAGTC
(SEQ ID NO:4) N=A,C,G or T
Ecompl ATGCACGAATTCCCTGACGCAGAGGGTGGA (SEQ ID NO:5) c-mplHindIII TGACAAGCTTACCTGACGCAGAGGGTGGACCCT
(SEQ ID NO:6) 4L-5' AATTCGGCAA (SEQ ID NO:7) 15 4L-3' CATGTTGCCG (SEQ ID NO:8) 5L-5' AATTCGGCGGCAA (SEQ ID NO:9) 5L-3' CATGTTGCCGCCG (SEQ ID NO:10) 8L-5' AATTCGGCGGCAACGGCGGCAA (SEQ ID NO:11) 8L-3' CATGTTGCCGCCGTTGCCGCCG (SEQ ID NO:12) 25 31-5' CGATCCATGGAGGTTCACCCTTTGCCT (SEQ ID NO:13) 31-3' GATCAAGCTTATGGGCACTGGCTCAGTCT (SEQ ID NO:14) 35-5' CGATACATGTTGCCTACACCTGTCCTG (SEQ ID NO:15) 35-3' GATCAAGCTTAAGGGTGAACCTCTGGGCA (SEQ ID NO:16) 39-5' CGATCCATGGTCCTGCTGCCTGCTGTG (SEQ ID NO:17) 35 39-3' GATCAAGCTTAAGGTGTAGGCAAAGGGTG (SEQ ID NO:18) 43-5' CGATCCATGGCTGTGGACTTTAGCTTGGGA (SEQ ID NO:19) 43-3' GATCAAGCTTAAGGCAGCAGGACAGGTGT (SEQ ID No:20) 45-5' CGATCCATGGACTTTAGCTTGGGAGAA (SEQ ID NO:21) 45-3' GATCAAGCTTACACAGCAGGCAGCAGGAC (SEQ ID NO:22) 45 49-5' CGATCCATGGGAGAATGGAAAACCCAG (SEQ ID NO:23) 49-3' GATCAAGCTTACAAGCTAAAGTCCACAGC (SEQ ID NO:24) 82-5' CGATCCATGGGACCCACTTGCCTCTCA (SEQ ID NO:25) 82-3' GATCAAGCTTACAGTTGTCCCCGTGCTGC (SEQ ID NO:26) CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 . 54 109-5' CAGTCCATGGGAACCCAGCTTCCTCCA ~SEQ ID NO:27) 109-3' GATCAAGCTTAAAGGAGGCTCTGCAGGGC (SEQ ID NO:28) 116-5' CGATCCATGGGCAGGACCACAGCTCAC (SEQ ID No:29) 116-3' GATCAAGCTTACTGTGGAGGAAGCTGGGTT (SEQ ID NO:30) 120-5' CGATCCATGGCTCACAAGGATCCCAATGCC (SEQ ID NO:31) lZ0-3' GATCAAGCTTATGTGGTCCTGCCCTGTGG (SEQ ID NO:32) 123-5' CGATCCATGGATCCCAATGCCATCTTCCTG (SEQ ID NO:33) 15 123-3' GATCAAGCTTACTTGTGAGCTGTGGTCCT (SEQ ID NO:34) 126-5' CGATCCATGGCCATCTTCCTGAGCTTCCAA (SEQ ID NO:35) 126-3' GATCAAGCTTAATTGGGATCCTTGTGAGCTGT (SEQ ID
NO:36) CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 '~ GF.l~F. S EouF~l~c F~ c;

TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAG~AGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC (SEQ ID NO:37);

TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGC
TCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCAC
AGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTG
TGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCT
GGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT
TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGC
AGAGCCTCCTTGGAACCCAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCT
GAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACC
CTCTGCGTCAGG (SEQ ID NO:38);

TCTCCCGCTCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:82) TCTCCCGCTCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG

CA 022340~9 1998-04-06 W O 97/lZ978 PCT~US96/15938 GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGC-TCCTTGGAACCCAGGGCAGGACCACAGCTCACAAGGATCCCAATGCC
ATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAG
GGTCCACCCTCTGCGTCAGG (SEQ ID NO:83) pMON28500 TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCAACATGGCGTCTCCCGCTCC
GCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC
AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGG
ACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGG
AGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC
CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGA
GCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGC
CATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGA
GGGTCCACCCTCTGCGTCAGG (SEQ ID NO:39);

TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGC
TCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCAC
AGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTG
TGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCT
GGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT
TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGC
AGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAA
TGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA
GGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:40);

TCCCCAGCGCCgCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCA-,ACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA

CA 022340~9 1998-04-06 TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACGGCGGCAACATGGC
GTCCCCAGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCAT
GTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGC
TGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACA
GGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG
GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTG
GGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAA
GGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG
ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:41);

GAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAA
TGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTG
CTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTG
GGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACC
CAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGC
TTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTC
TGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGA
GTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCA
(SEQ ID NO:61);

TTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAG
ATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTG
ATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCT
GGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCA
CAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTG
CTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAA
TTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAA
CTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCT
(SEQ ID NO:62);
PMoN30375 GTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACC
AAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGG
GGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGT
CTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACC
ACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAG
GTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAAC
ATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGAC
TCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCT
(SEQ ID NO:63);

GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC
ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 . 58 CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG
GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG
ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG
GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT
CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT
(SEQ ID NO:64);

GACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTG
GGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT
TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTG
CAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCC
AATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTT
GTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCG
CCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC
AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTG
(SEQ ID NO:65);

GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC
CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC
CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT
GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC
CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC
ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCNTCTCCGGCGCCGCCTGCTTGTGAC
CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG
TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG
(SEQ ID NO:66);
PMoN30379 GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTT
GGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCAC
AAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTC
CTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCT
CCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTC
CTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTG
CCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG
(SEQ ID NO:6/);

GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC
CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC
ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGAC
CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG
TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG

CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 . 59 GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC
CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC
CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT
(SEQ ID NO:68);

GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC
CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC
10 GGCGGCAACATG'J-GTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG
CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG
CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG
GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATG
GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGA
CAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAG
(SEQ ID NO:69) GCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTG
CGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATG
GCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCC
CATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTC
CTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAG
GCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGA
CAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTC
CTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACA
(SEQ ID NO:70);

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG
ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG
GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT

GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC
ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA
CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG
GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
40 (SEQ ID NO:71);

GCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA
GGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGCTCCGCCT
GCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGA
CTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGAC
TTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGA
GCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC
CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAG
AGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAAT
(SEQ ID NO:72).

CA 022340~9 l998-04-06 W O 97/12978 PCT~US96/15938 PROTF.TN SF.OUF.~C~.s PMON26458pep SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPhe (SEQ ID No:42);

PMON28548pep SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyvalMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyGlyAsnMetAla SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnGlyArgThrThrAlaHisLysAspPro AsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeu ValGlyGlySerThrLeuCysValArg (SEQ ID NO:43);

SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSe~ArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID No : 44);

SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis CA 022340~9 1998-04-06 W097l12978 PCT~S96/15938 ~ 61 ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnGlyArgThrThrAlaHisLysAspPro AsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeu ValGlyGlySerThrLeuCysValArg (SEQ ID NO:45);

SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyAsnMetAlaSer ProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHisVal LeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeuLeu ProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAlaGln AspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGlnLeu GlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeuLeu GlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAlaHis LysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPhe LeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:46);

SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyGlyAsnMetAla SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:47);

pMON32136 SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 ' 62 ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyGlyAsnGlyGly AsnMetAlaSerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArg AspSerHisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThr ProValLeuLeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGlu ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAla ArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnVal ArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLel;ProProGlnGlyArg ThrThrAlaHisLysAspproAsnAlaIlepheLeuserpheGlnHisLeuLeuArgGly LysValArgPheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:48);

pMON30373 GluValHisProLeuProThrProValLeuLeuProAlaValAspPheSerLeuGlyGlu TrpLysThrGlnMetGluGluThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeu LeuGluGlyValMetAlaAlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeu GlyGlnLeuSerGlyGlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThr GlnLeuproproGlnGlyArgThrThrAlaHisLysAspproAsnAlaIlepheLeuser PheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeu CysValArgGluPheGlyGlyAsnMetAlaSerProAlaProProAlaCysAspLeuArg ValLeuSerLysLeuLeuArgAspSerHisValLeuHisSerArgLeuSerGlnCysPro (SEQ ID NO:49);
pMON31074 Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu .ro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln Cys Pro Glu Val His Pro (SEQ ID NO:50);
pMON30375 Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 . 63 Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala HiS Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 5 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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro ( SEQ ID NO:51);
,.
10 pMON30376 Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser 15 Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala HiS Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 20 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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro (SEQ ID No:52);

25 pMON30377 Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu 3 0 Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala HiS Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 3 5 Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val HiS Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val ( SEQ ID NO: 53 );

40 pMON30378 Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu r 45 Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala HiS Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 50 Leu Arg Val: eu Ser Lys Leu Leu Arg Asp Ser HiS Val Leu HiS
Ser Arg Leu Ser Gln Cys Pro Glu Val HiS Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu ( SEQ ID NO: 5 4 );

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 pMON30379 5 Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly 10 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 Gln 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 Gln Met Glu Glu Thr Lys ~la Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu 15 Glu Gly Val Met Ala Ala Arg Gly Gln Leu (SEQ ID NO:55);

pMON30380 20 Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 25 Ser Arg Leu Ser Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg 30 Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu ( SEQ ID NO : 56);

pMON30381 35 Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln Cys Pro 40 Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln ~5 Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln ( SEQ ID NO : 57);

pMON30382 50 Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 CA 022340~9 1998-04-06 W O 97/12978 PCTAUS96/1~938 Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr (SEQ ID No:58);

pMON30383 Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys (SEQ ID NO:59);

pMON30384 Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn (SEQ ID No:6o);

The following examples will illustrate the invention in greater detail although it will be understood that the invention is not limited to these speci~ic examples.

F~AMPT.F~
Construction of the parental plasmid cont~; n; ng the first gene of the dimer template CA 022340~9 1998-04-06 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~ 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-mplNcoI (SEQ ID NO:4) and Reverse primer: Ecompl (SEQ ID NO:5). The c-mplNcoI (SEQ ID
NO:4) primer anneals to the c-mpl ligand gene (bases #279-311 based on c-mpl ligand se~uence 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-l and includes codon degeneracy for the Ala codon and the first four c-mpl ligand codons (Serl, Pro2, Ala3, & Pro4). The Ecompl (SEQ ID
NO:5) primer ~nneals to bases #720-737 of c-mpl ligand and encodes a EcoRI recognition site in-frame with the c-mpl ligand gene immediately following Argl53. The EcoRI site creates Glul54 and Phel55 codons following Argl53. 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 bp.). 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 se~uence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker (See CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 Hippenmeyer et al., Bio/Technology, 1037-1041, 1993). The human IL-3 signal peptide sequence, which had been subloned as a BamHI f.~gment into the unique BamHI site between the 110 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);
samHI
GGATCCACC~AGCCGCCTGCCCGTCCTGCTCCTGCTCCAACTCCTGGTCCGCCCCGC

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

F.~iMPT,F. 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-mplNcoI (SEQ ID NO:4) (forward primer) and c-mplH;n~TII (SEQ ID NO:6) (reverse primer). The c-mplNcoI (SEQ ID NO:4) primer is described in Example 1. The c-mplHindIII (SEQ ID NO:6) primer, which an~als to bases ~716-737 of c-mpl ligand, adds both a termination codon and a ~;n~TTI restriction enzyme site immediately following the final codon, Argl53.
Two types of PCR products are generated from the RT cDNA
samples, one with a deletion of the codons for amino acids CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96115938 ~ 68 112-115 and one without the deletion of these codons. The c-mpl 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 1). pMoN3934 is digested with NcoI and HindIII (ca. 3800 bp) and will accept the PCR products.
The plasmid, pMON32132, contians the DNA se~uence of (SEQ ID
NO:82) which encodes for amino acids 1-153 of c-mpl ligand (SEQ ID NO:~) 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 (~112-115) (SEQ ID NO:45) was also a result of this cloning.

EXAMPT .F. 3 Generation of PCR 5L dimer template containing ~112-115 in the secondlgene 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 NO:10).
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 se~uence 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 NO:43).

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 F.XAMPT.F. 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).

F.X.j~[PT.F. 5 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 NO:10).
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 se~uence of (SEQ ID NO:

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 ~ 70 40) 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)~

F.X~MPI.F. 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:ll) 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 seauence of (SEQ ID NO:41) and encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79) linker (8L) _o amino acids 1-153 c-mpl ligand (SEQ ID No:48).

F.XA~PT, F..S 7--18 Generation of novel c-mpl receptor agonists A. PCR aener~tion of novel c-mnl rece~tor 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).

CA 022340~9 1998-04-06 WO97/12978 PCT~S96/15938 31-5' (SEQ ID NO:13) and 31-3' (SEQ ID NO:14), 35-5' (SEQ ID
NO:15) 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
NO:20), 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 NO:25) 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
NO:30) ,120-5' (SEQ ID NO:31) and 120-3~ (SEQ ID NO:32), 123-5' (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. Sl~hclonin~ of novel c-mnl receDtor a~onists into m~mm~ n ex~ression 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.

CA 022340~9 l998-04-06 Example PCR PCR PCR Product Resulting # template Primer Breakpoint Linker Restriction Plasmid ~et Dige~t pMON
7 pMON28501 31 30-31 5LNcoI/HindIII pMON30373 8 pMON28501 35 34-35 5LAflIII/HindIII pMON30374 9 pMON28501 39 38-39 5LNcoI/HindIII pMON30375 pMON2;501 43 42-43 5LNcoI/HindIII pMoN3o376 11 pMON28501 45 44-45 5LNcoI/HindIII pMON30377 12 pMON28501 49 48-49 SLNcoI/HindIII pMON30378 13 pMON28501 82 81-82 SLNcoI/HindIII pMON30379 14 pMON28501 109 108-109 SLNcoI/HindIII pMON30380 lS pMON28501 116 115-116 5LNcoI/HindIII pMON30381 16 pMON28501 120 119-120 5LNcoI/HindIII pMoN30382 17 pMON28501 123 122-123 5LNcoI/HindIII pMON30383 18 pMON28501 126 125-126 SLNcoI/HindIII pMON30384 In a similar manner the dimer templates pMON28500, pMON32136 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 BafJ3 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 c-mpl responsive cell line and cloned into the multiple cloning site of the plasmid pcDNA3 (Invitrogen, San Diego Ca.). Baf/3 CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 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 ~++++~ level were assayed in the Baf-3/c-mpl cell proliferation assay.

Expres~ on and Bioactivity of c-mpl rece~tor aqonist pMON# c-mpl receptor BHK expression Baf-3/c-mpl agonist levels cell prolife-ation p~O~ / - C + n~
p V!O~ "_ _ / ,J / _ _ ~ . + n~
pvo\ __ / ~ n~
p~o~ ~ - / L/ -~ + n~
p~ON ' ~ -_ / _/ -~ ++ ne p~ON ' ~-_ / _/ -~. ++ ne pMON '~ - . + ne pMON _ -. / L/ -_ ++++ +
pMON~ -_ / L/_-__ ++++ +
pMON _0-: / L/:- + nd pMON _ _ - 3-_ / L/~ + nd 2. Bone marrow proliferation assay a. CD34+ Cell Purification:
Between 15-20 mL 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 Histopa~ue-1077 (Sigma) and centrifuged for 30 minutes at 300 RCF. The mononuclear interface layer is collected and washed in PBS. CD34+ cells CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 . 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 70% on average as determined by using flow cytometric analysis using anti CD3A 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 10 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 ~l of supernatant added to 1 mL cultures (approximately a 10% dilution). Cells are incubated at 37~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 ~m PGEl, 11 mM Glucose, 3% w/v BSA, in PBS, pH 7.4,) (Tomer et al., Blood 70(6): 1735-42 [1987]) resuspended in 500 ~l of MK buffer cont~;n-ng 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 0.5% 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 ~g/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

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 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 cellsJmL 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, 6.67~M FeCl2, 25~g/mL CaCl2, 25 ~g/mL L asparagine, 500 ~g/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 ~-lot formation. Cells are incubated at 37~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 (1:3), air dried and stored at -200C until st~ining. 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 approx. 25 cells), BFU MK (large, multi-foci colonies with >
25 cells) or mixed colonies (mixture of both positive and negative cells).

Various other examples will be apparent to the person skilled in t:l- art after reading the present disclosure without departing from the spirit and scope of the invention.
It is intended that all such other examples be included within the scope of the appended claims.

CA 022340~9 l998-04-06 WO 97/12978 PCT~US96/15938 ~QuAb~Ah. LISTING

(1) GENERAL INFORNATION:
(i) APPLICANT:
(A) NAME: G. D. Searle (B) STREET: P.O. Box 5110 (C) CITY: Chicago (D) STATE: Illinois (E) COUNAA~Y: United States of America (F) POSTAL CODE (ZIP): 60680 (G) TELEPHONE: (708) 470-6501 (H) TELEFAAY: (708) 470-6881 (A) NAME: Mon~Anto Company (B) STREET: 88 North Lindbergh Boulevard (C) CITY: St. Louis (D) STATE: Missouri (E) COUNAA~Y: United States of America (F) POSTAL CODE (ZIP): 63167 (G) TELEPHONE: (314) 694-3131 (H) TELEFAAY: (314) 694-5435 (ii) TITLE OF lNv~N~ oN: c-mpl ligand Receptor Agonists (iii) NUMLER OF ~Ab.yuAb~S: 83 (iv) C~ UAA~;K A~EADABLE FOA~A
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Vercion #1.30 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUMA~ER: US C-2908 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/004,824 (B) FILING DATE: 05-OCT-1995 (2) INFORNATION FOR SEQ ID NO: 1:
( i ) ~Ab~YUA~N~A~ CHARACTERISTICS:
(A) LENGTH: 332 amino acids (B) TYPE: amino acid (C) STR~-N~ N~ S: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Modified-site CA 022340~9 1998-04-06 (B) LOCATION:112 (D) OTHER INFORMATION:/note= "position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or N..."
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:113 (D) OTHER INFORMATION:/note= "position 113 is deleted or Pro, Phe, Ala, Leu, Ile, Trp, or Met"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:114 (D) OTHER INFORMATION:/note= "position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp or Met~
(ix) FEATURE:
(A) NANE/KEY: Modified-site (B) LOCATION:115 (D) OTHER INFORMATION:/note= "position 115 is deleted or Gln, 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 Asp Ser His Val Leu His Ser Arg Leu Ser Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Xaa Xaa Xaa Xaa Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Net Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala CA 022340~9 l998-04-06 WO 97/12978 PCTrUS96/l5938 Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 153 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:112 (D) OTHER INFORMATION:/note= ''position 112 is deleted or Leu, Ala,VAl, Ile, Pro, Phe, Trp or Met"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:113 CA 022340~9 l998-04-06 (D) OTHER INFORMATION:/note= "positoin 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp or Met"
_ (ix) FEATURE:
tA) NAME/KEY: Modified-site (B) LOCATION:114 (D) OTHER INFORMATION:/note= "position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp or Met"
(ix) FEATURE:
(A) NAME/REY: Modified-site (B) LOCATION:115 (D) OTHER INFORMATION:/note= "positon 115 is deleted or Gln, 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 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Xaa Xaa Xaa Xaa Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg (2) INFORMATION FOR SEQ ID NO: 3:
(i) ~:QU~NL~ CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STR~Nn~nN~.~s: unknown CA 022340~9 1998-04-06 WO 97/12978 PCT~US96/15938 (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) ~Q~ DESCRIPTION: SEQ ID NO: 3:
Gly Gly Gly Ser (2) INFORMATION FOR SEQ ID NO: 4:
(i) ~:QU~N~'~ CHARACTERISTICS:
(A) LENGTH: 4S base pairs (B) TYPE: nucleic acid .(C) STR~ S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic) n (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
ACGTCCATGG ~Nl-N-~N~C N~NC~l~CT TGTGCACTCC GAGTC

(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)~

(xi) ~QU~N~: DESCRIPTION: SEQ ID NO: 5:
ATG~AC~-AA~ TCCCTGACGC AGAGGGTGGA

(2) INFORMATION FOR SEQ ID NO: 6:
(i) ~QU~N~r: CHARACTERISTICS:
(A) LENGTH: 33 base pairs CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/1~938 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) ~:yU~:N~ DESCRIPTION: SEQ ID NO: 6:
TGACAAGCTT ACCTGACGCA GAGGGTGGAC CCT

(2) INFORMATION FOR SEQ ID NO: 7:
~N~ CHARACTERISTICS:
(A) LENGTH: l0 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = ~DNA (synthetic)"

(xi) ~:Qu~ DESCRIPTION: SEQ ID NO: 7:
AATTCGGCAA

(2) INFORMATION FOR SEQ ID NO: 8:
(i) ~:~u~N~ CHARACTERISTICS:
(A) LENGTH: l0 base pairs (B) TYPE: nucleic acid (C) sTRA~n~nN~s single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = ~DNA (synthetic)~

(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO: 8:
CATGTTGCCG

(2) INFORMATION FOR SEQ ID NO: 9:

CA 022340~9 l998-04-06 (i) ~:Q~N~ CHARACTERISTICS:
(A) LENGTH: 13 base pairs ~B) TYPE: nucleic acid ( C ) STR A NnEnNF~ C: S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)"

(xi) s~Q~N~: DESCRIPTION: SEQ ID NO: 9:
AAll~CGGCGG CAA

(2) INFORMATION FOR SEQ ID NO: lO:
(i) ~-QU~:N~ CHARACTERISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic) n (xi) ~Q~ DESCRIPTION: SEQ ID NO: 10:
CA~ ~CCG CCG

(2) INFORMATION FOR SEQ ID NO: 11:
(i) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRAN~N~:SS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)"

(xi) s~Qu~: DESCRIPTION: SEQ ID NO: 11:

CA 022340~9 1998-04-06 W 0 97/12978 PCT~US96/15938 AATTCGGCGG CAACGGCGGC AA

_ (2) INFORMATION FOR SEQ ID NO: 12:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CATGTTGCCG CCGTTGCCGC CG

(2) INFORMATION FOR SEQ ID NO: 13:
Q~N~ CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: other nucleic acid (A) D~-CCRTPTION: /desc = ~DNA (synthetic)~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CGATCCATGG AGGTTCACCC TTTGCCT

(2) INFORMATION FOR SEQ ID NO: 14:
(i) ~Ou~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

CA 022340~9 l998-04-06 WO 97/l2978 PCT~US96/15938 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GATCAAGCTT ATGGGCACTG GCTCAGTCT

(2) INFORMATION FOR SEQ ID NO: 15:
( i ) ~QU N~ CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(Xi) ~U~N~'~ DESCRIPTION: SEQ ID NO: 15:
CGATACATGT TGCCTACACC l~lC~lG

(2) INFORMATION FOR SEQ ID NO: 16:
(i) s~Qu~N~r: CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANn~n~FCS single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = ~DNA (synthetic)"

(Xi) S~QU N~ DESCRIPTION: SEQ ID NO: 16:
CGATCCATGG TCCTGCTGCC ~lG~l~lG

(2) INFORMATION FOR SEQ ID NO: 17:
(i) ~QU~:N~ CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)"

CA 022340~9 1998-04-06 (Xi) ~hUU~N~: DESCRIPTION: SEQ ID NO: 17:
CGATCCATGG ~l~C~l~G~l~GCC ~l~G~l~ G

(2) INFORMATION FOR SEQ ID NO: 18:
( i ) S~UU N~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GATCAAGCTT AAGGTGTAGG CAAAGGGTG

t2) INFORMATION FOR SEQ ID NO: 19:
QU~N~ CHARACTERISTICS:
(A) LENGTH: 3û base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic) n (xi) ~UU~N~ DESCRIPTION: SEQ ID NO: 19:
CGATCCATGG CTGTGGACTT TAG~l~lGGGA

(2) INFORMATION FOR SEQ ID NO: 20:
( i ) ~QU~N - ~: CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 022340~9 1998-04-06 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) ~:Qu~: DESCRIPTION: SEQ ID NO: 20:
GATCAAGCTT AAGGCAGCAG GACAGGTGT

(2) INFORMATION FOR SEQ ID NO: 2l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CGATCCATGG ACTTTAGCTT GGGAGAA

(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GATCAAGCTT ACACAGCAGG CAGCAGGAC

(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CGATCCATGG GAGAATGGAA AACCCAG

(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GATCAAGCTT ACAAGCTAAA GTCCACAGC

(2) INFORMATION FOR SEQ ID NO: 25:
(i) ~Q~N~: CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) ~:Q~N~ DESCRIPTION: SEQ ID NO: 25:
CGATCCATGG GACCCACTTG C~l~l ~A

(2) INFORMATION FOR SEQ ID NO: 26:

CA 022340~9 1998-04-06 WO 97/12978 PCTrUS96/15938 (i) ~hQu~N~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(Xi) ~hQ~ DESCRIPTION: SEQ ID NO: 26:
GATCAAGCTT ACAGTTGTCC CC~lG~lGC

(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
GATCAAGCTT AAAGGAGGCT CTGCAGGGC

(2) INFORMATION FOR SEQ ID NO: 28:
(i) ~Ou~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: s.ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)"

(xi) s~Q~N~ DESCRIPTION: SEQ ID NO: 28:

CA 022340~9 1998-04-06 W 0 97/12978 PCTrUS96/15938 GATCAAGCTT AAAGGAGGCT CTGCAGGGC

- (2) INFORMATION FOR SEQ ID NO: 29:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
CGATCCATGG GCAGGACCAC AGCTCAC

(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = ~DNA (synthetic)~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
GATCAAGCTT ACTGTGGAGG AAGCTGGGTT

(2) INFORMATION FOR SEQ ID NO: 3l:
(i) ~u~:~ CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetis)"

CA 022340~9 l998-04-06 WO 97/12978 PCT~US96/15938 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
GATCAAGCTT ACTGTGGAGG AAG~l~G~

(2) INFORMATION FOR SEQ ID NO: 32:
(i) ~Q~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) shQu~N~ DESCRIPTION: SEQ ID NO: 32:
GATCAAGCTT ATGTGGTCCT GCC~s~l~GG

(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (syntheitc)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
CGATCCATGG ATCCCAATGC CAl~llC~

(2) INFORMATION FOR SEQ ID NO: 34:
(i) ~QU~l~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 (xi) ~QU~ : DESCRIPTION: SEQ ID NO: 34:
GATCAAGCTT ACTTGTGAGC ~l~l~lC~

(2) INFORMATION FOR SEQ ID NO: 35:
(i) ~QU~ CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
CGATCCATGG CCA~ C~l~ GAGCTTCCAA

(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)"

(xi) ~uu~ DESCRIPTION: SEQ ID NO: 36:
GATCAAGCTT AATTGGGATC L~lu~lGAGCT GT

(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 022340~9 1998-04-06 WO 97/12978 PCT~us96/15938 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AA~lG~-llCG TGACTCCCAT

C~ ACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG

CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA

CTGGGACCCA CTTGCCTCTC A~l~CCC~l~C~l~ GGGCAGCTTT CTGGACAGGT CC~l~-l~C~l~C

CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAG~llC~lC CACAGGGCAG GACCACAGCT

CACAAGGATC CCAATGCCAT ~~ lGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT

TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTC

(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 927 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic)"

(xi) ~Q~ DESCRIPTION: SEQ ID NO: 38:
TCCC~CCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AA~ ~G TGACTCCCAT

~ ACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC AC~

CA 022340~9 l998-04-06 CTGC~ ~ TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CAGGACATTC TGGGAGCAGT GA~C~ll~lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA

CTGGGACCCA ~llGCC~ lC A~l~CC~lC'~lG GGGCAGCTTT CTGGACAGGT CC~l~ C~l~C300 ~Nl~GGGCCC TGCAGAGCCT CCTTGGAACC CAG~llC~lC CACAGGGCAG GACCACAGCT

CACAAGGATC CCAATGCCAT ~llC~lGAGC TTCCAACACC TGCTCCGAGG AAAG~l~GC~

TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCGG CAACATGGCG

l~lCCCG~lC CGC~l~-~l~ TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT

~lC~llCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC ~ GC~l~AC ACCTGTCCTG

CTG~l~lG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CAGGACATTC TGGGAGCAGT GACC~ll~l~ CTGGAGGGAG TGATGGCAGC ACGGGGACAA

CTGGGACCCA Cll~C-l~l~ AlCC~CuG GGGCAGCTTT CTGGACAGGT CC~ L ~1 C~ . C

~ll~GGGCCC TGCAGAGCCT CCTTGGAACC CAGGGCAGGA CCACAGCTCA CAAGGATCCC

AATGCCATCT TCCTGAGCTT CCAACACCTG CTCCGAGGAA AG~l~GC~ CCTGATGCTT

GTAGGAGGGT CCACC~l~lG CGTCAGG

~2) INFORMATION FOR SEQ ID NO 39 ~QU~N~ CHARACTERISTICS
tA) LENGTH 936 base pairs (B) TYPE nucleic acid (C) STRAN~ ~SS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = nDNA (synthetic)"

CA 022340~9 1998-04-06 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT

G~ lCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC AC~ lC~lG

CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CAGGACATTC TGGGAGCAGT GACC~ G CTGGAGGGAG TGATGGCAGC ACGGGGACAA

CTGGGACCCA CTTGCCTCTC ATCC~l~C~l~G GGGCAGCTTT CTGGACAGGT CCGTCTCCTC

~ll~GGGC~C TGCAGAGCCT CCTTGGAACC CAG~llC~lC CACAGGGCAG GACCACAGCT

CACAAGGATC CCAATGCCAT ~l~l~Cl~AGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT

TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCAA CATGGCGTCT

CCCG~lCCGC CTGCTTGTGA CCTCCGAGTC CTCAGTAAAC lG~ll~C~lGA CTCCCATGTC

CTTCACAGCA GACTGAGCCA GTGCCCAGAG GTTCACCCTT TGCCTACACC l~lC~-lGCTG

C~-l~l~lGG ACTTTAGCTT GGGAGAATGG AAAACCCAGA TGGAGGAGAC CAAGGCACAG

GACATTCTGG GAGCAGTGAC C~ll~lGCTG GAGGGAGTGA TGGCAGCACG GGGACAACTG

GGACCCACTT GCCTCTCATC CCTCCTGGGG CAG~lll~lG GACAGGTCCG 'l~lC~lC~

GGGGCC~lGC AGAGCCTCCT TGGAACCCAG ~ll~ lCCAC AGGGCAGGAC CACAGCTCAC

AAGGATCCCA ATGCCATCTT CCTGAGCTTC CAACACCTGC TCCGAGGAAA GGTGCGTTTC

CTGATGCTTG TAGGAGGGTC CACC~-~l~lGC GTCAGG

(2) INFORMATION FOR SEQ ID NO: 40:

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 939 base pairs - (B) TYPE: nucleic acid (C) STR~Nn~nNFss: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO: 40:
TCCCCAGCTC CAC~l~G~ G TGACCTCCGA GTCCTCAGTA AA~lG~luC~ TGACTCCCAT

C~ll~CACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC AC~l~iC~

GC~l~CTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CAGGACATTC TGGGAGCAGT GACC~~ lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA

CTGGGACCCA CTTGCCTCTC Al~CC~lC~l~ GGGCAGCTTT CTGGACAGGT ~ ~lC~lC

~ GGGCCC TGCAGAGCCT CCTTGGAACC CAG~ll-CC~C CACAGGGCAG GACCACAGCT

CACAAGGATC CCAATGCCAT ~ll~C'lGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT

TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCGG CAACATGGCG

TCTCCCGCTC CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT

C~ ACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC AC~ C~l~G

GC~l~GCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CAGGACATTC TGGGAGCAGT GACC~lc~l~G CTGGAGGGAG TGATGGCAGC ACGGGGACAA

CTGGGACCCA CTTGCCTCTC A~l~CC~lC~-~l~G GGGCAGCTTT CTGGACAGGT CC~l~l-~lC

CA 022340~9 l998-04-06 WO 97/12978 PCTrUS96/15938 CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT

CACAAGGATC CCAATGCCAT ~l~ C~lGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT

TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG

(2) INFORMATION FOR SEQ ID NO 41 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 948 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = nDNA (synthetic)"

(xi) SEQUENCE DESCRIPTION SEQ ID NO 41 TCCCCAGCGC CGC~lG~ G TGACCTCCGA GTCCTCAGTA AA ~l~G~l~lCG TGACTCCCAT

~ LlcAcA GCAGACTGAG CCAGTGCCCA GAGGTTCACC ~-lllGC~lAC A~-~l~lC~-lG

~lGC~l~l~ TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CAGGACATTC TGGGAGCAGT GACCCll~lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA

CTGGGACCCA CTTGCCTCTC ATCC~ G GGGCAGCTTT CTGGACAGGT CC~l~lC~lC

~ GGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT

CACAAGGATC CCAATGCCAT ~ C~lGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT

ll~l~ATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AA~ CGGCGG CAACGGCGGC

AACATGGCGT CCCCAGCGCC ~C~l~G~ GACCTCCGAG TCCTCAGTAA ACTGCTTCGT

GACTCCCATG ~l~C~ CACAG CAGACTGAGC CAGTGCCCAG AGGTTCACCC TTTGCCTACA

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 C~l~lC~l~C TGCCTGCTGT GGACTTTAGC TTGGGAGAAT GGAAAACCCA GATGGAGGAG

ACCAAGGCAC AGGACATTCT GGGAGCAGTG ACCCTTCTGC TGGAGGGAGT GATGGCAGCA

CGGGGACAAC TGGGACCCAC TTGCCTCTCA TCCCTCCTGG GGCAGCTTTC TGGACAGGTC

C~ C~lCC ~ GGGGCCCT GCAGAGCCTC CTTGGAACCC AG~lC~lCC ACAGGGCAGG

~ CTC ACAAGGATCC CAATGCCATC TTCCTGAGCT TCCAACACCT GCTCCGAGGA

AAG~l~C~ TCCTGATGCT TGTAGGAGGG TCCACCCTCT GCGTCAGG

(2) INFORMATION FOR SEQ ID NO: 42:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: l55 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) s~:~u~--~ DESCRIPTION: SEQ ID NO: 42:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Ar~ Val Leu Ser Lys Leu Leu l S l0 15 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu l00 105 ll0 CA 022340~9 l998-04-06 WO 97/12978 PCT~US96/15938 Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe (2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) ~:Qu~ DESCRIPTION: SEQ ID NO: 43:
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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 12û 125 Leu Ser Phe Gln 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 CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Ary Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg (2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 153 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) ~OLECULE TYPE: protein r (xi) ~Q~N~: DESCRIPTION: SEQ ID NO: 44:
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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu CA 022340~9 1998-04-06 WO 97/12978 PCT~US96/15938 Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg (2) INFORMATION FOR SEQ ID NO: 45:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 149 amino acids (B) TYPE: amino acid (C) STRA-N~N~:SS: llnknl, "
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln CA 022340~9 l998-04-06 W O 97/12978 PCTrUS96/15938 Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg lg5 (2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 312 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) S~Q~ ~ DESCRIPTION: SEQ ID NO: 46:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His V~l Leu His Ser Arg Leu Ser Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu CA 022340~9 l998-04-06 WO 97/12978 PCT~US96/15938 Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe 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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly l9S 200 20S
Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Lqu Val Gly Gly Ser Thr Leu Cys Val Arg (2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 313 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
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 Gln Cys Pro Glu Val CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln ~eu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln ~al Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 ~er Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu ~rg Asp Ser His Val Leu His Ser Arg Leu Ser Gln 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 Gln 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 Gln ~eu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln ~al Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg CA 022340~9 l998-04-06 (2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 316 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) 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 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Net Leu Val Gly Gly Ser Thr Leu Cys Val Arg (2) INFORNATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (Xi ) ~hUUhN~h DESCRIPTION: SEQ ID NO: 49:
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Net Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala CA 022340~9 l998-04-06 i ~le Phe Leu Ser Phe Gln 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 Gln Cys Pro (2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRAWDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) ~UUk~: DESCRIPTION: SEQ ID NO: 50:
Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 CA 022340~9 l998-04-06 W O 97/12978 PCT~US96/15938 Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro (2) INFORMATION FOR SEQ ID NO: 51:
(i) ~Q~ ~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu lOo 105 llO
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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro (2) INFORMATION FOR SEQ ID NO: 52:

CA 022340~9 1998-04-06 W O 97/12978 PCT~US96/15938 Qu~N~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr l 5 10 15 Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro (2) INFORMATION FOR SEQ ID NO: 53:
~Qu~-~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein CA 022340~9 l998-04-06 W O 97/12978 PCT~US96/lS938 - (xi) ~UU~N~ DESCRIPTION: SEQ ID NO: 53:
Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val (2) INFORMATION FOR SEQ ID NO: 54:
(i) ~uu~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B~ TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unkn~wn (ii) MOLECULE TYPE: protein (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO: 54:
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu l 5 10 15 CA 022340~9 l998-04-06 WO 97/12978 PCT~US96/15938 ~ly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu (2) INFORMATION FOR SEQ ID NO: 55:
(i) ~QukNC~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) ~u~ DESCRIPTION: SEQ ID NO: 55:
Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val CA 022340~9 l998-04-06 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 Gln 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 Gln 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 Gln Leu (2~ INFORMATION FOR SEQ ID NO: 56:
(i) ~QU~N~: CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STR~NnF~nN~.'::S: llnkn~ ."
(D) TOPOLOGY: llnkn( ...1 (ii) MOLECULE TYPE: protein (xi) ~QU~N~ DESCRIPTION: SEQ ID NO: 56:
Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln Met Glu Glu Thr Lys Ala CA 022340~9 l998-04-06 WO 97/12978 PCTrUS96/15938 Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu (2) INFORMATION FOR SEQ ID NO: 57:
(i) ~hyu~N~: CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRA~n~nNF.~S: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) 8~QU~N~: DESCRIPTION: SEQ ID NO: 57:
Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu CA 022340~9 l998-04-06 WO 97/12978 PCT~US96/15938 Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln -(2) INFORMATION FOR SEQ ID NO: 58:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY ~lnkn. ., (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr (2) INFORMATION FOR SEQ ID NO: 59:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 (B) TYPE: amino acid (C) STRAN~E~N~SS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) ~:QU~N~: DESCRIPTION: SEQ ID NO: 59:
Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Ary 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys (2) INFORMATION FOR SEQ ID NO: 60:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRAN~N~SS: unknown tD) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein -CA 022340~9 1998-04-06 (xi) ~;uu~ ; DESCRIPTION: SEQ ID NO: 60:
Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn (2) INFORMATION FOR SEQ ID NO: 61:
Qul~N~ ; CHARACTERISTICS:
( A ) LENGTH: 480 bas e pairs (B) TYPE: nucleic acid ( C ) sTRp~l~nFnNF:~s s ingle ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic) "

(xi ) ~;uu~ ; DESCRIPTION: SEQ ID NO: 61:
GAGGTTCACC ~ GC~ L~AC AC~: l ~ l C~: l G ~:~l GC~l~G~; l G TGGACTTTAG CTTGGGAGAA

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 ll6 TGGAAAACCC AGATGGAGGA GACCAAGGCA CAGGACATTC TGGGAGCAGT GACC~~ G

CTGGAGGGAG TGATGGCAGC ACGGGGACAA CTGGGACCCA ~~ GC~l~lC AlCC~lC~lG

GGGCAGCTTT CTGGACAGGT CC~~ C CTTGGGGCCC TGCAGAGCCT CCTTGGAACC

CA~ l~lC CACAGGGCAG GACCACAGCT CACAAGGATC CCAATGCCAT ~llC~lGAGC

TTCCAACACC TGCTCCGAGG AAAG~l~l TTCCTGATGC TTGTAGGAGG GTCCACCCTC

TGCGTCAGGG AA~l~lCGGCGG CAACATGGCG l~lC'C~GCGC CGCCTGCTTG TGACCTCCGA

GTCCTCAGTA AACTGCTTCG TGACTCCCAT ~l~C~lCACA GCAGACTGAG CCAGTGCCCA

(2) INFOR~ATION FOR SEQ ID NO 62 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 480 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = rDNA (Synthetic)~

(xi) ~Q~N~ DESCRIPTION SEQ ID NO 62 TTGCCTACAC ~ C~lGCT GC~lG~l~sG GACTTTAGCT TGGGAGAATG GAAAACCCAG

ATGGAGGAGA CCAAGGCACA GGACATTCTG GGAGCAGTGA CC~ G~l~ GGAGGGAGTG

ATGGCAGCAC GGGGACAACT GGGACCCACT TGCCTCTCAT CC~lC~l~GGG GCAG~

GGACAGGTCC ~ CCl~C~-l~ TGGGGCCCTG CAGAGCCTCC TTGGAACCCA G~ C~l~CCA

CAGGGCAGGA CCACAGCTCA CAAGGATCCC AATGCCATCT TCCTGAGCTT CCAACACCTG

CA 022340~9 1998-04-06 W 0 97/12978 PCT~US96/15938 CTCCGAGGAA AG~lGC~l~ CCTGATGCTT GTAGGAGGGT CCACC~l--lG CGTCAGGGAA

- TTCGGCGGCA ACAlGGC~lC lCCGGCGCCG CCTGCTTGTG AC~l~CCGAGT CCTCAGTAAA

C~lG ACTCCCATGT CCTTCACAGC AGACTGAGCC AGTGCCCAGA GGTTCACCCT

(2) INFORMATION FOR SEQ ID NO 63 (i) ~Qu~ CHARACTERISTICS
(A) LENGTH 480 base pairs (B) TYPE nucleic acid (C) STRANn~nN~ss single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = "DNA (synthetic) n (xi) SEQUENCE DESCRIPTION SEQ ID NO 63 ~ C ~lG~l~lGGA CTTTAGCTTG GGAGAATGGA AAACCCAGAT GGAGGAGACC

AAGGCACAGG ACA~ l~GG AGCAGTGACC ~11~1~1GG AGGGAGTGAT GGCAGCACGG

GGACAACTGG GACCCACTTG CCTCTCATCC ~lC~GGGGC AG~111~-lGG ACAGGTCCGT

C~lC~l-l~ GGGCC~lGCA GAGC~l~ll GGAACCCAGC llC~lCCACA GGGCAGGACC

ACAGCTCACA AGGATCCCAA TGCCATCTTC CTGAGCTTCC AACACCTGCT CCGAGGAAAG

~ C~lllCC TGATGCTTGT AGGAGGGTCC ACC~ GCG TCAGGGAATT CGGCGGCAAC

ATGGCGTCTC CGGCGCCGCC ~l~G~ll~l~AC CTCCGAGTCC TCAGTAAACT G~ll~C~l~GAC

TCCCATGTCC TTCACAGCAG ACTGAGCCAG TGCCCAGAGG TTCACCCTTT GCCTACACCT

(2) INFORMATION FOR SEQ ID NO 64 ( i ) sh~U~N~: CHARACTERISTICS

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 (A) LENGTH: 480 base pairs (B) TYPE: nucleic acid (C) STRANnFnN~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
GCTGTGGACT TTAGCTTGGG AGAATGGAAA ACCCAGATGG AGGAGACCAA GGCACAGGAC

ATTCTGGGAG CAGTGACCCT ~ lGGAG GGAGTGATGG CAGCACGGGG ACAACTGGGA

CCCACTTGCC TCTCATCCCT C~l~GGGCAG ~ GGAC AGGlCC~l~l~ C~lC~-l~lGGG

GCCCTGCAGA GC~lC~ll~G AACCCAGCTT CCTCCACAGG GCAGGACCAC AGCTCACAAG

GATCCCAATG CCATCTTCCT GAGCTTCCAA CACCTGCTCC GAGGAAAGGT GCGTTTCCTG

AlG~l~l~lAG GAGGGTCCAC C~~ GC~l~C AGGGAATTCG GCGGCAACAT GGC~l~lCCG

GCGCCGC~lG CTTGTGACCT CCGAGTCCTC AGTAAACTGC TTCGTGACTC CCAl~lC~

CACAGCAGAC TGAGCCAGTG CCCAGAGGTT CACC~l~llGC CTACACCTGT CCTGCTGCCT

(2) INFOR~ATION FOR SEQ ID NO: 65:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 480 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic) n (xi) ~OU~N~ DESCRIPTION: SEQ ID NO: 65:

CA 022340~9 1998-04-06 .

GACTTTAGCT TGGGAGAATG GAAAACCCAG ATGGAGGAGA CCAAGGCACA GGACATTCTG

GGAGCAGTGA CC~ lGCT GGAGGGAGTG ATGGCAGCAC GGGGACAACT GGGACCCACT

GC~ -'AT CC~l~C~l~GGG GCAG~ GGACAGGTCC ~ lC~lC~l~ TGGGGCCCTG

CAGAGCCTCC TTGGAACCCA G~ C~lCCA CAGGGCAGGA CCACAGCTCA CAAGGATCCC

AATGCCATCT TCCTGAGCTT CCAACACCTG CTCCGAGGAA AGGTGCGTTT CCTGATGCTT

GTAGGAGGGT CCACCCTCTG CGTCAGGGAA TTCGGCGGCA ACA~l~GGC~l~C TCCGGCGCCG

C~ lG ACCTCCGAGT CCTCAGTAAA ~-~l~llCGTG ACTCCCATGT CCTTCACAGC

AGACTGAGCC AGTGCCCAGA GGTTCACCCT TTGCCTACAC ~l~lC~l~GCT GC~lG~l~lG

t2) INFORMATION FOR SEQ ID NO 66 (i) ~Qu~ CHARACTERISTICS
(A) LENGTH 479 base pairs (B) TYPE nucleic acid (C) STRANn~nN~S single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = ~DNA (synthetic)~

(xi) SEQUENCE DESCRIPTION SEQ ID NO 66 GGAGAATGGA AAACCCAGAT GGAGGAGACC AAGGCACAGG ACAll~lGGG AGCAGTGACC

C~ ~lGG AGGGAGTGAT GGr~r~rGG GGACAACTGG GACCCACTTG C~ ATCC

CTCCTGGGGC AG~lll~lGG ACAG~lCC~l~ C~lC~lG GGGCCCTGCA GAGC~lC~ll GGAACCCAGC llC~l~CCACA GGGCAGGACC ACAGCTCACA AGGATCCCAA TGCCATCTTC

CA 022340~9 1998-04-06 WO 97/12978 PCT~US96/15938 CTGAGCTTCC AACACCTGCT CCGAGGAAAG ~lGC~ CC TGA'lG~ AGGAGGGTCC

ACC~L~lGCG TCAGGGAATT CGGCGGCAAC ATGGCTCTCC GGCGCCGCCT GCTTGTGACC

TCCGAGTCCT CAGTAAACTG ~ GACT CCCATGTCCT TCACAGCAGA CTGAGCCAGT

GCCCAGAGGT TCA~C~lllG CCTACACCTG TCCTGCTGCC TGCTGTGGAC TTTAGCTTG

(2) INFORMATION FOR SEQ ID NO 67 (i) s~Qu~N~ CHARACTERISTICS
(A) LENGTH 480 base pairs (B) TYPE nucleic acid (C) STRA~E~SS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = nDNA (synthetic) n ~Xi ) ~kQD~C~ DESCRIPTION SEQ ID NO 67 GGACCCACTT GCCTCTCATC CCTCCTGGGG CAG~ ~ GACAGGTCCG l~lC~lC~

GGGGCC~-l~C AGAGC-lC-l TGGAACCCAG ~lC~-lCCAC AGGGCAGGAC CACAGCTCAC

AAGGATCCCA ATGCCATCTT CCTGAGCTTC CAACACCTGC TCCGAGGAAA GGTGCGTTTC

CTGATGCTTG TAGGAGGGTC CACCCTCTGC GTCAGGGAAT ~l~CGGCGGCAA CATGGCGTCT

CCGGCGCCGC CTGCTTGTGA CCTCCGAGTC CTCAGTAAAC l~llC~lGA CTCCCATGTC

CTTCACAGCA GACTGAGCCA GTGCCCAGAG GTTCACCCTT TGCCTACACC ~l~lC~l~CTG

C~lG~l~lGG ACTTTAGCTT GG~-A~A~TGG AAAACCCAGA TGGAGGAGAC CAAGGCACAG

GACATTCTGG GAGCAGTGAC C~ll~lG~l~G GAGGGAGTGA TGGCAGCACG GGGACAACTG

(2) INFORMATION FOR SEQ ID NO 68 CA 022340~9 1998-04-06 (i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 480 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic) n (xi) ~ Qu~ DESCRIPTION: SEQ ID NO: 68:
GGAACCCAGC llC~lCCACA GGGCAGGACC ACAGCTCACA AGGATCCCAA TGCCATCTTC

CTGAGCTTCC AACACCTGCT CCGAGGAAAG GTGC~ll~lCC TGAlG~ll~ AGGAGGGTCC

AC~ ~CG TCAGGGAATT CGGCGGCAAC AlGGC~l~lC ~GGCGCCGC~ ~l~G~-~lu~l~AC

CTCCGAGTCC TCAGTAAACT G~lC~lGAC TCCCATGTCC TTCACAGCAG ACTGAGCCAG

TGCCCAGAGG TTCACCCTTT GCCTACACCT ~lC~l~-lGC ~l~l~l~GA CTTTAGCTTG

GGAGAATGGA AAACCCAGAT GGAGGAGACC AAGGCACAGG ACATTCTGGG AGCAGTGACC

~'1"1~ L G~'l'G~ AGGGAGTGAT GGCAGCACGG GGACAACTGG GACCCACTTG CCTCTCATCC

~lC~lGGGGC AG~~ lGG ACAG~lCC~l~ ~lC~lC~ll~ GGGCC~l~CA GAGC~lC~

(2) INFORMATION FOR SEQ ID NO: 69:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 480 base pairs (8) TYPE: nucleic acid (C) STRAN~:~N~:SS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = nDNA (synthetic) n CA 022340~9 1998-04-06 WO 97/12978 PCTrUS96/15938 (Xi) ~:QU~N~ DESCRIPTION SEQ ID NO 69 GGCAGGACCA CAGCTCACAA GGATCCCAAT GCCATCTTCC TGAGCTTCCA ACAC~lG~lC

CGAGGAAAGG TGC~~ C~l~ GAl~G~ ~lA GGAGGGTCCA CCCTCTGCGT CAGGGAATTC

GGCGGCAACA TGGCGTCTCC GGCGCCGCCT GCTTGTGACC TCCGAGTCCT CAGTAAACTG

C~l~ACT CCCATGTCCT TCACAGCAGA CTGAGCCAGT GCCCAGAGGT TCACC~l~llG

CCTACACCTG ~ G~l~GCC TGCTGTGGAC TTTAGCTTGG GAGAATGGAA AACCCAGATG

GAGGAGACCA AGGCACAGGA CATTCTGGGA GCAGTGACCC TTCTGCTGGA GGGAGTGATG

GCAGCACGGG GACAACTGGG ACCCACTTGC CTCTCATCCC TCCTGGGGCA G~ ~GA

CAG~lCC~l-- TC~ lGG GGCCCTGCAG AGCCTCCTTG GAACCCAGCT TCCTCCACAG

(2) INFORMATION FOR SEQ ID NO 70 (i~ SEQUENCE CHARACTERISTICS
(A) LENGTH 480 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = nDNA (synthetic)"

(xi) ~Q~N~ DESCRIPTION SEQ ID NO 70 GCTCACAAGG ATCCCAATGC CA~ C~l~G AGCTTCCAAC AC~ CC~ AGGAAAGGTG

~ A lG~ll~lAGG AGGGTCCACC CTCTGCGTCA GGGAATTCGG CGGCAACATG

G~l~lCCGG CGCCGCCTGC TTGTGACCTC CGAGTCCTCA GTAAACTGCT TCGTGACTCC

CA~l~lC~l~lC ACAGCAGACT GAGCCAGTGC CCAGAGGTTC ACC~ l~CC TACACCTGTC

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 G~lGC~l~ CTGTGGACTT TAGCTTGGGA GAATGGAAAA CCCAGATGGA GGAGACCAAG

GCACAGGACA TTCTGGGAGC AGTGACCCTT ~l~G~l~GGAGG GAGTGATGGC AGCACGGGGA

CAACTGGGAC CCA~ll~GC~l~ CTCATCCCTC CTGGGGCAGC lll~ GGACA G~l~CC~l~lC

~lC~llGGGG CCCTGCAGAG C~lC~ll~GGA ACCCAGCTTC CTCCACAGGG CAGGACCACA

(2) INFORMATION FOR SEQ ID NO 71 ( i ) ~QU ~:N~ CHARACTERISTICS
(A) LENGTH 480 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = ~DNA (synthetic) n (xi) ~yUrN~ DESCRIPTION SEQ ID NO 71 GATCCCAATG CCAl-~llC~l GAGCTTCCAA CAC~lG~lCC GAGGAAAGGT GC~l-llC~l~

ATGCTTGTAG GAGGGTCCAC CCTCTGCGTC AGGGAATTCG GCGGCAACAT GGC~l~lCCG

GCGCCGC~l~G CTTGTGACCT CCGAGTCCTC AGTAAACTGC TTCGTGACTC CCA~ C~

CACAGCAGAC TGAGCCAGTG CCCAGAGGTT CACC~~ lGC CTACACCTGT C~lG~lGC~l G~1~1~GACT TTAGCTTGGG AGAATGGAAA ACCCAGATGG AGGAGACCAA GGCACAGGAC

All~lGGGAG CAGTGACCCT ~ GGAG GGAGTGATGG CAGCACGGGG ACAACTGGGA

CCCACTTGCC TCTCATCCCT C~l~GGGCAG ~ lGGAC AG~l~CC~ C~l~C~l~ ~GG

GCC~l~GCAGA GC~l~C~ GG AACCCAGCTT CCTCCACAGG GCAGGACCAC AGCTCACAAG

CA 022340~9 1998-04-06 (2) INFORNATION FOR SEQ ID NO 72 (i) ~Q~N~ CHARACTERISTICS
(A) LENGTH 480 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = nDNA (synthetic)"

(xi) ~U~N~ DESCRIPTION SEQ ID NO 72 GCCATCTTCC TGAGCTTCCA ACAC~l~l~ CGAGGAAAGG ~l~GC~ lC~l GAlG~lu~l~A

GGAGGGTCCA CC~l~lGC~l~ CAGGGAATTC GGCGGCAACA 'l~GC~l~lCC CG~lCCGC~l G~ll~lGACC TCCGAGTCCT CAGTAAACTG ~llC~lGACT CCCATGTCCT TCACAGCAGA

CTGAGCCAGT GCCCAGAGGT TCACC~lllG CCTACACCTG TC~lG~l~GCC TGCTGTGGAC

TTTAGCTTGG GAGAATGGAA AACCCAGATG GAGGAGACCA AGGCACAGGA CAll~l~GGA

GCAGTGACCC ~ ~GA GGGAGTGATG GCAGCACGGG GACAACTGGG ACCCACTTGC

CTCTCATCCC lC~l~GGGCA G~-lll~lGGA CAG~l~CC~lC l-~lC~ll~G GGCC~lGCAG

AGCCTCCTTG GAACCCAGCT TCCTCCACAG GGCAGGACCA CAGCTCACAA GGATCCCAAT

(2) INFORMATION FOR SEQ ID NO 73 ( i ) ~:yU~N - '~ CHARACTERISTICS
(A) LENGTH 4 amino acids (B) TYPE amino acid (C) STRANDEDNESS unknown (D) TOPOLOGY unknown (ii) MOLECULE TYPE peptide W O 97/12978 PCT~US96/15938 (xi) ~uu N~ DESCRIPTION: SEQ ID NO: 73:
Gly Gly Gly Ser (2) INFORMATION FOR SEQ ID NO: 74:
(i) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STR~Nn~nN~S: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Gly Gly Gly Ser Gly Gly Gly Ser l 5 (2) INFORMATION FOR SEQ ID NO: 75:
(i) ~uu~ CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser l 5 l0 (2) INFORMATION FOR SEQ ID NO: 76:
~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) sTR~Nn~nN~cs unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide CA 022340~9 1998-04-06 W O 97tl2978 PCTtUS96/15938 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Ser Gly Gly Ser Gly Gly Ser l 5 (2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) ~Q~ DESCRIPTION: SEQ ID NO: 77:
Glu Phe Gly Asn Met Ala (2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANn~n~F~S: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
Glu Phe Gly Gly Asn Met Ala l 5 (2)~INFORMATION FOR SEQ ID NO: 79:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: l0 amino acids (B) TYPE: amino acid (C) STR~Nn~nN~S: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide W O 97/12978 PCTrUS96/15938 (xi) ~yU~N~: DESCRIPTION: SEQ ID NO: 79:
Glu Phe Gly Gly Asn Gly Gly Asn Met Ala (2) INFORMATION FOR SEQ ID NO: 80:
Qu~: CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRA~~ sS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) ~:~U~N~ DESCRIPTION: SEQ ID NO: 80:
Gly Gly Ser Asp Met Ala Gly (2) INFORMATION FOR SEQ ID NO: 81:
(i) ~:QU~N~: CHARACTERISTICS:
(A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = ~DNA (synthetic)"

(xi) ~ u~ DESCRIPTION: SEQ ID NO: 81:
GGATCCACCA TGAGCCGCCT GCCC~LC~l~G ~l~C~lG~l~CC AA~L~ G~l~ CCGCCCCGC

(2) INFORMATION FOR SEQ ID NO: 82:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 459 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA (synthetic)"

CA 022340~9 1998-04-06 (xi) ~QU~N~ DESCRIPTION SEQ ID NO 82 l~lCCCG~l~ CGCCTGCTTG TGACCTCCGA ~l~C~lCAGTA AA~l~G~ lCG TGACTCCCAT

~ ACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC ~ GC~lAC AC~

GC~lGLlG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CAGGACATTC TGGGAGCAGT GACC~ lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA

CTGGGACCCA ~lU~C~l~lC A~lCC~lC~l~G GGGCAGCTTT CTGGACAGGT CC~~ C~lC

~ GGGCCC TGCAGAGCCT CCTTGGAACC CAG~llC~lC CACAGGGCAG GACCACAGCT

CACAAGGATC CCAATGCCAT ~ilC~l~GAGC TTCCAACACC lG~l~CCGAGG AAAGGTGCGT

TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG

(2) INFORMATION FOR SEQ ID NO 83 yu~ CHARACTERISTICS
(A) LENGTH 936 base p~irs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = ~DNA (synthetic)~

(Xi) ~UU~N-~ DESCRIPTION SEQ ID NO 83 TCCCrAr~CTC CAC~lG~l~l~ TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT

CACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC ~lluGC~l~AC AC~l~lC~lG

~ C~l~l~G TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CA 022340~9 1998-04-06 W O 97/12978 PCTrUS96/15938 CAGGACATTC TGGGAGCAGT GACC~ll~lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA

~ CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CC~ C~lC

CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAG~llC~l~C CACAGGGCAG GACCACAGCT

CACAAGGATC CCAATGCCAT ~ C~lGAGC TTCCAACACC ~l~G~lCCGAGG AAAGGTGCGT

TTCCTGATGC TTGTAGGAGG GTCCACCCTC ~lGC~l~AGGG AATTCGGCAA CATGGCGTCT

CCCG~lCCGC ~l~ GA CCTCCGAGTC CTCAGTAAAC ~l~G~LlC~lGA CTCCCATGTC

CTTCACAGCA GACTGAGCCA GTGCCCAGAG GTTCACCCTT TGCCTACACC TGTCCTGCTG

CCTGCTGTGG ACTTTAGCTT GGGAGAATGG AAAACCCAGA TGGAGGAGAC CAAGGCACAG

GACATTCTGG GAGCAGTGAC C~ G~l~G GAGGGAGTGA TGGCAGCACG GGGACAACTG

GGACCCACTT GCCTCTCATC C~lC~l~GGGG CAG~lll~-L~G GACAGGTCCG l~lC~lC~ll GGGGCC~l~C AGAGC~lC~l TGGAACCCAG ~llC~lCCAC AGGGCAGGAC CACAGCTCAC

AAGGATCCCA ATGCCATCTT CCTGAGCTTC CAACACCTGC TCCGAGGAAA GGTGCGTTTC

CTGATGCTTG TAGGAGGGTC CACCCTCTGC GTCAGG

Claims (20)

WHAT IS CLAIMED IS:

1. A c-mpl receptor agonist polypeptide comprising, a modified c-mpl ligand amino acid sequence of the Formula:
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrPro ValLeuLeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGlu ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa XaaGlyArgThrThrAlaHisLysAspProAsnAlaIlePheLeuSerPheGlnHis LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal Arg (SEQ ID NO:2) 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 Gln, 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;

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 No:75);;
SerGlyGlySerGlyGlySer (SEQ ID NO:76);
GluPheGlyAsnMetAla (SEQ ID NO:77);
GluPheGlyGlyAsnMetAla ( SEQ ID NO:78);
GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79); and GlyGlySerAspMetAlaGly (SEQ ID NO: 80).

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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln Cys Pro Glu Val His Pro (SEQ ID NO:50);

Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val (SEQ ID NO:53);

Gly Glu Trp Lys Thr Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln Cys Pro Glu Val 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 Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu (SEQ ID NO:55);

Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu (SEQ ID NO:56);

Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln (SEQ ID NO:57);

Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr (SEQ ID NO:58);

Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys (SEQ ID NO:59); and Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn (SEQ ID NO: 60).

4. The c-mpl receptor agonist polypeptide as recited in claim 3 wherein said polypeptide is selected from the group consisting of:
Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu (SEQ ID NO;56); and Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln 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 Gln 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 Gln 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 Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln (SEQ ID NO:57).

5. 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.

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
ATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCT
GGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCA
CAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTG
CTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAA
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

CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT
(SEQ ID No: 64);

GACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTG
GGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT
TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTG
CAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCC
AATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTT
GTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCG
CCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC
AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTG
(SEQ ID NO:65);

GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC
CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC
CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT
GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC
CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC
ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCNTCTCCGGCGCCGCCTGCTTGTGAC
CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG
TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG
(SEQ ID No: 66);

GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTT
GGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCAC
AAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTC
CTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCT
CCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTC
CTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTG
CCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG
(SEQ ID NO:67);
GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC
CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC
ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGAC
CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG
TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG
GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC
CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC
CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT
(SEQ ID NO:68);
GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC
CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC
GGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG
CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG
CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG

GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATG
GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGA
CAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAG
(SEQ ID NO:69);

GCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTG
CGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATG
GCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCC
CATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTC
CTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAG
GCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGA
CAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTC
CTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACA
(SEQ ID NO:70);

GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG
ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG
GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT
CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT
GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC
ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA
CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG
GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
(SEQ ID NO:71); and GCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA
GGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGCTCCGCCT
GCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGA
CTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGAC
TTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGA
GCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC
CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAG
AGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAAT
(SEQ ID NO:72).

10. 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 GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC
CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC
GGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG
CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG
CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG
GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATG
GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGGCTTTCGGA
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 Ser17, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor; and a pharmaceutically acceptable carrier.

15. 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 for selective ex vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells; (b) culturing said separated stem cells with a selected culture medium comprising the polypeptide of claim 1, 2, 3 or 4; and (c) harvesting said cultured cells.

19. 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; (b) 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;
(d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.

20. Use of a hematopoietic protein of claim 1, 2, 3 or 4 for preparing a medicament for human gene therapy, comprising the steps of;
(a) removing stem cells from a patient;
(b) separating said stem cells from other cells;
(c) culturing said separated stem cells with a selected culture medium comprising the hematopoietic protein of claim 1, 2, 3 or 4;
(d) introducing DNA into said cultured cells;

(e) harvesting said transduced cells; and (f) transplanting said transduced cells into said patient.